Chromatin Structure & Function - Abcam

CHROMATIN STRUCTURE & FUNCTION 2006 Program & Abstract Book 

Chromatin 

Program & Abstract Book 

Structure&Function 

Punta Cana, Dominican Republic 

5 - 8 December 2006 

Organized By: 

Tony Kouzarides and Abcam

Punta Cana Prog 30/10/06 10:08 Page 2

Punta Cana Prog 30/10/06 10:08 Page 1 

Program & Abstract Book 

The third 

Chromatin 

Structure & Function 

Punta Cana, Dominican Republic 

5 - 8 December 2006 

Organizers: 

Tony Kouzarides 

(University of Cambridge) 

and Abcam 

Table of contents 

Conference Program . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .Page 2 

Poster Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .Page 6 

Abstracts - Oral . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .Page 18 

Abstracts - Poster . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .Page 51 

Resort Information . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .Page 179 

Disclaimer: Material contained within this booklet should be citied only with permission from the author(s). 

No live recording or photography is permitted during the oral or poster sessions. 

Copyright © 2006 Abcam, All Rights Reserved. The Abcam logo is a registered trademark. 

All information / detail is correct at time of going to print. 

1


Chromatin Structure & Function Punta Cana, Dominican Republic, 5 - 8 December 2006 

Conference Program 

Tuesday 5th December 

Meeting room - Allegro Plaza 

Keynote Speaker - introduced by Tony Kouzarides 

18:00 - 19:00 Steve Henikoff . . . . . . . . . . . . . . . . . . . . . . . .Page 18 

Epigenetic patterns generated by histone replacement 

Welcome reception and buffet at poolside. 

Allegro Live resort show 

Wednesday 6th December 

Chair: Jerry Workman 

09:00 - 09:30 Yang Shi . . . . . . . . . . . . . . . . . . . . . . . . . . . . .Page 19 

The identification of histone demethylases established the 

dynamic and reversible nature of histone methylation 

regulation 

09:30 - 09:45 Jesper Christensen . . . . . . . . . . . . . . . . . . . . .Page 20 

The retinoblastoma tumor suppressor binding protein RBP2 is 

a transcriptional repressor demethylating tri- and dimethylated 

lysine 4 on Histone H3 

09:45 - 10:00 Mischa Machius . . . . . . . . . . . . . . . . . . . . . . .Page 21 

Structural basis for CoREST-dependent demethylation of 

nucleosomes by the human LSD1 histone demethylase 

10:00 - 10:30 Ramin Shiekhattar . . . . . . . . . . . . . . . . . . . . .Page 22 

Functional and biochemical characterization of histone 

demethylase complexes 

Drinks break in hotel lobby 

11:00 - 11:30 Yi Zhang . . . . . . . . . . . . . . . . . . . . . . . . . . . . .Page 23 

Histone demethylation by the JmjC domain-containing proteins 

11:30 - 11:45 Francis Stewart . . . . . . . . . . . . . . . . . . . . . . . .Page 24 

Epigenetic aspects of lineage commitment 

2



11:45 - 12:00 Henk Stunnenberg . . . . . . . . . . . . . . . . . . . . .Page 25 

Title and abstract unavailable 

12:00 - 12:30 Ali Shilatifard . . . . . . . . . . . . . . . . . . . . . . . . . .Page 26 

H2B monoubiquitination and H3K4 methylation via COMPASS 

Lunch at the Beach Buffet Restaurant and free time 

Chair: Yang Shi 

16:00 - 16:30 Paulo Sassone-Corsi . . . . . . . . . . . . . . . . . . .Page 27 

A chromatin remodeling clock 

16:30 - 16:45 Sung Hee Baek . . . . . . . . . . . . . . . . . . . . . . .Page 28 

A Novel Link between SUMO Modification of a Chromatin 

Remodeling Complex and Cancer Metastasis 

16:45 - 17:00 Laszlo Tora . . . . . . . . . . . . . . . . . . . . . . . . . . .Page 29 

The simultaneously dimethylated Lys-9 and phosphorylated 

Ser-10 tails of histone H3 adopt different conformations 

during mitosis 

17:00 - 17:30 Tony Kouzarides . . . . . . . . . . . . . . . . . . . . . . .Page 30 

Characterisation of novel histone modifications 

18:00 - 21:00 Posters and buffet by the pool 

Allegro Live resort show 

Thursday 7th December 

Chair: Genevieve Almouzni 

09:00 - 09:30 Thomas Jenuwein . . . . . . . . . . . . . . . . . . . . . .Page 31 

Epigenetic control by histone methylation 

09:30 - 09:45 Roberta Benetti . . . . . . . . . . . . . . . . . . . . . . .Page 32 

The role of Dicer in the regulation of chromatin at telomeres 

3



09:45 - 10:00 Mareike Puschendorf . . . . . . . . . . . . . . . . . . .Page 33 

Ezh2 independent targeting of PRC1 proteins to paternal 

constitutive heterochromatin in mouse pre-implantation embryos 

10:00 - 10:30 Edith Heard . . . . . . . . . . . . . . . . . . . . . . . . . . .Page 34 

The nuclear and epigenetic dynamics of X-chromosome 

inactivation in the mouse 


11:00 - 11:30 Adrian Bird . . . . . . . . . . . . . . . . . . . . . . . . . . .Page 35 

MeCP2: molecular interactions and phenotypic stability in a 

mouse model of Rett Syndrome 

11:30 - 11:45 Jon Penterman . . . . . . . . . . . . . . . . . . . . . . . .Page 36 

DNA demethylation in Arabidopsis thaliana 

11:45 - 12:00 Francois Fuks . . . . . . . . . . . . . . . . . . . . . . . . .Page 37 

The Polycomb Group protein EZH2 is recruited to promoters by 

MECP2 

12:00 - 12:30 Shelley Berger . . . . . . . . . . . . . . . . . . . . . . . .Page 38 

Factor and histone covalent modifications in genome regulation 

Lunch at the Beach Buffet Restaurant and free time 

Chair: Ramin Shiekhattar 

16:00 - 16:30 Danny Reinberg . . . . . . . . . . . . . . . . . . . . . . .Page 39 

A molecular understanding of epigenetics 

16:30 - 16:45 Jessica Tyler . . . . . . . . . . . . . . . . . . . . . . . . . .Page 40 

The mechanistic basis for the requirement of promoter 

chromatin disassembly for transcriptional activation 

16:45 - 17:00 Gratien Prefontaine . . . . . . . . . . . . . . . . . . . . .Page 41 

Epigenetic mechanisms influencing pituitary gene expression 

17:00 - 17:30 Bob Kingston . . . . . . . . . . . . . . . . . . . . . . . . .Page 42 

Possible roles in silencing for piRNAs 

4



18:00 - 19:30 Posters and drinks by the pool 

Beach barbeque and live band on the sand 

Friday 8th December 

Chair: Edith Heard 

09:00 - 09:30 Michael Grunstein . . . . . . . . . . . . . . . . . . . . . .Page 43 

Deacetylation of histone H4 K16 regulates gene activity in yeast 

09:30 - 09:45 Ann Ehrenhofer-Murray . . . . . . . . . . . . . . . . .Page 44 

A role for the HDAC Rpd3 in establishing eurchromatinheterochromatin 

boundaries at yeast telomeres 

09:45 - 10:00 Wyatt Yue . . . . . . . . . . . . . . . . . . . . . . . . . . . .Page 45 

CARM1 and histone methylation - a structural study 

10:00 - 10:30 Sharon Dent . . . . . . . . . . . . . . . . . . . . . . . . . .Page 46 

Common and unique factors regulate Set1-mediated 

methylation of the Dam1 kinetochore protein and histone H3 


11:00 - 11:30 Genevieve Almouzni . . . . . . . . . . . . . . . . . . . .Page 47 

Chromatin assembly factors, histone H3 variants and cell cycle 

11:30 - 11:45 Dmitry Fyodorov . . . . . . . . . . . . . . . . . . . . . . .Page 48 

ATP-dependant deposition of Histone H3.3 by Drosophila CHD1 

in vivo 

11:45 - 12:00 Roberto Mantovani . . . . . . . . . . . . . . . . . . . . .Page 49 

The histone fold trimer NF-Y is required to define positive 

histone marks in CCAAT-promotors: a genome wide analysis 

12:00 - 12:30 Jerry Workman . . . . . . . . . . . . . . . . . . . . . . . .Page 50 

Histone modification and chromatin remodeling in transcription 

Lunch at the Beach Buffet Restaurant 

Conference ends 

5



Poster Index 

Abstract P1 Karl Agger . . . . . . . . . . . . . . . . . . . . . . . . . . . .Page 51 

The role of the polycomb group protein RYBP in oncogene 

induced senescence 

Abstract P2 Helena Ahlfors . . . . . . . . . . . . . . . . . . . . . . . .Page 52 

A novel player in T helper cell differentiation 

Abstract P3 Barbara Alberter . . . . . . . . . . . . . . . . . . . . . . .Page 53 

Histone modification pattern of the T lymphotropic Herpesvirus 

saimiri genome in latency 

Abstract P4 Marco Alvarez . . . . . . . . . . . . . . . . . . . . . . . . .Page 54 

Histone variant macroH2A is an epigenetic factor involved in 

the modulation of ribosomal gene expression during seasonal 

adaptation of carp fish 

Abstract P5 Terra Arnason . . . . . . . . . . . . . . . . . . . . . . . . .Page 55 

Rsp5 is required for nuclear shuttling of the Snf1 kinase 

complex in yeast 

Abstract P6 Stuart Atkinson . . . . . . . . . . . . . . . . . . . . . . . .Page 56 

Epigenetic mechanisms of pluripotency and differentiation 

Abstract P7 Joanne Attema . . . . . . . . . . . . . . . . . . . . . . . .Page 57 

Epigenetic features of hematopoietic stem cells using small 

numbers of highly purified primary cells 

Abstract P8 Kristin Baetz . . . . . . . . . . . . . . . . . . . . . . . . . .Page 58 

NuA4 is a cellular “Hub”: an integrative map of physical and 

genetic interactions mediated by the NuA4 histone 

acetyltransferase 

Abstract P9 Slobodan Barbaric . . . . . . . . . . . . . . . . . . . . .Page 59 

Chromatin remodeling activities at the yeast PHO84 promoter 

Abstract P10 Vivian Bardwell . . . . . . . . . . . . . . . . . . . . . . . .Page 60 

Polycomb group and SCF ubiquitin ligases are found in a novel 

BCOR complex that is recruited to BCL6 targets 

Abstract P11 Amrita Basu . . . . . . . . . . . . . . . . . . . . . . . . . .Page 61 

Computational prediction of histone and non-histone proteins 

6


Poster Index 

Abstract P12 Mark Bedford . . . . . . . . . . . . . . . . . . . . . . . . .Page 62 

Screening for the methylated proteome 

Abstract P13 Sukesh R. Bhaumik . . . . . . . . . . . . . . . . . . . .Page 63 

Regulation of transcriptional activation by mRNA cap-binding 

complex in vivo 

Abstract P14 Marjorie Brand . . . . . . . . . . . . . . . . . . . . . . . .Page 64 

The Ash2L/MLL2 methyltransferase complex is important for ß- 

globin transcription during erythroid differentiation 

Abstract P15 Lauren Buro . . . . . . . . . . . . . . . . . . . . . . . . . .Page 65 

Histone methylation patterns at interferon-gamma inducible 

gene loci 

Abstract P16 Jill Butler . . . . . . . . . . . . . . . . . . . . . . . . . . . . .Page 66 

CXXC-finger protein 1 regulates Dnmt1 protein expression 

Abstract P17 Jim Cakouros . . . . . . . . . . . . . . . . . . . . . . . . .Page 67 

Identification of a novel enzyme which regulates the kinetics of 

histone arginine methylation in Drosophila melanogaster 

Abstract P18 Raymond Camahort . . . . . . . . . . . . . . . . . . . .Page 68 

Genome-wide analysis of the budding yeast histone variant 

Cse4 reveals occupancy at a single centromeric nucleosome as 

well as additional non-centromeric locations 

Abstract P19 Dylan Carney . . . . . . . . . . . . . . . . . . . . . . . . .Page 69 

The RAG2 PHD Finger links the histone code to V(D)J 

recombination 

Abstract P20 Beverly Chilton . . . . . . . . . . . . . . . . . . . . . . . .Page 70 

Analysis of RUSH/SMARCA3 isoforms and their interactions 

with Egr-1 and c-Rel in the regulation of transcription 

Abstract P21 Alexandra Chittka . . . . . . . . . . . . . . . . . . . . . .Page 71 

Signalling by a novel p75 neurotrophin receptor interacting 

protein, SC1/PRDM4 

Abstract P22 Leslie Chu . . . . . . . . . . . . . . . . . . . . . . . . . . . .Page 72 

Inheritance of epigenetic chromatin states 

7



Abstract P23 Mair Churchill . . . . . . . . . . . . . . . . . . . . . . . . .Page 73 

Structural basis for the histone chaperone activity of Asf1 

Abstract P24 Jeffrey Craig . . . . . . . . . . . . . . . . . . . . . . . . . .Page 74 

What makes centromeres localise and cluster in interphase 

nuclei 

Abstract P25 Valerie Crusselle-Davis . . . . . . . . . . . . . . . . . .Page 75 

Regulation of beta-globin expression through the recruitment 

of chromatin modifying enzymes by TFII-I and USF 

Abstract P26 Eullia de Nadal . . . . . . . . . . . . . . . . . . . . . . . .Page 76 

Control of gene expression by the yeast Hog1 MAPK. 

Abstract P27 Foteini Davrazou . . . . . . . . . . . . . . . . . . . . . . .Page 77 

Molecular mechanism of histone H3K4me3 recognition by the 

PHD finger of ING2 

Abstract P28 Roger Deal . . . . . . . . . . . . . . . . . . . . . . . . . . .Page 78 

Repression of flowering in Arabidopsis requires histone H2A.Z 

deposition by a putative SWR1 complex 

Abstract P29 Laurent Delva . . . . . . . . . . . . . . . . . . . . . . . . .Page 79 

The Transcription Intermediary Factor 2 is required for 

zebrafish development 

Abstract P30 Luisa Di Stefano . . . . . . . . . . . . . . . . . . . . . .Page 80 

Lsd1 mutation in Drosophila disrupt normal level of H3K4 

methylation and affects viability and fertility 

Abstract P31 Stephan Diekmann . . . . . . . . . . . . . . . . . . . . .Page 81 

In vivo dynamic (FRAP, FCS) and neighbourhood relation (AB- 

FRET, FLIM) studies of human inner kinetochore proteins 

Abstract P32 Jeffrey Dilworth . . . . . . . . . . . . . . . . . . . . . . . .Page 82 

MEF2 helps establish muscle specific pattern of gene 

expression by recruiting Trithorax Group proteins to specific 

promoters 

Abstract P33 Ivana Djuretic . . . . . . . . . . . . . . . . . . . . . . . . .Page 83 

T-bet and Runx3 cooperate to activate Interferon gamma and 

silence Interleukin-4 in T helper-1 cells 

8


Poster Index 

Abstract P34 Tom Donndelinger . . . . . . . . . . . . . . . . . . . . . .Page 84 

Seeing cells in a new light: Improving resolution with a 

scientific approach to tissue processing 

Abstract P35 Bojan Drobic . . . . . . . . . . . . . . . . . . . . . . . . . .Page 85 

Characterization of Histone H3 kinases, MSK1 and MSK2 

Abstract P36 Danielle Ellis . . . . . . . . . . . . . . . . . . . . . . . . . .Page 86 

Histone acetylation of SRC and p21 promoters in response to 

histone deacetylase inhibitor treatment; implications of HDAC 

activity and SRC expression 

Abstract P37 Alexander Erkine . . . . . . . . . . . . . . . . . . . . . .Page 87 

Differential mechanisms of nucleosome displacement at yeast 

heat shock gene promoters 

Abstract P38 Ragnhild Eskeland . . . . . . . . . . . . . . . . . . . . .Page 88 

HP1 binding to chromatin methylated at H3K9 is enhanced by 

auxiliary factors 

Abstract P39 George Feehery . . . . . . . . . . . . . . . . . . . . . . .Page 89 

CpG methylated DNA standards and control primers for use in 

methyl sensitive PCR and bisulphite sequencing 

Abstract P40 Barna Fodor . . . . . . . . . . . . . . . . . . . . . . . . . .Page 90 

Identification of novel pericentric proteins by their localization 

Abstract P41 Maria Fousteri . . . . . . . . . . . . . . . . . . . . . . . . .Page 91 

Cockayne syndrome A and B proteins differentially regulate 

recruitment of chromatin remodeling and repair factors to 

stalled RNA polymerase II in vivo 

Abstract P42 Robert Gillespie . . . . . . . . . . . . . . . . . . . . . . .Page 92 

Retinoid regulated association of transcriptional coregulators 

and the polycomb group protein SUZ12 with the retinoic acid 

response elements of Hoxa1, RARß2, and Cyp26A1 in F9 

embryonal carcinoma cells 

Abstract P43 Clara Goday . . . . . . . . . . . . . . . . . . . . . . . . . .Page 93 

Chromatin modifications in germline chromosomes of sciarid flies 

9



Abstract P44 Aaron Goldberg . . . . . . . . . . . . . . . . . . . . . . .Page 94 

HIRA-dependent incorporation of histone H3.3 marks active 

genes in mouse embryonic stem cells 

Abstract P45 Elizabeth Goneska . . . . . . . . . . . . . . . . . . . . .Page 95 

Phosphorylation of the SQ H2A.X motif is required for proper 

meiosis and mitosis in Tetrahymena thermophila 

Abstract P46 Susana Gonzalo . . . . . . . . . . . . . . . . . . . . . . .Page 96 

Telomere epigenetic modifications: a control of telomere length 

and a stop on recombination 

Abstract P47 Tanya Gustafson . . . . . . . . . . . . . . . . . . . . . . .Page 97 

Epigenetic silencing of Singleminded-2 in breast cancer 

Abstract P48 Soon-Ki Han . . . . . . . . . . . . . . . . . . . . . . . . . .Page 98 

Role of plant CBP/p300-like genes in the regulation of 

flowering time 

Abstract P49 Christin Hanigan . . . . . . . . . . . . . . . . . . . . . . .Page 99 

Identification of an HDAC2 mutation in colorectal cancer and 

its consequences 

Abstract P50 

Troy Harkness . . . . . . . . . . . . . . . . . . . . . . . .Page100 



Abstract P51 Tiffany Hung . . . . . . . . . . . . . . . . . . . . . . . . .Page 101 

ING4 recognition of histone H3 trimethylated at lysine 4 

Abstract P52 David Johnson . . . . . . . . . . . . . . . . . . . . . . .Page 102 

E2F1 and GCN5 facilitate the recruitment of nucleotide excision 

repair factors to sites of UV-induced DNA damage 

Abstract P53 Paul Kalitsis . . . . . . . . . . . . . . . . . . . . . . . . .Page 103 

Nucleosome spacing analysis of repeat DNA regions in the 

mouse genome 

Abstract P54 Min-Jeong Kang . . . . . . . . . . . . . . . . . . . . . .Page 104 

Role of a RPD3/HDA1 family histone deacetylase in the regulation 

of phytochrome-mediated light respases in Arabidopsis 

10


Poster Index 

Abstract P55 Panagiota Karagianni . . . . . . . . . . . . . . . . . .Page 105 

ICBP90, a putative link between histone ubiquitination and cell 

cycle progression 

Abstract P56 Emmanuel Kas . . . . . . . . . . . . . . . . . . . . . . .Page 106 

Altering the structure and functional properties of 

heterochromatin with satellite-specific minor-groove binders 

Abstract P57 Chul Geun Kim . . . . . . . . . . . . . . . . . . . . . . .Page 107 

PIAS1 confers erythroid cell specific α-globin gene regulation 

by the CP2 transcription factor family 

Abstract P58 Keun Il Kim . . . . . . . . . . . . . . . . . . . . . . . . . .Page 108 

A novel link between SUMO modification of a chromatin 

remodeling complex and cancer metastasis 

Abstract P59 Sarah Kimmins . . . . . . . . . . . . . . . . . . . . . . .Page 109 

Methylation of Histone H3 at lysine 4 is dynamic and tightly 

regulated during male germ cell development 

Abstract P60 Robert Klose . . . . . . . . . . . . . . . . . . . . . . . . .Page 110 

JmjC-domain-containing proteins and histone demethylation 

Abstract P61 Christoph Koch . . . . . . . . . . . . . . . . . . . . . . .Page 111 

The landscape of activating histone modifications across 1% of 

the human genome 

Abstract P62 Ryoki Kujiki . . . . . . . . . . . . . . . . . . . . . . . . . .Page 112 

1alpha,25(OH)2D3-induced transrepression on 1alphahydroxylase 

gene promoter mediates chromatin remodeling 

through WINAC 

Abstract P63 Sharmistha Kundu . . . . . . . . . . . . . . . . . . . .Page 113 

SWI/SNF establishes transcriptional memory at the 

Saccharomyces cerevisiae GAL1 gene 

Abstract P64 Georg Kustatscher . . . . . . . . . . . . . . . . . . . .Page 114 

Metabolite-sensitive and metabolite-insensitive chromatin 

surfaces through the human histone macroH2A 

Abstract P65 Hyockman Kwon . . . . . . . . . . . . . . . . . . . . . .Page 115 

BAF53-dependent higher-order chromatin structure as the 

compartment of replication and repair foci 

11



Abstract P66 Monika Lachner . . . . . . . . . . . . . . . . . . . . . .Page 116 

Studying lysine methylation in non-histone proteins 

Abstract P67 Brian Larsen . . . . . . . . . . . . . . . . . . . . . . . . .Page 117 

Caspase 3 mediated DNA strand breaks contribute to genomic 

reorganization during skeletal muscle terminal differentiation 

Abstract P68 Richard Lawrence . . . . . . . . . . . . . . . . . . . . .Page 118 

Mechanisms controlling dynamic Swi6/HP1 binding in S. 

pombe facilitate de novo heterochromatin formation 

Abstract P69 Frederic Leduc . . . . . . . . . . . . . . . . . . . . . . .Page 119 

Presence of gamma-H2AX in elongating spermatids: 

involvement of NHEJ 

Abstract P70 Min Gyu Lee . . . . . . . . . . . . . . . . . . . . . . . . .Page 120 

Functional association of a trimethyl H3K4 demethylase and 

Ring6a/MBLR, a polycomb-like protein 

Abstract P71 Niraj Lodhi . . . . . . . . . . . . . . . . . . . . . . . . . . .Page 121 

Histone acetylation (H3K9) and methylation (H3K4) of the 

nucleosome over core promoter are associated with the 

induction of tobacco PR-1a gene 

Abstract P72 Mattias Mannervik . . . . . . . . . . . . . . . . . . . .Page 122 

An HDAC3/SMRTER/Ebi complex required for Snail repressor 

function in Drosophila development 

Abstract P73 Robert Martin . . . . . . . . . . . . . . . . . . . . . . . .Page 123 

Chromatin labeling and distribution in living cells 

Abstract P74 Peter McKeown . . . . . . . . . . . . . . . . . . . . . . .Page 124 

Chromatin components of the Arabidopsis thaliana nucleolus 

Abstract P75 Rosalind Meldrum . . . . . . . . . . . . . . . . . . . .Page 125 

Visualisation of DNA repair and chromatin dynamics 

Abstract P76 Brendon Monahan . . . . . . . . . . . . . . . . . . . .Page 126 

Purification and characterization of the fission yeast Swi/Snf 

and RSC chromatin remodeling complexes 

12


Poster Index 

Abstract P77 Antonin Morillon . . . . . . . . . . . . . . . . . . . . . .Page 127 

Transcriptional co-suppression in S. cerevisiae 

Abstract P78 Ashby Morrison . . . . . . . . . . . . . . . . . . . . . . .Page 128 

Mec1/Tel1-dependent phosphorylation of a chromatin 

remodeling complex influences the DNA damage checkpoint 

pathway 

Abstract P79 Raul Mostoslavsky . . . . . . . . . . . . . . . . . . . .Page 129 

Genomic instability and aging-like phenotype in the absence of 

mammalian SIRT6 

Abstract P80 Takahiro Nakayama . . . . . . . . . . . . . . . . . . .Page 130 

Drosophila GAGA factor promotes histone H3.3 replacement 

that prevents the heterochromatin spreading 

Abstract P81 Zuyao Ni . . . . . . . . . . . . . . . . . . . . . . . . . . . .Page 131 

The tumor suppressor BRG1 silences the distal silencers at 

interferon-responsive genes 

Abstract P82 Olivia Osborn . . . . . . . . . . . . . . . . . . . . . . . .Page 132 

Transcriptional targets of Af4 

Abstract P83 Julia Pagan . . . . . . . . . . . . . . . . . . . . . . . . . .Page 133 

A novel corepressor, BCOR-L1, functions through CTBP and 

class 2 HDACs 

Abstract P84 Maria Panchenko . . . . . . . . . . . . . . . . . . . . .Page 134 

Role of Jade-1 in the HAT HBO1 complex 

Abstract P85 Tej Pandita . . . . . . . . . . . . . . . . . . . . . . . . . .Page 135 

Mammalian ortholog of Drosophila MOF is critical for 

embryogenesis and DNA repair 

Abstract P86 Maëlle Pannetier . . . . . . . . . . . . . . . . . . . . . .Page 136 

Imprinting perturbation in mouse hepatocarcinoma: link 

between DNA methylation and histone methylation 

Abstract P87 Janet Partridge . . . . . . . . . . . . . . . . . . . . . . .Page 137 

Establishment and maintenance of centromeric 

heterochromatin in fission yeast are functionally separable 

13



Abstract P88 Kelly Perkins . . . . . . . . . . . . . . . . . . . . . . . . .Page 138 

Activated HIV-1 provirus forms a gene loop, connecting viral 

transcriptional initiation with termination 

Abstract P89 David Picketts . . . . . . . . . . . . . . . . . . . . . . . .Page 139 

SNF2L-mediated control of cell number in the developing brain 

Abstract P90 Romina Ponzielli . . . . . . . . . . . . . . . . . . . . . .Page 140 

Optimization of experimental design parameters of ChIP-onchip 

studies 

Abstract P91 Ryan Raisner . . . . . . . . . . . . . . . . . . . . . . . .Page 141 

Single nucleosome resolution mapping of the histone variant 

H2A.Z in a developing organism 

Abstract P92 Rama Natarajan . . . . . . . . . . . . . . . . . . . . . .Page 142 

Genome-wide analysis of histone lysine methylation variations 

caused by diabetic conditions in human monocytes 

Abstract P93 Edward Ramos . . . . . . . . . . . . . . . . . . . . . . .Page 143 

Global characterization and function of Gypsy-like endogenous 

insulators in Drosophila melanogaster 

Abstract P94 William Renthal . . . . . . . . . . . . . . . . . . . . . . .Page 144 

Class II histone deacetylases regulate the behavioral 

adaptations to chronic cocaine and stress 

Abstract P95 Karsten Rippe . . . . . . . . . . . . . . . . . . . . . . . .Page 145 

Activities of histone chaperone NAP1: Association states and 

interactions with histones, nucleosome assembly and effect on 

the chromatin fiber conformation 

Abstract P96 Charles Roberts . . . . . . . . . . . . . . . . . . . . . .Page 146 

The Swi/Snf chromatin remodeling complex regulates lineage 

specific transcription programs during development and 

impairment of this activity causes cancer 

Abstract P97 Paul Sadowski . . . . . . . . . . . . . . . . . . . . . . .Page 147 

Post-translational modification of the insulator protein, CTCF 

Abstract P98 Teresa Sanchez Alcaraz . . . . . . . . . . . . . . . .Page 148 

Role of USP7 and GMP synthetase in deubiquitination of 

human histone H2B 

14


Poster Index 

Abstract P99 Annette Scharf . . . . . . . . . . . . . . . . . . . . . . .Page 149 

Dynamics of histone modifications during chromatin assembly 

Abstract P100 Stefan Schoeftner . . . . . . . . . . . . . . . . . . . . .Page 150 

Screening for miRNAs regulating mammalian telomeres 

Abstract P101 Gunnar Schotta . . . . . . . . . . . . . . . . . . . . . .Page 151 

A genome-wide transition to H4K20 mono-methylation impairs 

stress-induced and programd DNA damage response in the 

mouse 

Abstract P102 David Schrump . . . . . . . . . . . . . . . . . . . . . . .Page 152 

Brother of the Regulator of Imprinted Sites (BORIS) recruits 

Sp1 to modulate NY-ESO-1 expression in lung cancer cells 

Abstract P103 Bonnie Scott . . . . . . . . . . . . . . . . . . . . . . . . .Page 153 

Evolution of centromere-binding proteins and their interactions 

with centromere DNA in Arabidopsis 

Abstract P104 David Shechter . . . . . . . . . . . . . . . . . . . . . . .Page 154 

Histone H2A arginine3 is mono- and symmetrically-di 

methylated by a complex of PRMT5 and the WD-repeat protein 

MEP50 in Xenopus laevis eggs 

Abstract P105 Yoichi Shinkai . . . . . . . . . . . . . . . . . . . . . . . .Page 155 

H3K9 methylation and germ cell development 

Abstract P106 Krishna Sinha . . . . . . . . . . . . . . . . . . . . . . . .Page 156 

Inhibition of the transcriptional activity of osterix by 

interactions with NO66, a jumonji family chromatin protein 

Abstract P107 Karen Smith . . . . . . . . . . . . . . . . . . . . . . . . .Page 157 

Identification and characterization of novel HDAC-associated 

proteins that regulate cancer cell growth 

Abstract P108 Matthew Smith . . . . . . . . . . . . . . . . . . . . . . .Page 158 

Chromatin- mediated silencing of immune response genes 

Abstract P109 Hae-Ryong Song . . . . . . . . . . . . . . . . . . . . .Page 159 

Coordination of transcriptional regulation and chromatin 

modification of Arabidopsis circadian clock genes 

15



Abstract P110 Stacey Southall . . . . . . . . . . . . . . . . . . . . . . .Page 160 

Structural studies of histone methyltransferases 

Abstract P111 Maike Stam . . . . . . . . . . . . . . . . . . . . . . . . . .Page 161 

Molecular analysis of chromatin changes involved in b1 

paramutation, an allele-dependent transfer of epigenetic 

information 

Abstract P112 Sean Taverna . . . . . . . . . . . . . . . . . . . . . . . .Page 162 

Connecting H3 methylation and acetylation: The role of Yng1 in 

transcription 

Abstract P113 Tage Thorstensen . . . . . . . . . . . . . . . . . . . . .Page 163 

The Arabidopsis SUVR proteins define a novel subgroup of 

SET domain proteins associated with the nucleolus 

Abstract P114 Christopher Topp . . . . . . . . . . . . . . . . . . . . .Page 164 

Unusually-sized centromeric RNAs associate with maize 

centromeric chromatin 

Abstract P115 Martin Tribus . . . . . . . . . . . . . . . . . . . . . . . . .Page 165 

Molecular mechanisms of histone variant H3.3 assembly by the 

motor protein CHD1 

Abstract P116 Christopher Vakoc . . . . . . . . . . . . . . . . . . . .Page 166 

A profile of histone lysine methylation generated by 

mammalian gene transcription 

Abstract P117 Claudius Vincenz . . . . . . . . . . . . . . . . . . . . .Page 167 

Visualizing polycomb group protein interactions with histones 

in vivo 

Abstract P118 Vikki Weake . . . . . . . . . . . . . . . . . . . . . . . . .Page 168 

The SAGA histone acetyltransferase complex functions in the 

development of neuronal connectivity in the Drosophila 

compound eye 

Abstract P119 Stephanie Williams . . . . . . . . . . . . . . . . . . . .Page 169 

Mechanistic insights into promoter chromatin disassembly 

Abstract P120 Jon Wilson . . . . . . . . . . . . . . . . . . . . . . . . . .Page 170 

Structural studies of SET domain methyltransferases 

16


Poster Index 

Abstract P121 Zhaodong Xu . . . . . . . . . . . . . . . . . . . . . . . .Page 171 

Remote elements critical for cytokine induced gene expression 

Abstract P122 Xiaofang Yang . . . . . . . . . . . . . . . . . . . . . . .Page 172 

Dissecting SWI/SNF ATP-dependent chromatin remodeling 

complex in Saccharomyces cerevisiae 

Abstract P123 Juan I. Young . . . . . . . . . . . . . . . . . . . . . . . .Page 173 

Post-transcriptional functions of MeCP2 

Abstract P124 Veronica Yu . . . . . . . . . . . . . . . . . . . . . . . . . .Page 174 

Over-expression of Cks proteins causes gene derepression in 

Saccharomyces cerevisiae 

Abstract P125 Rebekah Zinn . . . . . . . . . . . . . . . . . . . . . . . .Page 175 

hTERT is expressed in cancer despite promoter DNA 

methylation by preservation of unmethylated DNA and active 

chromatin around the transcription start site 

Additional poster submissions 

Abstract P126 Yoshimitsu Takahashi . . . . . . . . . . . . . . . . . .Page 176 

Degree of SUMO modification as a differential tag for targeting 

to specific chromosomal domains 

Abstract P127 Marna S. Costanzo . . . . . . . . . . . . . . . . . . . .Page 177 

The evolutionary conservation of chromatin modifying proteins 

in malaria 

Abstract P128 Philippe Prochasson . . . . . . . . . . . . . . . . . . .Page 178 

Functional characterization of the HIR corepressor complex 

17



Abstracts – Oral 

Steve Henikoff Abstract 1 

Epigenetic patterns generated by histone replacement 

Fred Hutchinson Cancer Research Center 1100 Fairview Avenue North, Seattle, WA 98109- 

1024, U.S.A. 

Histone H3 is deposited at replication, but it is replaced at active genes by the constitutive 

histone variant, H3.3. We have used chromatin affinity purification of biotin-tagged H3.3 to 

map histone replacement throughout the Drosophila genome. Replacement is especially 

prominent at active genes, corresponding to sites of abundant RNA polymerase II and 

methylated H3 lysine-4 throughout the genome. Active genes are depleted of histones at 

promoters and are enriched in H3.3 from upstream to downstream of transcription units. 

Histone replacement patterns differ between the dosage compensated X-chromosome and 

autosomes downstream of gene promoters, suggesting that dosage compensation is 

achieved by modulating transcriptional elongation. Histone replacement is low overall at the 

Bithorax Complex, but surprisingly, Polycomb Response Elements are sites of 

conspicuously high histone turnover, whose peaks precisely correspond to nuclease 

hypersensitive sites. We also observe high levels of histone turnover at the “poised” 

promoters of heat shock genes. We propose that the remodeling process responsible for 

histone replacement patterns at cis-regulatory elements maintains continuous accessibility 

of DNA to trans-acting factors, providing a simple general mechanism for cellular memory. 

18


Abstracts - Oral 

Yang Shi Abstract 2 

The identification of histone demethylases established the 

dynamic and reversible nature of histone methylation regulation 

In this presentation, I will discuss our continued efforts to catalog histone demethylases and 

to understand their roles in development and human diseases. 

19



Jesper Christensen Abstract 3 

The retinoblastoma tumor suppressor binding protein RBP2 

is a transcriptional repressor demethylating tri- and 

dimethylated lysine 4 on Histone H3 

Jesper Christensen 1 , Karl Agger 1 , Paul A. C. Cloos 1 , Diego Passini 1 , Klaus 

H. Hansen 1 and Kristian Helin 1, 2 

1 

Biotech Research & Innovation Centre, Fruebjergvej 3,2100 Copenhagen, Denmark; 

2 

Faculty of Health Sciences, University of Copenhagen, Blegdamsvej 3, 2200 Copenhagen, 

Denmark. 

The Retinoblastoma tumor suppressor protein, pRB, is a key regulator of cell-cycle 

progression, differentiation and senescence, and is often found deregulated in cancer. To 

repress transcription upon cell-cycle exit induced by differentiation or oncogene-induced 

senescence, pRB binds directly to members of the E2F transcription factor family and 

cellular factors, thereby bridging chromatin modifiers to E2F-regulated genes causing 

chromatin condensation. Here we show that the Jumonji domain containing protein, 

Retinoblastoma Binding Protein 2 (RBP2), is a transcriptional co-repressor, which modifies 

chromatin by demethylating tri- and dimethylated lysine 4 on histone 3 (H3K4), a chromatin 

mark present on active genes. Ectopic expression of RBP2 in human TIG3 fibroblasts 

induced a senescent-like phenotype with reduced H3K4 methylation. Similarly, ectopic 

expresion of RBP2 in U2OS cells strongly reduced H3K4 methylation when analyzed by 

immunofluorescence. Mutation of the Jumonji domain of RBP2 abolished the demethylation 

activity. Furthermore, purified recombinant RBP2 efficiently demethylated tri- or dimethylated 

H3K4 in vitro using purified calf thymus histones or HeLa cell nucleosomes as substrate for 

the enzyme reactions, while other histone methylation marks at H3K9, H3K27, H3K36 and 

H4K20 were unaffected. Finally, the enzymatic specificity of RBP2 was confirmed by testing 

tri-, di-, and monomethylated H3K4 peptides as substrate and subsequent mass 

spectrometry analysis of the reaction products. The biological function of RBP2 is currently 

not fully elucidated. However, considering the pRB binding and the demethylation activity of 

RBP2, a role for RBP2 in chromatin demethylation and repression of pRB regulated genes 

is a possibility and is currently being explored. 

20



Mischa Machius Abstract 4 

Structural Basis for CoREST-Dependent Demethylation of 

Nucleosomes by the Human LSD1 Histone Demethylase 

Mischa Machius, Maojun Yang, Christian B. Gocke, Xuelian Luo, 

Dominika Borek, Diana R. Tomchick, Zbyszek Otwinowski and Hongtao Yu 

University of Texas Southwestern Medical Center, 5323 Harry Hines Blvd., Dallas, TX, 

75390, U.S.A. 

Histone methylation regulates diverse chromatin-templated processes, including 

transcription. Many transcriptional corepressor complexes contain lysine-specific 

demethylase 1 (LSD1) and CoREST that collaborate to demethylate mono- and dimethylated 

H3-K4 of nucleosomes. We report the crystal structure of the LSD1-CoREST 

complex. LSD1-CoREST forms an elongated structure with a long stalk connecting the 

catalytic domain of LSD1 and the CoREST SANT2 domain. LSD1 likely recognizes a large 

segment of the H3 tail through a deep, negatively charged pocket at the active site and a 

shallow groove on its surface. CoREST SANT2 interacts with DNA. Disruption of the 

SANT2-DNA interaction diminishes CoRESTdependent demethylation of nucleosomes by 

LSD1. The shape and dimension of LSD1-CoREST suggest its bivalent binding to 

nucleosomes, allowing efficient H3-K4 demethylation. This spatially separated, multivalent 

nucleosome-binding mode may apply to other chromatin-modifying enzymes that generally 

contain multiple nucleosome-binding modules. 

21



Ramin Shiekhattar Abstract 5 

Functional and biochemical characterization of histone 

demethylase complexes 

Ramin Shiekhattar 

The Wistar Institute, 3601 Spruce Street, Philadelphia, PA 19104 

Schizosaccharomyces pombe contains two proteins, SWIRM1 and SWIRM2, with close 

homology to human histone H3 lysine 4 demethylase. Both proteins contain the amino 

oxidase catalytic domain and a recently described DNA interaction SWIRM domain. Our 

results indicate that while SWIRM2 is an essential gene, cells lacking SWIRM1 are viable. 

We found that SWIRM1 and SWIRM2 are stably associated in a multiprotein complex, but 

intriguingly, unlike their human counterpart, S. pombe SWIRM complex contains neither a 

histone deacetylase (HDAC) nor any detectable demethylase activity. Genome-wide 

chromatin immunoprecipitation unexpectedly showed the absence of both SWIRM proteins 

from heterochromatic domains. Instead, consistent with biochemical analyses, SWIRM1 and 

SWIRM2 co-localize to a common set of target gene promoters whose functions are 

implicated in diverse processes including mitochondrial metabolism and transcriptional 

regulation. Importantly, we show that SWIRM1 is not only required for optimum transcription 

of its target genes but also display a global role in regulation of antisense transcription. 

22



Yi Zhang Abstract 6 

Histone demethylation by the JmjC domain-containing 

proteins 

Yi Zhang 

Howard Hughes Medical Institute, University of North Carolina at Chapel Hill, Chapel Hill, 

NC 27599, U.S.A. 

Posttranslational histone modifications play an important role in regulating chromatin 

dynamics and function. One of the modifications, methylation, occurs on both lysine and 

arginine residues and participates in diverse range of biological processes including 

heterochromatin formation, X-chromosome inactivation, and transcriptional regulation. While 

acetylation, phosphorylation, and ubiquitylation are dynamically regulated by enzymes that 

catalyze the addition and removal of a particular modification, enzymes that are capable of 

removing methyl groups were not known until recently. Using a novel demethylase assay, we 

have identified a family of JmjC domain-containing histone demethylases. The mechanism 

of demethylation and biological significance of these demethylases will be discussed. 

23



Francis Stewart Abstract 7 

Epigenetic aspects of lineage commitment 

Glaser, S., Lubitz, S., Anastassiadis, K., Siebler, J., Schwenk, F. and 

Stewart, A.F. 

BioInnovationsZentrum, Technische Universitaet Dresden, Germany; Artemis 

Pharmaceuticals, Cologne, Germany 

In higher eukaryotes, somatic cells differ epigenetically from the pluripotent cells of early 

development. Consequently it is possible that epigenetic mechanisms play important roles 

in lineage commitment and cellular differentiation. In mammals, the simplest model 

suggests that the epiblast is pluripotent because its chromatin is epigenetically naive and 

lineage commitment restricts pluripotency via the imposition of epigenetic marks. A potential 

corollary to this model suggests that cellular identity in the adult is maintained, in part, by 

epigenetic mechanisms. 

Recent progress has highlighted the importance of three histone lysine methylations in 

epigenetics. Whereas methylation of histone 3 lysine 9 (H3 K9) and H3 K27 direct 

inheritable states of gene silencing, methylation of H3 K4 is associated with gene 

expression. Mammals have multiple enzymes for each of these methylations, including at 

least six for H3 K4. It is therefore possible that different gene expression programs are 

regulated by different methyltransferases. 

To explore these issues, we are studying two of the H3 K4 methyltransferases, Mll and Mll2, 

in mouse development. These two sister genes have arisen by a gene duplication and are 

closely related in many ways. However they regulate different genes. Notably Mll regulates 

the Hoxa complex whereas Mll2 the Hoxb complex. Based on experiments with conditional 

mutagenesis and studies in utero and in ES cells, we conclude that epigenetic mechanisms 

are not essential for lineage commitment decisions, rather they contribute to securing and 

co-ordinating decisions with notable effects on timing and the regulation of apoptosis. 

24



Henk Stunnenberg Abstract 8 

Title and abstract unavailable 

25



Ali Shilatifard Abstract 9 

H2B monoubiquitination and H3K4 methylation via COMPASS 

Ali Shilatifard 

Saint Louis University Cancer Center, Saint Louis University School of Medicine 

Saint Louis, MO 63104 

Chromosomal rearrangements and translocations play a major role in the pathogenesis of 

hematological malignancies. The trithorax related mixed lineage leukemia (MLL) gene 

located on chromosome 11q23 is rearranged in a variety of aggressive human B and T 

lymphoid tumors as well as acute myeloid leukemia (AML) in both children and adults. In 

order to better define the role of MLL in pathogenesis of leukemia, we have been studying 

the biochemical properties of MLL and MLL-related proteins from several different 

organisms. We have demonstrated that the MLL homologue in yeast, the Set1 protein, exist 

in a macromolecular complex we call COMPASS. COMPASS is a histone 

methyltransferases capable of mono- di and trimethylating the fourth lysine of histone H3. 

Previously, we demonstrated that the ubiquitin-conjugating enzyme Rad6 and its E3 ligase 

Bre1 and several other factors are required for COMPASS mediated methylation of H3K4 

through regulation of monoubiquitination of H2B at K123. Here, I will discuss our recent 

findings regarding the molecular mechanism and the role of H2B monoubiquitination in the 

regulation of H3K4 methylation by COMPASS. 

26



Paulo Sassone-Corsi Abstract 10 

A chromatin remodeling clock 

Jun Hirayama, Masao Doi, Saurabh Sahar, Benedetto Grimaldi, David 

Gauthier, Yasukazu Nakahata and Paolo Sassone-Corsi 

Department of Pharmacology, School of Medicine, University of California, Irvine. 

Circadian rhythms are the overt consequences of biological clocks, endogenous timers 

acting within cells. At the molecular level, circadian clocks are constituted by ‘clock genes’, 

some of which encode proteins able to feedback and inhibit their own transcription 

Circadian rhythms are regulated by clocks located in specific structures of the central 

nervous system – such as the suprachiasmatic nucleus (SCN) in mammals – but also by 

peripheral oscillators present in various other tissues. It is now established that an intrinsic 

circadian pacemaker functions in virtually each cell. Importantly, about 15% of all genes are 

expressed in a circadian manner. It is thereby conceivable to invoke large-scale events of 

chromatin remodeling in order to accommodate these global changes in gene expression. 

The molecular machinery that governs circadian rhythmicity comprises proteins whose 

interplay generates time-specific transcription of clock genes. The role of chromatin 

remodeling in a physiological setting such as the circadian clock has been unclear. We have 

shown that the protein CLOCK, a central component of the circadian pacemaker, has 

histone acetyltransferase (HAT) activity. CLOCK shares homology with acetyl-coenzyme A 

binding motifs within the MYST family of HATs. CLOCK displays high sequence similarity to 

ACTR, a member of SRC family of HATs, with which it shares also enzymatic specificity for 

histones H3 and H4. BMAL1, the heterodimerization partner of CLOCK, enhances HAT 

function. The HAT activity of CLOCK is essential to rescue circadian rhythmicity and 

activation of clock genes in Clock-mutant cells. Identification of CLOCK as a novel type of 

DNA-binding HAT reveals that chromatin remodeling is crucial for the core clock mechanism 

and identifies unforeseen links between histone acetylation and cellular physiology. 

27



Sung Hee Baek Abstract 11 

A Novel Link between SUMO Modification of a Chromatin 

Remodeling Complex and Cancer Metastasis 

Jung Hwa Kim 1,3 , Hyejin Nam 1,3 , Hee June Choi 1 , Bogyou Kim 1 , Ji Min 

Lee 1 , Ik Soo Kim 1 , Keun Il Kim 2 , and Sung Hee Baek 1 

1 

Department of Biological Sciences, Seoul National University, Seoul 151-746, South Korea, 

2 

Department of Biological Sciences, Sookmyung Women's University, Seoul 140-742, South 

Korea 

3 

These authors contributed equally 

Defining the functional modules with transcriptional regulatory factors that govern switching 

between repression and activation events is a central issue in biology. We have reported 

the dynamic role of a b-catenin/reptin chromatin remodeling complex to regulate a 

metastasis suppressor gene KAI1, which is capable of inhibiting the progression of tumor 

metastasis, and further which signaling factors confer repressive function on reptin and 

hence maintain a repressed state of KAI1 (Kim et al., Nature 434, 921-6; Kim et al., Nature 

Cell Biol. 8, 631-9). Biochemical purification of a reptin-containing complex has revealed 

the presence of specific deSUMOylating enzymes that reverse the SUMOylation of reptin 

that underlies its repressor function. DeSUMOylation of reptin alters the repressive function 

of reptin and its association with HDAC1. Further, SUMOylation status of reptin modulates 

the invasive activity in cancer cells with metastatic potential. This provides a clear definition 

of the functional model and a novel insight for linking SUMO modification to cancer 

metastasis. As a follow-up study, we will address novel findings on the function of newly 

identified histone methyltransferase as a component of reptin, linking chromatin remodeling 

process and cancer metastasis. 

28



Laszlo Tora Abstract 12 

The simultaneously dimethylated Lys-9 and phosphorylated 

Ser-10 tails of histone H3 adopt different conformations 

during mitosis 

Tora L., Eberlin A., Oulad-Abdelghani M., Robert F., Grauffel C., Spehner 

D., Wurtz J-M., Schultz P. and Dejaegere A. 

Institut de Genetique et de Biologie Moleculaire et Cellulaire (IGMBC), UMR 7104 CNRS, 

ULP, INSERM, INSERM U.596, Parc d’Innovation,1, rue Laurent Fries, BP 10142, 67404 

Illkirch Cedex, C.U. de Strasbourg, France 

Eukaryotic cells possess mechanisms for condensing and decondensing chromatin. 

Chromatin condensation is particularly evident during mitosis and cell death induced by 

apoptosis, whereas chromatin decondensation is necessary for replication, repair, 

recombination and transcription. Histones are among the numerous DNA binding proteins 

that control the level of DNA condensation, and post-translational modification of histone 

tails plays a critical role in the dynamic condensation/decondensation that occur at 

numerous cellular processes. Post-translational modifications, alone or in combination, can 

direct distinct downstream events. The association of lysine (K) 9 dimethylation (di-Me), a 

hallmark of the heterochromatin, with serine (S) 10 phosphorylation (P), a marker of 

mitosis, on the same histone H3 tail, as well as the idea of a structured histone-tail, has 

long been controversial. Interestingly, by using a specific antibody, we detect a histone H3 

tail conformation, which contains simultaneously K9(di-Me) and S10(P) that appears only 

between the late prophase and the early anaphase steps, being the strongest during 

metaphase. This H3 tail conformation is different from another state, where the K9(di-Me) 

S10(P) modifications are also simultaneously recognized, but more widely during mitosis. 

Furthermore, results obtained by confocal and electron microscopy suggest that the 

conformation of K9(di-Me) and S10(P) histone H3 tails changes during mitosis and can 

adopt at least two different conformations. This observation has also been confirmed by 

biostructural docking and molecular dynamics modelling as well as by competition tests, 

using various modified peptides. The localisation and the role of these different 

conformations in gene regulation and mitotic chromosome condensation will be discussed. 

29



Tony Kouzarides Abstract 13 

Characterisation of novel histone modifications 

Claire Pike, Chris Nelson, Paul Hurd, Andy Bannister and Tony Kouzarides 

The Gurdon Institute, Cambridge University, Tennis Court Road, Cambridge, U.K. 

We are investigating the mechanism of action of several novel modifications on histone H3. 

We have previously identified an enzyme FPR4 in yeast that can isomerise prolines in the tail 

of H3. A mammalian enzyme that can accomplish similar functions has been identified and is 

under characterisation. In addition, we have identified a new phosphorylation site on human 

H3 by mass spectrometry. Specific antibodies raised against this site are now being used to 

establish the kinase pathways that mediate this phosphorylation event. 

30



Thomas Jenuwein Abstract 14 

Epigenetic control by histone methylation 

Thomas Jenuwein 

IMP Vienna, Dr. Bohrgasse 7, Austria 

Epigenetic mechanisms, such as histone modifications, control eukaryotic development 

beyond DNA-stored information. We are analyzing histone lysine methylation in mammalian 

chromatin to further dissect its role(s) in chromosome organization, gene regulation, 

genome stability and overall epigenetic control. While there is under-representation of 

repressive histone marks in quiescent (resting), stem and regenerating cells, there is a 

selective accumulation of aberrant histone lysine methylation profiles in aging, ‘stressed’ 

and tumor cells. We have generated mutant mice that lack crucial HMTases, such as the 

Suv4-20h enzymes. In these Suv4-20h double-null mice, there is a genome-wide transition 

from H4K20 tri- to H4K20 mono-methylation, which appears to impair stress-induced and 

programd DNA damage response. In addition, we have screened chemical libraries (in 

collaboration with Boehringer Ingelheim, Ridgefield U.S.A.) and identified a small molecule 

inhibitor for the G9a HMTase. This novel compound, BIX-01294, is the first HMTase inhibitor 

that can be used to transiently modulate H3K9me2 levels in mammalian chromatin. Finally, 

we have been characterizing jumonjiC-containing proteins that represent hydroxylases with 

the potential to remove repressive H3K9me3 marks. Together, these approaches promise to 

yield new insights into the plasticity of cell fate decisions and may offer novel strategies to 

revert aberrant development. 

31



Roberta Benetti Abstract 15 

The role of Dicer in the regulation of chromatin at telomeres 

Roberta Benetti 1 *, Susana Gonzalo 1,4 *, Stefan Schoeftner 1 , Isabel Jaco 1 , 

Purificacion Muntildedoz 1 , Elizabeth Murchison 2 , Thomas Andl 3 , Peter 

Klatt 1 , Sarah Millar 3 , Gregory Hannon 2 and Maria A. Blasco 1 

1 

Telomeres and Telomerase Group, Molecular Oncology Program, Spanish National Cancer 

Centre (CNIO), Madrid E-28029, SPAIN; 2 Cold Spring Harbor Laboratory, NY 11724, U.S.A.; 

3 

Department of Dermatology, University of Pennsylvania, Philadelphia, PA 19104-6100, 

U.S.A.; 4 Radiation and Cancer Biology Division, Department of Radiation Oncology, 

Washington University School of Medicine, St. Louis, MO 63108, U.S.A. 

Dicer has been proposed to have a role in the maintenance of silencing at centromeres in 

several organisms, including mammals. Here we describe a role for Dicer in regulating 

mammalian telomeric chromatin. In particular, mouse ES cells and skin keratinocytes 

conditionally deleted for Dicer show aberrantly elongated telomeres compared to wild-type 

controls, concomitant with increased telomeric recombination. This occurs in the absence of 

changes in TRF1 and TRF2 expression and with decreased telomerase activity in Dicer-null 

cells. The long-telomere phenotype of Dicer-null cells is accompanied by an increased 

density of histone heterochromatic marks at telomeric chromatin, such as histone 3 lysine 9 

(H3K9) and histone 4 lysine 20 (H4K20) tri-methylation, and by decreased histone 

acetylation, supporting the idea of a silencing of telomeric chromatin in the absence of 

Dicer. In support of this, we observed a decreased abundance of telomeric RNA transcripts 

in Dicer-null cells. All together, these results demonstrate an unprecedented role for Dicer in 

the regulation of mammalian telomeric chromatin. Dicer has been proposed to have a role 

in the maintenance of silencing at centromeres in several organisms, including mammals. 

Here we describe a role for Dicer in regulating mammalian telomeric chromatin. In 

particular, mouse ES cells and skin keratinocytes conditionally deleted for Dicer show 

aberrantly elongated telomeres compared to wild-type controls, concomitant with increased 

telomeric recombination. This occurs in the absence of changes in TRF1 and TRF2 

expression and with decreased telomerase activity in Dicer-null cells. The long-telomere 

phenotype of Dicer-null cells is accompanied by an increased density of histone 

heterochromatic marks at telomeric chromatin, such as histone 3 lysine 9 (H3K9) and 

histone 4 lysine 20 (H4K20) tri-methylation, and by decreased histone acetylation, 

supporting the idea of a silencing of telomeric chromatin in the absence of Dicer. In support 

of this, we observed a decreased abundance of telomeric RNA transcripts in Dicer-null 

cells. All together, these results demonstrate an unprecedented role for Dicer in the 

regulation of mammalian telomeric chromatin 

32



Mareike Puschendorf Abstract 16 

Ezh2 independent targeting of PRC1 proteins to paternal 

constitutive heterochromatin in mouse pre-implantation 

embryos 

Mareike Puschendorf 

Friedrich Miescher Institute, Maulbeerstrasse 66, CH-4057 Basel Switzerland 

In mammals, fertilization triggers a cascade of events leading to the formation of a 

totipotent embryo from two highly specialized gametes. During this process both parental 

genomes undergo major epigenetic reprogramming, suggesting a potential causal 

relationship between the two events. Several immunofluorescence studies indicate that 

chromatin states of maternal and paternal genomes are initially highly asymmetric. 

Whereas the maternal genome inherits many distinct types of histone lysine methylation, 

the paternal genome is de novo methylated at different lysine residues in a highly spatially 

and temporally coordinated manner after the protamine to histone exchange. At the 

maternal genome, constitutive heterochromatin is labeled by modifications characteristic of 

the Suv39h pathway (such as H3K9 and H4K20 tri-methylation and binding of HP1b). 

Importantly, in proliferating somatic cells Suv39h function is required to maintain mitotic 

genome stability. Surprisingly, paternal constitutive heterochromatin in early embryos is 

devoid of the canonical Suv39h-dependent chromatin marks. Instead, we observe that 

various proteins of the Polycomb Repressive Complex 1 (PRC1) are targeted to constitutive 

heterochromatin of only the paternal genome. By using embryos maternally and zygotically 

deficient for Ezh2, we demonstrate that the parental-origin-specific labeling is independent 

of Ezh2 function and H3K27 tri-methylation. PRC1 binding to paternal heterochromatin is 

stably transmitted over several mitotic divisions suggesting the existence of a memory of 

parental identity of constitutive heterochromatin in pre-implantation embryos. 

33



Edith Heard Abstract 17 

The nuclear and epigenetic dynamics of X-chromosome 

inactivation in the mouse 

Edith Heard 

Mammalian Developmental Epigenetics Group, CNRS UMR 218 - Nuclear Dynamics and 

Genome Plasticity Curie Institute, 26 rue d’Ulm, 75248 Paris Cedex 05, France 

In female mammals, one of the two X chromosomes is converted from the active euchromatic 

state into inactive heterochromatin during early embryonic development. This process, known 

as X-chromosome inactivation, results in the transcriptional silencing of over a thousand 

genes and ensures dosage compensation between the sexes. X inactivation is a dramatic 

example of mammalian epigenetics, involving differential regulation of two homologous 

chromosomes within the same nucleus, in a mitotically heritable but developmentally 

reversible manner. We are interested in the mechanisms and kinetics of this process in early 

mouse embryos and differentiating embryonic stem (ES) cells. X inactivation is a highly 

dynamic process during early development (Okamoto et al, 2004) and we are interested in 

defining the epigenetic marks that underlie its initiation and its maintenance. Given the monoallelic 

character of X inactivation, we are also investigating the role of sub-nuclear compartmentalization 

in this process, both at the level of the master control locus of X inactivation, 

the Xic, and the non-coding Xist transcript it produces, that is responsible for inducing 

transcriptional silencing in cis. In this context, we have recently discovered that the two Xics 

transiently co-localise just prior to random monoallelic up-regulation of Xist and the onset of 

X inactivation (Bacher et al, 2006). This co-localisation seems to be important for ensuring 

that X inactivation is triggered when more than one Xic is present. Recent evidence will be 

presented for a new region of the Xic that seems to be critical for bringing the two loci together 

in trans and that is characterized by specific histone modifications. 

Refs: 

Okamoto,I., Otte,A., Allis,C.D., Reinberg,D. and Heard,E. (2004) Epigenetic dynamics of 

imprinted X inactivation during early mouse development. Science, 303, 644-649 

Bacher,C., Guggiari,M., Brors,B., Augui,S., Avner,P., Eils,R. and Heard,E. (2006) Transient 

colocalization of X-inactivation centres accompanies the initiation of X inactivation. Nature 

Cell Biology, 8, 293-239. 

34



Adrian Bird Abstract 18 

MeCP2: molecular interactions and phenotypic stability in a 

mouse model of Rett Syndrome 

Jacky Guy 1 , Xinsheng Nan 1 , Jianghui Hou 2 , Skirmantas Kriaucionis 1 and 

Adrian Bird 1 

1 

Wellcome Trust Centre for Cell Biology, The University of Edinburgh, The Kings Buildings, 

Edinburgh EH9 3JR, U.K., 2 Molecular Medicine Centre, The University of Edinburgh, 

Western General Hospital, Edinburgh EH4 2XU, U.K. 

Rett Syndrome (RTT) is a profound neurological disorder that almost exclusively affects 

girls. More than 80% of patients carry a new mutation in one copy of the X-linked MECP2 

gene and this is now established as the primary cause of the condition. Overt symptoms 

show delayed onset in girls between 6 and 18 months of age and include developmental 

delay, loss of purposeful limb use and breathing abnormalities. As there is no obvious 

neurodegeneration in post-mortem brains of RTT patients, the question of reversibility 

arises and is of obvious relevance for therapeutic approaches to RTT. We earlier created a 

mouse model for RTT that lacks an intact Mecp2 gene and mimicks several features of the 

disorder including late inset. Using a mouse with an Mecp2 allele that can be conditionally 

activated, we are asking whether neuronal defects in the young adult can be rectified if 

MeCP2 is provided after abnormal neuronal morphology and symptoms have arisen. Can 

switching on MeCP2 in these animals reverse the phenotype, or is it too late In addition to 

these physiological studies, we have identified the Swi/Snf motor protein ATRX as an 

MeCP2 binding partner. Mutations in both MECP2 and ATRX genes cause X-linked mental 

retardation and we have preliminary evidence for interdependence in the mouse brain. 

35



Jon Penterman Abstract 19 

DNA demethylation in Arabidopsis thaliana 

Jon Penterman 1 , Daniel Zilberman 2 , Jin Hoe Huh 1 , Tracy Ballinger 2,3 , 

Steven Henikoff 2,3 , Robert Fischer 1 

1 

Department of Plant and Microbial Biology, University of California, Berkeley, California 

94720, U.S.A. 2 Basic Sciences Division, Fred Hutchinson Cancer Research Center, Seattle, 

Washington, 98109, U.S.A. 3 Howard Hughes Medical Institute 

Cytosine DNA methylation is an epigenetic modification that functions in a number of 

processes, one of which is genome defense against transposons and repetitive elements. In 

Arabidopsis thaliana DNA is methylated by methyltransferases whose specificity is 

determined by parental methylation patterns, histone modifications, and/or small RNAs. 

Here we show that methylation at many loci throughout the genome is actively removed by 

a DNA demethylation pathway. The DEMETER-LIKE (DML) DNA glycosylases, which excise 

5-methylcytosine and initiate the base excision DNA repair pathway, mediate this process. 

Using genome-tiling arrays, we detected nearly two hundred discrete loci that are 

demethylated in a DML-dependent manner. We find that DML demethylation primarily 

occurs at the 5’ and 3’ ends of genes, a pattern opposite to the overall distribution of wildtype 

DNA methylation. Our results show that DML-dependent DNA demethylation is a 

fundamental pathway that edits the Arabidopsis methylation profile. We believe that DML 

demethylation provides a protective buffer against the methylation pathway of Arabidopsis, 

which might indirectly enable Arabidopsis to have a robust defense pathway for repressing 

transposons and repetitive elements. 

36



Francois Fuks Abstract 20 

The Polycomb Group protein EZH2 is recruited to promoters 

by MECP2 

Emmanuelle Vire 1 , Helene Denis 1 , Esteban Ballestar 2 , Yvan de Launoit 3 , 

Manel Esteller 2 and Francois Fuks 1 

1 

Free University of Brussels, Faculty of Medicine, Laboratory of Cancer Epigenetics, 808 

route de Lennik, 1070 Brussels, Belgium; 2 CNIO, Cancer Epigenetics Group, C/ Melchor 

Fernandez Almagro 3,28029-Madrid, Spain; 3 Institut de Biologie de Lille, 1 rue Calmette, 

59021 Lille, Cedex, France 

Polycomb Group (PcG) proteins and DNA methylation are fundamental epigenetic systems 

involved in gene silencing. Recently we have uncovered a close connection between these 

two systems: the PcG protein EZH2 can control DNA methylation (1). 

Here we show that conversely, CpG methylation can influence EZH2 function through the 

methyl-CpG-binding protein MECP2. We demonstrate that EZH2 interacts physically with 

MECP2 in vivo. Chromatin immunoprecipitations indicate that the presence of MECP2 is 

required for binding of EZH2 to target promoters. Genome-wide location analysis with 

antibodies against these proteins are under way to explore whether there is a cross-talk 

between EZH2 and MECP2 across promoters within the genome. 

Our results suggest that MECP2 may act as a molecular scout for PcG recruitment to 

chromatin. They could shed light on the poorly understood mechanisms by which 

mammalian Polycomb Group proteins are targeted to promoters. 

Ref. 

(1) Vire E, Brenner C, Deplus R, Blanchon L, Fraga M, Didelot C, Morey L, Van Eynde A, 

Bernard D, Vanderwinden JM, Bollen M, Esteller M, Di Croce L, de Launoit Y, Fuks F. 

Nature. 2006 Feb 16:871-4. 

37



Shelley Berger Abstract 21 

Factor and histone covalent modifications in genome regulation 

Shelley L. Berger 

The Wistar Institute, Philadelphia, PA, 19104, U.S.A. 

Genomic structure and function is regulated in part through covalent post-translational 

modifications (PTMs) of factors and histones, including acetylation (ac), methylation (me), 

phosphorylation (ph), ubiquitylation (ub), and sumoylation (su). There are an enormous 

number of factor and histone PTMs. To make sense of this bewildering complexity, we 

focuses on patterns, temporal sequences, and cross-talk between PTMs in the yeast S. 

cerevisiae and mammalian cells. 

In mammals we study PTMs of DNA-bound transcription factors, using the tumor 

suppressor and transcription factor p53 as a model. We currently focus on methylation by 

the SET domain methyltransferase Smyd2. Smyd2 methylates p53 at K370, adjacent to the 

previously identified methylation site at K372. K372me is associated with transcriptional 

activation by p53, and inhibits K370me, which in contrast, is associated with transcriptional 

repression by p53. Thus, there is regulatory cross-talk between activating and repressing 

lysine methylation. Our most recent results indicate that demethylation occurs within p53. 

A methylation/demethylation pathway will be discussed in detail. 

38



Danny Reinberg Abstract 22 

A molecular understanding of epigenetics 

Danny Reinberg 

NYU-School of Medicine, NY, U.S.A. 

Epigenetics encompasses changes in gene expression profiles that occur without 

alterations in the genomic DNA sequence of a cell. This arises from the dynamic processes 

that structure regions of chromosomal DNA through a range of compaction in eukaryotes. 

The altered pattern of gene expression is pivotal to cellular differentiation and development 

and is inherited by daughter cells thereby maintaining the integrity, specifications, and 

functions for a given cell type. Aberrancies in this epigenetic process give rise to 

perturbations that are also inherited and disruptive to normal cellular properties. The histone 

proteins that package DNA into chromatin are subject to post-translational modifications 

generating different chromatin structures. While euchromatin has a relaxed structure 

permissive to transcription, constitutive heterochromatin is densely packed and inaccessible 

to transcription factors. On the other hand, facultative heterochromatin is repressive, but can 

be altered in its properties to become active. Our goals are to identify the molecular 

mechanisms controlling the formation of facultative heterochromatin and the epigenetic 

parameters that ensure its propagation through cell divisions. 

39



Jessica Tyler Abstract 23 

The mechanistic basis for the requirement of promoter 

chromatin disassembly for transcriptional activation 

Jessica K Tyler 1 , Stephanie Williams 1 , Melissa Adkins 1 , Christine English 1 

and Mair Churchill 2 

1 

Department of Biochemistry and Molecular Genetics, 2 Department of Pharmacology, 

University of Colorado at Denver and Health Sciences Center, Aurora CO 80045 

Nucleosomes appear to be disassembled from the promoters of transcriptionally active 

genes in Eukaryotic species spanning from yeast to humans. We have previously shown 

that this promoter chromatin disassembly is essential for transcriptional activation of the 

budding yeast PHO5 and PHO8 genes. Promoter chromatin disassembly is mediated by 

the highly conserved histone H3/H4 chaperone Anti-silencing function 1 (Asf1) during 

activation of the PHO5 and PHO8 genes upon phosphate depletion and during activation of 

the ADY2 and ADH2 genes upon glucose removal. Using PHO5 as our model system to 

learn how promoter chromatin disassembly activates gene expression, we have discovered 

that Asf1-mediated promoter chromatin disassembly is required for recruitment of TBP and 

RNA polymerase II, but not for recruitment of the Pho4 and Pho2 activators. Furthermore, 

accumulation of SWI/SNF and SAGA at PHO5 required promoter chromatin disassembly. 

We have also uncovered a novel requirement for SWI/SNF and SAGA in chromatin 

disassembly to facilitate activator recruitment to the nucleosome-buried binding site in the 

PHO5 promoter that is distinct from the stable recruitment of SWI/SNF and SAGA after 

chromatin disassembly. 

Towards addressing the mechanism whereby Asf1 mediates chromatin disassembly, we 

have solved the crystal structure of Asf1 in complex with histones H3/H4 to 1.7 Angstrom 

resolution; this is the first time histone proteins have been “seen” outside of the nucleosome 

or octamer structures (see Mair Churchill’s abstract for more details). Using mutants 

designed from the Asf1-H3/H4 structure, we show that Asf1 needs to bind to the H3:H3 

dimerization surface of the H3/H4 heterodimer in order to achieve chromatin disassembly 

from the PHO5 promoter. Furthermore, binding of Asf1 to the C-terminal beta strand of 

histone H4, which in itself induces an 180˚ flipping-out of this region of H4 as compared to 

the nucleosome, is also required for chromatin disassembly by Asf1. From these results we 

propose a “strand capture” model for chromatin disassembly. 

40



Gratien Prefontaine Abstract 24 

Epigenetic mechanisms influencing pituitary gene 

expression. 

Prefontaine G.G., Lunyak, V. and Rosenfeld M.G. 

Department of Medicine, University of California, San Diego, 9500 Gilman Drive, CMM- 

West #345, La Jolla, CA, 92093-0648 

During development, tissue-specific transcription factors direct covalent modifications of 

chromatin, laying down the foundation for regulated gene transcription. These factors act 

through cis-acting elements located in promoter proximal and distal elements. The pituitary 

gland provides an excellent model system for studying epigenetic changes in gene 

regulation. Common pluripotent primordial ectodermal cells differentiate to produce a 

pituitary gland composed of 5 major cell types characterized by types of hormone they 

produce and secrete, including: lactotropes, somatotropes, thyrotropes, corticotropes and 

gonadotropes. I have performed a comprehensive analysis of the CpG DNA methylation 

status of the growth hormone (GH) promoter in mouse pituitary. Early in development the 

embyonic murine GH promoter was largely CpG methylated. Later postnatally, the GH 

promoter was demethylated in a subset of cell types. I have linked the DNA demethylation 

of the promoter, genetically and spatially with the occupancy of a cell-type specific factor. 

Furthermore, using a combination of techniques, I show the CpG methylation status of the 

promoter associated with specific histone modifications. These results have allowed us to 

propose a model where the GH gene is differentially repressed by polycomb (long term 

silencing) and HP1 (short term repression) proteins in distinct pituitary cell types. Using 

BAC recombination to substitute a fluorescent gene into the GH gene locus and to delete 

upstream distally located regulatory elements, I have created multiple transgenic mice that 

demonstrate the importance of a GH enhancer or locus control region and a repetitive 

element that are critical for proper regulation of the GH locus. 

41



Bob Kingston Abstract 25 

Possible roles in silencing for piRNAs 

Bob Kingston, Anita Seto, Nelson Lau, Jinkuk Kim, David Bartel, 

Jonathan Dennis and Caroline Woo 

Massachusetts General Hospital, 185 Cambridge Street, CPZN7250, Boston MA 02114, U.S.A. 

Maintaining a silent state requires the targeting of epigenetic regulatory complexes to 

specific genes. Small noncoding RNAs have been proposed to play a role in this targeting, 

based on studies in numerous model organisms. Because these RNAs appeared to be 

enriched meiotic cells in model organsims, we made extracts from Rat testes to try to 

identify RNAs that might be involved in silencing in mammals. From these extracts, we 

purified a mammalian complex that might function in transcriptional gene silencing (TGS). 

This complex, called piRC, contains small RNAs and Riwi, the rat homolog to human Piwi. 

The RNAs, frequently 29–30 nt in length, are called Piwi-interacting RNAs (piRNAs), 94% of 

which map to 100 small (



Michael Grunstein Abstract 26 

Deacetylation of histone H4 K16 regulates gene activity in 

yeast 

Wei Xie, Amy Wang and Michael Grunstein 

Department of Biological Chemistry and the Molecular Biology Institute, Boyer Hall, UCLA, 

Los Angeles, CA. 90095, U.S.A. 

We have found that of all the H4 sites of acetylation, K16 when deacetylated is uniquely 

involved in the silencing of heterochromatin through its interaction with the silencing protein 

Sir3. This site is also uniquely involved in gene activation in euchromatin. Surprisingly, while 

histone hyperacetylation is generally correlated with gene activity, it is the hypoacetylation of 

K16 that is associated with gene activity genome wide. This occurs in part through its 

recruitment of the bromodomain containing transcription factor, Bdf1. We describe here the 

enzymes which deacetylate and acetylate K16 and their recruitment during gene activity to 

dynamically regulate transcription. 

43



Ann Ehrenhofer-Murray Abstract 27 

A role for the HDAC Rpd3 in establishing eurchromatinheterochromatin 

boundaries at yeast telomeres 

Stefan Ehrentraut and Ann E. Ehrenhofer-Murray 

Universität Duisburg-Essen, 45117 Essen, Germany 

Eukaryotic genomes are organized into euchromatic and heterochromatic regions. Cells 

need to ensure that the respective chromatin states are restricted to their location in order 

to prevent inappropriate gene expression. 

In Saccharomyces cerevisiae, spreading of the telomeric Sir2/ Sir3/ Sir4 heterochromatin 

complex is prevented by the activity of the HAT complex SAS-I, which acetylates lysine 16 

of histone H4, a residue that is required in the deacetylated state for the SIR complex to 

bind to chromatin. In sas2-delete(D) cells, SIR spreads to more centromere-proximal 

positions and causes repression of subtelomeric genes. However, the SAS2 deletion is not 

lethal in yeast. 

Here, we performed a genetic screen to identify factors that become lethal in the absence of 

Sas2. Surprisingly, we found that the absence of the HDAC Rpd3 was synthetically lethal in 

combination with sas2D. The lethality was specific for rpd3D and sas2D in that no other 

HDAC deletion was lethal with sas2D, and no other HAT deletion was lethal with rpd3D. 

Furthermore, the lethality depended on the components of the Rpd3(L) complex and on all 

SAS-I components. Our observations suggest parallel functions of the two protein 

complexes despite their opposing enzymatic activities. 

Significantly, we found that the lethality of sas2D rpd3D cells was caused by inappropriate 

spreading of SIR complexes, because the lethality was suppressed by sir2/3/4D. In line with 

this, we found by ChIP analysis that Sir2 was more abundant at telomeres and in 

subtelomeric regions in rpd3D cells than in wild-type. Furthermore, subtelomeric genes 

were more repressed in rpd3D cells than in wild-type as measured by qRT-PCR, and the 

repression was abrogated in the absence of SIRs. 

Altogether, our data show a novel and unexpected role for Rpd3 in preventing spreading of 

SIR complexes into euchromatic regions, indicating that Rpd3 exerted a boundary function 

at yeast telomeres. 

44



Wyatt Yue Abstract 28 

CARM1 and Histone Methylation - a Structural Study 

W.W. Yue 1 , V. Thompson Vale 1 , M. Hassler 1 , S. Kisakye-Nambozo 1 , 

M. Roe 1 , T. Kouzarides 2 and L.H. Pearl 1 

1 

Section of Structural Biology, Institute of Cancer Research, 237 Fulham Road, London 

SW3 6JB, UK; 2 Gurdon Institute and Department of Pathology, Tennis Court Road, 

Cambridge CB2 1QN, U.K. 

Covalent histone modifications have important roles in transcriptional regulation by effecting 

changes in the chromatin structure. Coactivator-associated arginine methyltransferase 1 

(CARM1) methylates histone H3 at Arg17 and Arg26 upon hormonal activation of nuclear 

receptors. It enhances transcriptional activation through synergistic interactions with 

coactivators such as GRIP-1 and the histone acetyltransferase CBP. 

CARM1 contains a conserved protein arginine methyltransferase (PRMT) catalytic domain 

flanked by unique N- and C-terminal extensions. We have determined the 2.8 Å structures 

of the CARM1 catalytic domain alone, and in complex with the cofactor product AdoHcy. 

The catalytic domain consists of a cofactor binding region and a nine-stranded ß-barrel. 

The binding of AdoHcy allows the ordering of the N-terminal helix α1, which forms 

extensive contacts with AdoHcy. This almost completely buries the cofactor and limits the 

accessibility of the arginine substrate to a narrow channel. Helix α1 also forms the upper 

ridge of a proposed substrate binding groove lining the entrance to the active site. The 

bottom ridge of this groove is formed by the first 10 residues of the CARM1 C-terminal 

extension, which is not present in other PRMT structures. 

Using in vitro methylation and pull-down assays, we have demonstrated that the N- and C- 

terminal extensions are important in contributing to the enzyme activity of CARM1. In 

addition, CARM1 activity towards histone H3 Arg17 is potentiated by pre-acetylation at 

Lys18, suggesting a possible cross-talk mechanism between histone acetylation and 

methylation. We are in the process of determining the structure of a CARM1-cofactorsubstrate 

complex, in order to establish the molecular basis for the substrate specificity of 

CARM1 towards histone H3, which is unique among PRMTs. 

45



Sharon Dent Abstract 29 

Common and unique factors regulate Set1-mediated 

methylation of the Dam1 kinetochore protein and histone H3 

Sharon Y.R. Dent, John A. Latham and Ke Zhang 

Univ. Texas M.D. Anderson Cancer Center, Dept. Biochemistry and Molecular Biol., Unit 

1000, 1515 Holcombe Blvd, Houston, Texas 77030, U.S.A. 

We reported last year that deletion of the SET1 methyltransferase gene suppresses defects 

in chromosome segregation caused by mutations in the IPL1 aurora kinase in yeast (Zhang 

et al. Cell 122, 2005). Mutations in other components of the COMPASS complex also 

suppress the ipl1-2 mutation, but mutations in PAF1 or H2B K123 do not. These results 

indicate that Set1 and the COMPASS complex normally oppose functions of Ipl1, but that 

these effects are independent of the functions of Set1 in transcription initiation and 

elongation that are mediated by Paf1 and H2B ubiquitylation. Moreover, we determined 

ipl1-2 suppression is not related to loss of H3 K4 methylation in set1 mutant cells. Rather, 

Set1 is required for methylation of a kinetochore protein, Dam1, and Dam1 methylation at 

K233 limits phosphorylation of neighboring serines. We have now defined the effects of 

mutations in individual COMPASS components on Dam1 methylation in vivo. We have also 

begun to define upstream factors that are required for methylation of both H3 K4 and 

Dam1, and as well as unique factors required for methylation of each of these substrates. 

Our findings demonstrate that Set1 has important functions in mitosis, and they suggest 

that antagonism between lysine methylation and serine phosphorylation is a fundamental 

mechanism for controlling protein function. 

46



Geneviève Almouzni Abstract 30 

Chromatin assembly factors, histone H3 variants and cell cycle 

Dominique Ray-Gallet, Sophie Polo, Jean-Pierre Quivy, Anja Groth, 

Danièle Roche and Geneviève Almouzni 

UMR 218 CNRS, Institut Curie – Recherche, 26 rue d’Ulm, F-75248 Paris cedex 05, France 

The ordered assembly of chromatin produces a nucleoprotein template capable of 

regulating the expression and maintenance of the genome functions. 

Factors have been isolated from cell extracts that stimulate early steps in chromatin 

assembly in vitro. One such factor, chromatin assembly factor-1 (CAF-1), facilitates 

nucleosome formation coupled to DNA synthesis. It is thought to participate in a marking 

system at the crossroads of DNA replication and repair to monitor genome integrity and to 

define particular epigenetic states. We have begun to approach its critical importance during 

early development in Xenopus laevis and using mammalian cell systems. In addition, we 

have now identified a chromatin assembly pathway independent of DNA synthesis. The 

HIRA protein appears critical for this pathway in Xenopus egg extracts. Notably, CAF-1 was 

part of the the histone H3 complex, H3.1 complex (replicative form) and HIRA of the H3.3 

complex (replacement form) (Tagami et al, 2004, Nakatani et al, 2004). A major goal in our 

laboratory is now to better integrate the function of these factors in vivo during development 

and also in connection with replication, repair and control of histone pools. 

We will discuss our recent findings on this topic and the interrelationships with other 

assembly factors. 

Refs. 

Groth A., Ray-Gallet D., Quivy J.P., Lukas J., Bartek J. & Almouzni G. (2005) Human Asf1 

regulates the flow of S-phase histones during replicational stress. Mol. Cell, 17, 301-311. 

Polo S. & Almouzni G. (2006) Chromatin assembly : a basic recipe with various flavors. 

Current Opinion in Genetics and Development, 16, 104-111. 

Gérard A., Koundrioukoff S., Ramillon V., Sergère J.C., Mailand N., Quivy J.P. & Almouzni G. 

(2006) The replication kinase Cdc7-Dbf4 promotes the interaction of the p150 subunit of 

Chromatin Assembly Factor 1 with proliferating cell nuclear antigen. EMBO Reports , 7, 817-823. 

Polo S., Roche D. & Almouzni G. (2006) Evidence for new histone incorporation marking 

sites of UV-repair in human cells. Cell (in press). 

Loyola A., Bonaldi T., Roche D., Imhof A. & Almouzni G. (2006) Modifications on histone H3 

variants before chromatin assembly potentiate their final epigenetic state. Mol. Cell (in press). 

47



Dmitry Fyodorov Abstract 31 

ATP-dependent deposition of Histone H3.3 by Drosophila 

CHD1 in vivo 

Alexander Konev 1 , Martin Tribus 2 , Alexandra Lusser 2 and Dmitry Fyodorov 1 

1 

Albert Einstein College of Medicine, Bronx, NY 10461, U.S.A.; 2 Innsbruck Medical 

University, Innsbruck, A-6020, Austria. 

The assembly of nucleosomes in vitro is mediated by the concerted action of two groups of 

factors – histone chaperones, such as CAF-1, NAP-1, Asf1 and HIRA, and ATP-utilizing 

enzymes, such as ACF/CHRAC, RSF and CHD1. However, the respective roles of these 

factors in the chromatin assembly process in vivo remain controversial. For instance, it is 

possible that ATP-dependent factors are dispensable for the histone deposition and 

participate only in the spacing of the nascent nucleosomes. 

Drosophila CHD1, in conjunction with the histone chaperone NAP-1, can mediate the 

assembly of periodic arrays of nucleosomes in vitro. To elucidate the biological function of 

CHD1 we generated mutant alleles of Chd1. Homozygous null Chd1 female flies survive to 

adulthood and lay fertilized eggs, but the embryos die before hatching. We have analyzed 

the chromosome structure in developing Chd1 embryos and found that the absence of 

CHD1 impedes decondensation of paternal sperm chromatin and consequently results in 

the development of haploid embryos. 

Sperm decondensation is the earliest developmental instance of genome-scale chromatin 

assembly. The male pronucleus undergoes profound chromatin reorganization, as paternal 

protamines are replaced with maternal histones. This process occurs in a replicationindependent 

manner and involves H3.3 variant but not the canonical H3. We found that in 

the absence of maternal CHD1, H3.3 fails to become incorporated into male chromatin. 

Thus, we demonstrate that CHD1 directly mediates the loading of H3.3 onto DNA in vivo. 

The function of CHD1 in histone H3.3 deposition, combined with the recent finding that the 

delivery of H3.3 to the male pronucleus is dependent on the chaperone HIRA, support a 

molecular model in which CHD1 utilizes HIRA-delivered histones to assemble H3.3- 

containing nucleosomes. The combined action of CHD1 and HIRA defines a novel pathway 

for the replication-independent deposition of variant histones into chromatin. 

The nucleosome assembly in the male pronucleus takes place in the transcriptionally silent 

phase of Drosophila embryonic development. However, both H3.3 and CHD1 are expected 

to function in transcription-coupled nucleosome assembly. Therefore, we analyzed H3.3 

incorporation into chromatin during later developmental stages, after the onset of zygotic 

transcription. We discovered that elimination of maternal CHD1 blocks the assembly of 

H3.3-containing nucleosomes in transcriptionally active chromatin. 

Our work provides the first conclusive evidence that ATP-dependent mechanisms are 

utilized for histone deposition during chromatin assembly in vivo. Hence, molecular motor 

proteins, such as CHD1, function not only in remodeling of existing nucleosomes but also in 

de novo nucleosome assembly from DNA and histones. 

48



Roberto Mantovani Abstract 32 

The histone-fold trimer NF-Y is required to define positive 

histone marks in CCAAT-promoters: a genome-wide analysis. 

Michele Ceribelli, Giacomo Donati, Diletta Dolfini, Giulio Pavesi, 

Alesaandra Viganò and Roberto Mantovani 

Dipartimento di Scienze Biomolecolari, U. di Milano, Via Celoria 26 20133 Milano, Italy 

The CCAAT box is a promoter element, bound by the NF-Y trimer, composed of an H2A- 

H2B-like dimer and a sequence-specific subunit, NF-YA. To gain an unbiased view of NF-Y 

binding in vivo, we performed ChIP on chips with anti-NF-YB antibodies on 3 platforms. (i) 

CpG islands arrays identified 300 genes targeted by NF-Y. Surprisingly, 41% of NF-Y sites 

are not in promoters, but in introns or at distant 3’ or 5’ loci (1). (ii) A more sensitive oligobased 

chip containing 179 human promoters indicated that NF-Y binds to 40/54% of 

promoters, essentially all containing one -or more- CCAAT boxes (2). (iii) We performed 

location analysis on the Nimblegen tiling arrays of chromosomes 20/21/22. This method 

proved to be highly specific, as none of 24 negative locations scored positive, but missed 

some 30% of sites. Consistent with the CpG array, only a minority of the sites -15/20%- are 

in promoters. A positive correlation with active histone –H3-Acetylation and H3-K4- 

trimethylation- was established in parallel ChIP on Chip experiments on the same platform. 

ChIP analysis of cells infected with a dominant negative NF-YA expressing Adenovirus 

showed a remarkable decrease in NF-YB local promoter binding and in the above 

mentioned histone modifications, as well as H3-K79-dimethylation. Similar results were 

obtained transfecting cells with siRNA for NF-YB. Consequently, elimination of NF-Y binding 

leads to transcriptional impairment. These data establish NF-Y as a crucial factor in the 

formation of a positive epigenetic environment around the transcription start sites. 

Refs 

1) Testa et al. J. Biol Chem. 280, 13606-13615 (2005). 

2) Cerebelli et al. Cell Cycle, In press (2006). 

49



Jerry Workman Abstract 33 

Histone modification and chromatin remodeling in transcription 

Bing Li, Kenneth Lee, Mark Chandy, Michael Carey and Jerry Workman 

Stowers Institute for Medical Research 1000 East 50th Street Kansas City, MO 64110 U.S.A. 

Nucleosome remodeling and histone modification play crucial roles in the process of gene 

transcription. Sequence-specific DNA binding transcription activators recruit the SAGA 

histone acetyltransferase complex to gene promoters during activation. Targeted acetylation 

of promoter nucleosomes by the SAGA complex marks them for subsequent displacement 

by the Swi/Snf nucleoosome remodeling complex. This generates nucleosome free regions 

where the general transcription factors and RNA polymerase II can form a preinitiation 

complex. Following initiation the elongating RNA polymerase associates with 

acetyltransferases that co-transcriptionally acetylate nucleosomes in the coding region. This 

acetylation is recognize by the bromo-domain containing RSC complex which remodels 

nucleosomes to assist polymerase passage. The Set2 histone methyltransferase also 

travels with the polymerase and co-transcriptionally methylates histone H3. This methylation 

is subsequently recognized by the chromodomain containing Rpd3S histone deacetylase 

complex which removes the co-transcriptional histone acetylation marks returning the 

stability of nucleosomes within the coding region. 

50


Abstracts – Poster 

Karl Agger 

Abstract P1 

The role of the polycomb group protein RYBP in oncogene 

induced senescence 

Karl Agger 1 , Paul Cloos 1 , Michael Lees 1 and Kristian Helin 1,2 

1 

Biotech Research & Innovation Centre, Fruebjergvej 3 Copenhagen, Denmark; 

2 

Faculaty of Health Sciences, University of Copenhagen, Blegdamsvej 3, 2200 

Copenhagen, Denmark 

Abstracts - Poster 

Several links between deregulated chromatin modifying activities and cancer have been 

described. In our laboratory special attention has been applied to the role of PcG genes in 

the regulation of cell cycle progression and cancer because of their well- established 

oncogenic potential. In an attempt to identify additional proteins involved in PcG related 

chromatin modification we performed multiple Yeast two hybrid (YTH) screens using PcG 

genes as baits. From the results of these screens we constructed a protein interaction map 

of the human Polycomb group proteins. We chose to focus on one of the identified 

interactions between the PcG gene RYBP and the HMT SUV4-20H1. This interaction is 

interesting because it links a new repressive enzymatic activity to the PcG protein family. To 

further characterise the interaction we confirmed the binding between the two proteins by 

co-immunoprecipitation. We found that RYBP and SUV4-20H1 over-expression induced 

heterochromatin foci in U2OS cells. These foci resemble Senescence associated 

heterochromatin foci (SAHF). SAHFs are dapi dense heterochromatin foci that appears in 

cells undergoing senescence, a proliferative arrest that provides a barrier to malignant 

transformation and contributes to the antitumor activity of certain chemotherapies. We found 

that both RYBP and SUV4-20H1 can induce senescence in diploid fibroblasts upon overexpression. 

Additionally we found that ectopic RYBP activity can induce SAHF-like 

structures and the protein RYBP co-localise with these. Hence, RYBP could be directly 

involved in the formation of SAHF at the chromatin level and additionally be involved in 

senescence induction. 

51



Helena Ahlfors 

A novel player in T helper cell differentiation 

Abstract P2 

Helena Ahlfors 1 , Soile Tuomela 1,2 , Tiina Henttinen 1 , Riikka Lund 1 and Riitta 

Lahesmaa 1 

1 

Turku Centre for Biotechnology, University of Turku and Åbo Akademi University, Turku, 

Finland, 2 Turku Graduate School for Biomedical Sciences, Turku, Finland 

Naïve CD4+ T helper lymphocytes can differentiate into two distinct subsets, termed as Th1 

and Th2, according their cytokine expression profiles. These subsets of T helper cells are 

responsible for specific immune functions; Th1 cells contribute to cell-mediated 

inflammatory immunity, while Th2 cells are responsible for humoral responses. Defects in 

the T helper cell differentiation may result in the pathogenesis of various immune mediated 

diseases such as asthma and allergies. We have carried out genome wide gene expression 

profiling during various stages of Th cell polarization to Th1 or Th2 cells to identify potential 

new players involved in the process. In this study we have focused on one Th2-specific 

transcription factor (acronym TF2). The differential expression of TF2 observed with 

microarray experiments was confirmed both at mRNA and protein level during the early Th1 

and Th2 cell polarization. TF2 is clearly induced by interleukin-4 at early time points both on 

mRNA and protein level, and its expression remains at increased level throughout the early 

differentiation process. As STAT6 plays a key role in Th2 cell differentiation, we investigated 

whether downregulation of STAT6 would regulate the expression of TF2. We transfected 

primary human Th cells with plasmid-based constructs of STAT6 siRNAs contaning a 

marker for cell selection and cultured the cells in polarizing conditions. Here we show that 

downregulation of STAT6 by siRNA downregulates the expression of TF2. Characterization 

of putative STAT6 binding sites in the promoter region of TF2 is in progress. In addition, we 

designed and optimized vector-based siRNAs to downregulate the expression of TF2. 

These constructs were transfected to primary human Th cells to elucidate the dosedependent 

effects of TF2 in T helper cell differentiation, growth, proliferation and survival. 

After 24 and 48 hours and 7 days of polarization the cytokine production of the transfected 

cells was analyzed. Interestingly, the knockdown of TF2 changed the cytokine production 

profiles of cells polarized to Th1 and Th2 directions. Samples from the early time points 

were hybridized into Illumina beadarrays for genome wide detection of the genes regulated 

by TF2. Studies in progress aim at further characterization of the role of TF2 in Th cell 

differentiation. 

52



Barbara Alberter 

Abstract P3 

Histone modification pattern of the T lymphotropic 

Herpesvirus saimiri genome in latency 

Barbara Alberter and Armin Ensser 

Institut für Klinische und Molekulare Virologie, Friedrich-Alexander Universität Erlangen- 

Nürnberg, D-91054 Erlangen, Germany 

Herpesvirus saimiri (HVS) is the prototypic γ2-herpesvirus. Its circular dsDNA genome (155 

kb) consists of an AT-rich coding region harbouring at least 77 open reading frames (orf) 

and a non-coding region which is made up of tandem repetitive elements with high GC 

content. After infection HVS establishes latency in its natural host, the new world primate 

Saimiri sciureus. Experimentally infected and thereby growth transformed human T cells 

also retain latent HVS genomes. Here, only the viral orf1 (bicistronic transcript, StpC and 

Tip) and orf73 (LANA homolog) are transcribed. StpC and Tip are essential for the 

transformation of T cells to antigen-independent growth and the orf73/LANA supports the 

episomal maintenance of the viral genome in those cells. It is not known how gene 

expression of latent genes on the one hand and repression of the major group of lytic 

genes on the other hand are epigenetically regulated in γ2-herpesviruses. In this study, we 

performed chromatin immunoprecipitation with seven different acetylation or methylation 

specific antibodies followed by quantitative SYBR Green PCR to profile the histone 

modifications in the herpesviral genome. Four studied promoters of lytic genes carry 

repressive marks, whereas the orf73 promoter revealed a variable modification pattern. As 

expected, the promoter of orf1 was found to be wrapped up in permissive chromatin, but to 

our surprise the most permissive chromatin structure was revealed in the non-coding 

repetitive elements at the ends of the genome. 

53



Marco Alvarez 

Abstract P4 

Histone variant macroH2A is an epigenetic factor involved in 

the modulation of ribosomal gene expression during 

seasonal adaptation of carp fish 

Pinto R., Bouvet P. 1 , Dimitrov S. 2 , Molina A., Vera M.I. and Alvarez M. 

Departamento de Ciencias Biologicas, Universidad Andres Bello and Millennium Institute for 

Fundamental and Applied Biology, Santiago, Chile, 1 

Ecole Normale Superieure, Lyon, 

France, 2 Institut Albert Bonniot, Grenoble, France 

Cyprinus carpio acclimatization is a process mediated through molecular mechanisms that 

coordinate a homeostatic state in response to cyclic environmental changes. Consequently, 

molecular and cellular functions are reprogramd during seasonal adaptation of the fish. This 

“phenotypic plasticity” is the result of fine and coordinated regulation of gene expression. 

MacroH2A is a histone variant associated with epigenetic mechanisms of gene silencing. 

Previously, we have reported that high levels of macroH2A correlate with hypermethylation of 

the carp rDNA gene promoter during winter, concomitant with a lower transcription level of 

ribosomal genes. Altogether these observations seem to suggest that histone variant 

macroH2A could be involved into the seasonal regulation mechanisms of the carp ribosomal 

biogenesis. 

In the present work, by means of chromatin immunoprecipitation (CHIP), we demonstrated 

that macroH2A is present in the rDNA cistron during the cold season. Furthermore, realtime 

PCR analyses of these experiments confirmed that macroH2A is mainly enriched in 

the promoter region of the rDNA compared to summer season. Moreover, we tested the 

nuclease accessibility of carp rDNA promoter and here we show that the enzyme 

accessibility decrease during winter suggesting a more compact state of the chromatin. In 

conclusion, we postulate that an epigenetic mechanism like histone replacement by its 

variants, particularly the replacement of H2A by macroH2A, plays a central role in the 

regulation of ribosomal genes expression in carp fish. 

FONDECYT 1040197; DI-UNAB 37-04 

54



Terra G. Arnason 

Abstract P5 



Terra G. Arnason, Megan D. Dash, Gerald F. Davies and Troy A. A. Harkness 

Department of Anatomy and Cell Biology, University of Saskatchewan, Saskatoon, SK, Canada 

Chromatin assembly in yeast is regulated by a complex molecular network governed in part 

by the ubiquitin ligases Rsp5p, the Anaphase Promoting Complex (APC) and the SCF. We 

have shown that Rsp5p, localized exclusively to the plasma membrane and adjacent to 

vacuoles, triggers nuclear APC activity by blocking the activity of APC inhibitors, such as 

the SCF. The APC then initiates replication-independent, but CAF-I-dependent, chromatin 

assembly. Here, we demonstrate another mechanism leading to Rsp5p-dependent APC 

activity. Mutation to RSP5 leads to increased histone H3 phosphorylation and decreased 

histone H3 acetylation at elevated temperatures. We show that the histone H3 kinase, 

Snf1p, is required for the rsp5 phenotype. Interestingly, we previously demonstrated that the 

Snf1 kinase complex, which shuttles across the nuclear membrane, is required for APC 

activity. Thus, we propose that Rsp5p is required for the transit of Snf1p across the nuclear 

membrane. In support of this theory, we show that GFP-tagged Snf1p, Snf4p (activator 

subunit) and Gal83p (localizing subunit) all fail to localize to the nucleus upon carbon stress 

in rsp5 mutant cells. Similarly, the GFP-tagged Snf1p target, Mig1p, failed to exit the nucleus 

in rsp5 mutants. We next asked whether Snf4p, which requires ubiquitination for stability 

and function, requires Rsp5p or any of the Rsp5p associated E2 enzymes. In ubiquitin 

coimmunoprecipitation (CoIP) experiments, we recovered GST-Snf4p bound to ubiquitin, but 

not GST alone. We observed that carbon stress induced an increase in ubiquitinated GST- 

Snf4p in wild type cells. When ubiquitin was CoIPed from ubc4p ubc5p cells, GST-Snf4p 

was again recovered, but we failed to observe induction of ubiquitinated GST-Snf4p upon 

carbon stress. The influence of i) Rsp5p, ii) the Snf1p and Rsp5p interacting protein, Rod1p, 

and iii) Ubc7p, an E2 that physically interacts with Rsp5p, on Snf4p ubiquitination will be 

discussed. 

55



Stuart P. Atkinson 

Abstract P6 

Epigenetic mechanisms of pluripotency and differentiation 

Stuart P. Atkinson and Anna Golebiewska 

Institute of Human Genetics, International Centre for Life, Central Parkway, Newcastle upon 

Tyne, NE1 3BZ. U.K. 

A great deal of recent research has concentrated on the epigenetic basis of pluripotency of 

human and mouse embryonic stem cells (ESCs) and studies have also suggested that the 

differences between various cell types may be due to differences in global epigenetic 

profiles. Understanding the epigenetic differences between pluripotent cell, such as human 

ESCs, and differentiated cell lines may allow further understanding of the role epigenetics 

plays in development. Human embryonal carcinoma (EC) cells are first utilised as a model. 

The chromatin environment of promoters of genes involved in pluripotency, self renewal and 

early stages of differentiation were studied in great detail in undifferentiated EC cells as 

well as after ATRA-mediated differentiation. An active chromatin configuration, such as high 

levels of H3K4 methylation and histone acetylation, was observed in EC cells at the 

promoters of genes involved in maintenance of pluripotency and self-renewal (e.g. Sox2, 

Oct4, and Nanog). A similar pattern was observed at the promoters of genes involved in 

early stages of differentiation (e.g. Gata2, Pax6) with additional H3K27 methylation 

suggesting that such genes were primed for expression, but still kept in repressed state in 

pluripotent EC cells. During differentiation, such promoter configurations become more 

active linking this to expression of such genes in differentiated ECs, whereas more 

repressed pattern was observed at promoters of genes involved in pluripotency and selfrenewal 

(e.g. presence of H3K9me2). These studies and the growing knowledge of the 

epigenetic state of hES cells could potentially be used to devise reprogramming strategies 

to induce ‘stem-like’ phenotypes in differentiated cells through the modulation of epigenetic 

mechanisms. In the future these studies may facilitate the production of isogenic tissues for 

regenerative therapy without the ethical and logistical problems associated with therapeutic 

cloning in humans. 

56



Joanne L. Attema 

Abstract P7 

Epigenetic features of hematopoietic stem cells using small 

numbers of highly purified primary cells 

Joanne L. Attema 1 , Peter Papathanasiou 1 , E. Camilla Forsberg 1 , Jian Xu 2 , 

Stephen T. Smale 2 and Irving L. Weissman 1 

1 

Institute of Stem Cell Biology and Regenerative Medicine, Departments of Pathology and 

Developmental Biology, Stanford University School of Medicine, Stanford, California, U.S.A; 

2 

Howard Hughes Medical Institute, Molecular Biology Institute and Department of 

Microbiology, Immunology, and Molecular Genetics, University of California, Los Angeles, 

California, U.S.A. 

Hematopoietic stem cells (HSC) are endowed with the ability to produce all blood cell 

lineages through their capacity to self-renew and differentiate to descendent progenitors 

with restricted potential. Recent studies have shown that HSC express many different 

lineage-affiliated genes at low levels, leading to the hypothesis that epigenetic marks may 

exist at these loci for their critical expression during differentiation. We investigated this by 

examining histone and DNA modifications at lineage-affiliated genes in prospectively 

purified hematopoietic stem and progenitor cells. Here, we describe a method that allows for 

the analysis of DNA and associated histones from as few as 50,000 primary cells. We 

found that histone modifications and unmethylated CpG dinucleotides co-localize across 

defined regulatory regions of key lineage-affiliated genes in HSC that were either 

maintained or lost in the committed progenitors consistent with their expression. These data 

support a model in which epigenetic histone modifications are present at lineage-affiliated 

genes in HSC and could serve as an epigenetic-based mechanism that underlies 

multipotency. 

57



Kristin Baetz 

Abstract P8 

NuA4 is a cellular “Hub”: an integrative map of physical and 

genetic interactions mediated by the NuA4 histone 

acetyltransferase 

Leslie Mitchell, Wendan Chen, Maria Gerdes, Jean-Philippe Lambert, 

Daniel Figeys and Kristin Baetz 

Ottawa Institute of Systems Biology, Department of Biochemistry, Microbiology and 

Immunology, University of Ottawa, Ottawa, Ontario, Canada K1H 8M5 

NuA4 is the only essential histone acetyltransferase in the budding yeast Saccharomyces 

cerevisiae and has established roles in transcription, DNA repair and faithful chromosome 

segregation. NuA4 acetylates histone H4, H2A and the histone variant Htz1, however the 

exact molecular mechanisms by which NuA4 mediates its cellular processes are poorly 

understood. Nor is it known if NuA4 has additional cellular roles or acetylation targets. To 

better understand the cellular functions of NuA4 we have exploited the biochemical and 

genetic amenabilities of the seven non-essential subunits of NuA4 – Eaf1, Eaf3, Eaf5, 

Eaf6, Eaf7, Yaf9 and Yng2. Physical and genetic interactions centered on the non-essential 

subunits of NuA4 were mapped at high resolution using systematic proteomic and genomic 

methods. Physical interactions were identified using large scale affinity purification of NuA4 

complex in all seven non-essential NuA4 mutant backgrounds to establish the contribution 

of each subunit to NuA4 complex integrity and their role in mediating interactions with non- 

NuA4 proteins. Genetic interactions for each of the non-essential NuA4 subunits were 

uncovered using genome-wide synthetic genetic array technology. An extended network, 

consisting of more then 300 genetic and physical interactions, was found to connect NuA4 

to a wide range of cellular functions and has identified several novel cellular roles for NuA4, 

including golgi-vacuole protein transport. The NuA4 network is helping to define roles for 

each of the non-essential subunits of NuA4 and we determined that there is a direct 

correlation between a subunit’s contribution to NuA4 complex integrity and the extent of 

genetic interactions identified. For example unlike most of the non-essential subunits, Eaf1 

is crucial to NuA4 complex stability and displays greater than100 genetic interactions 

suggesting that Eaf1 may be a scaffold protein for the NuA4 complex. Our extensive 

network indicates that NuA4 is a “hub” of fundamental importance in the cell. 

58



Slobodan Barbaric 

Abstract P9 

Chromatin remodeling activities at the yeast PHO84 promoter 

B. Silic 1 , T. Luckenbach 2 , S. Stuerzl 2 ,P. Korber 2 and S. Barbaric 1 

1 

Laboratory of Biochemistry, Faculty of Food Technology and Biotechnology, University of 

Zagreb, 10000 Zagreb, Croatia and 2 Adolf-Butenandt-Institut, Universitaet Muenchen, 

Schillerstr. 44, 80336 Muenchen, Germany 

The yeast PHO84 promoter, which is coregulated with the well studied PHO5 and PHO8 

promoters in response to phosphate availability, is the strongest promoter of the PHO family, 

containing five binding sites for the specific activator Pho4. Under repressive conditions there 

is a short hypersensitive region in the promoter containing two closely positioned Pho4 

binding sites, but upon induction the promoter chromatin structure is altered, so that at least 

one nucleosome upstream and one downstream from the hypersensitive region are 

remodeled. Remodeling of chromatin structure leads to histone depletion from the promoter 

region. The rate of histone eviction and consequently the rate of promoter activation are 

strongly delayed in mutants deleted for either Snf2 or Gcn5. Nonetheless, after prolonged 

induction full activation is achieved, but in the absence of Snf2 chromatin remodeling is only 

partial, resulting in displacement of the downstream but not of the upstream nucleosome. 

Therefore, eviction of these two nucleosomes requires different chromatin remodeling 

activities. 

In contrast to Snf2, Gcn5 is not required for efficient remodeling of both nucleosomes upon 

full induction, either in the presence or absence of Snf2. Similarly to Gcn5, the absence of 

Ino80 also causes delay in the promoter activation without affecting the final extent of 

chromatin remodeling and the same is true for the histone chaperone Asf1. Taken together, 

Gcn5, Ino80, or Asf1 affects only the rate of remodeling at the PHO84 promoter, similar as 

we previously found for the PHO5 promoter. However, with respect to the requirement for Snf2, 

the PHO84 promoter chromatin structure possesses hybrid characteristics compared to the 

two coregulated promoters: the PHO8 is essentially dependent on Snf2 and at the PHO5 only 

the rate of chromatin remodeling is reduced in the absence of Snf2. 

We have also examined a possible role of the histone variant H2A.Z in regulation of chromatin 

remodeling at PHO promoters. Interestingly, the absence of H2A.Z has only a slight effect on 

activation of the PHO5 promoter, but causes a strong delay in the activation kinetics of the 

PHO84 promoter. This provides additional evidence that chromatin remodeling process at the 

two coregulated promoters involves different mechanisms. 

59



Vivian Bardwell 

Abstract P10 

Polycomb group and SCF ubiquitin ligases are found in a 

novel BCOR complex that is recruited to BCL6 targets 

Micah Gearhart, Connie Corcoran, Joseph Wamstad and Vivian Bardwell 

Department of Genetics, Cell Biology and Development and the Cancer Center, University 

of Minnesota, Minneapolis, MN 55455, U.S.A. 

The corepressor BCOR potentiates transcriptional repression by the proto-oncoprotein 

BCL6 and suppresses the transcriptional activity of a common mixed-lineage leukemia 

fusion partner, AF9. Mutations in human BCOR cause male lethal, X-linked oculofaciocardiodental 

syndrome. We identified a BCOR complex containing Polycomb group (PcG) and 

Skp-Cullin-F-box subcomplexes. The PcG proteins include RING1, RYBP, NSPC1, a 

Posterior Sex Combs homolog, and RNF2, an E3 ligase for the mono-ubiquitylation of H2A. 

BCOR complex components and mono-ubiquitylated H2A localize to BCL6 targets, 

indicating that the BCOR complex employs PcG proteins to expand the repertoire of 

enzymatic activities that can be recruited by BCL6. This also suggests that BCL6 can target 

PcG proteins to DNA. In addition, the BCOR complex contains components of a second 

ubiquitin E3 ligase, namely, SKP1 and FBXL10 (JHDM1B). We show that BCOR coimmunoprecipitates 

isoforms of FBXL10 which contain a JmjC domain that recently has been 

determined to have histone H3K36 demethylase activity. The recruitment of two distinct 

classes of E3 ubiquitin ligases and a histone demethylase by BCOR suggests that BCOR 

uses a unique combination of epigenetic modifications to direct gene silencing. 

60



Amrita Basu 

Abstract P11 

Computational prediction of histone and non-histone proteins 

A. Basu 1 , Y. Zhao 2 ,S.Hake 1 , K. Rose 3 , B. Ueberheide 3 , D. Hunt 3 ,C.D. 

Allis 1 and E. Segal 4 

1 

Laboratory of Chromatin Biology, The Rockefeller University, New York NY 10021,U.S.A., 

2 

Department of Biochemistry, UT Southwestern Medical Center at Dallas, Dallas, TX 

75390,U.S.A., 3 Department of Chemistry, University of Virginia, Charlottesville, VA 22904, 

U.S.A., 4 Department of Computer Science and Applied Mathematics, The Weizmann 

Institute of Science, Rehovoth 76100, Israel 

Acetylation, a well-studied post-translational modification,.plays essential regulatory roles in 

a broad spectrum of biological processes, notably gene regulation. Although many studies 

have been contributed on the molecular mechanism of acetylation dynamics, the intrinsic 

features of substrate site specificity are still elusive and remain to be critically defined to a 

point where predictions of unknown acetylation sites can be made with reasonable 

accuracy. Since many enzymatic processes that modify histones have been identified and 

characterized, it is highly probable that these histone-modifying enzymes target nonhistones 

in mammalian cells, as illustrated by the p300/CBP requirement for p53 and HIV1 

acetylation in human cells (1,2). Whether or not these enzymes target the substrate in a 

specific manner and whether sequence composition surrounding the modified amino acids 

plays an important role remains to be elucidated. In this work, using a sequence based 

bioinformatics approach, we show that there is a bias towards specific amino acids 

surrounding an acetylated lysine that may result from HAT recognition in histones and 

nuclear non-histone proteins. We hope that our approaches can be used as a predictive 

method in identifying acetylated substrates in the human proteome. 

Refs: 

(1) Gu W and Roeder R.G, Activation of p53 sequence-specific DNA binding by acetylation 

of the p53 C-terminal domain. Cell 1997. 90: pp. 595–606. 

(2) Kiernan R.E. et al., HIV-1 tat transcriptional activity is regulated by acetylation. EMBO J, 

1999, 18: pp. 6106–6118. 

61



Mark T. Bedford 

Screening for the methylated proteome 

Abstract P12 

Donghang Cheng and Mark T. Bedford 

University of Texas M.D. Anderson Cancer Center, Department of Carcinogenesis 

PO Box 389, 1808 Park Road 1-C, Smithville, TX 78957, U.S.A. 

The coactivator associated arginine methyltransferase, CARM1, is recruited by many 

different transcription factors as a positive regulator. To understand the mechanism by which 

CARM1 functions, we sought to isolate its substrates. We developed a small-pool screening 

approach for this purpose and identified CA150, SAP49, SmB and U1C as splicing factors 

that are specifically methylated by CARM1. We further showed that CA150, a molecule that 

links transcription to splicing, interacts with the tudor domain of the spinal muscular atrophy 

protein SMN, in a CARM1-dependent fashion. Experiments with an exogenous splicing 

reporter and the endogenous CD44 gene revealed that CARM1 promotes exon skipping in 

an enzyme-dependent manner. The identification of splicing factors that are methylated by 

CARM1, and protein-protein interactions that are regulated by CARM1, strongly implicate 

this enzyme in the regulation of alternative splicing and points towards its involvement in 

spinal muscular atrophy pathogenesis. 

62



Sukesh R. Bhaumik 

Abstract P13 

Regulation of transcriptional activation by mRNA cap-binding 

complex in vivo 

Pratibha Bajwa, Abhijit Shukla, Nadia Stanojevic and Sukesh R. Bhaumik 

Department of Biochemistry and Molecular Biology, Southern Illinois University School of 

Medicine, Carbondale, IL-62901, U.S.A. 

Eukaryotic gene regulation is largely controlled at the level of transcriptional activation by 

gene-specific activators which function by stimulating the assembly of general transcription 

factors to form a preinitiation complex (PIC) at the promoters of the active genes. 

Interestingly, we show here that mRNA cap-binding complex (CBC) dramatically stimulates 

formation of the PIC assembly and subsequently transcription in vivo. However, CBC is 

recruited to the active gene following formation of the PIC assembly. These results reveal 

that PIC and CBC are intimately coupled via their reciprocal synergism, providing a novel 

regulatory pathway of transcriptional activation by CBC in a positive feedback mechanism. 

63



Marjorie Brand 

Abstract P14 

The Ash2L/MLL2 methyltransferase complex is important for 

ß-globin transcription during erythroid differentiation 

Patrick Lai 1 , Jeffrey A. Ranish 2 , Celina Demers 1 , Gaetan Juban 3 , Francois 

Morle 3 , Ruedi Aebersold 2,4 , F. Jeffrey Dilworth 1 , Mark Groudine 5 and 

Marjorie Brand 1 

1 

Sprott Center for Stem Cell Research, Ottawa Health Research Institute, Ottawa, ON, 

Canada, 2 Institute for Systems Biology, Seattle, WA, USA, 3 Centre de Genetique Moleculaire 

et Cellulaire, Villeurbanne, France, 4 Institute for Molecular Systems Biology, Zurich, 

Switzerland, 5 Fred Hutchinson Cancer Research Center, Seattle, WA, U.S.A. 

The mammalian ß-globin locus is comprised of five clustered ß-like globin genes, whose 

developmental-specific expression is regulated by the distal locus control region (LCR), 

located dozens of kilobases upstream of the genes. Methylation of histone H3 at lysine 4 

(H3-K4) occurs at the ßMaj-globin gene during erythroid differentiation and is dependent 

upon the hematopoietic-specific transcription factor NF-E2-p45. However, the enzyme 

responsible for H3-K4 methylation at the ß-globin locus is unknown. 

Here, we set out to identify methyltransferase(s) interacting with NF-E2-p45 in erythroid 

cells using immunoprecipitation and mass spectrometry. Strikingly we found that NF-E2- 

p45 associates with 2 distinct histone H3 methyltransferases: G9a which modifies lysine 9, 

and MLL2 which is specific for lysine 4. Interestingly, in vitro these 2 methyltransferases 

display competing activities, which appear to be regulated by the acetylation status of their 

target, suggesting a new element to the histone-code. Using chromatin 

immunoprecipitation, we then showed that, during erythroid differentiation, the MLL2 

complex is recruited to the ß-globin LCR, 38kb upstream of the ßMaj-Globin gene. 

Interestingly, MLL2 (but not the Ash2L subunit of the MLL2 complex) is also recruited 

across the entire ß-globin locus from the LCR to the transcribed ß-Majglobin area, 

suggesting that the H3-K4 methyltransferase is transported across the ß-globin locus via a 

spreading mechanism. Finally, in contrast to the recent proposition that trimethylation of H3- 

K4 and H3-K9 co-exists within the active ßMaj-globin gene, we demonstrate that these 2 

modifications are mutually exclusive, trimethylated H3-K4 being instead strongly correlated 

to acetylated H3-K9. In fact, we found that the ß-Majglobin transcribed area (methylated on 

H3-K4 and acetylated on H3-K9) is flanked by regions that are enriched in trimethyl-H3-K9, 

while being poorly trimethylated on H3-K9 itself. This is in agreement with earlier 

suggestions that one role of trimethyl-H3-K4 is to prevent spreading of the repressive H3- 

K9 trimethyl mark. Thus altogether our results provide new insights into the regulation of 

transcription via recruitment and spreading of the H3-K4 methyltransferase MLL2 to specific 

genes during development. 

64



Lauren Buro 

Abstract P15 

Histone methylation patterns at interferon-gamma inducible 

gene loci 

Lauren Buro and Melissa Henriksen 

Department of Biological Sciences, Fordham University, Bronx, NY 10458, U.S.A. 

Here we examine the lysine methylation profile of histone H3 (H3) at several interferon 

gamma (IFN-() induced, STAT1 dependent gene loci. STATs are a family of transcription 

factors that, in response to a variety of extracellular ligands, are rapidly and transiently 

recruited from their latent state in the cytoplasm to the nucleus where they drive 

transcription of target genes, affecting growth, differentiation, homeostasis and the immune 

response. The accessibility of the activated STAT to its DNA binding site and the 

succeeding gene expression are intimately tied to chromatin structure. Post-translational 

modifications of the residues in the amino terminal tails of the lysine residues of histones 

define the functional state of the chromatin. Methylation of lysine residues of H3 is 

described as either activating or repressive of gene expression, with the activity correlated 

to the position of the lysine residue (K4 or K9), the level of methylation (mono, di or 

trimethylation) and the location of the nucleosome itself (promoter or transcribed regions, 

euchromatin or heterochromatin). Chromatin immunoprecipitation followed by real-time 

PCR revealed K4 dimethylation (K4M2) and K4 trimethylation (K4M3) primarily in the 

control regions of all the IFN-( inducible genes assayed, independent of transcriptional 

activity. That such methylation was not dynamic suggests a role for these modifications only 

in poising these genes for transcription. Further experiments to determine if K4M2 and/or 

K4M3 are permissive for gene induction are underway. H3 K9 trimethylation (K9M3), a 

modification reportedly associated with transcription elongation was not observed in the 

coding regions of the IFN-gamma inducible genes. 

65



Jill S. Butler 

Abstract P16 

CXXC-finger Protein 1 regulates Dnmt1 protein expression 

Jill S. Butler and David G. Skalnik 

Indiana University School of Medicine, Herman B Wells Center for Pediatric Research 

1044 W. Walnut Street, R4 Room 324, Indianapolis, IN 46202, U.S.A. 

Cytosine methylation and histone tail modifications are two epigenetic modifications that 

influence gene expression. Elucidation of epigenetic regulation is becoming increasingly 

important as deregulation of epigenetic processes is observed in many diseases, including 

cancer. The CXXC1 gene encodes CXXC finger protein 1 (CFP1), a transcriptional activator 

that specifically binds unmethylated CpG dinucleotides. This DNA binding activity of CFP1 

makes it unique in that most CpG binding proteins bind methylated CpG dinucleotides and 

facilitate heterochromatin formation. CFP1 has recently been identified as a component of 

the mammalian SET1 histone H3 lysine 4 methyltransferase complex (Lee and Skalnik, JBC 

(2005) 280:41725-31). Disruption of CXXC1 in mice results in an early embryonic lethal 

phenotype (Carlone and Skalnik, MCB (2001) 21:7601-6), and embryonic stem (ES) cells 

lacking CFP1 exhibit multiple epigenetic defects including altered histone modifications and 

reduced global cytosine methylation (Carlone, et al, MCB (2005) 21:4881-91). DNA 

methyltransferase 1 (Dnmt1) is the major source of maintenance DNA methyltransferase 

activity in mammalian cells and is primarily responsible for copying cytosine methylation 

patterns during DNA replication. Dnmt1 protein level and DNA methyltransferase activity 

are decreased by ~50% in CXXC1-/- ES cells and are rescued by stable expression of 

murine CFP1. Northern blot analysis along with real-time PCR experiments revealed 

Dnmt1 transcript level is elevated ~ 40% in CXXC1 -/- ES cells. Additionally, 

immunoprecipitation experiments revealed an interaction between CFP1 and Dnmt1 in vivo. 

Regulation of Dnmt1 protein level by CFP1 is the first example of reduced Dnmt1 protein 

without direct disruption of Dnmt1 gene function. The functional significance of this novel 

intersection of epigenetic regulatory proteins is currently under investigation. 

66



Jim Cakouros 

Abstract P17 

Identification of a novel enzyme which regulates the kinetics 

of histone arginine methylation in Drosophila melanogaster 

D. Cakouros, T. Daish and S. Kumar 

Hanson Institue, Department of Haematology, Frome Road, Adelaide, Australia 

The sequential modifications of histones form the basis of the histone code which 

translates into either gene activation or repression. The dynamic fluctuations of histone 

methylation occurs in response to specific signals, regulated by the interplay of histone 

methyltransferases and histone demethylases. Nuclear receptors recruit a cohort of histone 

modifying enzymes in response to ligand binding and regulate proliferation, differentiation 

and programd cell death (PCD). In Drosophila, coactivators for the nuclear ecdysone 

receptor (EcR/UsP) have not been extensively examined. We have identified a novel 

cofactor for the EcR which is normally involved in amino acid catabolism. This enzyme 

contains two enzymatic domains in its amino and carboxyl termini respectively, binds 

EcR/UsP directly and potentiates ecdysone mediated transcription. We found that it can 

inhibit H3-R17 dimethylation mediated by the drosophila methyltransferase CARMER 

(DART4) by directly binding the amino tail of histone H3 specifically and this inhibition is 

dependant on the cofactors ketoglutarate and NADH. In drosophila cells this protein 

regulates the kinetics of H3-R17 dimethylation and knockdown by RNAi can compromise 

ecdysone mediated transcription and PCD. The in vivo function of this enzyme is currently 

being examined in flies. 

67



Raymond Camahort 

Abstract P18 

Genome-wide analysis of the budding yeast histone variant 

Cse4 reveals occupancy at a single centromeric nucleosome 

as well as additional non-centromeric locations 

Raymond Camahort 1,2 , Bing Li 1 , Brian Fleharty 1 , Jerry L. Workman 1 , Chris 

Seidel 1 , Jennifer L. Gerton 1,2 

1 

Stowers Institute for Medical Research, Kansas City, MO 64110, U.S.A., 2 Department of 

Biochemistry and Molecular Biology, 4011 Wahl Hall East, 3901 Rainbow Blvd., University 

of Kansas Medical Center, Kansas City, KS 66160, U.S.A. 

Cse4 is the S.cerevisiae centromeric H3 histone variant, also known as CENP-A. This 

histone variant is incorporated into nucleosomes that are located at centromeres in budding 

yeast and are required for proper kinetochore assembly and chromosome segregation. 

Centromeres in budding yeasts are defined by a specific 125 bp sequence that contains the 

elements CDEI, CDEII, and CDEIII. We demonstrate the localization of Cse4 to the sixteen 

centromeres of budding yeast chromosomes, as expected, and additionally the surprising 

result that Cse4 nucleosomes are located at other regions in the yeast genome, mainly 

repetitive regions including telomeres, rDNA, and Ty elements. To verify the localization of 

Cse4 to these regions, we fused Cse4 to a transcriptional activation domain and 

demonstrate that this chimeric protein can activate transcription from the rDNA and Ty 

elements. Using high resolution quantitative PCR we demonstrate that there is a single 

Cse4-containing nucleosome at centromeres in vivo and this nucleosome is positioned over 

centromere sequences. Finally, using purified components, we assemble canonical and 

Cse4-containing nucleosomes in vitro and demonstrate that they can assemble with high 

efficiency on non-centromeric DNA. Our results demonstrate that Cse4 alone is not 

sufficient to nucleate a kinetochore. We suggest that Cse4, in addition to its critical role at 

the centromere, may participate in repressing DNA metabolic processes such as 

transcription and recombination. 

68



Dylan Carney 

Abstract P19 

The RAG2 PHD finger links the histone code to V(D)J 

recombination 

Dylan Carney, Alex Kuo, Tina Christakos and Or Gozani 

Gilbert Biological Sciences, 371 Serra Mall, Stanford, CA 94306, U.S.A. 

The PHD finger (Plant Homeodomain) module is a signature chromatin-associated domain 

that is found throughout eukaryotic proteomes and is mutated in several human diseases. 

One such PHD-containing protein, the Recombination Activating Gene 2 (RAG2), is 

necessary for the V(D)J recombination reaction that is responsible for generating 

immunoglobulin and T-Cell Receptor (TCR) diversity among lymphocytes. Mutations within 

the RAG2 PHD finger have been implicated in Omenns Syndrome, which is a result of an 

inability to effectively carry out V(D)J recombination. Preliminary work has shown that the 

RAG2 PHD domain binds specifically to histone H3 trimethylated at lysine 4 (H3K4me3). 

Furthermore, we have shown that the very same mutation (W453R) implicated in several 

cases of Omenns Syndrome abrogates this binding. Our work suggests that the recognition 

of H3K4me3 by the RAG2 PHD finger plays an important role in V(D)J recombination and 

thus proper immune system function. 

69



Beverly S. Chilton 

Abstract P20 

Analysis of RUSH/SMARCA3 isoforms and their interactions 

with Egr-1 and c-Rel in the regulation of transcription 

Aveline Hewetson and Beverly S. Chilton 

Department of Cell Biology & Biochemistry, Texas Tech University Health Sciences Center, 

3601 4th Street – MS6540, Lubbock, TX 79430, U.S.A. 

RUSH, a SWI/SNF-related regulator of chromatin, was cloned from rabbit endometrium. Steroiddependent 

alternative splicing yields two distinct isoforms (alpha and beta). Alpha has seven highly 

conserved DNA-dependent ATPase domains, and beta has four. Although both isoforms are capable of 

DNA binding, no studies have addressed their individual involvement in any biological process. RUSH is 

highly conserved in eukaryotes and was independently identified as HIP116 (human) and P113 (mouse). 

These names were replaced by the gene name, SMARCA3. In addition to alternative splicing, 

progesterone-induced transcriptional activation is mediated by a bipartite progesterone receptor (PRE) 

half-site/overlapping Y-box combination (-38/-26) in the proximal promoter (-162/+90). Activation by 

progesterone is achieved via the PRE half-site, and attenuated by NF-Y binding to the Y-box. Repression 

is also achieved at two GC-rich sequences in the proximal promoter and a distal RUSH site (-616/-611). 

At each GC-rich site, specific binding of Sp3 was 15-17-fold more abundant than Sp1. The rate of 

complex disassociation (off rate) for the distal A site (-131/-126) with the consensus Sp binding 

sequence (GGGGCGGGG) was half that of the proximal B site (-62/-53) with a variant (GGGGCGGAG) 

sequence. MatInspector (Genomatix) analysis revealed the complexity of the stronger A site. A putative 

Egr-1/Sp/MAZ/MZF1 site on the positive strand overlaps a putative c-Rel site on the negative strand 

(matrix similarity values ( 0.91). Supershift assays with nuclear extracts from progesterone-treated 

animals confirmed the binding of each candidate protein to the composite A site. In contrast, only Sp3 

binds the A site in nuclear extracts from estrous animals, and protein binding to the B site is negligible. At 

the RUSH site, isoform-specific binding was demonstrated with supershift assays. Exclusive binding of 

alpha was confirmed with isoform-specific antibodies and nuclear extract from progesterone-treated 

animals. Exclusive binding of beta to the same site was confirmed with isoform-specific antibodies and 

nuclear extract from estrous animals. TransSignal TF-TF Interaction Arrays showed strong physical 

associations between alpha and DNA-bound Egr-1 and c-Rel. No physical interactions occurred between 

alpha and DNA-bound Sp1, MZF1 or NF-Y. Supershift assays confirmed alpha interacts physically with 

Egr-1 and c-Rel bound to DNA at the A site, and conversely Egr-1 and c-Rel interact with alpha bound to 

DNA at the distal RUSH site. ChIP assays confirmed alpha interacts physically with Egr-1 and c-Rel at 

each site in the transcriptionally active promoter. RUSH/Egr-1 and RUSH/c-Rel interactions were 

visualized by confocal microscopy. The proposed model for this interaction includes the fact that the 

authentic RUSH site is separated from the composite A site by 500-bp of 5(-sequence with a series of 

alternating pyrimidine-purine (CA) elements, which are anisotropically flexible and promote DNA bending. 

Thus progesterone acts via the PRE to induce transactivation. Fine-tuning the magnitude of this 

response includes isoform-specific autorepression in which newly synthesized alpha binds DNA in a 

sequence selective manner, and interacts physically with ligand-bound Egr-1 and c-Rel in the proximal 

promoter. Conversely, when the gene is silent (estrous), beta replaces alpha, and the Egr-1 and c-Rel 

partners are unavailable for binding interactions. Alternative splicing is a powerful means of generating 

macromolecular complexity. Understanding the functional capabilities of alternatively spliced isoforms of 

specific genes that are transcription factors with intrinsic helicase activity will enhance our understanding 

of isoform-distinctive mechanisms of transcriptional regulation. 

70



Alexandra Chittka 

Abstract P21 

Signalling by a novel p75 neurotrophin receptor interacting 

protein, SC1/PRDM4 

Alexandra Chittka 1* , Juan Carlos Arevalo 2 , Maria Rodriguez-Guzman 1 , 

Pilar Perez 3 , Moses V. Chao 2 and Michael Sendtner 1 

1 

*MRC centre for developmental neurobiology, King’s College, Guy’s Campus, London 

Bridge, London SE1 1UL, 1 Institute for Clinical Neurobiology, University of Wuerzburg, 

97080 Wuerzburg, Germany, 2 Molecular Neurobiology Program, Skirball Institute of 

Biomolecular Medicine, New York University School of Medicine, New York, NY 10016, 

3 

Instituto de Microbiologia Bioquimica, CSIC/Departamento de Microbiologia y Genetica, 

Universidad de Salamanca, 37007 Salamanca, Spain 

Schwann cell factor 1 (SC1), a p75 neurotrophin receptor-interacting protein, is a member of 

the positive regulatory/suppressor of variegation, enhancer of zeste, trithorax (PR/SET) 

domain-containing zinc finger protein family, and it has been shown to be regulated by 

serum and neurotrophins. SC1 shows a differential cytoplasmic and nuclear distribution, 

and its presence in the nucleus correlates strongly with the absence of bromodeoxyuridine 

(BrdU) in these nuclei. Here, we investigated potential transcriptional activities of SC1 and 

analyzed the function of its various domains. We show that SC1 acts as a transcriptional 

repressor when it is tethered to Gal4 DNA-binding domain. The repressive activity requires 

a trichostatin A-sensitive histone deacetylase (HDAC) activity, and SC1 is found in a 

complex with HDACs 1, 2, and 3. Transcriptional repression exerted by SC1 requires the 

presence of its zinc finger domains and the PR domain. Additionally, these two domains are 

involved in the efficient block of BrdU incorporation by SC1. The zinc finger domains are 

also necessary to direct SC1’s nuclear localization. Lastly, SC1 represses the promoter of a 

promitotic gene, cyclin E, suggesting a mechanism for how growth arrest is regulated by 

SC1. 

Currently, we are searching for additional target genes of SC1/PRDM4 to address the 

mechanistic role it plays during the development of the nervous system. 

71



Leslie Chu 

Inheritance of epigenetic chromatin states 

Abstract P22 

Leslie Chu and Joachim Li 

University of California San Francisco, 600 16th Street, Genetech Hall, Rm S376, San 

Francisco, CA 94158 U.S.A. 

In eukaryotes, DNA is packaged into either euchromatin or heterochromatin. Euchromatin 

is classically defined as actively transcribed regions of DNA, while heterochromatin is 

defined as transcriptionally silenced regions of DNA. This regional silencing requires the 

formation of a highly compact chromatin structure. Once established, the silent chromatin 

structure is stably maintained throughout the cell cycle and inherited in each subsequent 

generation. The inheritance of silent chromatin is surprisingly stable, given the stresses 

placed on chromatin through out the cell cycle. Previous work has shown that in each cell 

cycle, DNA replication disrupts nucleosomes, the fundamental unit of chromatin. This 

suggests that DNA replication also disrupts the silent chromatin structure. During each cell 

cycle, however, transcriptional silencing remains intact. These studies imply that if DNA 

replication disrupts silent chromatin, then the restoration of silencing is tightly coupled to 

replication. Our studies focus on understanding if DNA replication disrupts silencing, what 

serves as the molecular memory for silent chromatin and identifying proteins required for 

the inheritance of transcriptional silencing. 

Using Saccharomyces cerevisiae, we show that Sir1, a heterochromatin establishment 

protein, and Asf1, a nucleosome deposition factor, are required for the inheritance of 

HMLalpha transcriptional silencing. We also show that progression through S phase, in the 

absence of Sir1 and Asf1, disrupts transcriptional silencing. This finding demonstrates that 

an S phase event, possibly DNA replication, perturbs silencing. We used chromatin 

immunoprecipitation to analyze the chromatin structure of HMLalpha. Interestingly, when 

silencing is not inherited (lost with progression through one cell cycle), an intermediate 

chromatin structure is formed. This intermediate structure contains both euchromatin and 

heterochromatin features. These remaining heterochromatin features may serve as a 

molecular memory to restore the silent chromatin structure. These results suggest that S 

phase disrupts transcriptional silencing, resulting in an intermediate chromatin structure. 

This intermediate chromatin structure may provide the molecular memory that directs Sir1 

and Asf1 restorative silencing activity, resulting in the faithful inheritance of silencing. 

72



Mair Churchill 

Abstract P23 

Structural basis for the histone chaperone activity of Asf1 

Mair E.A. Churchill, Christine M. English, Melissa W. Adkins, Joshua J. 

Carson and Jessica K. Tyler 

Department of Pharmacology, Department of Biochemistry and Molecular Genetics, U. 

Colorado Health Sciences Center, U.S.A. 

Asf1 is a highly conserved chaperone of histones H3/H4 that assembles or disassembles 

chromatin during transcription, replication, and repair. The structure of the globular domain 

of Asf1 bound to H3/H4 determined by X-ray crystallography to a resolution of 1.7 shows 

how Asf1 binds the H3/H4 heterodimer, enveloping the C-terminus of histone H3 and 

physically blocking formation of the H3/H4 heterotetramer. Unexpectedly, the C-terminus of 

histone H4 that forms a mini-beta sheet with histone H2A in the nucleosome, undergoes a 

major conformational change upon binding to Asf1 and adds a beta strand to the Asf1 betasheet 

sandwich. Interactions with both H3 and H4 were required for Asf1 histone 

chaperone function in vivo and in vitro. The Asf1-H3/H4 structure suggests a strandcapture 

mechanism whereby the H4 tail acts as a lever to facilitate chromatin disassembly / 

assembly that may be used ubiquitously by histone chaperones. 

73



Jeffrey Craig 

Abstract P24 

What makes centromeres localise and cluster in interphase 

nuclei 

Irina Solovei 1 , Claudia Weierich 1 , Tatyana Karamysheva 1 , Paul Canham 2 , 

K.H. Andy Choo 3 , Thomas Cremer 1 and Jeffrey M. Craig 2 

1 

Biozentrum (LMU), Grosshaderner Str. 2, Planegg-Martinsried, 82152 Germany, 

2 

Epigenetics Research Laboratory and 3 Chromosome and Chromatin Research Laboratory, 

Murdoch Childrens Research Institute, Royal Children’s Hospital, Flemington Road, 

Parkville, Victoria 3052, Australia 

Centromeres lie at the heart of chromosomes where they choreograph the multiple events 

of cell division. Centromeres are also a prime example of intergenerational epigenetic 

inheritance – their chromatin structure is preserved from one generation to the next. They 

contain large regions of heterochromatin and this has been shown to play a major role in 

establishing structured nuclear domains which can control gene expression. In mammals, 

these domains are composed of clusters of centromeres and are often located close to the 

nuclear periphery. Furthermore, centromere are also localise to the periphery of their 

individual chromosome territories. The roles of centromeres per se and of heterochromatin 

in this nuclear localisation and clustering are unclear. To separate out these roles we have 

looked at the nuclear localisation of human centromeres without tandemly-repetitive DNA. 

These neocentromeres contain only small domains of pericentric heterochromatin. We have 

looked at the three dimensional position of neocentromeres in the nuclei of human cell lines 

using in situ hybridisation and immunolofluorescence using antibodies to centromere 

proteins. We found that consistently, neocentromeres exhibited the same localisation as 

repetitive centromeres within a territory and within the whole nucleus. However, ongoing 

findings are showing that neocentromeres may not cluster with other centromeres in the 

interphase nucleus. These results imply that large regions of heterochromatin exert more 

influence on centromere clustering and the creation of large heterochromatic domains in 

interphase and less influence on positioning with chromosomes and nuclei. 

74



Valerie J. Crusselle-Davis 

Abstract P25 

Regulation of beta-globin expression through the recruitment 

of chromatin modifying enzymes by TFII-I and USF 

Valerie J. Crusselle-Davis, Archana Anantharaman, Tihomir Dodev and 

Jurg Bungert 

Department of Biochemistry and Molecular Biology, Powell Gene Therapy Center, Center for 

Mammalian Genetics, and Shands Cancer Center, University of Florida, Gainesville, FL 

32610 U.S.A. 

The human beta-globin locus contains five functional genes which are arranged in the order 

of their developmental expression. Gene proximal cis-regulatory DNA elements and 

interacting proteins restrict expression of the genes to the embryonic, fetal, or adult stages 

of erythropoiesis. In addition, the relative order of the genes with respect to the locus 

control region also contributes to the temporal regulation of the genes. To more fully 

understand adult beta-globin gene regulation, we examined the downstream promoter and 

found that transcription factors TFII-I and USF interact with elements within this region in 

erythroid cells. TFII-I was found to act as a repressor of beta-globin expression while USF 

proteins were found to act as activators of beta-globin expression. It is becoming 

increasingly clear that one role DNA binding proteins play is to recruit co-activators or corepressors 

that modify histones or mobilize nucleosomes at regulatory sites. Therefore, to 

understand the mechanism behind the regulation of beta-globin expression by TFII-I and 

USF we investigated the recruitment of chromatin modifying enzymes to the beta-globin 

gene locus by these proteins in both an embryonic and adult environment. TFII-I was found 

to interact with HDAC3 exclusively in embryonic environment. Suz12, a component of the 

Polycomb group complexes 2,3, and 4 which contains histone methylase activity, was also 

found to interact at the beta-globin promoter in an embryonic environment but not in an 

adult environment suggesting a role in repression. USF was found to interact with 

activators in an adult environment. The role of USF in beta-globin expression was also 

further investigated in transgenic mice which express a dominant-negative protein to USF 

exclusively in erythroid cells. 

75



Foteini Davrazou 

Abstract P26 

Molecular mechanism of histone H3K4me3 recognition by 

the PHD finger of ING2 

Pedro V. Peña 1 , Foteini Davrazou 1 , Xiaobing Shi 2 , Kay L. Walter 2 , Vladislav 

V. Verkhusha 3 , Or Gozani 2 , Rui Zhao 4 and Tatiana G. Kutateladze 1 

1 

Department of Pharmacology, University of Colorado Health Sciences Center, Aurora, 

Colorado 80045, U.S.A. 2 Department of Biological Sciences, Stanford University, Stanford, 

California 94305, U.S.A. 3 Department of Anatomy and Structural Biology, Albert Einstein 

College of Medicine, Bronx, New York 10461, U.S.A. 4 Department of Biochemistry and 

Molecular Genetics, University of Colorado Health Sciences Center, Aurora, Colorado 

80045, U.S.A. 

The PHD (plant homeodomain) finger is found in many chromatin remodeling complexes 

however its function remains unknown. We found that a subset of PHD fingers targets trimethylated 

H3 histone (H3K4me3) tail representing a novel family of protein-effectors that 

recognize this epigenetic mark1,2. We have determined the structure of the PHD finger of 

ING2 (inhibitor of growth) tumor suppressor in complex with a histone H3K4me3 peptide 

and characterized its specificity toward post-translationally modified histone tails. The 

H3K4me3 peptide is bound in an extended conformation in a deep and extensive binding 

site consisting of elements that are conserved among other PHD fingers. The 

trimethylammonium group of Lys 4 is recognized by aromatic residues of the domain, 

whereas the intermolecular hydrogen-bonding and complementary surface interactions, 

involving five peptide residues, account for the PHD finger’s high specificity and affinity. 

Substitution of the binding site residues disrupts H3K4me3 interaction in vitro and impairs 

the ability of ING2 to induce apoptosis, suggesting a novel tumor suppressive mechanism. 

Strong binding of other human ING and yeast YNG PHD fingers indicates that the 

recognition of the H3K4me3 histone code is a general function of this protein family. 

76



Eullia de Nadal 

Abstract P27 

Control of gene expression by the yeast Hog1 MAPK 

Eullia de Nadal, Meritxell Zapater, Glria Mas, Nuria Noriega, lex Vendrell, 

Sergi Regot and Francesc Posas 

Cell Signaling Unit, Departament de Cincies Experimentals i de la Salut, Universitat 

Pompeu Fabra (UPF), E-08003 Barcelona, Spain 

Mitogen-activated protein kinase (MAP) cascades are common signaling modules found in 

both higher and lower eukaryotic cells. Budding yeast has several MAP kinase cascades 

one of which contains a relative of the p38 family of stress activated MAP kinases. This 

kinase coordinates cellular responses to increases in external osmolarity by inducing 

diverse osmo-adaptative response. Recent genome-wide transcriptional studies revealed 

that a great number of genes are regulated by osmotic stress in a Hog1 dependent manner, 

suggesting a key role for the MAP kinase in stress-induced gene expression. However, there 

is not a uniform mechanism by which stress-activated MAP kinase modulates gene 

expression. It has been reported that MAPK can modify gene regulation by direct 

phosphorylation of transcription factors, activators and repressors, such Smp1 and Sko1 

proteins. Apart from the role of Hog1 in the modification of transcription factors, this kinase 

is associated specifically to chromatin in stress responsive promoters. Binding of the MAPK 

is critical for RNA Pol II and chromatin remodeling factors recruitment to osmostress 

responsive promoters and for gene expression. These data suggest a new dimension to 

gene regulation by signaling kinases and it prompted us to further study alternative 

mechanisms by which the MAP kinase Hog1 could regulate osmostress gene expression. 

77



Roger Deal 

Abstract P28 

Repression of flowering in Arabidopsis thaliana requires 

histone H2A.Z deposition by a putative SWR1 complex 

Roger B. Deal, Christopher N. Topp, Elizabeth C. McKinney and Richard 

B. Meagher 

Department of Genetics (Deal, McKinney, Meagher) and Department of Plant Biology 

(Topp), University of Georgia, Athens, GA 30602, U.S.A. 

In addition to the bulk histones that package the nascent genome during S phase, 

eukaryotes also encode variant histones that are deposited independently of DNA 

replication and serve to functionally specialize particular chromatin regions. The histone 

variant H2A.Z is universally conserved and has been implicated in a wide variety of 

chromatin-mediated processes including transcriptional activation and euchromatin 

maintenance in yeast, and heterochromatin formation in metazoans. In budding yeast and 

humans H2A.Z is deposited into chromatin through the action of a conserved protein 

complex known as SWR1 or SRCAP, respectively. Here we show that the Arabidopsis 

thaliana homologs of two components of this complex, ACTIN-RELATED PROTEIN 6 

(ARP6) and the Snf2 protein PHOTOPERIOD-INDEPENDENT EARLY FLOWERING 1 

(PIE1), are part of a single protein complex and are each required for deposition of H2A.Z 

into chromatin at multiple loci. One of these loci is the FLOWERING LOCUS C (FLC) gene, 

a central repressor of the transition from vegetative to reproductive development. Loss of 

H2A.Z from chromatin in arp6 and pie1 mutants results in reduced FLC expression and 

premature flowering, indicating that H2A.Z is required for transcriptional activation of FLC to 

levels that inhibit flowering. Collectively these results support the existence of a SWR1-like 

complex in Arabidopsis thaliana and show that, similar to its role in yeast, H2A.Z can serve 

to potentiate transcriptional activation in plants. 

78



Laurent Delva 

Abstract P29 

The Transcription Intermediary Factor 2 is required for 

zebrafish development 

Julia Zhuravleva 1 , Bernard Thisse 2 , Christine Thisse 2 , Jean-Noël Bastie 1 

and Laurent Delva 1 

1 

Inserm U517, Faculté de Médecine, Université de Bourgogne, 7, bd Jeanne d’Arc, 21000 

Dijon, France, 2 Institut de Génétique et de Biologie Moléculaire et Cellulaire, 

CNRS/Inserm/ULP, 1, rue Laurent Fries, BP10142, 67404 Illkirch Cedex, France 

The TIF2 (Transcription Intermediary Factor 2) gene, encoding a histone acetyl transferase, 

belongs to the p160 family. TIF2 interacts with liganded nuclear receptors, enhancing 

transcription activity of the receptors. Because of partial genetic compensation effect, knock 

out TIF2 mice did not inform completely about the role of TIF2 in development. Therefore, 

we decided to use zebrafish as an animal model. tif2 is ubiquitously expressed in zebrafish 

development. tif2-knock down zebrafish embryos present embryonic alterations such as a 

smaller tail size, abnormalities in the notochord and mesenchyme. In addition, tif2 

morphants did not harbor posterior intermediate cell mass (ICM) indicating putative 

hematopoietic differentiation defects. To better understand the nature of the defects 

observed in tif2 morphants, we performed molecular analysis of various differentiation 

markers by using whole-mount RNA in situ hybridization. Our results suggest the 

involvement of tif2 in embryonic development, particularly in primitive hematopoiesis. 

Furthermore, we observed that loss-of-function of zebrafish tif2 results in massive apoptosis 

in the mesenchyme. 

79



Luisa Di Stefano 

Abstract P30 

Lsd1 mutation in Drosophila disrupt normal level of H3K4 

methylation and affects viability and fertility 

Di Stefano L, Moon NS, Ji JY and Dyson N. 

Massachusetts General Hospital Cancer Research Center and Harvard Medical School, 

Charlestown, Massachusetts, U.S.A. 

Covalent modifications of histone tails have fundamental roles in determining the chromatin 

structure and in regulating gene expression. One such modification, histone lysine 

methylation was considered irreversible until the recent discovery of histone demethylases. 

Two distinct classes of histone lysine demethylases have been characterized so far, the 

Jumonji-domain containing proteins and Lsd1. In mammalian cells, Lsd1 was shown to 

specifically demethylate mono and di-methyl histone H3 lysine 4 and, when associated with 

the androgen receptor, to act on dimethyl-H3K9. Lsd1 is highly conserved between 

organisms from yeast to human but its role has yet to be studied in vivo. Here we describe 

the effects of Lsd1 mutation in Drosophila. We find that mutation of dLsd1 strongly affects 

global level of methylation of mono and dimethyl H3K4 revealing that specificity towards 

these residues is conserved throughout evolution. In contrast the global levels of dimethyl- 

H3K9 are not affected in dLsd1 mutant suggesting either a functional difference to the 

human counterpart or that this regulation is restricted to specific tissues. As a consequence 

of dLsd1 mutation, animal viability is strongly affected in a gender specific manner. dLsd1 

mutant flies are sterile and ovary development is strongly impaired. dLsd1 mutation strongly 

suppresses positional effect variegation (PEV) and affects gene expression. Taken together 

our results support an important role for histone H3K4 lysine demethylation in the regulation 

of chromatin structure and gene transcription in Drosophila development. 

80



Stephan Diekmann 

Abstract P31 

In vivo dynamic (FRAP, FCS) and neighbourhood relation 

(AB-FRET, FLIM) studies of human inner kinetochore 

proteins 

S. Diekmann, P. Hemmerich, S. Orthaus, S. Weidtkamp-Peters and 

C. Hoischen 

Molecular Biology, FLI, Beutenbergstr. 11, D-07745 Jena, Germany 

The kinetochore specifies a DNA/protein assembly at the surface of chromosomes that 

plays an essential role in faithful segregation of the genetic material. To gain a precise 

dynamic understanding of this complex, the mobility of GFP-tagged inner kinetochore 

proteins CENP-A, CENP-B, CENP-C, CENP-H, CENP-I, and hMis12 were analyzed in living 

human cells using fluorescence recovery after photobleaching (FRAP) and fluorescence 

correlation spectroscopy (FCS). In interphase cells, CENP-A, CENP-B, CENP-C, CENP-H 

and CENP-I are stable components of the kinetochore over hours while hMis12 rapidly and 

completely exchanges within seconds. FCS detected soluble pools of kinetochore proteins 

in interphase cells with protein-specific diffusion coefficients indicating the absence of preassembled 

kinetochore protein subcomplexes. During mitosis CENP-A, CENP-C, CENP-H, 

and CENP-I remain stably associated with the kinetochores, while CENP-B becomes mobile 

and, strikingly, hMis12 becomes completely immobilized at the kinetochores. Thus, unlike all 

other chromatin binding complexes analysed so far, the kinetochore is not maintained by a 

constant flux of rapidly exchanging components but rather by a static assembly mechanism. 

Alterations in the mobility of specific kinetochore proteins such as Mis12, however, appear 

to be associated with the changing functional properties of kinetochores during mitosis. 

Many of the proteins involved in kinetochore formation are known, however, little information 

is available on molecular structures and complex architecture although structural 

phenomena seem to play an important role for kinetochore function. In addition to their 

dynamic behaviour, we also determined the neighbourhood relation (in the < 10 nm range) 

of the inner kinetochore proteins CENP-A, CENP-B, CENP-C and CENP-I as well as 

histones in living human HEp-2 cells by energy transfer (FRET and FLIM). The data can be 

well explained by a centromeric chromatin 30 nm fiber model. Our results elucidate the 

architecture of the human inner kinetochore complex. 

81



Jeffrey Dilworth 

Abstract P32 

MEF2 helps establish muscle specific pattern of gene 

expression by recruiting Trithorax Group proteins to specific 

promoters 

Shravanti Rampalli, Marjorie Brand and F. Jeffrey Dilworth 

Sprott Stem Cell Research Center, Ottawa Health Research Institute, Ottawa, Ontario, K1H 

8L6., Canada 

Pluripotency of stem cells is proposed to be due to the fact that all genes that have the 

potential to be transcribed lie in open chromatin. As cells divide and differentiate, epigenetic 

marking acts as a cellular memory to decide which genes should be expressed, or 

repressed through chromatin condensation. These epigenetic signals are generated by 

polycomb (repression) and trithorax (activation) group proteins which mark genes by 

methylating hisone H3 on lysine residue 9/27 and 4 respectively. In an effort to understand 

how the muscle specific pattern of gene expression is established during differentiation, we 

set out to determine whether trithorax proteins play a role in establishing muscle specific 

gene expression. Initially, we used chromatinimmunoprecipitation (ChIP) to show that upon 

differentiation, several specific muscle specific genes are marked by H3K4 trimethylation. To 

determine how trithorax proteins are targeted to these promoters, we used coimmunoprecipitation 

and found that the Mef2 family of transcriptional regulators interact with 

the trithorax group protein Ash2L. In addition, pull-down studies suggest that in cellular 

extracts from muscle cells, only specific Mef2 isoforms (Mef2C and Mef2D but not Mef2A) 

interact with Ash2L. Importantly, this interaction is significantly increased by pre-treating 

Mef2C, or Mef2D with p38 kinase in the presence of ATP, suggesting that phosphorylation 

of Mef2 proteins enhances its binding to Ash2L. Finally, we demonstrate by ChIP that the 

timing of Histone H3K4 trimethylation (epigenetic mark established by Ash2L complex) 

during differentiation of muscle cells coincides with the binding of Mef2 to several muscle 

specific promoters. Thus, it appears that the transcriptional activator Mef2 is helping 

establish the muscle specific pattern of gene expression though epigenetic marking of 

specific genes. 

82



Ivana Djuretic 

Abstract P33 

T-bet and Runx3 cooperate to activate Interferon gamma 

and silence Interleukin-4 in T helper-1cells 

Ivana Djuretic 1 , Ditsa Levanon 2 , Varda Negreanu 2 , Yoram Groner 2 , Anjana 

Rao 1 and K. Mark Ansel 1 

1 

Harvard Medical School and the CBR Institute for Biomedical Research, Boston, MA, USA 

02115, U.S.A., 2 The Weizmann Institute of Science, Rehovot, Israel 76100 

Cell differentiation requires activation of lineage-appropriate genes and silencing of lineageinappropriate 

genes. Naive CD4+ T cells can differentiate down one of the two effector 

pathways: T helper-1 (Th1) or T helper-2 (Th2). T-bet, a T-box family transcription factor, is a 

central regulator during Th1 differentiation because it directly activates Th1-specific genes 

such as Interferon gamma (Ifng), and silences Th2-specific genes, such as Interleukin-4 

(Il4). Here we demonstrate that T-bet induces another transcription factor, Runx3, with which 

it cooperates in both the activation of Ifng and silencing of Il4. Although each factor was 

capable of functioning on their own, optimal gene activation and rtpression depended on 

the presence of both T-bet and Runx3. The mechanim of repression likely involves a direct 

co-operation on DNA, as the two factors were able to form a complex on the Il4 silencerderived 

probe in vitro. Current studies are aimed in further defining the nature of T- 

bet/Runx3 cooperation, including their ability to cause context-dependent gene activation 

and repression. In addition, since Il4 silencing has been associated with the appearance of 

H3K27 methylation, an important goal of this study will be to dettermine if direct 

cooperation of T-bet and Runx3 mediates the process of H3K27-methylation dependent 

silencing in the Il4 locus as well as other relevant loci. 

83



Tom Donndelinger 

Abstract P34 

Seeing cells in a new light: Improving resolution with a 

scientific approach to tissue processing 

Tom Donndelinger and Elizabeth Oldenkamp 

BI-Biomics, 1512 12th Ave Rd, Nampa, Idaho, U.S.A. 

One of the greatest shortcomings of fluorescent microscopy is the lack of resolution. 

Fluorescence studies alone cannot be solid proof of sub-cellular localization. Although this 

problem will never be entirely overcome, our methods for tissue fixation combined with 

fluorescent technology return more conclusive results for localization studies with resolution 

up to 2400x. We also show that superimposition of fluorescent images with H&E stained 

images creates a much more informative and cohesive picture of cell biology than 

traditional images with DAPI counterstains. Further, our studies have revealed 

unprecedented nuclear and nucleolar detail as well as multiple novel cellular processes that 

have never been seen through light microscopy. This technology, while a simple upgrade to 

an existing procedure, is revealing data that could seriously alter currently accepted 

scientific paradigms. 

84



Bojan Drobic 

Abstract P35 

Characterization of Histone H3 kinases, MSK1 and MSK2 

Bojan Drobic and James R. Davie 

CancerCare MB, Manitoba Institute of Cell Biology, 675 McDermot Ave., Winnipeg, 

Manitoba, R3E 0V9, Canada 

Stimulation of the Ras-MAPK signal transduction pathway by growth factors (EGF) or 

phorbol esters (TPA) in parental (10T1/2) and oncogene (H-ras)-transformed (Ciras-3) 

mouse fibroblasts induces rapid phosphorylation of histone H3. Phosphorylation of H3 

occurs on Ser10 and Ser28 in the NH2-terminal tail. This phosphorylation event is 

implicated in the regulation of immediate early genes such as c-fos and c-jun. Constitutive 

activation of the Ras-MAPK pathway in ras-transformed mouse fibroblasts increases 

phosphorylation of H3 at Ser10 and Ser28 and we have shown that this increase is due to 

enhanced activity of histone H3 kinase, mitogen- and stress-activated protein kinase 1 

(MSK1). Characterization of the MSK complex will be undertaken in 10T1/2, Ciras-3 and 

HEK293. Preliminary results demonstrate that MSK1 is associated with SWI/SNF ATPase 

(Brg1) and histone H3 acetyltransferase (HAT) [PCAF], as well as with c-Fos/c-Jun, p65 

sub-unit of NFKB, 14-3-3 proteins, but not with HDAC1. Furthermore, chromatin remodeling 

activity of the MSK complex will be investigated by isolating the MSK complex from 10T1/2, 

Ciras-3 and HEK293 (cycling, serum-starved, TPA/EGF treated) cells and performing HAT 

and mononucleosome disruption assays. Preliminary HAT assay results suggest that the 

MSK complex contains HAT activity. Dynamics of MSK1/2 association with c-fos and c-jun 

promoters will be investigated via ChIP assay. Since the Ras-MAPK signaling is frequently 

deregulated in cancer (30% of human cancers contain aberrant ras), characterization of the 

Ras-MAPK activated MSK complex and its associating activities (chromatin remodeling and 

modifying activities) could provide a basis for the assessment of MSK as a novel 

therapeutic target to treat cancer. 

85



Danielle Ellis 

Abstract P36 

Histone acetylation of SRC and p21 promoters in response 

to histone deacetylase inhibitor treatment; implications of 

HDAC activity and SRC expression 

Danielle Ellis 1 and Keith Bonham 2 

1 

Department of Biochemistry, University of Saskatchewan. and 2 Cancer Research Unit, 

Health Research Division, Saskatchewan Cancer Agency, 20 Campus Drive, Saskatoon, 

SK, Canada, S7N 4H4 

Histone deacetylase (HDAC) inhibitors (HDIs), such as trichostatin A (TSA), are well 

documented for their ability to induce apoptosis, growth arrest and differentiation in a variety 

of neoplasms. These effects are mediated through the activation and/or repression of gene 

expression. Some classic examples of this phenomenon include the transcriptional 

activation of p21WAF1 and the transcriptional repression of SRC. Currently, it is believed 

that HDAC inhibitors act at the histone level to alter chromatin dynamics through the 

inactivation of HDACs thereby resulting in histone hyperacetylation and increased 

transcriptional activation. However, transcriptional repression of gene expression is not so 

easily explained by this model. Indeed, changes in the acetylation status of histones 

associated with genes repressed by HDAC inhibitors, such as SRC, have not been reported. 

Therefore, we carried out a systematic investigation of the changes in histone H3 and H4 

acetylation status at the promoter regions of two genes differentially affected by HDAC 

inhibitors. Treatment of HT29 colon cancer cells with TSA led to similar changes in the 

acetylation of discreet H3 and H4 lysine residues at the SRC1A, SRC1alpha and p21 

promoter regions. Differential promoter specific acetylation changes were also observed; 

whereby, the SRC1alpha and p21WAF1 promoter regions demonstrated differential changes 

in acetylation as compared to SRC1A. The observations that SRC is repressed by HDIs and 

demonstrates rapid changes in histone acetylation upon HDI treatment suggests that an 

HDAC(s) may be localized at the SRC promoter regions and may be involved in SRC 

activation. Through the use of RNA interference, we observed that the knockdown of each 

class I HDAC (HDAC 1, 2, 3 and 8) did not repress SRC but actually resulted in increased 

SRC expression. Taken together these results suggest that histone acetylation is not a 

simple predictor of promoter activity and class I HDACs have an inhibitory affect on SRC 

expression. Further study of class II HDACs may aid in elucidating the mechanism by which 

SRC is repressed by HDIs. 

86



Alexander Erkine 

Abstract P37 

Differential mechanisms of nucleosome displacement at 

yeast heat shock gene promoters 

T. Y. Erkina and A. M. Erkine 

Division of Basic Biomedical Sciences, Sanford School of Medicine, University of South 

Dakota, 414 E. Clark St., Vermillion, SD 57069-2390, U.S.A. 

Chromatin remodeling at promoters of co-regulated and highly inducible heat shock genes 

HSP12, HSP82, and SSA4 is characterized by robust histone displacement during induction 

of transcription. These promoters reach their maximally histone stripped state via two 

distinct chromatin remodeling pathways: one associated (for the HSP12 and HSP82 

promoters) and the other one not associated (for the SSA4 promoter) with robust histone 

H3 specific acetylation. This observation implies that a histone acetylated platform 

recognized by bromodomain containing chromatin remodeling complexes is not uniformly 

required for the robust histone displacement at gene promoters during induction of 

transcription. SNF2 deletion causes elimination of histone displacement from the HSP12 

promoter but not from the HSP82 and SSA4 promoters. Out of three analyzed heat shock 

genes only HSP12 is characterized by inducible binding of HSF to the promoter, while 

HSP82 and SSA4 have HSF preloaded before heat shock. The SNF2 deletion prevents 

HSF binding to the HSP12 promoter. Knowing that HSF cannot bind to chromatinized DNA 

we speculate that in wild type cells the SWI/SNF complex determines sliding or relocation of 

nucleosomes along the HSP12 promoter allowing HSF to establish promoter binding, while 

in the snf2 mutant strain nucleosomes lose their dynamic behavior and block HSF loading, 

thus eliminating histone displacement. 

87



Ragnhild Eskeland 

Abstract P38 

HP1 binding to chromatin methylated at H3K9 is enhanced 

by auxiliary factors 

Ragnhild Eskeland, Anton Eberharter and Axel Imhof 

Adolf-Butenandt Institut, University of Munich, Schillerstr. 44, 80336 Muenchen Germany 

A large portion of the eukaryotic genome is packaged into transcriptionally silent 

heterochromatin. Several factors have been identified that play important roles during the 

establishment and maintenance of this condensed form. Methylation of lysine 9 within 

histone H3 and the subsequent binding of the chromo-domain protein HP1 is thought to 

initiate heterochromatin formation in vivo and to propagate a heterochromatic state through 

several cell divisions. Here we analysed the binding of HP1 to methylated chromatin in a 

fully reconstituted system. In contrast to its strong binding to methylated peptides, HP1 only 

weakly binds to methylated chromatin. However, the addition of recombinant SU(VAR) 

proteins such as ACF1 or SU(VAR)3-9 facilitates HP1 binding to chromatin methylated at 

H3K9. We propose that HP1 has multiple target sites that contribute to its recognition of 

chromatin only one of them being H3K9me. These findings have implications for the 

mechanisms of how specific chromatin modifications are recognized in vivo. 

88



George Feehery 

Abstract P39 

CpG methylated DNA standards and control primers for use 

in methyl sensitive PCR and bisulphite sequencing 

George R. Feehery, Pierre-Olivier Esteve, Hang Gyeong Chin and 

Sriharsa Pradhan 

New England Biolabs, 240 County Road, Ipswich, MA 01938-2723. U.S.A. 

Methylation-specific PCR (MSP), is a technology for the sensitive detection of gene 

methylation in the vertebrate genome. The procedure employs an initial bisulfite reaction 

that modifies the genomic DNA, or converts unmethylated cytosines to uracils while 5- 

methylcytosines remain unaltered. The bisulphate modified DNA is amplified by PCR with 

specific primers designed to distinguish methylated from unmethylated sequences. Because 

this is a sensitive PCR-based assay, the use of DNA and primer controls are necessary to 

determine the quality of the bisulfite conversion, the identification of artifacts such as 

primer-dimer pairing, and mispriming to undesired target DNA that can cloud the 

interpretation of results. 

To create a methylation-positive DNA control, we have enzymatically methylated Hela, NIH- 

3T3, and Jurkat genomic DNA with a prokaryotic CpG Methylase (M.SssI). All the cytosine 

residues (C) within the double-stranded dinucleotide recognition sequence 5CG3 are 

methylated cytosine (C5). To test the extent of CpG methylation, we challenged the various 

modified DNAs with [3H]AdoMet and an excess of bacterial methyltransferase M.SssI. No 

further incorporation of tritiated AdoMet was observed in the DNA substrates even after 

extensive overnight incubation. MSP-PCR using 10 different primer sets also revealed 

complete methylation of all CpG dinucleotides. Bisulphite sequencing of methylated DNA 

displayed ~99.5% of CpG methylation of candidate genes. 

We created a reduced methylation DNA control by incubating Jurkat cells with a 2M 

concentration of the methyltransferase inhibitor 5-aza-2-deoxycytidine (5-Azadc) for eight 

days. The genomic DNA derived from cells treated with this drug exhibited some lower 

molecular weight smearing when visualized on a 0.8% agarose gel. Bisulfite conversion and 

sequencing of a section of intergenic (IGS) repetitive DNA (rDNA) that is normally 

methylated revealed significant CpG demethylation. Used in conjunction CpG methylated 

and unmethylated genomic DNA controls may serve as powerful tools in the investigation of 

DNA methylation in the genome. 

89



Barna Fodor 

Abstract P40 

Identification of novel pericentric proteins by their localization 

Barna D. Fodor, Mario Richter, Manuela Scaranaro and Thomas Jenuwein 

Research Institute of Molecular Pathology (IMP) Dr. Bohrgasse 7, A-1030 Vienna, Austria 

The eukaryotic genome is organized into distinct euchromatic and heterochromatic 

subdomains. Heterochromatin has fundamental roles in the structural organization of 

chromosomes, genome stability, and controlling epigenetic programs. Genetic screens in S. 

pombe and Drosophila have identified a number of genes called suppressors of variegation 

[Su(var)s] most of which encode components of heterochromatin. These screens exploit 

the transcriptional silencing effect of heterochromatin on juxtaposed reporter genes, which 

is compromised in Su(var) mutants (Schotta et al. 2003). Another principle to discriminate 

heterochromatin components is their characteristic localization in cells. In mouse cells 

pericentric heterochromatin forms typical (DAPI dense) foci. Co-localization of epitope 

tagged factors to these foci can easily be detected. Localization screens based on this 

principle, were also successful in identifying heterochromatin associated proteins (Dellaire 

et al. 2003). However, the potential of this approach was not yet fully exploited. 

In this study we applied genetrapping to identify novel components of heterochromatin. The 

transduced genetrap constructs randomly integrate in the genome, generating translational 

fusions of endogenous loci and the GFP transgene. We observed characteristic localization 

patterns of the expressed GFP fusion product for a number of clones, proving the utility of 

our experimental design. The interesting clones were picked, and expanded. The identity of 

the trapped genes can be determined by 5’- and 3’-races. We are now extending our studies 

to a number of cell lines with a variety of genetrap constructs. 

90



Maria Fousteri 

Abstract P41 

Cockayne syndrome A and B proteins differentially regulate 

recruitment of chromatin remodeling and repair factors to 

stalled RNA polymerase II in vivo 

Anne Jensen and Leon H.F. Mullenders 

Department of Toxicogenetics, Leiden University Medical Center, Einthovenweg 20, 2333 

RC Leiden, The Netherlands 

Restoration of UV-inhibited transcription requires removal of transcription-blocking DNA 

lesions by transcription-coupled repair (TCR), a specialized repair pathway that removes UV 

induced photolesions from transcriptional active genes. In mammals TCR is dependent on 

CSA and CSB proteins, however, their function and interactions are not well understood. 

CSA is a WD-40 repeat protein and a component of an E3-ubiquitin ligase, while CSB is a 

DNA-dependent ATPase that shares homology with SW2/SNF2 chromatin remodelers. 

Mutations in any of the two genes that encode for CSB and CSA lead to a rare recessive 

neuro-developmental progeroid-like disorder, Cockayne syndrome (CS). 

Currently, information is lacking on the exact composition and molecular interactions of an 

active TCR complex. Our aim was to improve understanding of TCR in vivo by isolation and 

analysis of lesion-stalled transcription elongation complexes. We have used in vivo 

crosslinking and ChIP and we examined the recruitment of specific repair and chromatin 

remodeling factors to a UV-stalled RNAPII in TCR-proficient and -deficient human cells. The 

protocol was modified in such a way that it enabled the analysis of co-immunoprecipitated 

proteins that reside in close proximity on damage containing chromatin fragments. Our 

study revealed that CSB and CSA display differential roles in recruitment of TCR-specific 

factors and that assembly for TCR in vivo occurs without disruption of the UV-stalled 

RNAPIIo. CSB fulfills a key role as coupling factor in the assembly of a chromatin-bound 

TCR complex that involves histone acetyltransferase p300, nucleotide excision repair (NER) 

proteins and CSA-DDB1 E3-ubiquitin ligase complex with the COP9 signalosome. CSA is 

dispensable for attraction of NER proteins, yet is required to recruit XAB2, the nucleosomal 

binding protein HMGN1 and transcription cleavage factor TFIIS. This approach highlights 

the essential roles of CS proteins in TCR complex formation and provides a molecular link 

between damage recognition, chromatin remodelling and NER. 

91



Robert Gillespie 

Abstract P42 

Retinoid regulated association of transcriptional coregulators 

and the polycomb group protein SUZ12 with the retinoic acid 

response elements of Hoxa1, RARß2, and Cyp26A1 in F9 

embryonal carcinoma cells 

Robert F. Gillespie and Lorraine J. Gudas 

Molecular Biology Program of Weill Graduate School of Medical Sciences and 

Pharmacology Department of Weill Medical College of Cornell University, New York, New 

York 10021, U.S.A. 

Hox gene expression is activated by retinoic acid (RA) binding to Retinoic Acid Receptor- 

Retinoid X Receptor (RAR-RXR) heterodimers bound at RA response elements (RAREs) of 

target genes. Hox genes are also repressed by polycomb group proteins (PcG), though how 

these proteins are targeted is unclear. We used chromatin immunoprecipitation assays to 

investigate the association of RXRα, cofactors, and the PcG protein SUZ12 with the Hoxa1, 

RARß2, and Cyp26A1 RAREs in F9 embryonal carcinoma cells during RA treatment. We 

demonstrate that the association of RARγ-RXRα with RAREs before and during RA 

treatment remains relatively constant. pCIP, p300, and RNA polymerase II levels at target 

RAREs also varied by gene, though RA increased the associaton of these proteins with 

RAREs. Conversely, SUZ12 was associated with all RAREs studied and this association was 

attenuated by RA. Upon RA removal, SUZ12 re-associated with RAREs. H3ac, H3K4me2, 

and H3K27me3 marks were simultaneously detected at target loci, indicative of a bivalent 

domain chromatin structure. During RA mediated differentiation, H3K27me3 levels decreased 

at target RAREs whereas H3ac and H3K4me2 levels remained constant. These studies 

provide insight into the dynamics of association of coregulators with RAREs and 

demonstrate a novel link between RA signaling and PcG repression. 

92



Clara Goday 

Abstract P43 

Chromatin modifications in germline chromosomes of 

sciarid flies 

P.G. Greciano and C. Goday 

Ramiro de Maeztu 9, Centro de Investigaciones Biologicas, CSIC, 28040 Madrid, Spain 

A classic example of programd chromosome elimination and genomic imprinting is found 

in sciarid flies (Diptera, Sciaridae), where whole chromosomes of paternal origin are 

selectively discarded from the genome during development. In early germ cells a single 

paternal X chromosome is eliminated in embryos of both sexes and in male meiotic cells 

the whole paternal complement is discarded. In sciarids, differential acetylation of histones 

H3 and H4 occurs between chromosomes of different parental origin, both in early germ 

nuclei and in male meiotic cells. We here investigated histone methylation modifications 

between chromosomes in germline cells of Sciara ocellaris. In early germ nuclei, maternal 

chromosomes show high levels of di- and trimethylated histone H3 at lysine 4, whereas 

this histone modification is not detected in paternal chromosomes. In male meiosis, only 

the eliminated paternal chromosomes exhibit high levels of di- and trimethylated histones 

H3 at lysine 4 and dimethylated H4 at lysine 20. In early germ nuclei, RNA polymerase II 

associates to maternally-derived chromosomes but lacks phosphorylation of the carboxyterminal 

domain on serine 2. The results suggest that histone H3 methylation at lysine 4 

does not correlates with transcriptional activity in early Sciara germline nuclei. Our results 

supports that specific covalent chromatin modifications such as histone 

acetylation/methylation are involved in the imprinted behaviour of germline chromosomes 

in Sciara. 

93



Aaron D. Goldberg 

Abstract P44 

HIRA-dependent incorporation of histone H3.3 marks active 

genes in mouse embryonic stem cells 

Aaron D. Goldberg 1 , Fyodor D. Urnov 4 , Ileana M. Cristea 2 , Chingwen 

Yang 3 , Jeffrey Miller 4 , Carlos Ramos 5 , Marco Seandel 5,6 , Daylon James 5 , 

Sandra B. Hake 1,7 , Peter J. Scambler 8 , Brian T. Chait 2 , Philip D. Gregory 4 , 

Shahin Rafii 5,6,9 and C. David Allis 1 

1 

Laboratory of Chromatin Biology, 2 Laboratory of Mass Spectrometry and Gaseous Ion 

Chemistry, and 3 Gene Targeting Resource Center, The Rockefeller University, 1230 York 

Avenue, New York, NY, 10021, U.S.A. 4 

Sangamo BioSciences, Inc. Pt. Richmond Tech 

Center 501, Canal Blvd, Suite A100 Richmond, California 94804, U.S.A. 5 

Department of 

Genetic Medicine, 6 Department of Medicine, Division of Hematology-Medical Oncology, 

Weill Medical College of Cornell University, 1300 York Avenue, New York, New York 10021, 

U.S.A. 7 

Adolf-Butenandt-Institut, Molekularbiologie, Ludwig-Maximiliams-Universitaet, 

Schillerstr. 44, 80336 Muenchen, Germany. 8 

Molecular Medicine Unit, Institute of Child 

Health, London WC1N 1EH, U.K. 9 

Howard Hughes Medical Institute, Weill Medical College 

of Cornell University, 1300 York Avenue, New York, New York 10021, U.S.A. 

The histone H3 variant H3.3 has been shown to be associated with transcriptionally active genes in 

multiple organisms, and to be retained in some loci after transcription has ceased. (Ahmad and 

Henikoff 2002; Schwartz and Ahmad 2005) It is tempting to speculate that H3.3 associates with 

transcriptionally permissive chromatin in differentiating mammalian cells, and that this localization 

has functional importance in the establishment or maintenance of gene activation. However, it is 

difficult to address these questions due to the lack of available tools or antibodies to distinguish 

H3.3 from the other H3 variants H3.2 or H3.1. Here we describe a rapid and efficient method that 

uses engineered zinc finger protein nucleases and a short, selection-less, promoter-less targeting 

construct to introduce an enhanced yellow fluorescent protein (EYFP) sequence into the C-terminal 

coding exon of the endogenous histone H3.3B gene in mouse embryonic stem (ES) cells. In work 

in progress, we have used these heterozygous H3.3B-EYFP tagged ES cells to show that H3.3 is 

excluded from H3K9me3-rich pericentric heterochromatin on metaphase chromosomes, and 

partially but incompletely co-localizes with regions of H3K4me3. Through chromatin 

immunoprecipitation (ChIP) assays, we show that H3.3 is significantly enriched at the promoters, 

depleted at the transcriptional start sites, and enriched into the 3’ ends of pluripotency genes Oct4 

and Nanog in undifferentiated ES cells. As a control for the specificity of H3.3 localization, we have 

also used our method to introduce point mutations in H3.3B, generating EYFP-tagged H3.2 

expressed from the endogenous histone H3.3B locus. Unlike H3.3, H3.2-EYFP is diffusely 

localized throughout metaphase chromosomes, and is not excluded from pericentric 

heterochromatin. Finally, we have used our method to visualize the subcellular localization of H3.3 

in ES cells homozygous for a knockout of the putative H3.3 chaperone HIRA, demonstrating that 

H3.3 incorporation into metaphase chromosomes is HIRA-dependent. These studies confirm and 

extend studies carried out in other non-mammalian models and provide a “proof-of-principle” of an 

approach permitting the tagging of key chromatin constituents for analyses in mammalian cells. 

94



Elizabeth Goneska 

Abstract P45 

Phosphorylation of the SQ H2A.X motif is required for proper 

meiosis and mitosis in Tetrahymena thermophila 

Xiaoyuan Song* 1,3 , Elizabeth Goneska* 1,2 , Qinghu Ren* 1,4 , Sean D. Taverna 2 , 

C. David Allis 2 and Martin A. Gorovsky 1 

1 

Department of Biology, University of Rochester, Rochester, NY, U.S.A., 2 Laboratory of 

Chromatin Biology, The Rockefeller University, New York, NY, U.S.A., 3 School of Medicine, 

UCSD, La Jolla, CA, U.S.A., 4 The Institute for Genomic Research, Rockville, MD, 

U.S.A.,*These authors contributed equally to this work 

Phosphorylation of the H2A.X C-terminal SQ motif is required for efficient DNA doublestrand 

break (DSB) repair in diverse organisms. Here we show that H2A.X, one of the two 

major H2As in Tetrahymena, is phosphorylated at serine 134 in response to DSBs induced 

by chemical agents and during prophase of meiosis I. Using strains containing a mutation 

(S134A) that abolishes this phosphorylation, we demonstrate that phosphorylation of the 

SQ motif is required for normal micronuclear meiosis and mitosis, and to a lesser extent, for 

normal amitotic macronuclear division. H2A.X phosphorylation is also important for 

Tetrahymena cells to recover from exogenous DNA damage, and its absence, while not 

lethal, leads to extensive accumulation of DSBs in both micro- and macronuclei. 

95



Susana Gonzalo 

Abstract P46 

Telomere epigenetic modifications: a control of telomere 

length and a stop on recombination 

Susana Gonzalo 1,3 , Isabel Jaco 1 , Roberta Benetti 1 , Gunnar Schotta 2 , Peter 

Klatt 1 , Thomas Jenuwein 2 and María A. Blasco 1 

1 

Telomeres and Telomerase Group. Molecular Oncology Program. Spanish National Cancer 

Centre (CNIO). Madrid E-28029, Spain., 2 Research Institute of Molecular Pathology (IMP). 

The Vienna Biocenter. A-1030 Vienna, Austria, 3 Department of Radiation Oncology. 

Washington University School of Medicine. St. Louis, MO 63108, U.S.A. 

Telomeres protect chromosome ends from being recognized as double strand breaks. Alterations 

in the binding of telomere proteins or erosion of telomeric DNA below a critical threshold lead to 

telomere instability, activation of the DNA damage response pathway, cell cycle arrest, and 

senescence or apoptosis. To avoid growth inhibition, cells maintain telomere length either by 

telomerase or alternative lengthening of telomeres mechanism (ALT). Recently, we and other 

investigators have demonstrated that telomere metabolism can also be regulated epigenetic 

modifications. Our studies identified a number of repressive chromatin-modifying activities that 

participate in the assembly of telomeric chromatin in mouse cells. In particular, the abrogation of 

histone methyltransferases (HMTases) Suv39h1 and h2, results in defective trimethylation of 

telomeric histone H3 at lysine 9 (H3K9me3), leading to telomere elongation. These observations, 

together with a report showing telomere erosion upon heterochromatin protein 1 (HP1) overexpression 

in human cells, was the first demonstration that epigenetic alterations of mammalian 

telomeres can lead to telomere length deregulation. Furthermore, loss of DNA methyltransferases 

Dnmt1 or Dnmt3a/3b, resulting in DNA hypomethylation of subtelomeres, or loss of 

Retinoblastoma (Rb) family function, affecting trimethylation of telomeric histone H4 at lysine 20 

(H4K20me3) and global DNA methylation, lead to telomere elongation. Most recently, we have 

observed decreased telomeric H4K20me3 and telomere length deregulation in cells deficient for 

Suv4-20h1 and h2 HMTases, suggesting that these enzymes are responsible for this chromatin 

modification at telomeres. Based on the available data, we propose the hypothesis that the 

acquisition of a heterochromatic “condensed” structure at the telomere restricts the access of 

telomere elongating machineries in order to ensure telomere homeostasis. The “opening” of 

telomeric chromatin by loss of repressive chromatin marks or an excessive heterochromatinization 

by up-regulation of these marks could facilitate or further restrict the accessibility of telomere 

elongating activities, leading to telomere elongation or erosion, respectively. In fact, we have 

recently found an increased frequency of telomeric sister chromatid exchange (T-SCE) events in 

cells lacking Dnmt1 or Dnmt3a/3b, and also in cells lacking HMTases Suv39h1/h2 or Suv4- 

20h1/h2, by the Chromosome Orientation FISH technique (CO-FISH). This technique is a read-out 

of recombination among sister telomeres, a hallmark of activation of ALT mechanism of telomere 

elongation. In summary, we conclude that modifications of telomeric chromatin serve to control 

telomere length and impose a stop on recombination. 

96



Tanya Gustafson 

Abstract P47 

Epigenetic silencing of Singleminded-2 in breast cancer 

Tanya Gustafson 1 , Keelan Anderson 1 , Mike Kladde 2 and Weston Porter 1 

1 

Texas A&M College of Veterinary Medicine, Department of Integrative Biosciences, College 

Station, TX 77843, 2 Texas A&M University, Department of Biochemistry and Biophysics, 

College Station, TX 77843, U.S.A. 

Epigenetic gene regulation has been identified as a crucial event in breast cancer 

development. Epigenetic mechanisms of regulation include covalent modifications of 

histones and DNA methylation. These events occur throughout all stages of breast 

tumorigenesis and are a more common alternative to deletions or mutations for inactivating 

breast tumor suppressor genes. The human gene Singleminded-2 (Sim2) is a member of 

the basic helix-loop-helix Per-Arnt-Sim (bHLH/PAS) family of transcription factors, which 

includes genes responsible for maintenance of circadian rhythms (Per), sensors of hypoxia 

(Hif1α) and environmental contaminants (AhR). We have recently shown that Sim2 has 

tumor suppressor activity in the breast. Sim2 is expressed highly in normal breast cell lines, 

but expression is lost in invasive breast cancer cells. The purpose of this work is to 

elucidate the epigenetic mechanisms of Sim2 silencing in breast cancer cells. 

To determine whether Sim2 was silenced through epigenetic mechanisms, highly invasive 

breast cancer cells (MDA435) and normal breast epithelial cells (MCF10A) were treated 

with a demethylating agent and a histone deacetylase inhibitor. Sim2 expression was 

increased by treatment in MDA435 cells, but did not change in MCF10A cells. Bisulfite 

sequencing of the CpG islands in MCF10A, MCF7 (mildly invasive breast cancer cells) and 

MDA435 cells revealed that methylation of a large island in exon 1 correlates with Sim2 

expression levels. Chromatin immunoprecipitation demonstrated that the Sim2 promoter is 

hyperacetylated in MCF10A cells and heterochromatic in MDA435 cells. The novel 

technique, MAP-IT, has been used to simultaneously characterize DNA methylation and 

chromatin structure on individual molecules. Overexpression of DNA methyltransferases 

has led to methylation, decreased expression and decreased histone acetylation of the 

Sim2 gene. The data support Sim2 silencing through epigenetic mechanisms during breast 

cancer progression. 

97



Soon-Ki Han 

Abstract P48 

Role of plant CBP/p300-like genes in the regulation of 

flowering time 

Soon-Ki Han 1,3 , Ju-Dong Song 2,3 , Yoo-Sun Noh 2,3 and Bosl Noh 1,3 

1 

Environmental Biotechnology National Core Research Center, Gyeongsang National 

University, Jinju 660-701, Korea. 2 Department of Biological Sciences, Seoul National 

University, Seoul 151-742, Korea. 3 Global Research Laboratory for Flowering at SNU and 

UW, Seoul National University, Seoul 151-742, Korea 

CREB-binding protein (CBP) and its homolog p300 possess histone acetyltransferase (HAT) 

activity and function as key transcriptional coactivators in the regulation of gene expression 

that controls differentiation and development in animals. However, the role of CBP/p300-like 

genes in plants is not yet elucidated. Here, we show that Arabidopsis CBP/p300-like genes 

promote flowering through affecting the expression of a major floral repressor FOWERING 

LOCUS C (FLC). Although animal CBP/p300 generally function as coactivators, Arabidopsis 

CBP/p300-like proteins are required for the negative regulation of FLC. This CBP/p300- 

mediated FLC repression might involve reversible protein acetylation independent of histone 

modification within FLC chromatin. 

98



Christin Hanigan 

Abstract P49 

Identification of an HDAC2 mutation in colorectal cancer and 

its consequences 

Christin L Hanigan, Manon van Engeland, James Eshleman and 

James Herman 

Cancer Biology Program, Department of Oncology, The Sidney Kimmel Comprehensive 

Cancer Center at Johns Hopkins, Baltimore, MD 21231, U.S.A., Department of Pathology, 

University Maastricht, 6200 MD Maastricht, The Netherlands 

Histone deacetylases are an important component of chromatin remodeling machinery 

associated with transcriptional repression. Specifically, these proteins have been shown to 

aid in silencing tumor suppressor genes. While these proteins regulate a number of genes 

and cellular processes, we are only beginning to understand what genes and processes 

regulate them. One recent study showed mutations in the Wnt signaling pathway can lead 

to an upregulation of a histone deacetylase, HDAC2. Microsatellite instability can lead to 

increased mutation rates in genes that contain mono, di, and tri-nucleotide repeat tracts. 

We have found taht HDAC2 has a poly(A) tract in exon1, which in some MSI+ colon cancer 

cells is mutated leading to a frameshift and premature stop codon. We have observed the 

apoptotic resistance fo pharmacologically inhibiting HDACs in cell lines lacking HDAC2. We 

propose that HDAC2’s regulation of APAF-1 may play a role in this resistance. We have 

found HDAC2 on the APAF-1 promoter and see up-regulation of APAF-1 at the mRNA level 

only in cell lines that have functional HDAC2 and are responsive to HDAC inhibition. We 

suggest that SAHA treatment up-regulates APAF-1 as one mechanism of inducing cell 

death. Cell lines that have lost HDAC2 already have compensated for high levels of APAF- 

1 and are resistant to HDAC inhibition induced cell death. 

99



Troy Harkness 

Abstract P50 



Terra G. Arnason, Megan D. Dash, Gerald F. Davies and Troy A. A. 

Harkness 

Department of Anatomy and Cell Biology, University of Saskatchewan, Saskatoon, SK, Canada 

Chromatin assembly in yeast is regulated by a complex molecular network governed at 

least in part by the ubiquitin ligases Rsp5p, the Anaphase Promoting Complex (APC) and 

the SCF. We have shown that Rsp5p, localized exclusively to the plasma membrane and 

adjacent to vacuoles, triggers nuclear APC activity by blocking the activity of APC inhibitors, 

such as the SCF. The APC then initiates replication-independent, but CAF-I-dependent, 

chromatin assembly. Here, we demonstrate another mechanism leading to Rsp5pdependent 

APC activity. Mutation to RSP5 leads to increased histone H3 phosphorylation 

and decreased histone H3 acetylation at elevated temperatures. We show that the histone 

H3 kinase, Snf1p, is required for the rsp5 phenotype. Interestingly, we previously 

demonstrated that the Snf1 kinase complex, which shuttles across the nuclear membrane, 

is required for APC activity. Thus, we propose that Rsp5p is required for the transit of Snf1p 

across the nuclear membrane. In support of this theory, we show that GFP-tagged Snf1p, 

Snf4p (activator subunit) and Gal83p (localizing subunit) all fail to localize to the nucleus 

upon carbon stress in rsp5 mutant cells. Similarly, the GFP-tagged Snf1p target, Mig1p, 

failed to exit the nucleus in rsp5 mutants. We next asked whether Snf4p, which requires 

ubiquitination for stability and function, requires Rsp5p or any of the Rsp5p associated E2 

enzymes. In ubiquitin coimmunoprecipitation (CoIP) experiments, we recovered GST-Snf4p 

bound to ubiquitin, but not GST alone. We observed that carbon stress induced an increase 

in ubiquitinated GST-Snf4p in wild type cells. When ubiquitin was CoIPed from ubc4∆ ubc5∆ 

cells, GST-Snf4p was again recovered, but we failed to observe induction of ubiquitinated 

GST-Snf4p upon carbon stress. The influence of i) Rsp5p, ii) the Snf1p and Rsp5p 

interacting protein, Rod1p, and iii) Ubc7p, an E2 that physically interacts with Rsp5p, on 

Snf4p ubiquitination will be discussed. 

100



Tiffany Hung 

Abstract P51 

ING4 recognition of histone H3 trimethylated at lysine 4 

Tiffany Hung, Xiaobing Shi and Or Gozani 

Stanford University, 371 Serra Mall, Stanford, CA 94305, U.S.A. 

ING4 is a candidate tumor suppressor protein and member of the evolutionarily conserved 

ING (Inhibitor of Growth) family of chromatin-regulatory proteins. ING4 is a native subunit 

of an HBO1 histone acetyltransferase (HAT) complex and is thought to link HAT activity and 

tumor suppression. Here we present in vitro and in vivo evidence that the PHD finger (plant 

homeodomain) module of ING4 specifically recognizes histone H3 trimethylated at lysine 4 

(H3K4me3). This modification is an epigenetic hallmark of active transcription, and we are 

testing the model that recognition of H3K4me3 by the ING4 PHD finger is important for 

gene activation via an increase in HBO1-dependent acetylation at nearby nucleosomes. 

101



David Johnson 

Abstract P52 

E2F1 and GCN5 facilitate the recruitment of nucleotide 

excision repair factors to sites of UV-induced DNA damage 

Ruifeng Gou, David L. Mitchell, Thomas R. Berton and David G. Johnson 

The University of Texas M.D. Anderson Cancer Center, Department of Carcinogenesis, Science 

Park-Research Division, PO Box 389, 1808 Park Road 1-C, Smithville, TX 78957, U.S.A. 

The E2F1 transcription factor regulates the expression of genes involved in cell cycle 

progression, apoptosis, differentiation, and DNA repair. In addition, emerging data suggests 

that E2F1 has a transcription-independent function in response to at least some forms of 

DNA damage. We find that E2F1 localizes to sites of DNA damage caused by ultraviolet 

(UV) radiation and that this requires the ATR kinase and phosphorylation of E2F1 at serine 

31. In the absence of E2F1 the recruitment of nucleotide excision repair (NER) factors, such 

as XPC and XPA, to sites of UV damage is impaired. This correlates with a defect in DNA 

repair and an increased sensitivity to UV-induced apoptosis in cells lacking E2F1. The GCN5 

histone acetyltransferase also accumulates at sites of UV-induced DNA damage. Moreover, 

GCN5 associates with E2F1 in response to UV exposure and knocking down E2F1 

expression impairs co-localization of GCN5 with damaged DNA. Like E2F1, GCN5 is also 

important for the efficient recruitment of repair proteins to sub-nuclear regions containing UV 

damage. These findings indicate that phosphorylation mediated by ATR converts E2F1 into a 

DNA repair factor that localizes to UV damaged DNA. E2F1 then promotes DNA repair by 

recruiting GCN5, and in turn, the NER machinery to sites of damage. 

102



Paul Kalitsis 

Abstract P53 

Nucleosome spacing analysis of repeat DNA regions in the 

mouse genome 

Paul Kalitsis, Sheena Rigby and K.H. Andy Choo 

Murdoch Children’s Research Institute, Royal Children’s Hospital, Melbourne 3052, Australia 

Mammalian genomes consist of highly repeated satellite DNAs that are found in gene-poor 

regions such as centromeres and telomeres. In the mouse genome the proximal telomere is 

within 11 kb of the centromeric minor satellite DNA. These chromosome structures have 

separate and unique roles in chromosome segregation and stability. To investigate the 

relationship between these two domains we have examined the primary chromatin structure 

by partially digesting mouse chromatin with micrococcal nuclease from a variety of cell lines 

and tissues. Each digested chromatin extract was run on an agarose gel and hybridised 

with a representative repeat probe from telomeric, centromeric and peri-centromeric 

domains. Furthermore, we examined the effects on nucleosomal spacing in cell lines that 

were treated with chemical agents that perturb chromatin structure and differentiate cells. 

103



Min-Jeong Kang 

Abstract P54 

Role of a RPD3/HDA1 family histone deacetylase in the 

regulation of phytochrome-mediated light respases in 

Arabidopsis 

Min-Jeong Kang 1,3 , Soon-Ki Han 2,3 , Yoo-Sun Noh 1,3 and Bosl Noh 2,3 

1 

Department of Biological Sciences, Seoul National University, Seoul 151-742, Korea. 

2 


University, Jinju 660-701, Korea. 3 Global Research Laboratory for Flowering at SNU and 


Posttranslational acetylation of histone N-terminal tail is well known to influence gene 

transcription by changing chromatin structures: it relaxes the association of histone proteins 

with DNA, displacing nucleosomes from the promoters of genes. Lately, histone acetylation 

has also been thought to provide surfaces which transcription activators or repressors 

recognize and bind to. Histone acetylation is reversibly regulated by histone deacetylases 

(HDACs) that are categorized into three major groups; the RPD3/HDA1 superfamily, the 

SIR2 family, and the HD2 family. Arabidopsis has 10 members of HDACs belonging to the 

RPD3/HDA1 superfamily. In order to address the biological roles of the RPD3/HDA1 family 

HDACs in plants, we isolated the loss of function mutants of Arabidopsis HDACs by a 

reverse genetics and have characterized their phenotypes. We found the mutation in one of 

the HDACs causes hypersensitivity to the light-mediated inhibition of hypocotyl elongation. 

Genetic analyses showed the mutation in the photoreceptor phyB is epistatic to the hdac 

mutation in the hypocotyl elongation. More data indicating the role of the HDAC in the 

regulation of light responses will be presented. 

104



Panagiota Karagianni 

Abstract P55 

ICBP90, a putative link between histone ubiquitination and 

cell cycle progression 

Panagiota Karagianni, Jun Qin and Jiemin Wong 

Department of Molecular and Cellular Biology, Baylor College of Medicine, One Baylor 

Plaza, Houston TX 77030, U.S.A. 

ICBP90 was initially identified as a transcription factor that can regulate Topoisomerase IIα 

expression. The primary sequence of ICBP90 contains a Ubiquitin like, a SET and RING 

Associated (SRA), a PHD and two RING domains. It was recently shown that ICBP90 

exhibits auto-ubiquitination activity. Since a murine protein with high sequence identity with 

ICBP90, Np95, has been shown to bind chromatin through the SRA domain as well as 

ubiquitinate core histones in vitro, we wanted to examine if ICBP90 can also function as a 

ubiquitin ligase for histones. We have found that ICBP90 can bind to chromatin and 

ubiquitinate histone H3 both in vitro as well as in transfected cells, in a RING domaindependent 

fashion. In fact, ICBP90 can poly-ubiquitinate H3 in vitro, which provides, to our 

knowledge, the first paradigm of histone H3 poly-ubiquitination. By using mass 

spectrometry we have mapped Lysine 79 of histone H3 as the site of covalent ubiquitin 

linkage. ICBP90 has been previously shown to be required for G1/S as well as G2/M 

transition. We are currently addressing the potential role of ICBP90-mediated histone 

ubiquitination in cell cycle progression. 

105



Emmanuel Kas 

Abstract P56 

Altering the structure and functional properties of 

heterochromatin with satellite-specific minor-groove binders 

Roxane Blattes, Guillaume Susbielle, Caroline Monod and Emmanuel Kas 

LBME - UMR5099 CNRS/UPS, IBCG, 118 route de Narbonne, 31062 Toulouse cedex 9, France 

Repeated AT-rich sequences constitute an enormous target for DNA minor-groove binders 

such as oligopyrrole polyamides and diamidines. These sequence arrays are highly 

enriched in pericentric heterochromatin, but also in the genomes of certain pathogenic 

parasitic microorganisms, or portions thereof as in the kinetoplast DNA of trypanosomes. 

We have previously shown that targeting of these sequences is easily achieved in vivo and 

is extraordinarily specific. The D1 protein of Drosophila melanogaster is associated with the 

359-bp 1.688 g/cm3 satellite III (SAT III) repeats that are cleaved by topoisomerase II (topo 

II) in vivo. We show that synthetic minor-groove binders that selectively target SAT III 

sequences alter the nuclear localization of topo II or interfere with its enzymatic activity to 

induce modifications of position-effect variegation (PEV). P9, a satellite-specific polyamide, 

affects a D1-dependent pathway that directs topo II to the SAT III array and also perturbs 

the association of HP1 with heterochromatin. In contrast, synthetic diamidines uncouple the 

topo II/D1 interaction and cause a massive D1-independent relocalization of topoisomerase 

II to AT-rich heterochromatin. This mobilization in turn results in a suppression of PEV. We 

propose that synthetic and natural AT-specific minor groove binders act coordinately with 

topo II to effect assembly of specialized nucleoprotein structures such as heterochromatin. 

The antiparasitic diamidines used here also target AT-rich DNA sequences in their target 

microorganisms. Their effects on the localization of topo II provide keys to understanding 

their mechanism of biological action, which most likely reflects an anti-genome activity that 

interferes with essential nucleoprotein complexes involving AT-rich sequences. 

106



Chul Geun Kim 

Abstract P57 

PIAS1 confers erythroid cell specific α-globin gene regulation 

by the CP2 transcription factor family 

Ho Chul Kang, Kyung Sook Choi, Hyen Seok Heo, Tae Ho Jeon and 

Chul Geun Kim 

Department of Life Science, Hanyang University, Seoul 133-791, Korea 

CP2c, a member of CP2 transcription factor family genes, was discovered initially in mouse 

as a transcription factor that binds to and stimulates transcription from the α-globin 

promoter. We previously demonstrated that ubiquitously expressed CP2c exerts potent 

erythroid-specific transactivation of α-globin through interactions with CP2b, which is 

identical to CP2a except that it has an additional 36 amino acids encoded by an extra exon, 

and protein inhibitor of activated STAT1 (PIAS1) (Kang et al., 2005). Indeed, significant 

reduction of α-globin expression was observed in RNAi-mediated knockdown of CP2c, 

CP2b or PIAS1 in erythroid cells, indicating that these three factors are indispensable 

components for α-globin expression. However, the mechanisms by which how these three 

factors confer erythroid-specific activation of α-globin in vivo are unsolved. Here we report 

that PIAS1 confers erythroid cell specific α-globin gene regulation by the CP2 transcription 

factor family. We find that CP2c is mostly present in the cytoplasm, whereas both CP2b and 

PIAS1 are solely in the nucleus and CP2a is in the cytoplasm. Interestingly, overexpression 

of CP2b or PIAS1 induces nuclear translocation of CP2c. Furthermore, the strong DNA 

binding activity of CP2c which was destabilized by CP2b is restored by supplementation of 

PIAS1 into the EMSA reaction, suggesting that PIAS1 induces the DNA/CP2b/CP2c/PIAS1 

quaternary complex formation in the nucleus. We confirmed that all three factors interact 

with each other using their two discrete binding domains, and proteins containing minimally 

two tethered CP2b/CP2c binding domains of PIAS1 are sufficient to maintain the high level 

of DNA binding and transcriptional activities of CP2b/CP2c. Taken together, our data 

suggest that PIAS1 commands the protein-protein interaction, subcellular localization, and 

DNA binding abilities of the CP2 transcription factor family to confer erythroid cell specific 

α-globin gene regulation. 

107



Keun Il Kim 

Abstract P58 

A novel link between SUMO modification of a chromatin 

remodeling complex and cancer metastasis 

Sung Hee Baek 1 , Jung Hwa Kim 1 , Hee June Choi 1 , Bogyou Kim 1 , Ji Min 

Lee 1 , Ik Soo Kim 1 and Keun Il Kim 2 

1 

Department of Biological Sciences, Seoul National University, Seoul 151-746, South 

Korea, 2 Department of Biological Sciences, Sookmyung Womens University, Seoul 140- 

742, South Korea 

Defining the functional modules with transcriptional regulatory factors that govern switching 

between repression and activation events is a central issue in biology. We have reported 

the dynamic role of a b-catenin/reptin chromatin remodeling complex to regulate a 

metastasis suppressor gene KAI1, which is capable of inhibiting the progression of tumor 

metastasis, and further which signaling factors confer repressive function on reptin and 

hence maintain a repressed state of KAI1 (Kim et al., Nature 434, 921-6; Kim et al., Nature 

Cell Biol. 8, 631-9). Biochemical purification of a reptin-containing complex has revealed 

the presence of specific deSUMOylating enzymes that reverse the SUMOylation of reptin 

that underlies its repressor function. DeSUMOylation of reptin alters the repressive function 

of reptin and its association with HDAC1. Further, SUMOylation status of reptin modulates 

the invasive activity in cancer cells with metastatic potential. This provides a clear definition 

of the functional model and a novel insight for linking SUMO modification to cancer 

metastasis. As a follow-up study, we will address novel findings on the function of newly 

identified histone methyltransferase as a component of reptin, linking chromatin remodeling 

process and cancer metastasis. 

108



Sarah Kimmins 

Abstract P59 

Methylation of Histone H3 at lysine 4 is dynamic and tightly 

regulated during male germ cell development 

Véronik Auger, Maren Godmann and Sarah Kimmins 

Departments of Animal Science and Pharmacology and Therapurtics, McGill University, 

Montreal, Canada 

Idiopathic male infertility is associated with genetic and epigenetic abnormalities. Histones 

can undergo epigenetic modifications on the N-terminus including methylation, acetylation 

and phosphorylation, among others. These histone modifications are hypothesized to signal 

changes in chromatin structure leading to altered gene expression and recruitment of 

regulatory transcription complexes. To date there is little information on the distribution and 

significance of histone modifications in spermatogenesis. We have identified the cellular 

localization patterns and developmental regulation of histone H3 mono-, di-, and trimethylation 

at lysine 4 (K4), and the epigenetic modifiers implicated in the, removal and 

reading of these epigenetic marks namely, LSD1, MBD2a/b, and HDAC1, during 

mammalian spermatogenesis. Testis were collected from the first wave of spermatogenesis 

in mice at postnatal days 6 (type A spermatogonia), 8 (type A and B spermatogonia), 10 

(pre-leptotene and leptotene), 12, (zygotene), 14 (early pachytene), and 20 (late pachytene) 

for immunolocalization and Western blot analysis. Patterns of distribution were further 

confirmed using highly specific cell isolation and staging methods. Histone H3-K4 mono-, 

di- and tri-methylation is dynamic and widely distributed in spermatogenic cell types and is 

associated with euchromatic regions. This strongly suggests that as in other tissues, in the 

testis, methylation of histone H3 at lysine 4 serves key functions in the regulation of gene 

transcription. Expression of LSD1 is tightly regulated during germ cell differentiation. 

Remarkably, in comparison to somatic tissues, LSD1 is preferentially expressed in the testis. 

Interaction studies reveal unique transcriptional regulatory complexes associated with H3- 

K4 methylation in the testis including the association of LSD1 and MBD2b in a complex with 

HDAC1, presumably forming transcriptional repressor complexes. These studies serve to 

enhance our understanding of epigenetic control of the transcriptional program governing 

male germ cell differentiation in normal and pathological states. 

109



Robert Klose 

Abstract P60 

JmjC-domain-containing proteins and histone demethylation 

Robert J. Klose and Yi Zhang 

Howard Hughes Medical Institute, Lineberger Comprehensive Cancer Center, Department of 

Biochemistry and Biophysics, University of North Carolina at Chapel Hill, U.S.A. 

Histone methylation plays important roles in regulation of gene expression, maintenance of 

genome integrity, and epigenetic inheritance. A wealth of understanding is available about 

the histone methyltransferase enzymes which place these covalent modifications, but, until 

recently, enzymes capable of reversing histone methylation remained elusive and histone 

methylation was thought to be a relatively static modification. The identification of lysine 

specific demethylase1 (LSD1) revealed that histone methylation could be dynamically 

regulated in a manner similar to histone acetylation and phosphorylation. Recently the 

Jumonji C (JmjC) domain has been shown to possesses Fe(II)/alpha-ketoglutarate 

dependent histone demethylase activity. Bioinformatic analysis of the extended JmjCdomain 

containing family of proteins has enabled us to categorize these proteins into seven 

evolutionarily conserved groupings based on homology within the JmjC-domain and overall 

protein domain architecture. By analysing the predicted co-factor binding sites within 

individual JmjC-domain groupings we have been able to utilize a targeted approach to 

characterize additional JmjC-domain containing histone demethylases. Based on our 

analysis we recently identified a novel histone demethylase, JHDM3A, which has the 

capacity to reverse the tri-methyl lysine modification mark on histone H3K9/36. Our 

continued functional analysis has revealed additional histone demethylases within the JmjCdomain 

containing family of proteins. 

110



Christoph M. Koch 

Abstract P61 

The landscape of activating histone modifications across 1% 

of the human genome 

Christoph M. Koch, Robert M. Andrews, Paul Flicek, Shane C. Dillon, 

Ulas Karaoz, Gayle K. Clelland, Sarah Wilcox, Dave M Beare, Joanna C. 

Fowler, Phillippe Couttet, Keith D. James, Gregory C. Lefebvre, Alexander 

W. Bruce, Oliver M. Dovey, Peter D. Ellis, Pawandeep Dhami, Cordelia F. 

Langford, Cordelia F. Langford, Zhiping Weng, Ewan Birney, Nigel P. 

Carter, David Vetrie and Ian Dunham 

The Wellcome Trust Sanger Institute and European Bioinformatics Institute Wellcome Trust 

Genome Campus, Hinxton, Cambridge, U.K., Bioinformatics Program and Biomedical 

Engineering Department Boston University, 24/44 Cummington St., Boston, MA 02215, U.S.A. 

The NHGRI has established a pilot project (ENCODE) to explore computational and 

experimental methods to develop an encyclopaedia of DNA elements in the human 

genome. Initially the project targets 1% of the genome chosen according to the criteria 

outlined at http://www.genome.gov/10506161. We have constructed a microarray 

representing the 44 ENCODE regions consisting of 24005 PCR fragments with an average 

size of ~1 kb. It covers ~80% of the targeted regions including repetitive elements where 

possible. We are using this microarray to assay DNA samples enriched for sequences 

involved in specific biological processes and functions generated by chromatin 

immunoprecipitation (ChIP). ChIP experiments are being performed with a variety of 

antibodies for specific histone modifications in a lymphoblastoid cell line (GM06990), an 

erythroleukemia cell line (K562) , foetal lung fibroblastoid cell lines (IMR90, HFL-1), cervix 

carcinoma cell line (HeLaS3), a T-cell line (MOLT4) and a chimpanzee cell line PTR8. We 

correlate maps of histone modifications with a range of genomic DNA features including 

C+G content, genes/exons, repeat elements, SNP density and regions of conserved DNA 

sequence identified by comparative sequencing across multiple species as well as the 

expression profiles of the cell lines. Preliminary analysis reveals strong enrichments of 

acetylated histone H3 and di- and tri-methylated histone H3 (H3K4me2 and H3K4me3) at 5 

ends of transcriptional start sites. Mono-methylated histone H3 (H3K4me1) was found to be 

widely distributed and not exclusive focussed to transcriptional start sites similar to 

acetylated histone H4 (H4ac). While comparing enrichments between different cell lines we 

found a correlation of the expression status of genes and the absence or presence of 

H3K4me3 at the transcriptional start site/promoter. Active promoters in each cell line show a 

robust enrichment of H3K4me3 while inactive promoter do not. 

111



Ryoki Kujiki 

Abstract P62 

1alpha,25(OH)2D3-induced transrepression on 1alphahydroxylase 

gene promoter mediates chromatin remodeling 

through WINAC 

R. Fujiki 1 , M. Kim 1 , Y. Sasaki 1 , H. Kitagawa 1 and S. Kato 1,2 

1 

Laboratory of Nuclear Signaling, Institute of Molecular and Cellular Biosciences, University 

of Tokyo, Tokyo, Japan, 2 ERATO, Japan Science and Technology Agency 

Vitamin D receptor (VDR) is a nuclear receptor (NR) regulating bone metabolism and 

calcium homeostasis. VDR modulates many target genes in a ligand-dependent manner. 

VDR-mediated gene expression requires a large number of co-regulator complexes. 

Recently, we identified WINAC as a novel VDR co-regulater complex that reconfigures 

nucleosomal array around vitamin D response element (VDRE) (Cell, 113, 905, 2003). 

While mechanism of transactivation by NRs is well understood, the molecular basis of the 

ligand-induced repression remains to be uncovered. 

To address this issue, we focused on the 1a,25(OH)2D3-induced transrepression 

mechanism of 25(OH) D3 1a-hydroxylase [1a(OH)ase] gene, encoding a key enzyme of 

vitamin D metabolism. The transactivation function by VDIR, a transcriptional factor 

recognizing negative VDRE in 1a(OH)ase promoter (1anVDRE) (EMBO J. 23, 1598, 2004), 

was found to be suppressed by liganded VDR and/or WSTF, a WINAC major component. In 

co-immunoprecipitation assay, WSTF physically interacts with both unliganded VDR and 

acetylated histones. ChIP assay showed that this interaction presumably allowed DNAunbound 

VDR to associate with the 1anVDRE region prior to ligand binding. Using mouse 

embryonic fibloblasts from VDR knockout mice, we further found that VDR is required for 

the association of WSTF with 1a(OH)ase promoter, implying 1anVDRE-specific association 

of WSTF is defined by VDR. Next, we showed in vitro that the association of WSTF with 

acetylated histones is required for assembly of liganded VDR with VDIR bound to the 

1anVDRE. WSTF bromodomain was then mapped as interaction surface to acetylated 

histone H3. Interestingly, WSTF with deleted bromodomain acts as a dominant negative 

mutant in the transrepression of 1a(OH)ase gene. All together, WSTF association with both 

unliganded VDR and acetylated nucleosomes, appears to be indispensable for this ligandinduced 

transrepression (EMBO J. 24, 3881, 2005). Thus, we have identified a novel 

mechanism of ligand-induced tranrepression by NRs that links transrepression and 

promoter histone acetylation. 

112



Sharmistha Kundu 

Abstract P63 

SWI/SNF establishes transcriptional memory at the 

Saccharomyces cerevisiae GAL1 gene 

Sharmistha Kundu, Peter J. Horn and Craig L. Peterson 

Program in Molecular Medicine, University of Massachusetts Medical School, 373 

Plantation St., Biotech 2, Suite 210, Worcester, 01605, U.S.A. 

Chromatin remodeling enzymes that chemically modify histones or DNA have long been 

linked with transcriptional memory in diverse eukaryotes. We show that the ATP-dependent 

chromatin remodeling enzyme, SWI/SNF is essential for establishing transcriptional memory 

in the yeast, Saccharomyces cerevisiae. We observe memory in the regulation of the yeast 

GAL1 gene expression. Though SWI/SNF is dispensable for inducing GAL1 transcription, its 

recruitment allows much rapid kinetics of GAL1 reinduction, after being transitorily 

repressed with glucose. This phenomenon requires the ATPase activity of SWI/SNF and is 

epigenetically inherited by daughter cells. Significantly, SWI/SNF appears to antagonize the 

ISWI complexes to establish transcriptional memory at GAL1. Upon further examination we 

find that deleting the ISW2 complex specifically rescues growth defect of swi2- cells in 

galactose and also rescues expression of another SWI/SNF dependent gene. Further 

examples of SWI/SNF - ISW2 antagonism are presented. 

113



Georg Kustatscher 

Abstract P64 

Metabolite-sensitive and metabolite-insensitive chromatin 

surfaces through the human histone macroH2A 

Georg Kustatscher, Judith Sporn, Michael Hothorn, Bjoern Fritz, Miriam 

Bortfeld, Klaus Scheffzek and Andreas Ladurner 

Gene Expression and Structural Biology Unit, EMBL Heidelberg, Germany 

Small-molecule metabolites play an important role in the regulation of gene expression. We 

discovered that human chromatin might be a receptor for NAD-metabolites produced by Sir2 

deacetylases. We are now studying whether this histone might provide a regulatory 

connection between metabolism and vertebrate chromatin. 

Unlike other deacetylases, Sir2 and its metazoan homologues use NAD to carry out the 

chemically straightforward deacetylation reaction. Sir2 may thus respond to the redox state 

of a cell. In fact, evidence links Sir2 to metabolism, chromatin and aging by mediating the 

benefits of life-extension through caloric restriction in yeast, C. elegans and Drosophila. The 

mammalian orthologue, SirT1, is induced under caloric restriction and regulates fat storage 

and the insulin pathway through PPARgamma and FOXO deacetylation. Sir2/SirT1 might 

thus play a conserved role in the metabolic control of gene expression and in 

heterochromatin formation. 

We find that its metabolite, O-acetyl-ADP-ribose (AAR), binds the macro domain of the 

human histone macroH2A1.1. The novel crystal structure of the protein-nucleotide complex 

reveals how chromatin may be a direct target for endogenous metabolites. Further, 

macroH2A1 is subject to alternative splicing, where two mutually exclusive exons produce 

two distinct proteins. Crucially, these exons encode a region critical for ligand-binding. In 

fact, the splice variant cannot bind AAR. Structural plasticity between the isoforms thus 

results in proteins that adopt the same fold, but show binary sensitivity to AAR. Human 

chromatin may thus bear metabolite-sensitive and metabolite-insensitive surfaces. 

We present in vivo evidence, including human tumors, that the two macroH2A1 isoforms 

show cell-type specific expression, in particular with regard to proliferation. Could there be 

metabolic control of gene activity through macroH2A-containing chromatin and how is this 

linked to proliferation and gene repression in mammals 

114



Hyockman Kwon 

Abstract P65 

BAF53-dependent higher-order chromatin structure as the 

compartment of replication and repair foci 

Ki Won Lee 1 , Su Jin Kwon 1 , Phan Kyu Park 1 , Jae Yong Kim 1 , Yunhee Kim 

Kwon 2 , and Hyockman Kwon 1 

1 

Department of Bioscience and Biotechnology, Hankuk University of Foreign Studies, 

Yongin 449-791, Republic of Korea, 2 Department of Biology, Kyunghee University, Seoul 

130-701, Republic of Korea 

It is becoming evident that higher-order chromatin structure plays a critical role in many 

aspects of gene regulation in interphase mammalian nuclei. The emerging view is that 

chromosomes are compartmentalized into discrete chromosome territories in which 

chromatins are packaged into compact chromosomal subdomains with diameters of 100 to 

450 nm. Chromosomal subdomain may define the functional compartment as well as the 

structural compartment. In mammalian cell nucleus, DNA replication and DNA doublestrand 

break repair occur at discrete sub-nuclear structure called replication foci and repair 

foci, respectively. Interestingly, the average sizes of replication and repair foci, ~1 Mb, are 

similar each other, suggesting that they could represent different functional states of 

chromosomal subdomains. However, this possibility has not been fully appreciated yet. 

Previously, we showed that BAF53 is required for the higher-order chromatin structure. 

BAF53 knockdown resulted in the expansion of chromosome territories and the remarkable 

increase in the micrococcal nuclease sensitivity of chromatin. Here we found that BAF53 

knockdown suppressed the formation of replication foci and repair foci. Although DNA 

replication proceeded normally in the BAF53-knockdowned cells, the early S-phase 

replication foci were not observed. Instead, a diffused pattern of BrdU incorporation was 

found in the nucleoplasm. Interestingly, the mid and late S-phase replication foci remained 

intact. Reduction of H3-K9 dimethylation foci in the nucleoplasm in the BAF53- 

knockdowned cells supports the specific disappearance of the early S-phase replication 

foci. In addition, the formation of H2AX foci in response to DNA damage by adriamycin was 

largely reduced in the BAF53-knockdowned cells. Activation of ATM appeared unchanged in 

the BAF53-knockdowned cells. Taken together, these results raised the possibility that 

replication foci and repair foci are originated from the same structural entity such as 

chromosomal subdomain whose formation requires BAF53. We discussed our results based 

on the multi-loop subcompartment model for chromosomal subdomain. 

115



Monika Lachner 

Abstract P66 

Studying lysine methylation in non-histone proteins 

Monika Lachner 1 , Kristie L. Rose 2 , Jeffrey Shabanowitz 2 , Karl Mechtler 3 , 

Thomas Jenuwein 3 , Donald F. Hunt 3 and C. David Allis 1 

1 

The Rockefeller University, 1230 York Avenue, New York, NY 10021, U.S.A., 2 Department of 

Chemistry, University of Virginia, Charlottesville, VA 22901, U.S.A., 3 Research Institute of 

Molecular Pathology, Dr. Bohrgasse 7, 1030 Wien, Austria 

While the impact of histone lysine methylation on chromatin structure and function has been 

extensively studied, there is little known about the occurrence and function of this posttranslational 

modification in non-histone proteins. This obvious lack of understanding 

sparked our interest in exploring lysine methylation in non-histone proteins. 

In order to identify novel lysine methylation sites in non-histone proteins, we employed an 

approach that combines immunoprecipitation with a pan-methyl-lysine antibody and massspectrometry. 

These experiments provided several interesting candidate proteins that are 

involved in a variety of cellular processes. For a more detailed analysis we chose two of 

these proteins (Eset and mAM), since they have a well-documented function. Eset and 

mAM form an enzymatic complex that specifically trimethylates histone H3 on lysine 9. 

Interestingly, immunoprecipitations with the pan-methyl-lysine antibody suggested that both 

complex members are lysine-methylated proteins. At this point, we have confirmed this 

finding and we have been able to map one methylation site in mAM. Experiments are 

currently underway that will address the identity of the respective methyltransferase as well 

as the potential functional implications of this post-translational modification (e.g. 

modulation of the enzymatic activity of the complex, regulation of protein-protein 

interactions). 

We anticipate that further investigation of lysine methylation in non-histone proteins will 

demonstrate the importance of this post-translational modification in numerous biological 

processes. 

116



Brian D. Larsen 

Abstract P67 

Caspase 3 mediated DNA strand breaks contribute to 

genomic reorganization during skeletal muscle terminal 

differentiation 

Brian D. Larsen 1,2 and Lynn A. Megeney 1,2 

1 

Ottawa Health Research Institute, Centre for Stem Cell Research, Molecular Medicine 

Program, Ottawa Hospital General Campus, Ottawa ON, Canada. 2 Department of Cellular 

and Molecular Medicine, University of Ottawa, Ottawa ON, Canada 

Skeletal muscle differentiation is dependent on chromatin remodelling and genome wide 

reorientation of gene expression programs. However, the factors controlling this transition 

have not been fully elucidated. One mechanism known to influence the differentiation 

process is the activity of pro-apoptotic proteins. The apoptotic process is itself associated 

with genomic reprogramming events i.e. DNA strand breaks, suggesting that similar 

mechanisms may influence the genomic transition during cell differentiation. Here we 

explored the role of strand break formation in skeletal muscle differentiation. In situ nick 

translation (ISNT) was used to detect DNA strand breaks during differentiation of an in vitro 

model system (C2C12 myoblast cell line). Transient DNA strand breaks were detected early 

during differentiation between 12 and 24 hours. The formation of DNA strands breaks 

coincided with caspase 3 activation, a requirement for skeletal muscle differentiation. 

Moreover, treatment of differentiating C2C12 myoblasts with the caspase 3 inhibitor (z- 

DEVD-fmk) blocked the detection of DNA strand breaks by ISNT coincident with an 

inhibition of differentiation/myotube formation. A well characterized function of caspase 3 is 

the activation of the caspase activated nuclease (CAD) through proteolytic cleavage of its 

inhibitor (ICAD). Western blot analysis revealed cleavage of the long isoform of ICAD in 

C2C12 cells at 12 and 24 hours following low serum induction of differentiation; these 

results suggest the stand breaks are mediated through caspase 3 activity. The formation of 

transient DNA strand breaks implicates the necessity of a DNA repair mechanism during 

the differentiation process. As such we monitored the phosphorylation status of the histone 

variant H2AX, a well characterized marker of DNA double strand breaks, during myoblast 

differentiation. Phosphorylation of H2AX coincided with the formation of DNA strand 

breaks during myoblast differentiation and also appeared to be dependent on caspase 3 

activity. These results suggest a role for pro-apoptotic proteins in regulating gene expression 

and chromatin remodelling to promote myoblast differentiation. 

117



Richard Lawrence 

Abstract P68 

Mechanisms controlling dynamic Swi6/HP1 binding in S. 

pombe facilitate de novo heterochromatin formation 

Richard J. Lawrence and Thomas A. Volpe 

Department of Cell and Molecular Biology, Feinberg School of Medicine, Northwestern 

University, 303 E. Chicago Avenue, Chicago, IL 60611, U.S.A. 

Heterochromatin plays a critical role in genome stability and organization, and has long 

been regarded as statically condensed and inert. Contrarily, recent evidence suggests that 

heterochromatin is maintained by dynamic binding of heterochromatin protein 1 (HP1). 

However, the mechanisms, if any, that mediate dynamic HP1 binding and the in vivo 

function of HP1 mobility are unknown. We are studying a jmjC domain protein that 

modulates binding of the HP1 homolog, Swi6, to heterochromatin in S. pombe thereby 

antagonizing epigenetic stability. Interestingly, the protein lacks histone demethylase activity. 

In the absence of this protein a reporter gene in centromere heterochromatin is more 

enriched with Swi6 and in vivo binding studies indicate that Swi6 is more tightly bound to 

heterochromatin. Paradoxically, this “hyper-heterochromatization” promotes epigenetic 

heterochromatin stability but inhibits de novo heterochromatin nucleation. These results 

ultimately suggest that Swi6 binding is actively antagonized by this protein; thus, it functions 

to mobilize a pool of Swi6 that targets heterochromatin nucleation, most likely through a 

novel mechanism that is independent of histone demethylase activity. 

118



Frederic Leduc 

Abstract P69 

Presence of gamma-H2AX in elongating spermatids: 

involvement of NHEJ 

Frederic Leduc, Veronique Lauziere, Marilyne Joly, Leila Jaouad and 

Guylain Boissonneault 

Faculte de Medecine, Departement de Biochimie, 3001, 12eme avenue Nord, Sherbrooke, 

Qc J1H 5N4, Canada 

The histone variant H2AX is involved in early DNA damage response and controls the 

recruitment of DNA repair proteins at sites of double-stranded breaks. In testis, the active, 

phosphorylated form of H2AX (gamma-H2AX) is thought to be involved in two processes 

namely the inactivation of sex chromosomes (at the sex vesicle) and the control of genome 

integrity during meiotic recombination. We used confocal microscopy applied to both 

immunofluorescence and TUNEL (terminal deoxynucleotidyl transferase dUTP nick-end 

labeling) on squash preparations of seminiferous tubules. Stage-specific sections were 

obtained according to their light absorption pattern. Here, we demonstrate the presence of 

gamma-H2AX in the whole population of elongating spermatids in mouse (steps 8-9), 

coincident with the onset of transient DNA strand breakage and chromatin remodeling as 

shown by the hyperacetylation of histone H4. These results strongly suggest that a 

complex DNA repair system is recruited and required during the chromatin remodeling steps 

in elongating spermatids. Given their haploid character, we hypothesize that the nonhomologous 

end-joining (NHEJ) is responsible for the DNA repair during step 8 through 13. 

The presence of NHEJ-associated factors is being investigated. 

Funded by Canadian Institutes of Health Research (Grant# MOP-74500) 

119



Min Gyu Lee 

Abstract P70 

Functional association of a trimethyl H3K4 demethylase and 

Ring6a/MBLR, a polycomb-like protein 

Min Gyu Lee 1 , Jessica Norman 1 , Anne llvarsonn 2 , Joel C Eissenberg 2 , Ali 

Shilatifard 2 and Ramin Shiekhattar 1 

1 

The Wistar Institute, 3601 Spruce Street, Philadelphia, PA 19104 U.S.A., 2 Department of 

Biochemistry and Molecular Biology, Saint Louis University Health Sciences Center, Saint 

Louis, MI 63104, U.S.A. 

Histone methylation is a post-transcriptional mark regulating chromatin structure and gene 

regulation. Once deemed irreversible, recent findings have identified two classes of 

enzymes capable of demethylating lysine residues. BHC110/LSD1, the catalytic heart of 

multiple co-repressor complexes, was the first of such demethylases shown to reverse 

dimethyl histone H3K4. However, due to intrinsic limitations of BHC110/LSD1 mode of 

action, it is unable to remove trimethyl H3K4 marks. Here we show that JARID1d, a member 

of a second class of histone demethylases containing JmjC-domain, can specifically 

demethylate trimethyl H3K4. Detailed mapping analysis revealed that besides the JmjCdomain, 

the BRIGHT and zinc-finger-like C5HC2 domains are required for maximum 

catalytic activity. Importantly, isolation of native JARID1d complexes from human cells 

revealed the association of the demethylase with a polycomb-like protein Ring6a/MBLR. 

Ring6a/MBLR not only directly interacts with JARID1d but also regulates its enzymatic 

activity. We show that JARID1d occupies human Engrailed 2 promoter and regulates its 

expression and H3K4 methylation levels. Finally, we show that the single Drosophila 

homolog of JARID1d, little imaginal discs (Lid), is also a trimethyl H3K4 demethylase 

attesting to the cross-species conserved function for this family of histone demethylases. 

120



Niraj Lodhi 

Abstract P71 

Histone acetylation (H3K9) and methylation (H3K4) of the 

nucleosome over core promoter are associated with the 

induction of tobacco PR-1a gene 

Niraj Lodhi, C. P. Chaturvedi, Suraiya A. Ansari, Rakesh Srivastava, 

Samir V. Sawant and Rakesh Tuli 

National Botanical Research Institute India, Rana Pratap Marg, Lucknow, India 226001 

The PR-1a gene encodes one of the major defense related protein in tobacco (Nicotiana 

tabacum) and it is tightly regulated at the transcription level. Detailed studies on PR-1a 

promoter in tobacco leaves have shown that it is induced specifically on pathogen attack or 

after Salicylic Acid (SA) induction. Our work suggested that the core promoter region of PR- 

1a gene plays an important role in determining SA induced transcription. We have mapped 

a nucleosome which spanned the core promoter of PR-1a gene. The nucleosome spanned 

the region between -103 to +55 relative to the transcription initiation site of the PR-1a gene. 

We carried out Chomatin Immunoprecitation (ChIP) with H3K9 acetylation specific and 

H3K4 methylation specific antibodies to identify the role of acetylation and methylation of 

nucleosome over the core promoter region. Our results revealed that the nucleosome over 

the core promoter results in a repressive chromatin architecture which is remodeled by 

histone modifications concomitant with the gene activation after SA induction. The SA 

induction leads to shifting of nucleosome from the core promoter which allows the assembly 

of the pre-initiation complex and initiation of transcription. The results related to our new 

findings will be presented at the conference. 

121



Mattias Mannervik 

Abstract P72 

An HDAC3/SMRTER/Ebi complex required for Snail 

repressor function in Drosophila development 

Mattias Mannervik, Dai Qi and Mattias Bergman 

Stockholm University, Wenner-Gren Institute, Dept. of Developmental Biology, 

Arrheniuslaboratories E3, Stockholm, Sweden 

The zinc-finger transcription factor Snail is critical for Drosophila embryo development by 

preventing expression of neuroectoderm-specific genes in the mesoderm. We found that in 

embryos devoid of maternal Ebi protein, Snail repressor function is impaired. Drosophila Ebi 

and its mammalian homolog TBL1 are WD40 proteins with a divergent F-box domain. 

Previous studies have linked Ebi and TBL1 to two different pathways; ubiquitin conjugation 

through SCF-type ligases, and N-CoR/SMRT/HDAC3-mediated transcriptional repression. In 

ebi mutant embryos, Snail target genes are de-repressed in the mesoderm. De-repression 

of a Snail-dependent reporter gene in ebi mutant embryos, and genetic interactions with 

snail support a requirement for Ebi in Snail function. Snail-mediated repression was 

previously shown to depend on another co-repressor, CtBP. We found that both CtBP and 

Ebi can interact with Snail protein in vitro, but through different interaction domains. A 

minimal Ebi-interaction domain that fails to bind CtBP constitutes a potent repression 

domain in both S2 cells and in transgenic embryos. This suggests that Snail uses Ebi as corepressor 

independently of CtBP. The repression activity of this domain can be attenuated 

either by knockdown of HDAC3 or by TSA treatment, indicating an involvement of histone 

deacetylation. By contrast, inhibition of proteasome activity does not affect Snail-mediated 

repression. We suggest that Ebi as part of a SMRTER/HDAC3 co-repressor complex is 

required for Snail function in Drosophila, and that histone deacetylation is part of the 

mechanism by which Snail represses transcription. 

122



Robert Martin 

Chromatin labeling and distribution in living cells 

Abstract P73 

Martin, R.M., Leonhardt, H. and Cardoso, M.C. 

Max Delbrueck Center for Molecular Medicine, Robert Roessle Str. 10, 13125 Berlin, 

Germany and Ludwig Maximilians University Munich,Department of Biology II, 82152 

Planegg-Martinsried, Germany 

Live cell fluorescence microscopy experiments often require visualization of the nucleus and 

the chromatin to determine the nuclear morphology or the localization of nuclear 

compartments. We compared five different DNA dyes, TOPRO-3, TOTO-3, propidium iodide, 

Hoechst 33258, and DRAQ5, to test their usefulness in live cell experiments with continuous 

imaging and photobleaching in widefield epifluorescence and confocal laser scanning 

microscopy. In addition, we compared the DNA stainings with fluorescent histones as an 

independent fluorescent label to mark chromatin. From the dyes tested, only Hoechst and 

DRAQ5 could be used to stain DNA in living cells. However, DRAQ5 had several 

advantages, namely low photobleaching, labeling of the chromatin compartments 

comparable to that of H2B-GFP fusion proteins, and deep red excitation/emission 

compatible with available genetically encoded fluorescent proteins such as C/G/YFP or 

mRFP. The DNA dye DRAQ5 is well suited for chromatin visualization in living cells and can 

easily be combined with other fluorophores with blue to orange emission. It could be used in 

a variety of cells of different species including primary cultures after a few minutes 

incubation in the culture medium. 

Furthermore the effect on histones and other chromatin proteins will be discussed. DRAQ5 

is a useful molecular tool for cell biology that allows a fast and non invasive labeling as well 

as microscopic visualization of DNA structures in living cells. 

Ref: 

Martin, R. M., Leonhardt, H., and Cardoso, M. C. (2005) Cytometry A 67, 45-52 

123



Peter McKeown 

Abstract P74 

Chromatin components of the Arabidopsis thaliana nucleolus 

Peter C. McKeown, Alison F. Pendle and Peter J. Shaw 

Department of Cell and Developmental Biology, John Innes Centre, Norwich Research Park, 

Colney Lane, Norwich, Norfolk U.K., NR4 7UH, John Innes Centre, Colney Lane, Norwich, 

Norfolk, NR4 7UH, U.K. 

Eukaryote DNA is packaged into chromatin through association with highly basic histone 

proteins. The presence of histone variants and multiple covalent modifications engender a 

‘histone code’ which controls the expression of the packaged DNA. This chromatin is not 

purely informational – it also has a structural existence within the nucleus. This is welldemonstrated 

in the largest nuclear structure, the nucleolus, which forms through the 

chromatin-regulated expression of rRNA genes and acts as the site of pre-ribosome 

formation and many RNA processing pathways. However, few distinctive chromatin marks 

have been identified in the nucleolus, in contrast to other structures such as centromeres, 

telomeres and sites of DNA breaks. 

We have now identified a group of nucleolar histones and histone modifications in the 

model plant, Arabidopsis thaliana thaliana, through a combination of techniques. Previously, 

we used mass spectrometry of extracted nucleoli to analyse the organellar proteome 

(Pendle et al., Mole. Biol. Cell, 16:260-269, 2005) identifying several nucleolar histones in 

the process. We have now used further MS techniques to determine how these proteins are 

modified, and demonstrated nucleolar specificity with GFP-fusion proteins. 

Immunofluorescence has identified other modifications, and confirmed the presence of a 

nucleolus-specific linker histone which may bind inactive rRNA genes and acetylated 

histone H2B which colocalises with sites of RNA polymerase I-mediated transcription. Such 

chromatin components may represent a source of nucleolus-specific information additional 

to the standard histone code. 

We conclude that the nucleolus is a good model for assessing the links between the 

informational content of the histone code and the its effects upon nuclear structures, and 

are currently determining the roles of the chromatin marks identified in both rRNA 

transcription and nucleolar structure. 

124



Rosalind Meldrum 

Abstract P75 

Visualisation of DNA repair and chromatin dynamics 

Rosalind Meldrum 

School of Biosciences, University of Birmingham, Edgbaston, Birmingham B15 2TT U.K. 

The area of research describing interactions between chromatin structure and the DNA 

damage response is receiving an increasing amount of attention. It is likely that DNA 

damage has a considerable influence on the physical structure and movements of 

chromatin. It is with out doubt that new high-resolution microscopy techniques, that can 

visualise detailed geometrical and spatial changes that take place in the cell in response to 

DNA damage, will play a critical role in this area of research. 

The most useful approaches to detecting and visualising chromatin dynamics are those 

where DNA damage can be induced in predetermined patterns and target specific 

structures in a cell nucleus. 

To be able to see the details of structural and dynamic changes it is desirable to induce and 

visualise DNA damage with very high resolution. 

UV lesions can be induced with nanoscale resolution in cell nuclear DNA by triple-photon 

infra-red absorption. The number or lesions induced is much smaller than when total cell 

irradiation by a mercury lamp is used or when localised damage is induced by irradiation of 

cells with UV light through a micro-filter. Because the UV photoproducts can be induced in a 

defined pattern, during a period of incubation following irradiation, distinctive movement of 

the damage DNA is seen to take place and the damage distributed over the cell nucleus 

forms individual clusters of lesions. The characteristics of the clustering of lesions are being 

investigated in relation to cell cycle, histone modification, ATP-dependent remodelling and 

incorporation of histone variants. A further interesting observation revealed that higher 

levels of damage immobilise and preserve the induced pattern for some hours following 

irradiation. 

Ref: 

R.A.Meldrum, S.W.Botchway, C.W.Wharton, G.J.Hirst. (2003) Nanoscale induction of UV 

photoproducts in cellular DNA by 3-photon near infra-red absorption EMBO Reports 4, 12 

1144-1149 

125



Brendon Monahan 

Abstract P76 

Purification and characterization of the fission yeast Swi/Snf 

and RSC chromatin remodeling complexes 

Brendon J. Monahan 1 , Judit Villen 2 , Samuel Marguerat 3 , Jurg Bahler 3 , 

Steve Gygi 2 and Fred Winston 1 

1 

Department of Genetics, Harvard Medical School, Boston MA, U.S.A, 2 Taplin Biological 

Mass Spectrometry Facility, Department of Cell Biology, Harvard Medical School, Boston 

MA, U.S.A., 3 Wellcome Trust Sanger Institute, Cambridge, U.K. 

The Swi/Snf type of ATP-dependent chromatin remodeling complexes are molecular motors 

that modify chromatin structure thereby regulating transcription and mediating other cellular 

processes such as DNA repair. Two evolutionarily conserved and distinct subclasses of 

Swi/Snf, named Swi/Snf and RSC, have been extensively studied in Saccharomyces 

cerevisiae. Here we present the purification and characterization of the Swi/Snf and RSC 

chromatin remodeling complexes from the fission yeast Schizosaccharomyces pombe. S. 

pombe, which is as closely related evolutionary to humans as it is to S. cerevisiae, is of 

interest as its chromatin shares several important similarities to mammalian chromatin that 

do not occur in S. cerevisiae and also provides a basis for valuable comparative analysis. 

The S. pombe Swi/Snf and RSC complexes were purified using tandem affinity purification 

(TAP) methodology and components identified by mass spectrometry. The S. pombe 

Swi/Snf complex is composed of 12 subunits, of which six are shared with the 14-member 

RSC complex. Deletion and tetrad analysis has shown that the core subunit genes in RSC 

are essential for growth whereas deletion mutants of the paralogous Swi/Snf genes were 

viable. Four of the six genes shared between the two complexes are essential for cell 

growth, the exceptions being the two arp (actin related protein) genes, arp4+ and arp9+. To 

investigate the global effect Swi/Snf has on S. pombe gene expression, whole genome 

expression analysis was done using deletion mutants of two core Swi/Snf complex subunit 

genes, snf22 and snf5. Overall, the expression levels of approximately 2.5% of S. pombe 

genes were altered greater than 2-fold in the swi/snf mutants. Interestingly, genes involved 

in sugar uptake and iron homeostasis were significantly enriched in the up-regulated gene 

set. This work has established the foundation for further detailed analysis into the role of 

these chromatin remodeling complexes in transcription activation and repression and other 

cellular processes in fission yeast. 

126



Antonin Morillon 

Transcriptional co-suppression in S. cerevisiae 

Abstract P77 

Julia Berreta, Benjamin Pernot-Cornu and Antonin Morillon 

CGM-CNRS, Gif/Yvette, France 

Cosuppression has been defined as high gene copy number-triggered, homologydependent, 

gene silencing and may have evolved in eukaryotic cells as a defensive strategy 

against viral infections and to control activity of transposons. In S. cerevisiae, cosuppression 

was first unveiled in 2002 (Y. Jiang, 2002) and was shown to act at the 

transcriptional level controlling the Ty1 retrotransposon. As the main actors of 

Transcriptional Gene Silencing are not conserved in baker yeast, co-suppression must be 

mediated by an original pathway which we try to characterize in this work. We hypothesize 

that it is mediated by regulatory RNAs which may inhibit Ty1 expression. We show that the 

expression of Ty1 is dependent upon the presence of several proteins involved in RNA 

degradation, in particular the 5-3 exonuclease Xrn1. Furthermore, the drop of Ty1 

expression correlates with the accumulation of an antisense non coding RNA 

corresponding to the 5 Long Terminal Repeat (LTR) region of Ty1. Through ChIP 

experiments, we show that the decrease of Ty1 RNA does not correlate to a reduction of 

RNAPII occupancy on the Ty1 gene suggesting that Ty1 RNA is affected at a posttranscriptional 

step. Finally, it has been shown that the retrotransposon is very sensitive to 

the histone levels and to Chromatin Remodeling Complex activity. We will discuss our 

preliminary results on nucleosome positions and histone modifications on Ty1 elements 

upon co-suppression conditions. 

127



Ashby Morrison 

Abstract P78 

Mec1/Tel1-dependent phosphorylation of a chromatin 

remodeling complex influences the DNA damage 

checkpoint pathway 

Ashby J. Morrison 1 , Jung-Ae Kim 2 , Maria D. Person 3 , Jessica Highland 1 , 

Tammy S. Wehr 1 , Sean Hensley 1 , Jianjun Shen 1 , Sean R. Collins 5 ,Jeff 

Delrow 4 , Nevan J. Krogan 5 , James E. Haber 2 and Xuetong Shen 1 

1 

Department of Carcinogenesis, Science Park Research Division, University of Texas M.D. 

Anderson Cancer Center, Smithville, Texas 78957, 2 Rosentiel Center and Department of 

Biology Brandeis University, Waltham, Massachusetts 02454, 3 College of Pharmacy, 

Division of Pharmacology & Toxicology, University of Texas at Austin, Austin, Texas 78712, 

4 

Division of Basic Sciences Fred Hutchinson Cancer Research Center, Seattle, Washington 

98109, 5 Department of Cellular and Molecular Pharmacology, University of California San 

Francisco, San Francisco, California 94143 U.S.A. 

The Mec1/Tel1 kinases in yeast, ATM/ATR in mammals, play central roles in coordinating 

the DNA damage response by phosphorylating proteins involved in DNA repair and 

checkpoint pathways. Recently, ATP-dependent chromatin remodeling complexes, such as 

the yeast INO80 complex, which were originally characterized as transcriptional regulators, 

have also been implicated in the DNA damage response. Here, we show that the Ies4 

subunit of the INO80 complex is phosphorylated in a Mec1/Tel1-dependent manner during 

exposure to DNA damaging agents. The phosphorylation status of Ies4 does not 

significantly affect transcription or DNA repair processes, such as homologous 

recombination. However, DNA damage checkpoint pathways are influenced by the 

phosphorylation status of Ies4. These findings establish a chromatin remodeling complex 

as a functional component in the Mec1/Tel1 DNA damage signaling pathway that modulates 

checkpoint responses, and suggest that post-translational modification of chromatin 

remodeling complexes may regulate their involvement in distinct nuclear processes. 

128



Raul Mostoslavsky 

Abstract P79 

Genomic instability and aging-like phenotype in the absence 

of mammalian SIRT6 

Raul Mostoslavsky 1 , David B. Lombard 1,2 , Katrin F. Chua 1 , Jennifer Kim 1 , 

Lionel Gellon 4 , Bruce Demple 4 , George Yancopoulos 3 and Frederick W. Alt 1 

1 

Howard Hughes Medical Institute, The Children’s Hospital, CBR Institute for Biomedical 

Research, and 2 Department of Genetics, Harvard Medical School, Boston, Massachusetts 

021151, 3 Department of Pathology, Brigham and Women’s Hospital, Boston, Massachusetts 

021152, 4 Regeneron Pharmaceuticals, Inc., 777 Old Saw Mill River Road, Tarrytown, New 

York 10591-67073, 5 Department of Genetics and Complex Diseases, Harvard School of 

Public Health, Boston Massachussets 021154 

In yeast, the histone deacetylase/ADP-ribosyltransferase Sir2 inhibits DNA recombination, 

promoting longevity. Seven mammalian Sir2 homologs, termed SIRT1-SIRT7, have been 

described. Here we show that SIRT6 is a nuclear, chromatin-associated protein, expressed 

in multiple tissues. At the cellular level, SIRT6 promotes resistance to DNA damage and 

suppresses genomic instability, in association with a role in Base Excision Repair (BER). 

SIRT6-deficient mice are small and at 2-3 weeks of age develop abnormalities that include 

acute lymphocyte depletion, loss of subcutaneous fat, lordokyphosis and severe metabolic 

defects, eventually dying at about 4 weeks. 

We conclude that one function of SIRT6 is to promote normal DNA repair, and that SIRT6 

loss in mice leads to defects in lymphocyte homeostasis and abnormalities that overlap with 

aging-associated degenerative processes. Recent progress in understanding the role of 

SIRT6 in DNA repair and metabolism will be discussed. 

129



Takahiro Nakayama 

Abstract P80 

Drosophila GAGA factor promotes histone H3.3 replacement 

that prevents the heterochromatin spreading 

Takahiro Nakayama, Kenichi Nishioka, Yi-Xin Dong, Tsukasa Shimojima 

and Susumu Hirose 

Department of Developmental Genetics, National Institute of Genetics, and Department of 

Genetics, SOKENDAI, Mishima, Shizuoka-ken 411-8540, Japan 

Drosophila white gene that is normally located euchromatin region governs a red eye 

phenotype. When the white gene is juxtaposed with centromeric heterochromatin regions by 

chromosomal rearrangement, its expression is subject to variable in a heritable silencing 

manner, giving rise to white mottled eye color. This phenomenon termed position effect 

variegation (PEV) provides evidence for a crucial role of chromatin structure in gene 

expression. Previously, Karch group showed that the Trithorax-like gene encoding GAGA 

factor is a dominant enhancer of PEV, suggesting that the GAGA factor plays a role in the 

active maintenance of white under heterochromatin environment. However, little is known 

about the molecular mechanism. Here, we demonstrate that the GAGA factor binds to a site 

just downstream of the white gene and this binding site is necessary and sufficient to block 

heterochromatin spreading. Interestingly there are a dip of histone H3 Lysine 9 methylation 

and a peak of H3 Lysine 4 methylation at this site. Furthermore, the GAGA factor promotes 

chromatin remodeling and replacement of histone H3 with H3.3 through recruitment of 

HIRA at this site, and maintains white expression under the heterochromatin environment. 

Based on these findings, we propose that the GAGA factor- dependent replacement of 

Lysine 9-methylated histone H3 by H3.3 counteracts the spreading of silent chromatin. 

130



Zuyao Ni 

Abstract P81 

The tumor suppressor BRG1 silences the distal silencers at 

interferon-responsive genes 

Zuyao Ni*, Mohamed A I Abou El Hassan*, Zhaodong Xu, Tao Yu, and 

Rod Bremner 

Toronto Western Research Institute, University Health Network, 399 Bathurst Street, 

Toronto, Ontario, M5T 2S8, Departments of Lab Medicine and Pathobiology and 

Ophthalmology and Vision Science, University of Toronto, Ontario, Canada 

Communication between distal regulatory DNA elements is essential for control of many 

nuclear processes in higher eukaryotes. Long-range gene regulation has been associated 

primarily with step-wise activation of differentiation genes over days. Recently, our group 

implicated long-range effects in rapid IFNgamma (IFNg)-mediated gene induction. However, 

the mediators of such long-range effects are unknown. Previously, we showed that the 

tumor suppressor and chromatin remodeling factor BRG1 has a primary role in regulating 

IFNg-responsive genes, so we hypothesized it may act through remote elements. Here, we 

show that BRG1 constitutively binds distal regulatory sites and binding increases upon IFNg 

treatment. BRG1 is required for transcription factor (STAT1 and IRF1) binding, histone 

acetylation, and chromatin remodeling at the distal 

IFNg sensitive sites at several target loci. At both CIITA and SOCS1 loci distal elements 

loop and interact with target promoters and/or with each other in a BRG1 dependent 

manner. To determine the functional relevance of these events in the appropriate context, 

we constructed a 200 kb bacterial artificial chromosome (BAC) reporter vector containing 

the entire CIITA locus. Remarkably, deleting any one of several distal elements induced 

basal CIITA activity to levels similar to those seen in IFNg-treated cells, even in the absence 

of BRG1. Thus, multiple remote elements cooperate to silence CIITA, and IFNg-overcomes 

their effect, derepressing CIITA in a BRG1-dependent manner. Negative regulation of distal 

silencing complexes is a novel mechanism of action of BRG1. 

(* Equal contributors) 

131



Olivia Osborn 

Transcriptional targets of Af4 

Abstract P82 

Osborn O, Oliver P.L., Bitoun E. and Davies K.E. 

Dept of Physiology Anatomy & Genetics, Oxford University 

The putative transcription factor Af4, known to be translocated in acute leukaemia, is also 

expressed in the Purkinje cells of the cerebellum. The robotic mouse mutant has an ataxic 

gait, is smaller than its littermates and shows distinct and progressive pattern of Purkinje 

cells loss from 8 weeks of age. 

This phenotype is caused by a gain of function mutation (V280A) in Af4 which prevents its 

normal rapid turnover by the proteosome and consequently its accumulation in numerous 

tissues as well as the brain. The cause of the cell loss in robotic and the normal function of 

Af4 is, however, unknown. 

Chromatin immunoprecipitation coupled with microarrays have been used with the aim of 

finding the direct targets of this transcription factor along with expression analysis to 

determine the downstream targets that contribute to the ataxic phenotype. 

132



Julia Pagan 

Abstract P83 

A novel corepressor, BCOR-L1, functions through CTBP and 

class 2 HDACs 

Julia K. Pagan 1,2 , Jeremy Arnold 1 , Kim J. Hanchard 1 , Mathew J.K. Jones 1,2 , 

Derek J. Richard 1 , Alistair Forrest 3 , Amanda Spurdle 1 , Eric Verdin 4 , Merlin 

Crossley 5 , Georgia Chenevix- Trench 1 , David B. Young* 1 and Kum Kum 

Khanna* 1 

1 

Queensland Institute of Medical Research, 300 Herston Rd, Herston 4029, Queensland, 

Australia, 2 

School of Medicine, Central Clinical Division, University of Queensland, Royal 

Brisbane Hospital, Herston 4029, Queensland, Australia, 3 Institute for Molecular Bioscience, 

University of Queensland, Brisbane, QLD 4072, Australia, 4 Gladstone Institute of Virology 

and Immunology, University of California San Francisco, San Francisco, California, United 

States of America, 5 School of Molecular and Microbial Biosciences, G08, University of 

Sydney, New South Wales 2006, Australia 

Corepressors play a crucial role in negative gene regulation and are defective in several 

diseases. BCoR is a corepressor for the BCL6 repressor protein. Here we describe and 

functionally characterize BCoR-L1, a homolog of BCoR corepressor. When tethered to a 

heterologous promoter, BCoR-L1 is capable of strong repression. Most corepressors 

function by associations with histone deacetylase (HDAC) activity. BCoR-L1 coprecipitates 

with Class II HDACs; HDAC4, HDAC5 and HDAC7, suggesting they are involved in its role 

as a transcriptional repressor. BCoR-L1 interacts with the CtBP corepressor through a 

CtBP-interacting motif in its amino-terminus. Abrogation of the CtBP binding site within 

BCoR-L1 partially relieves BCoR-L1-mediated transcriptional repression. Furthermore, 

BCoR-L1 is located on the E-Cadherin promoter, a known CtBP regulated promoter, and is 

involved in repression of E-Cadherin. 

133



Maria V. Panchenko 

Role of Jade-1 in the HAT HBO1 complex 

Abstract P84 

Rebecca L. Foy, Vipul C. Chitalia, Herbert T. Cohen and Maria V. Panchenko 

Boston University School of Medicine, Evans Biomedical Research Center, MA 02118- 

2393, U.S.A. 

Regulation of global chromatin acetylation is important in processes requiring chromatin 

remodeling, including DNA replication. We demonstrated that PHD zinc finger protein Jade- 

1 is localized to the nucleus, activates rates of transcription when tethered to a 

heterologous promoter, and is associated with endogenous HAT activity. It has been recently 

reported (J. Coté’s lab, 2006) that Jade-1 co-purifies with a novel HAT complex, consisting 

of three additional proteins, including HBO1, ING4/5 and Eaf6. In mammalian cells, HBO1 

provides the basal level of global histone H4 acetylation, is required for DNA replication, 

and its activity is regulated during the cell cycle. We thus investigated a functional role for 

Jade-1 in the HBO1 complex. We demonstrated that while overexpression of HBO1 did not 

alter levels of endogenous histone H4 acetylation, co-transfection of even low sub-sufficient 

amounts of Jade-1 resulted in a dramatic, up to a 50-fold upregulation of histone H4 

acetylation, strongly suggesting that Jade-1 plays a crucial role in HBO1-mediated 

acetylation of nucleosomal histones. Interestingly, while PHD fingers were indispensable for 

Jade-1 to synergize with HBO1, they were dispensable for Jade-1-HBO1 physical 

interactions. We proposed that Jade-1 might promote histone acetylation by binding 

chromatin via its PHD fingers and targeting the HAT HBO1 complex to the proximity of 

histone substrates. Because Jade-1 partner HBO1 is involved in DNA replication, we 

investigated an effect of cell cycle progression on endogenous Jade-1 expression and 

nuclear distribution. We found that Jade-1 nuclear distribution and presumably chromatin 

association is strongly regulated by cell growth arrest and during cell cycle progression. In 

addition, Jade-1 undergoes phosphorylation followed by dephosphorylation in synchronized 

cycling cells. The data suggest a role for cdks in this posttranslational modification of Jade- 

1 and implicates a potential mechanism for the regulation of HBO1 HAT activity during the 

cell cycle and DNA synthesis. 

134



Tej Pandita 

Abstract P85 

Mammalian ortholog of Drosophila MOF is critical for 

embryogenesis and DNA repair 

Arun Gupta 1 , Geraldine Guerin-Peyrou 2 , Girdhar G. Sharma 1 , Manjula 

Agarwal 1 , Raj K. Pandita 1 , Raju Kucheralapati 3 , Thomas Ludwig 2 and 

Tej K. Pandita 1 

1 

Washington University School of Medicine, Saint Louis, MO 63108; 2 College of Physicians 

and Surgeons, Columbia University, New York, NY 10032, 3 Harvard Medical School, Boston, 

MA 02115, U.S.A. 

Human ortholog (hMOF) of the Drosophila MOF gene (males absent on the first) is histone 

H4 lysine K16-specific acetyltransferase. It is involved in transcription as it is a component of 

a functional dosage compensation complex required for male killing in Drosophila and DNA 

damage response. Cellular exposure to ionizing radiation enhances hMOF-dependent 

acetylation of its target substrate, lysine 16 of histone H4. Depletion of hMOF results in 

abrogation of ATM autophosphorylation, ATM kinase activity and DNA repair as well as 

increases cell killing after IR exposure. Based on these preliminary studies, we hypothesize 

that hMOF is involved in the regulation of DNA damage-induced ATM activation. In addition, 

MOF function is indispensable for development, because Mof-deficiency in mouse embryos 

results in early embryonic lethality which cannot be overcome by inactivation of p53 or ATM. 

Mice with haploinsufficiency of mMof in Atm null background are smaller in size and die 

early mostly because of leukemia. MOF over expression results in enhanced oncogenic 

transformation and is over expressed in most of the human tumors. These results 

demonstrate that ‘MOF’ is not only required for early embryonic development but plays a 

critical role in cell growth during oncogenic transformation in absence of Atm. We will 

discuss the role of MOF in regulation of DNA double strand break repair. 

135



Maëlle Pannetier 

Abstract P86 

Imprinting perturbation in mouse hepatocarcinoma: link 

between DNA methylation and histone methylation 

Maëlle Pannetier, Katia Delaval, Alexandre Wagschal and Robert Feil 

Institut de Génétique Moléculaire de Montpellier, CNRS UMR 5535 et Université Montpellier 

II, 1919 route de Mende, 34293 Montpellier cedex 5, France 

Genomic imprinting is an essential mechanism in development that gives rise to mono-allelic 

expression of genes depending on the parental origin of the allele. This mono-allelic 

expression is controlled by imprinting control regions (ICRs), short CpG-rich sequences that 

are differentially methylated. In addition to differential DNA methylation, allelic histone 

modifications, like lysine methylation and acetylation, are observed. We found that 

trimethylation of H3K27, H3K9 and H4K20 are associated with the DNA-methylated allele, 

whereas dimethylation of H3K4 and acetylation of H3 are associated with the unmethylated 

one. Cancer cells are characterized by prominent epigenetic dysregulation, including altered 

DNA methylation patterns, chromatin modifications and loss of genomic imprinting. Using a 

murine model of hepatocarcinoma, we study such epigenetic alterations at two imprinted 

domains located on chromosome 7: the Kcnq1 and Igf2/H19 domains. Transgenic mice 

showing liver-specific expression of a c-myc transgene were crossed with P53 knock-out 

mice. Thus, c-myc overexpression, in a hemizygous state for P53, gives rise to liver tumours 

at about 9 months of age. Moreover, these mice have a paternal chromosome 7 from M. 

Spretus and a maternal one from M. Domesticus, allowing us to discriminate the maternal 

allele from the paternal one. In this context, we determine if imprinting is perturbed in thusinduced 

hepatocarcinomas, studying DNA methylation and histone modifications. 1/4 of 

tested tumours showed a gain of DNA methylation at the Igf2/H19 ICR, whereas 1/16 

showed a loss of DNA methylation at the Kcnq1 ICR. Analysis of histone modifications by 

ChIP will allow us to determine how DNA and histone modifications are correlated during 

epigenetic perturbation. 

136



Janet Partridge 

Abstract P87 

Establishment and maintenance of centromeric 

heterochromatin in fission yeast are functionally separable 

Janet F. Partridge, Jennifer L. DeBeauchamp, Michael Hadler, Dagny L. 

Ulrich and Victoria J.P. Noffsinger 

Dept. Biochemistry, St. Jude Children’s Research Hospital, Memphis, TN. U.S.A. 

Both the establishment and maintenance of centromeric heterochromatin in fission yeast 

require the RITS complex. Comprised of centromeric siRNAs, the chromodomain protein 

Chp1, Argonaute (Ago1) and Tas3, RITS couples the cellular RNAi pathway with assembly 

of constitutive heterochromatin. However, it remains unclear if mechanisms governing RITSdependent 

establishment of centromeric heterochromatin differ from its maintenance. Here, 

we generate a Tas3 protein, mutated in a highly conserved GW-rich Argonaute-binding 

domain, which cannot bind Ago1. This mutant exhibits near normal maintenance of 

centromeric heterochromatin, but cannot support its establishment. We show that Ago1 can 

be maintained at centromeres through binding siRNA, but to establish centromeric 

heterochromatin, Ago1 must bind Tas3-Chp1. Our results support a model whereby 

recruitment of RITS to centromeres is initiated by Chp1 binding to K9-methylated histone 

H3, with the RNAi pathway responsible for maintenance of RITS at centromeres. 

137



Kelly Perkins 

Abstract P88 

Activated HIV-1 provirus forms a gene loop, connecting viral 

transcriptional initiation with termination 

Kelly J. Perkins, Marina Lusic 2 , Mauro Giacca 2 and Nick J. Proudfoot 1 

1 

Sir William Dunn School of Pathology, South Parks Road, University of Oxford, Oxford OX1 

3RE, U.K., 2 Laboratory of Molecular Medicine, International Center for Genetic Engineering 

and Biotechnology (ICGEB), Padriciano, 99-34012 Trieste, Italy 

We are investigating high order chromatin structure and factor recruitment to integrated HIV- 

1 provirus following re-activation from latency. Both chromosome conformation capture (3C) 

and chromatin immunoprecipitation (ChIP) techniques are being utilized. 3C analysis of promonocytic 

cell line U937 cell line U1/HIV-1 (U1) chromatin induced by TPA treatment or 

exogenous viral Tat expression indicated that activated provirus exists in a transcriptiondependent 

loop conformation that juxtaposes the 5 long terminal repeat (LTR) promoter and 

3LTR terminator regions. Based on ChIP analysis, RNA polymerase II (Pol II) and factors 

involved in transcriptional initiation and elongation (Cdk9 and USF) were detected at both 

ends of the HIV-1 provirus, supporting the existence of LTR-LTR interaction upon activation 

of proviral reservoirs. To determine whether proviral loop formation occurs before or after 

Cdk9-mediated Pol II CTD phosphorylation, we are currently performing 3C and ChIP 

analysis on chromatin treated with flavopiridol, a specific inhibitor of Cdk9 kinase activity. 

We propose that when latent integrated HIV-1 proviral DNA is transcriptionally activated in 

reponse to cellular or Tat-mediated stimulation, a structural formation is created where the 

flanking LTRs reside in close spatial proximity. This permits “cross-talk” between initiation 

and termination factors and so may allow efficient recycling of transcriptional machinery. 

This is the first study to identify the spatial arrangement of active integrated HIV-1 provirus 

and may provide valuable insight into the physical properties of HIV-1 proviral reservoirs 

upon transcriptional activation. 

138



David Picketts 

Abstract P89 

SNF2L-mediated control of cell number in the developing 

brain 

Darren J. Yip 1,3 , Stephen Rennick 1,3 , Adriana de Maria 1 , Josée Coulombe 1 , 

Michael Rudnicki 1,2 and David J. Picketts 1,3 

1 

Molecular Medicine Program, Ottawa Health Research Institute, 501 Smyth Road, Ottawa, 

ON, 2 Department of Cellular and Molecular Medicine and 3 Department of Biochemistry, 

Microbiology and Immunology, University of Ottawa, Ottawa, ON 

Epigenetic modification of the genome is becoming recognized as a major point of 

regulation governing many developmental processes. Certainly, defective epigenetic 

regulation is already implicated in a wide range of genetic disorders including mental 

retardation, autoimmune disorders, and cancer. We have shown that the ISWI family of 

chromatin remodelling proteins are abundantly expressed during mammalian brain 

development and thus, may be key participants in establishing chromatin domains that are 

characteristic of neurons. Here we used a conditional gene-targeting approach to inactivate 

the murine Snf2l gene in order to assess its role in neurodevelopment. Heterozygous 

females (Snf2lf/x) were crossed to mice expressing Cre-recombinase under the control of 

the GATA-1 promoter. SNF2L-null male mice were viable and born at classic Mendelian 

ratios. These mice displayed no overt developmental or behavioral abnormalities; however, 

the loss of Snf2l resulted in a 2-fold increase in the brain weight to body weight ratio. This 

was accompanied by a concomitant increase in cell number in the hippocampus and 

cerebral cortex ranging from 0.5-2-fold in the six distinct cortical layers and a 2-fold increase 

in all hippocampal strata. Using a combination of in situ BrdU labeling experiments and 

primary neuroprogenitor cultures we demonstrate that the increased cell number results 

from a delay in terminal differentiation. Indeed, neuroprogenitor cultures isolated from 

knockout animals continue to incorporate BrdU twice as long as wild type cells when 

induced to differentiate. As a result, fewer cells stain positive for neuronal or astrocytic 

markers at earlier times (4 days) but are equivalent by the end of the 7-day differentiation 

time course. Taken together, our results suggest that Snf2l has an important role in 

regulating the switch from proliferation to differentiation as a mechanism to control cell 

number and brain size during neuronal development. Moreover, we propose that Snf2l 

regulation of brain size occurs through direct effects on genes involved in cell cycle exit 

and/or terminal differentiation. 

139



Romina Ponzielli 

Abstract P90 

Optimization of experimental design parameters of ChIP-onchip 

studies 

Romina Ponzielli 1 Paul C. Boutros 1,2,3 , Sigal Katz 1 , Igor Jurisica 1,3,4 and 

Linda Z. Penn 1,2 

1 

Division of Cancer Genomics and Proteomics, Ontario Cancer Institute, University Health 

Network, Toronto, Canada M5G 2M9, 2 Department of Medical Biophysics, University of 

Toronto, Canada, 3 Division of Signaling Biology, Ontario Cancer Institute, University Health 

Network, Toronto, Canada M5G 2M9, 4 Department of Computer Science, University of 

Toronto, Canada 

Chromatin immunoprecipitation (ChIP) is an antibody-based technique for determining 

transcription-factor binding in vivo. ChIP-chip technology combines the sensitivity and 

specificity of ChIP with high-throughput analysis, by exploiting microarray platforms spotted 

with promoter region DNA. In yeast, this technique has been used to map transcriptionregulatory 

networks; in mammals, ChIP-on-Chip has determined transcription-factor binding 

in normal and cancerous cells and tissues. While ChIP-on-Chip studies are yielding useful 

results, their bioinformatic analysis is not yet fully realized. In this study we characterize 

both the statistical and experimental-design features of ChIP-on-Chip. 

To profile the signal-to-noise characteristics of ChIP-on-Chip data we performed a series of 

validation studies. First, we compared the performance of different amplification methods. 

Second, we determined optimal sample-to-array allocation. Third, we profiled the effect of 

dye-bias. Fourth, we evaluated effects of array batch variability. Finally, we determined the 

importance of antibody purity for successful ChIP-on-Chip studies. These studies 

encompass over 100 arrays, the data from which was exploited in a large-scale empirical 

study of statistical pre-processing methods. We assessed 84 distinct analysis methods for 

sensitivity, stability, and selectivity. Through these analyses we have optimized the major 

design parameters of ChIP-on-Chip studies. Our rigorous characterization of ChIP-on-Chip 

data is a key step towards exploiting this important technology for the rapid elucidation of 

regulatory networks. 

140



Ryan Raisner 

Abstract P91 

Single nucleosome resolution mapping of the histone variant 

H2A.Z in a developing organism 

Ryan M Raisner and Hiten D Madhani 

UCSF Department of Biochemistry and Biophysics, U.S.A. 

The conserved histone variant H2A.Z is an essential protein in higher organisms that is 

required early in development. It is poorly understood where H2A.Z is localized in 

metazoans, and furthermore what it’s essential biological function is. It has been shown by 

several groups that in the budding yeast Saccharomyces cerevisiae, H2A.Z is present 

throughout the genome, and more specifically it marks the promoter regions of the majority 

of genes, regardless of their transcription rate. More recently, there has been evidence that 

H2A.Z can selectively mark active genes in vertebrates. We have chosen the model 

organism Danio rerio (zebrafish) to study the distribution of H2A.Z using chromatin 

immunoprecipitation along with high resolution tiling microarrays at a range of genes, 

including the developmentally important Hox gene clusters at several stages of the 

developing animal. In addition to H2A.Z, we have also profiled the chromatin modifications 

H3 tri-methyl lysine 27 and tri-methyl lysine 4 at the same regions at single nucleosome 

resolution. These modifications have been shown recently to be present predominantly at 

transcription factor promoters in pre-differentiated cells. Accordingly, their coincidence with 

H2A.Z, along with the dynamics of all three chromatin marks at the Hox gene clusters 

throughout development is of great general interest. Our localization studies of H2A.Z in the 

developmentally tractable vertebrate system of zebrafish will allow us to better understand 

the nature of the activity of H2A.Z in the context of a complex developing organism. 

141



Rama Natarajan 

Abstract P92 

Genome-wide analysis of histone lysine methylation 

variations caused by diabetic conditions in human monocytes 

Feng Miao 1 , Xiwei Wu 2 , Lingxiao Zhang 1 , Yate-Ching Yuan 2 , Arthur D. 

Riggs 3 and Rama Natarajan 1 

Departments of Diabetes 1 , Biomedical Informatics 2 and Biology 3 , Beckman Research 

Institute of City of Hope, 1500 East Duarte Road, Duarte, CA 91010, U.S.A. 

Histone modifications in chromatin, particularly histone lysine methylations, play key 

epigenetic roles in gene expression and constitute a new layer of transcription regulation. 

Aberrant methylation patterns that change chromatin structure can promote dysregulated 

gene expression associated with disease progression. Current approaches for acquiring 

genome-wide information of histone modifications involve the use of chromatinimmunoprecipitation 

linked to DNA arrays (ChIP–arrays). This method generates data sets 

of chromatin status and provides snapshot views of the cell at the layer of histone 

methylation. 

Diabetic conditions such as high glucose (HG) can alter key pathologic pathways and 

genes. However, their impact on cellular histone lysine methylation is not known. We 

hypothesized that chronic HG can induce aberrant changes in histone H3 lysine-4 and 

lysine-9 dimethylation (H3K4me2 and H3K9me2), key histone marks normally associated 

with active and repressed genes respectively. We used antibodies to H3K4me2 and 

H3K9me2 in ChIP-arrays to compare their profiles and variations in human THP-1 

monocytes cultured in normal glucose (NG) and HG separately. We used human 12K cDNA 

and 12K CpG arrays representing coding and promoter regions respectively. After statistical 

analyses of the ChIP-microarray data, we identified key candidate genes relevant to 

diabetes that displayed changes in H3K4me2 and H3K9me2 in HG relative to NG and also 

validated them with follow-up conventional ChIPs. Relevance to diabetes was further 

demonstrated by examining these modifications by ChIPs in peripheral blood monocytes 

isolated from patients with type 1 diabetes relative to normal controls. In addition, regular 

mRNA profiling with the cDNA arrays revealed both anticipated and unanticipated 

correlations between mRNA expression, H3K4me2 and H3K9me2 levels. These results 

showing HG-and diabetes-induced variations in histone methylation genome-wide suggest 

that the diseased cells may have distinct epigenomes and also provide new insights into 

diabetic changes in the context of histone methylation. This may lead to an epigenetic 

memory of transcription history responsible for the “metabolic memory” and sustained 

clinical complications of diabetes. 

142



Edward Ramos 

Abstract P93 

Global characterization and function of Gypsy-like 

endogenous insulators in Drosophila melanogaster 

Edward Ramos and Victor Corces 

Johns Hopkins Univ, Dept of Biol Mudd Hall, 3400 N Charles St, Baltimore, MD 21218, 

U.S.A. 

Higher-order chromatin organization is essential for the proper expression of eukaryotic 

genes. The chromatin domains established through this organization are thought to regulate 

gene expression by controlling intra-chromosomal domain communication. The gypsy 

chromatin insulator proteins of Drosophila melanogaster have been implicated in the 

establishment of these domains. However, the mechanism governing the genomic 

organization and maintenance of these elements is not well understood. To better 

understand the role of insulators in chromatin organization, we investigated the function of 

the insulator protein Su(Hw) at the global level. To this end we used a combination of 

traditional molecular, biochemical and genetic approaches along with novel computational 

tools for our studies. We have thoroughly characterized a number of endogenous Su(Hw) 

binding sites and determined, through gel shift binding assays, direct interaction between 

predicted Su(Hw) binding sites and Su(Hw) insulator protein. In addition, in vivo experiments 

demonstrated that these predicted endogenous Su(Hw) binding sites co-localize with 

Su(Hw) protein on polytene chromosomes. Finally and most importantly, these endogenous 

Su(Hw) binding sites function as bona fide insulators as they can prevent communication 

between enhancers and promoters as demonstrated in an enhancer blocking assay. Using 

computational analysis we are in the process of mapping these Su(Hw) binding site to the 

entire Drosophila genome to determine the global function of the Su(Hw) protein during fly 

development and everyday function. It is our hope to find a functional correlation between 

our endogenous insulator binding sites and global genomic regulation 

143



William Renthal 

Abstract P94 

Class II histone deacetylases regulate the behavioral 

adaptations to chronic cocaine and stress 

William Renthal 1 , Arvind Kumar 1 , Vaishnav Krishnan 1 , Scott J. Russo 1 , 

David E. Theobald 1 , Kwang-Ho Choi 1 , Nadia Tsankova 1 , Rachel Neve 2 , 

Eric N. Olson 3 and Eric J. Nestler 1 

1 

Department of Psychiatry, UT Southwestern Medical Center, 2 Department of Psychiatry, 

McLean Hospital, Harvard Medical Center, 3 Department of Molecular Biology, UT 

Southwestern Medical Center, U.S.A. 

Addiction and depression are chronic psychiatric disorders in which long-lasting changes in 

gene expression are thought to contribute to the behavioral pathologies. Previous studies 

from our lab have identified alterations in histone acetylation in rodent models of addiction 

and depression that may contribute to the longevity of these disorders. Since 

neurotransmission is highly dependent on calcium signaling, we focused on the calciumregulated 

class II histone decacetylases (HDACs) as potential mediators of the addiction 

and depression-induced changes in chromatin structure. Calcium influx results in the rapid 

phosphorylation and nuclear exportation of these HDACs, which in turn leads to a 

subsequent increase of histone acetylation on target genes. Mice lacking the class II 

HDACs, HDAC5 or HDAC9, are completely normal in a variety of behavioral tests from 

learning and memory to acute cocaine reward. However, after chronic cocaine treatments 

or chronic stress, they demonstrate increased sensitivity to both stimuli. These mice seem 

to hyper-adapt, suggesting that proper balance of histone acetylation is important in the 

behavioral adaptations to chronic but not acute stimuli. We have also found that cocaine 

and stress regulate HDAC5 phosphorylation in wild type mice, which corresponds with 

changes in histone acetylation of HDAC5 target genes that were identified by gene 

expression microarrays and ChIP on chip technology. We are currently investigating the 

role of these HDAC5 target genes individually using viral-mediated gene transfer into 

specific nuclei in the brain. These studies together with analogous findings in models of 

chronic cardiac stress, implicate class II HDACs in the molecular machinery needed for 

adapting to a variety of chronic environmental stimuli. 

144



Karsten Rippe 

Abstract P95 

Activities of histone chaperone NAP1: Association states and 

interactions with histones, nucleosome assembly and effect 

on the chromatin fiber conformation 

Karsten Rippe, Jacek Mazurkiewicz, Felix Kepert and Katalin Fejes Toth 

Kirchhoff-Institut fur Physik, Molecular Biophysics Group, Ruprecht-Karls-Universitat 

Heidelberg, Im Neuenheimer Feld 227, D-69120 Heidelberg, Germany 

The nucleosome assembly protein 1 (NAP1) is a histone chaperone that functions as a 

carrier of histones during nuclear import, nucleosome assembly and chromatin remodeling. 

We have examined (i) the association states of NAP1 alone and in complexes with histones 

[1], (ii) the process of mononucleosome assembly mediated by NAP1 on DNA fragments of 

146 and 207 bp length containing a 5 S rDNA nucleosome positioning sequence [2], and 

(iii) how the interaction of NAP1 with core and linker histones affects the chromatin fiber 

organization [3]. Taken together these results provide complementary insight into the 

mechanisms, by which NAP1 exerts its different biological activites. 

Refs: 

1. Fejes Toth, K., Mazurkiewicz, J. & Rippe, K. (2005). J. Biol. Chem. 280, 15690-15699. 

2. Mazurkiewicz, J., Kepert, J.F. & Rippe, K. (2006). J. Biol. Chem. 281, 16462-16472. 

3. Kepert, J.F., Mazurkiewicz, J., Heuvelman, G., Fejes Toth, K. & Rippe, K. (2005). J. Biol. 

Chem. 280, 34063-34072. 

145



Charles Roberts 

Abstract P96 

The Swi/Snf chromatin remodeling complex regulates 

lineage specific transcription programs during development 

and impairment of this activity causes cancer 

Xi Wang, Miriam B. Werneck, Courtney G. Sansam, Michael S. Isakoff 

and Julia A. Evans 

Harvey Cantor and Charles W. M. Roberts. Dana-Farber Cancer Inst., Boston, MA, U.S.A. 

Chromatin remodeling complexes control nucleosome positioning and are dynamic 

regulators of transcription. While the biochemical role of these complexes in nucleosome 

mobilization is beginning to be elucidated, little understanding exists with respect to the role 

of the complexes in coordinated control of gene expression programs such as those 

required for lineage specific development. Snf5/Ini1/Baf47/Smarcb1 is a core member of the 

Swi/Snf chromatin remodeling complex and is a potent tumor suppressor that is inactivated 

in aggressive childhood cancers. As many transcription factors involved in oncogenic 

transformation are master regulators of lineage specific development, we hypothesized that 

the Swi/Snf complex, which also regulates transcription, may serve a similar role and that 

this activity may underlie its role in tumor suppression. We therefore sought to identify the 

developmental function of Snf5 and to elucidate the mechanistic basis of its tumor 

suppressor activity. Since the progressive stages of T cell development are well 

characterized and because Snf5 loss gives rise to T cell lymphomas, we chose to 

investigate the role of Snf5 in T cell development. We found that deletion of Snf5 in mice 

leads to aberrant gene expression in T cells, abnormal developmental progression with 

imbalanced CD4:CD8 bifurcation and culminates in a specific developmental block. This 

dysfunction and imbalance is coupled with the extremely rapid formation of mature CD8+ T 

cell lymphomas in 100% of mice. This developmental role is highly specific for alpha-beta T 

cells as we show that Snf5 is largely dispensable for T cells of the gamma-delta lineage and 

that it possesses no tumor suppressor activity within the gamma-delta or B cell lineages. 

Lastly, we show that the tumor suppressor activity is likely derived from the role of Snf5 in 

restricting cell cycle progression/proliferation and promoting differentiation. Loss of Snf5 

leads to aberrant stimulation of the cell cycle by causing down-regulation of p16Ink4a and 

activation of E2F targets which in turn directly drive replication. These changes promote 

cancer formation. Collectively, our data demonstrate a role for the Swi/Snf chromatin 

remodeling complex in controlling lineage specific developmental programs and provide 

insight into how loss of this control leads to oncogenic transformation. 

146



Paul Sadowski 

Abstract P97 

Post-translational modification of the insulator protein, CTCF 

Melissa MacPherson, Linda Beatty, Wenjing Zhou and Paul Sadowski 

Department of Medical Genetics and Microbiology, University of Toronto, Toronto 

M5S 1A8 Canada 

The CTCF protein is a highly conserved zinc finger protein that has been implicated in many 

aspects of gene regulation and nuclear organization. It acts as a repressor of some genes 

such as the c-myc gene but it activates others such as the ß-amyloid precursor protein 

gene. It also plays a key role in regulating genomic imprinting of the IGF2 and H19 genes 

by binding to a differentially methylated insulator sequence that lies between the two genes. 

The CTCF protein may be over expressed or mutated in some cancers. It is posttranslationally 

modified by poly(ADP) ribosylation and is phosphorylated by casein kinase II. 

We now report that CTCF is also post-translationally modified by the ubiquitin-like proteins, 

SUMO. We have co-transfected HEK293 cells with plasmids encoding tagged versions of 

CTCF and SUMOs 1, 2 or 3 followed by immunoprecipitation and western blotting to reveal 

that CTCF is SUMOylated by SUMOs 1, 2 and 3. The reaction is dependent upon the C- 

terminal diglycine amino acids of SUMOs 1 and 2 and is sensitive to the SUMO proteases 

SENP1 and SENP5 (vectors supplied by Dr. E. Yeh). The SUMOylation reaction is 

insensitive to 3-aminobenzamide, an inhibitor of poly (ADP) ribose polymerase. Thus far, we 

have identified one strong SUMO acceptor site in the C-terminus of CTCF. SUMOylation of 

CTCF also occurs efficiently in vitro and SUMOylation does not affect CTCF’s ability to bind 

to DNA that contains CTCF-binding consensus sequences from the H19 insulator. 

We are studying the interrelationship of SUMOylation of CTCF with other post-translational 

modifications of the protein and its influence upon the multitude of transcriptional activities 

of this protein. 

(Supported by the Canadian Institutes of Health Research) 

147



Teresa Sanchez Alcaraz 

Abstract P98 

Role of USP7 and GMP synthetase in deubiquitination of 

human histone H2B 

Teresa Sanchez Alcaraz 1 , Melissa Holowaty 1 , Yi Sheng 2 , Cheryl 

Arrowsmith 2 and Lori Frappier 1 

1 

Department of Medical Genetics and Microbiology and 2 Banting and Best Department of 

Medical Research, University of Toronto, Toronto, Canada 

One of the post-translational modifications that histones can undergo is monoubiquitination. 

This modification occurs predominantly on H2A and H2B but is poorly understood in terms 

of its effect on chromatin structure and function, as well as the enzymes controlling the 

ubiquitination/deubiquitination process. In mammalian cells, histone H2B has been shown 

to be ubiquitinated by Mdm2 and RFN20/40 but the ubiquitin-specific protease (USP) that 

reverses this modification is not known. We have been studying the structure and function of 

USP7 in human cells (also called HAUSP), which is the target of herpes simplex and 

Epstein-Barr virus proteins (ICP0 and EBNA1) and plays roles in p53 regulation (2005 Mol. 

Cell 18, 25; 2006 Nature Struc. Mol. Biol. 13, 285). To more completely determine how 

USP7 affects cellular processes, we investigated protein interactions of USP7 using an 

affinity column approach. We found that the most predominant interaction of USP7 is with 

human GMP synthetase. Interestingly, recent studies in Drosophila indicate that H2B is 

deubiquitinated by USP7 in complex with GMP synthetase (2005 Mol Cell 17, 695). We 

found that the USP7-GMP synthetase interaction occurs through the USP7 N-terminal 

domain, which is also responsible for binding p53, Mdm2 and EBNA1. We have shown that 

purified USP7 has some capacity to cleave ubiquitin from monoubiquitinated H2B isolated 

from human cells, but that this activity is greatly stimulated by purified GMP synthetase. 

This effect was specific for H2B, as deubiquitination of polyubiquitinated p53 by USP7 was 

not affected by GMP synthetase. Consistent with these observation, cellular levels of 

ubiquitinated H2B were decreased when USP7 was silenced in Hela cells by a hairpin RNA. 

The results suggest that a complex of USP7 and GMP synthetase is responsible for 

deubiquitination of H2B in higher eukaryotes. 

148



Annette Scharf 

Abstract P99 

Dynamics of histone modifications during chromatin 

assembly 

Annette ND Scharf, Ana Villar-Garea and Axel Imhof 

Adolf Butenandt Institute, Schillerstrasse 44, 80336 Munich, Germany 

Histone tails undergo a variety of posttranslational modifications that change their 

interaction with DNA and nuclear proteins. Combinations of modifications are thought to 

serve as a ‘histone code’. Changes in combinatorial histone modifications mark unique 

downstream events such as gene expression and DNA repair. Moreover histone 

modifications are thought to be transmitted through mitosis and therefore serve as 

mediators of cellular memory. However the way histone modifications are passed on from 

one cell generation to the other remains elusive. Using well-established MALDI-TOF and 

ESI MS/MS techniques our goal is to describe the changes in histone modifications during 

chromatin assembly. Therefore we use a Drosophila melanogaster assembly extract derived 

from preblastoderm embryos to assemble chromatin in vitro. Here, we find that there is a 

gradual deacetylation of H4K5 and H4K12 during the assembly, and that the deacetylation 

is dependent on ATP. We also confirm that the deacetylation is sensitive to HDAC inhibitors. 

We are currently investigating these findings in vivo by means of stable isotope labeling with 

amino acids in cell culture (SILAC). 

149



Stefan Schoeftner 

Abstract P100 

Screening for miRNAs regulating mammalian telomeres 

Stefan Schoeftner 1 , Susana Gonzalez 1 , Manuel Serrano 1 , Gregory J. 

Hannon 2 and Maria A. Blasco 1 

1 

Spanish National Cancer Center (CNIO), 28029 Madrid, Spain; 2 Cold Spring Harbor 

Laboratory, Cold Spring Harbor, New York 11724, U.S.A. 

Mammalian telomeres are specialized chromatin structures at the ends of chromosomes 

that are essential for genomic stability. Telomeres shorten witch age due to the so called 

“end-replication problem”, a process underlying ageing. 

Telomere length is regulated by proteins binding directly to telomeric TTAGGG tandem 

repeats but also by chromatin modifying enzymatic activities such as histone lysine 

methyltransferases (HMTases) and DNA methyltransferases (DNMTs). 

Recently, microRNAs (miRNAs) were identified to play a central role in regulating animal 

development and physiology. miRNAs are ~21-26 nucleotide RNAs that regulate their target 

RNA by triggering endonuclease driven cleavage or translational repression. Mammalian 

miRNAs are generated from PolII transcribed precursor RNAs and processed into an active 

form by cleavage by the RNase III enzymes Drosha and Dicer. Importantly, mouse ES cells 

lacking Dicer show dramatically elongated telomeres with altered chromatin structure*. This 

finding suggests that miRNAs could be directly or indirectly involved in the regulation of 

telomere function and contribute to telomere associated diseases. 

In order to identify miRNAs involved in the regulation of telomeres we are taking advantage 

of a miRNA library comprising a set of 150 predicted miRNAs. 

The miRNA containing vectors were transiently transfected in to a Hela cell line carrying a 

luciferase reporter gene in close proximity to a telomere. miRNA induced changes in 

telomeric or subtelomeric chromatin result in altered expression of the reporter gene, a 

phenomenon known as “telomere position effect” (TPE). Additionally, the miRNA library was 

transfected into a standard Hela cell line to study changes in telomere length and global 

chromatin structure using high throughput quantitative fluorescence hybridisation (Q-FISH) 

and quantitative immunofluorescence (Q-IF). 

The poster outlines the primary screening as well functional validation of candidate miRNAs 

regulating telomere length and telomeric chromatin. 

*Benetti et al., unpublished data 

150



Gunnar Schotta 

Abstract P101 

A genome-wide transition to H4K20 mono-methylation 

impairs stress-induced and programd DNA damage response 

in the mouse 

Gunnar Schotta, Roopsha Sengupta, Stefan Kubicek, Stephen Malin, 

Michaela Pagani, Monika Kauer, Alexsandra Espejo, Mark Bedford, 

Meinrad Busslinger and Thomas Jenuwein 

Research Institute of Molecular Pathology (IMP), The Vienna Biocenter, A-1030 Vienna, 

Austria; University of Texas, M.D. Anderson Cancer Center, Science Park Research 

Division, PO box 389, Smithville, Texas 79857, U.S.A. 

H4K20 methylation is a broad epigenetic modification that has been linked with gene 

silencing, heterochromatin formation and response of the chromatin template to 

environmental signals. To analyze its function during mammalian development, we have 

disrupted the two Suv4-20h HMTases in the mouse. Whereas Suv4-20h1 null mutants 

display perinatal lethality, Suv4-20h2 deficient mice develop normally. Using conditional 

alleles, we generated Suv4-20h double null (dn) mice, in which nearly all H4K20me3 and 

H4K20me2 states are lost, resulting in a genome-wide transition to H4K20me1 chromatin. 

Suv4-20h dn mouse embryonic fibroblasts (MEFs) display impaired proliferation, have 

reduced S-phase ratios and enter crisis at early passage numbers. The DNA damage 

checkpoint protein 53bp1 selectively associates with H4K20me2 nucleosomes, and 

recruitment of 53bp1 to DSBs is significantly impaired in Suv4-20h dn cells. Importantly, 

Suv4-20h dn B-cells are also defective in class switch recombination, which reflects a 

developmentally programd pathway for DSB-mediated antibody isotype diversification. Thus, 

an H4K20me1 chromatin is insufficient to ensure mammalian genome function and 

H4K20me2 is a major signal in processing stress-induced and programd DNA damage 

response in the mouse. 

151



David Schrump 

Abstract P102 

Brother of the Regulator of Imprinted Sites (BORIS) recruits 

Sp1 to modulate NY-ESO-1 expression in lung cancer cells 

Yang Kang, Julie A. Hong, G. Aaron Chen, Dao M. Nguyen and David S. 

Schrump 

Thoracic Oncology Section, Surgery Branch, Center for Cancer Research, National Cancer 

Institute, Bethesda, MD, U.S.A. 

Epigenetic alterations during malignant transformation facilitate de-repression of a variety of 

germ cell-restricted genes, such as NY-ESO-1, which encodes a cytoplasmic protein that is 

immunogenic in cancer patients. Recently we reported that the paralogous zinc finger 

proteins- CTCF and BORIS, directly contribute to transcriptional regulation of NY-ESO-1 in 

lung cancer cells. To further examine mechanisms that mediate NY-ESO-1 expression, we 

performed software-guided analysis of the NY-ESO-1 promoter region, which revealed 

several potential Sp1 binding motifs. Promoter-reporter assays demonstrated that deletions, 

which sequentially eliminated the putative BORIS/CTCF recognition sequence and a 

prototypic Sp1 binding site markedly decreased NY-ESO-1 promoter activity. Transient 

transfection experiments using promoter-reporter constructs, electromobility shift assays, 

and chromatin immunoprecipitation experiments revealed that NY-ESO-1 promoter activity 

coincided with increased occupancy of the proximal Sp1 binding site in lung cancer cells. 

Mutations within the Sp1 recognition sequence specifically eliminated binding of Sp1 to this 

motif in vitro. siRNA-mediated inhibition of Sp1 expression coincided with markedly 

diminished NY-ESO-1 promoter activity in lung cancer cells. In contrast, abrogation of CTCF 

expression resulted in pronounced augmentation of NY-ESO-1 promoter activity. Coimmunoprecipitation 

experiments indicated that Sp1 physically interacts with BORIS but not 

with CTCF in vivo. Collectively, these findings suggest that NY-ESO-1 expression is 

governed by distinct transcriptional complexes during pulmonary carcinogenesis, and that 

BORIS recruits Sp1 to augment NY-ESO-1 expression in lung cancer cells. 

152



Bonnie Scott 

Abstract P103 

Evolution of centromere-binding proteins and their 

interactions with centromere DNA in Arabidopsis 

Bonnie Scott, Song Luo, Sarah Hall and Daphne Preuss 

Howard Hughes Medical Institute and Department of Molecular Genetics and Cell Biology, 

GCIS W519, University of Chicago, 929 E. 57th Street, Chicago, IL 60637, U.S.A. 

During cell division, proper chromosome segregation involves assembling a kinetochore 

protein complex at the centromere region of every chromosome. A fundamental yet 

unresolved question is how inner kinetochore proteins initially choose the site for 

kinetochore assembly and subsequently maintain its identity as a centromere throughout 

the cell cycle. The challenge in understanding these phenomena is that inner kinetochore 

proteins, namely centromere-binding proteins (CENP) A and C, share high sequence 

similarity between plants, yeast, and animals, yet the centromere DNA to which they 

associate lacks a conserved sequence and is instead composed of repetitive DNA, 

including rapidly evolving satellite DNA. These observations have led to the hypothesis that 

CENPs evolve adaptively across species to maintain protein-DNA interactions at the 

centromere for chromosome inheritance. The goal of this study is to identify structural 

features within plant centromere-binding proteins that are required for species-specific 

centromere function in vivo. To accomplish this, we have first cloned CENP-A and CENP-C 

sequences from increasingly diverged Arabidopsis species and used phylogenetic tools to 

understand their evolutionary history and identify putative regions important for speciesspecificity. 

Second, we are expressing these CENP sequences into corresponding 

Arabidopsis thaliana mutants to define the degree of natural variation able to rescue CENP 

function at the centromere. 

153



David Shechter 

Abstract P104 

Histone H2A arginine3 is mono- and symmetrically-di 

methylated by a complex of PRMT5 and the WD-repeat 

protein MEP50 in Xenopus laevis eggs 

David Shechter 1 , Raghu Chitta 2 , Eileen Woo 3 , Brian Chait 3 , Jeffrey 

Shabanowitz 2 , Donald F. Hunt 2 and C. David Allis 1 

1 

The Laboratory of Chromatin Biology, Rockefeller University, New York, NY 10021; 

2 

Chemistry Department, University of Virginia, Charlottesville, VA 22904; 3 The Laboratory of 

Mass Spectrometry and Gaseous Ion Chemistry, Rockefeller University, New York, NY 

10021, U.S.A. 

A wealth of emerging literature demonstrate that post-translational modification of histones, the 

protein components of nucleosomes which are the fundamental repeating unit of chromatin, serve 

to regulate a wide variety of DNA-templated processes, notably gene regulation. Methylation of 

arginines in histones has been observed in many contexts, including gene activation and 

repression [1]. Histone arginine methylation has been correlated with events during development, 

especially in germ cells. Here we report the identification of the predominant histone 

methyltransferase in extracts of the large eggs of Xenopus laevis. The activity is composed of, at 

minimum, a complex of the methyltransferase PRMT5 (also known as Hsl7 in Xenopus and 

yeast) and the WD-Repeat Protein MEP50. It specifically catalyzes mono- and symmetric-di 

methylation on free histone H2A on arginine 3, as determined by mass spectrometry and by 

immunoblotting with H2A/H4R3me-specific antibodies. The complex methylates H2A and H4 in 

nucleosomes at a much lower activity. The methylation activity towards H2A is inhibited by H2A 

Serine-1 phosphorylation, although we did observe H2AS1phos and H2AR3Me on the same 

protein molecule by mass spectrometry. AMI-1, a general small molecule inhibitor of PRMTs, 

inhibits this complex at approximately 100µM. This activity and H2AR3Me2S modification is 

abundant in the egg and disappears rapidly during development. In conclusion, H2AR3 

methylation is a major histone modification in the early development of Xenopus laevis. 

Experiments are in progress to gain insight into the biological function(s) of PRMT5/MEP50 

complex in the context of early development. We also are exploring whether MEP50 serves to 

present H2A to PRMT5, analogous to the function for the WD-Repeat Protein WDR5 in MLL 

(mixed lineage leukemia) complex isolated from human cells as recently described by our 

laboratory [2, 3]. 

Refs 

1. Bedford, M.T. and S. Richard, Arginine methylation an emerging regulator of protein function. 

Mol Cell, 2005. 18(3): p. 263-72. 

2. Dou, Y., et al., Regulation of MLL1 H3K4 methyltransferase activity by its core components. Nat 

Struct Mol Biol, 2006. 13(8): p. 713-9. 

3. Ruthenburg, A.J., et al., Histone H3 recognition and presentation by the WDR5 module of the 

MLL1 complex. Nat Struct Mol Biol, 2006. 13(8): p. 704-12. 

154



Yoichi Shinkai 

H3K9 methylation and germ cell development 

Abstract P105 

Makoto Tachibana 1 , Masami Nozaki 2 , Naoki Takeda 3 and Yoichi Shinkai 1 

1 

Institute for Virus Research, Kyoto University, 2 Research Institute for Microbial Disease, Osaka 

University, 3 Center for Animal Resources and Development, Kumamoto University, Japan 

It is known that epigenetic cellular memories are dynamically reprogramd during germ cell 

development. G9a is one of major histone H3 lysine 9 (H3K9) methyltransferases at 

euchromatin. To elucidate how G9a and G9a-mediated H3K9 methylation are crucial for 

germ cell development, we established germ-lineage specific G9a deficient mice. In the 

absence of G9a, male and most female mice are sterile. Even TNAP (tissue non-specific 

alkaline phosphatase) promoter-driven Cre enzyme supposedly inactivates a G9a gene (and 

G9a-mediated H3K9 methylation) before the migration stage into testis, spermatogenesis in 

G9a-deficient mice until a gonia type stage seems to be intact. However, spermatocytes 

later a pachytene stage are missing. Furthermore, abnormal meiotic-prophase progression 

and synaptonemal complex formation are frequently observed in G9a-deficient 

spermatocytes. Oogenesis is also impaired around the pachytene stage. DNA microarray 

analysis described that multiple genes are activated or up-regulated in G9a-deficient 

testicular cells. These results emphasize a crucial function for G9a (and G9a-mediated 

H3K9 methylation) in controlling germ cell development and meiotic-prophase progression. 

155



Krishna Sinha 

Abstract P106 

Inhibition of the transcriptional activity of osterix by 

interactions with NO66, a jumonji family chromatin protein 

Krishna M Sinha, Xin Zhou, Chi Zhang, Lingna Zhang and Benoit de 

Crombrugghe 

Department of Molecular Genetics, UT M. D. Anderson Cancer Center, Houston, 

TX 77030, U.S.A. 

Osterix (Osx) is a critical osteoblast-specific transcription factor required for bone formation. 

Genetic inactivation of Osx in mice leads to complete arrest of osteoblast differentiation, 

although chondrocyte differentiation and cartilage formation are normal. However, the 

mechanism of Osx function during osteoblast differentiation is not well understood. 

In the present study, the Jumonji (JmjC) domain containing protein, NO66, was identified as 

an Osx-interacting protein using an Osx expressing stable osteoblast cell line by tandem 

immunoaffinity chromatography and mass spectrometry. Co-immunoprecipitation and GSTpulldown 

assays showed that NO66 physically interacts with Osx. NO66 interacts through 

its JmjC domain with the transcription activation domain of Osx. In situ RNA hybridization 

experiments revealed that NO66 and Osx are co-expressed in skeletal elements of hind 

limbs and forelimbs, in vertebrae, ribs, and craniofacial bones during mouse embryonic 

development. In DNA transfection assays, Osx strongly stimulates the activity of a 1kb 

osteocalcin promoter and that of an osteoblast specific 2.3-kb Col1a1 promoter. Cotransfection 

of NO66 results in a strong inhibition of the Osx-dependent activity of these 

promoters, whereas the activity of several other promoters was not affected by NO66. In 

addition to this, transfection of NO66 in osteoblasts also inhibits the activity of the 

endogenous osteocalcin gene. Furthermore, knockdown of Obelix expression in osteoblasts 

by specific siRNAs results in increased expression of several osteoblast-specific marker 

genes including Col1a1, Osteocalcin and Bone Sialoprotein (BSP) without apparent 

changes in the level of Osx expression. 

Overall our data suggest the hypothesis that NO66 is a negative regulator of Osx and, 

hence, of osteoblast differentiation or function. The presence of a JmjC domain in NO66 

raises the intriguing possibility that inhibitory effect of NO66 is mediated by demethylation of 

specific lysine residues of histones within the chromatin environment of osteoblasts. 

156



Karen Smith 

Abstract P107 

Identification and characterization of novel HDAC-associated 

proteins that regulate cancer cell growth 

Karen T. Smith, Skylar A. Martin-Brown, Laurence Florens, Michael P. 

Washburn and Jerry L. Workman 

The Stowers Institute for Medical Research, Kansas City, MO 64110 U.S.A. 

Histone deacetylase (HDAC) inhibitors are one class of chemotherapeutic drugs. However, 

these inhibitors exhibit undesirable side effects in cancer patients due to their inability to 

discriminate among the several structurally similar HDACs in humans. HDACs reside in 

multi-subunit protein complexes and some of these HDAC-associated proteins are needed 

for full activity of the HDACs themselves. We propose that a single HDAC can be inhibited 

in the cell by targeting HDAC-associated proteins rather than the HDACs themselves. We 

have taken a proteomics approach to identify proteins differentially associated with human 

HDAC1 and HDAC3 in 293T cells. Preliminary experiments have identified several known 

members of HDAC-containing complexes and many novel HDAC-interacting proteins. 

Importantly, many of these proteins differentially associate with HDAC1 or HDAC3. Several 

of these newly identified HDAC1 and HDAC3-interacting proteins have a connection to 

chromatin or cell growth, while others have not yet been described. We are currently 

confirming these interactions in 293T cells and testing the conservation of these 

interactions in cancer cell lines. We then will test if these HDAC-interacting proteins are 

important for their associated HDAC’s activity. Proteins that have a significant effect on 

HDAC1 or HDAC3 activity will then be abrogated in cancer cell lines to determine if loss of 

these HDAC-associated proteins can inhibit cancer cell growth. 

157



Matthew Smith 

Abstract P108 

Chromatin - mediated silencing of immune response genes 

Matthew Smith, Jian Wu and Kenneth Wright 

University of South Florida College of Medicine, Department of Molecular Medicine and 

Moffitt Cancer Center Tampa, FL, U.S.A. 

The transcriptional repressor PRDI-BF1 (Blimp-1) is required for terminal differentiation of 

plasma cells, T-cell homeostasis and silencing of interferon beta expression upon viral 

induction. The repressive functions of PRDI-BF1 are mediated through interactions with 

additional co-factors, such as Groucho, HDAC1/2 and G9a. Here, we demonstrate a role for 

PRDI-BF1 in the maturation of dendritic cells (DCs). Considerable phenotypic differences 

exist between immature and mature DC populations. Immature DCs function in immune 

surveillance via antigen uptake and processing, while mature DCs present antigen in the 

context of MHC Class II molecules to immune effector molecules. This phenotypic transition 

involves an orchestrated change in gene expression profiles, exemplified by the downregulation 

of CIITA and MRC1 upon maturation. CIITA, encoded by MHC2TA, is the master 

regulator of MHC Class II transcription which is known to be silenced upon maturation. 

MRC1 (CD206) recognizes extracellular pathogens via oligosaccharide domains and is 

capable of activating the innate immune response. PRDI-BF1 is induced upon DC 

maturation (antigen encounter) and inversely correlates with expression of MHC2TA and 

MRC1 mRNA. Using in vivo genomic footprinting and chromatin immunoprecipitation 

experiments, we show that transcriptional co-activators are displaced by PRDI-BF1 at the 

promoter regions of MHC2TA and MRC1. Upon maturation, these regions demonstrate 

hallmarks of silent chromatin, such as decreased acetylation of histones H3 and H4, 

decreased methylation of histone H3 lysine 4 and increased di-methylation of histone H3 

lysine 9. Furthermore, this silencing is mediated by the G9a histone methyltransferase and 

the heterochromatin protein, HP1 gamma. These results provide novel insight into the 

molecular mechanisms underlying the maturation process in DCs. 

158



Hae-Ryong Song 

Abstract P109 

Coordination of transcriptional regulation and chromatin 

modification of Arabidopsis circadian clock genes 

Hae-Ryong Song 1,3 , Ju-Hee Jeong 1,3 , Bosl Noh 2,3 and Yoo-Sun Noh 1,3 

1 

Department of Biological Sciences, Seoul National University, Seoul 151-742, Korea., 

2 


University, Jinju 660-701, Korea. 3 Global Research Laboratory for Flowering at SNU and 


Circadian clock genes are regulated through a transcriptional-translational feedback loop. In 

Arabidopsis, LHY and CCA1 transcripts are highly expressed in the early morning. 

Translated LHY and CCA1 proteins repress the expression of TOC1 transcript which peaks 

in the evening. TOC1 protein elevates the expression of LHY and CCA1 mRNAs, forming a 

negative feedback loop that is believed to constitute the oscillatory mechanism of the clock. 

Recently the rhythmic oscillation of mouse clock genes, mPER1 and mPER2, was shown to 

be correlated with the regular alteration of chromatin structure through histone 

acetylation/deacetylation. However, little is known about the chromatin modificationmediated 

transcriptional regulation of Arabidopsis circadian clock genes. Here we propose 

a possibility that Arabidosis clock-associated genes, LHY, CCA1, and TOC1 might be 

regulated by rhythmic histone modifications. Our results show that certain type of histone 

modifications either has positive or negative correlations with the expression of LHY, CCA1, 

and TOC1 transcripts. Therefore, the rhythmic transcription of these clock genes might 

depend on regular histone modifications within their chromatin and the fine-tuning of the 

feedback loop comprising an oscillator in plants might be accomplished by an ordered 

modification of histones. 

159



Stacey Southall 

Structural studies of histone methyltransferases 

Abstract P110 

Stacey M. Southall and Jonathon R. Wilson 

Chromatin Regulation Team, Section of Structural Biology, Institute of Cancer Research, 

Chester Beatty Laboratories, 237 Fulham Road, Chelsea, London, SW3 6JB 

Histone lysine methylation is an important epigenetic marker that can profoundly influence 

delineation of chromatin regions determining patterns of gene expression and consequently 

defining cell state. This precisely targeted modification is catalysed by a family of histone 

methyltransferases (HMTs) containing the evolutionarily conserved SET domain. Our focus 

is on the methylation of lysine 20 of histone H4. Multiple SET-containing methyltransferases 

are able to specifically mono-, di- and/or tri-methylate this lysine residue. Although the 

precise downstream effects of these modifications have not been determined, it is clear that 

each methylation state has a different physiological role. Structural analysis of SET domain 

methyltransferases has given insight into the molecular mechanism of methyltransfer, 

however to fully understand epigenetic regulation both in the normal cell and in disease we 

need to obtain a better understanding of how different HMTs are targeted to the same 

residue and how their activity is regulated at the molecular level. 

Our strategy is to apply biochemical, biophysical and structural techniques to different H4- 

K20 HMTs such as nuclear receptor-binding SET domain-containing proteins (NSD family) 

and the Suv4-20h family. It is now understood that many cancers have an epigenetic 

component and so modifying enzymes such as HMTs are likely to become drug targets. Our 

studies will therefore help to inform the rational design of such drugs. 

160



Maike Stam 

Abstract P111 

Molecular analysis of chromatin changes involved in b1 

paramutation, an allele-dependent transfer of epigenetic 

information 

Maike Stam, Max Haring, Rechien Bader and Marieke Louwers 

SILS, University of Amsterdam, Kruislaan 318, 1098 SM Amsterdam, The Netherlands 

We investigate the molecular mechanism underlying paramutation, a mitotically and 

meiotically heritable change in gene expression induced by allele interactions in trans. 

Paramutation is observed in a wide variety of plants and more recently in fungi and 

mammals, emphasizing its significance. We study paramutation at the b1 locus in maize, 

where it affects the pigmentation phenotype. The low expressed B’ epiallele imposes its low 

transcription rate onto the high expressed B-I epiallele in trans. This change correlates with 

changes in DNA methylation and chromatin structure. 

A regulatory element, containing seven tandem repeats located ~100 kb upstream of the b1 

coding region, is essential for b1 paramutation and functions as an enhancer for the B-I 

epiallele. We hypothesize a physical interaction between the repeats and the b1 promoter, 

and are currently using 3C technology (Dekker et al., 2002) to identify spatial, long range, in 

cis interactions within the 100 kb b1 chromatin domain. The regions spanning the repeat 

junctions show differential DNA methylation (only methylated in B’) and nuclease sensitivity 

(nuclease hypersensitive in B-I), suggesting differential binding of chromatin factors. DNA 

methylation analyses of mutants preventing paramutation indicate that the repeat junctions 

play an important role in trans-inactivation, while the regions flanking the junctions play a 

role in enhancement of b1 expression. ChIP experiments using an anti-H3K4me2 antibody 

indicates that both the B’ and B-I coding regions are transcription competent throughout 

development. Upon transcriptional enhancement of b1, the repeat region shows 

H3K9ac/14ac in high expressing B-I, and H3K9me2 in low expressing B’ tissue, indicating a 

developmentally regulated crosstalk between the transcription activation machinery and the 

heritable B-I and B’ epigenetic states. Intriguingly, B’ is H3K27 dimethylated at the promoter 

and coding region throughout development, suggesting a role for H3K27me2 in the 

heritability of the B’ expression state. We are currently using mutants to dissect the role of 

histone modifications in paramutation. 

161



Sean Taverna 

Abstract P112 

Connecting H3 methylation and acetylation : The role of 

Yng1 in transcription 

Sean D. Taverna 1 , Serge Ilin 3 , Richard S. Rogers 4,5 , Jason C. Tanny 1 , 

Heather Lavender 6 , Haitao Li 3 , Lindsey Baker 1 , John Boyle 4,5 , Lauren P. 

Blair 6 , Brian T. Chait 2 , Dinshaw J. Patel 3 , John D. Aitchison 4,5 , Alan J. 

Tackett 6 and C. David Allis 1 

1 

Laboratory of Chromatin Biology, The Rockefeller University, New York, NY 10021, U.S.A., 

2 

Laboratory of Mass Spectrometry and Gaseous Ion Chemistry, The Rockefeller University, 

New York, NY 10021, U.S.A., 3 Structural Biology Program, Memorial Sloan-Kettering Cancer 

Center, New York, NY 10021, U.S.A., 4 Institute for Systems Biology, Seattle, WA 98103, 

U.S.A., 5 Department of Cell Biology, University of Alberta, Edmonton, AB T6G 2H7, 

Canada, 6 Department of Biochemistry and Molecular Biology, University of Arkansas for 

Medical Sciences, Little Rock, AR 72205, U.S.A. 

Post-translational histone modifications participate in modulating the structure and function 

of chromatin. Promoters of transcribed genes are enriched with K4 trimethylation and 

hyperacetylation on the N-terminal tail of histone H3. Recently, PHD finger proteins, like the 

Yng1 component in the NuA3 HAT complex, were shown to interact with H3K4me3, 

indicating a biochemical link between K4 methylation and hyperacetylation. Using a 

combination of mass spectrometry, biochemistry, and NMR, we detail the Yng1 PHD- 

H3K4me3 interaction and the importance of NuA3-dependent acetylation at K14. 

Furthermore, genome-wide ChIP-Chip analysis demonstrates co-localization of Yng1 and 

H3K4me3 in vivo. Disrupting the K4me3-binding capacity of Yng1 altered K14ac and 

transcription at certain genes, thereby demonstrating direct in vivo evidence of sequential 

trimethyl-binding, acetyltransferase activity, and gene regulation by NuA3. Our data support 

a general mechanism of transcriptional control through which histone acetylation upstream 

of gene activation is promoted partially through availability of H3K4me3, “read” by binding 

modules in select subunits. 

162



Tage Thorstensen 

Abstract P113 

The Arabidopsis SUVR proteins define a novel subgroup of 

SET domain proteins associated with the nucleolus 

Tage Thorstensen, Andreas Fischer 1 , Silje V. Sandvik, Sylvia S. Johnsen, 

Paul E. Grini, Gunter Reuter 1 and Reidunn B. Aalen 

Department of Molecular Biosciences, University of Oslo P.O. Box 1041 Blindern, N-0316 Oslo, 

Norway, 1 Institute of Genetics, Biologicum, Martin Luther University Halle Halle, Germany 

The methylation pattern of the different lysine residues on histone tails has been shown to 

be part of the so called “histone code” and to be important in the regulation of eukaryotic 

gene expression and chromatin structure. The proteins responsible for the majority of this 

methylation contain the evolutionary conserved SET domain. SET domain proteins related 

to the Drosophila SU(VAR)3-9 protein have been associated with gene repression and heterochromatinization. 

There are 10 SUVH and 5 SUVR genes encoding proteins similar to 

SU(VAR)3-9 in Arabidopsis, and 4 SUVH proteins have been shown to control 

heterochromatic silencing by its HMTase activity and by directing DNA methylation. The 

SUVR proteins differ from the SUVH proteins in their domain structure, and we show that 

the closely related SUVR1, SUVR2 and SUVR4 proteins contain a novel WIYLD domain at 

their N-terminus, and a SUVR specific region preceding the SET domain. Green fluorescent 

protein (GFP)-fusions of these SUVR proteins preferably localize to the nucleolus, 

suggesting involvement in regulation of rRNA expression, in contrast to other SET-domain 

proteins studied so far which are associated with heterochromatin. The subnuclear 

localization of SUVR proteins is regulated by alternative splicing and we found that the 

SUVR4 protein is a histone lysine methyltransferase (HKMTase) with preference for 

monomethylated histone H3K9 in vitro. 

163



Christopher Topp 

Abstract P114 

Unusually-sized centromeric RNAs associate with maize 

centromeric chromatin 

Christopher N. Topp 1 and R. Kelly Dawe 1,2 

1 

Department of Plant Biology and 2 Department of Genetics, University of Georgia, Athens, 

GA 30602 

Centromeres control the strict inheritance of genetic information, yet are themselves 

genetically ill-defined. The repetitive DNA sequences of most eukaryotic centromeres are 

disposed to rapid evolutionary change, differing among even recently diverged species. 

Currently the best definition for a functional centromere is epigenetic: the presence of a 

centromere-specific histone variant, CENH3. When highly overexpressed, CENH3 can 

apparently nucleate de novo kinetochore formation at ectopic locations (Heun et al 2006). 

However the normal mechanisms that specify CENH3 deposition and the composition of 

centromeric chromatin remain unclear. Here we demonstrate that unusually-sized small, 

non-coding centromeric RNA species are physically associated with maize centromeric 

chromatin, as assayed by chromatin immunoprecipitation with CENH3. We discuss the 

implications of these data for centromere function and evolution. 

164



Martin Tribus 

Abstract P115 

Molecular mechanisms of histone variant H3.3 assembly by 

the motor protein CHD1 

Martin Tribus 1 , Alexander Konev 2 , Valerie Podhraski 1 , Dmitry Fyodorov 2 

and Alexandra Lusser 1 

1 

Division of Molecular Biology, Biocenter, Innsbruck Medical University, A-6020 Insbruck, 

Austria, 2 Department of Cell Biology, Albert Einstein College of Medicine, Bronx, NY 

10461, U.S.A. 

The Drosophila Snf2-related ATPase CHD1 functions as an ATP-dependent chromatin 

assembly factor. We have previously shown that CHD1 mediates the assembly of extended 

periodic nucleosome arrays in a purified system containing recombinant histone chaperone 

NAP-1, purified Drosophila core histones and relaxed circular DNA. 

We have begun to examine the biological functions of CHD1. To this end, we generated 

three mutant alleles of Chd1 in Drosophila. Two mutations result in a loss of CHD1 protein, 

while the third allele gives rise to a C-terminally truncated polypeptide. We discovered that 

all three alleles are maternal effect embryonic lethal mutations as embryos laid by 

homozygous mutant females die before hatching. Intriguingly, we discovered that the 

absence of maternal CHD1 blocks the incorporation of the histone variant H3.3 into the 

paternal chromatin and thus results in the exclusion of the paternal genome from zygote 

formation. Furthermore, CHD1 is required for H3.3 deposition in transcriptionally active 

chromatin of late syncytial embryos. 

It was recently shown that the histone chaperone HIRA is required for delivery of H3.3 into 

the male pronucleus in Drosophila. 

Here we present evidence that CHD1 can utilize HIRA as a chaperone to mediate the 

assembly of H3.3 containing nucleosomes in vitro. We could not detect direct or histone 

mediated interactions between recombinant CHD1 and HIRA. However, coimmunoprecipitation 

from Drosophila embryonic extracts revealed a weak association of 

CHD1 with HIRA. Thus, our in vivo and in vitro data uncover a CHD1 and HIRA-dependent 

pathway for the assembly of the histone variant H3.3. 

CHD1 contains two chromodomains that have recently been shown to be H3-methylK4 

binding modules. Binding of CHD1 to methylated H3 is thought to be critical for the function 

of CHD1 as an elongation factor. To examine the contribution of the chromodomains of 

CHD1 to its transcription-independent function in chromatin assembly during early 

embryonic development, we have generated transgenes that carry point mutations at 

conserved amino acid residues critical for H3-methylK4 binding. We will present data that 

show that the chromodomains are required for some but not all in vivo functions of CHD1. 

165



Christopher Vakoc 

Abstract P116 

A profile of histone lysine methylation generated by 

mammalian gene transcription 

Christopher R. Vakoc, Mira M. Sachdeva, Hongxin Wang, and Gerd A. Blobel 

Children’s Hospital of Philadelphia,Division of Hematology, Philadelphia, PA 19104, and the 

University of Pennsylvania School of Medicine, Philadelphia, PA 19104, U.S.A. 

Complex patterns of histone methylation encode distinct functions within chromatin. Lysine 

methylation displays the highest degree of complexity among known covalent histone 

modifications, with each site of methylation regulating the association of different effector 

molecules. We and others previously reported that tri-methylation of lysine 9 of histone H3 

occurs at both silent heterochromatin and at the transcribed region of active mammalian 

genes, suggesting that the extent of histone lysine methylation involved in mammalian gene 

activation is not completely defined. To identify additional sites of histone methylation that 

respond to mammalian gene activity, we describe here a comparative assessment of all six 

known positions of lysine methylation and relate them to gene transcription. For our 

studies we used the highly expressed housekeeping gene PABPC1 that spans more than 19 

kb thus permitting high-resolution comparison of histone lysine methylation landscapes. In 

addition, we used genes that can be transcriptionally induced or repressed. We observed 

high trimethylation of H3K4, H3K9, H3K36, andH3K79 in actively transcribed regions, 

consistent with previous findings. H4K20 mono-methylation, a modification previously linked 

with repression, we identified as a mark of transcription elongation in mammalian cells. In 

contrast, H3K27 monomethylation, a modification enriched at peri-centromeric 

heterochromatin, was observed broadly distributed throughout all euchromatic sites 

analyzed, with selective depletion in the vicinity of the transcription start site at active genes. 

Together, these results underscore that similar to other described methyl-lysine 

modifications, H4K20 and H3K27 mono-methylation are versatile and dynamic with respect 

to gene activity, suggesting the existence of novel site-specific methyltransferases and 

demethylases coupled to the transcription cycle. 

166



Claudius Vincenz 

Abstract P117 

Visualizing polycomb group protein interactions with histones 

in vivo 

Claudius Vincenz and Tom Kerppola 

1150 West Medical Ctr. Dr., 4574 MSRBII, Ann Arbor, MI 48109, U.S.A. 

The Polycomb Group proteins consist of about three dozen structurally unrelated proteins 

that were originally identified by their ability to produce a common phenotype in Drosophila. 

More recent mechanistic studies have identified chromatin as the target for the activity of 

these proteins. Specifically, these proteins both methylate lysine 27 of H3 as well as bind to 

this post-translational modification to repress transcription of the underlying DNA. This 

repression is maintained through cell division producing a molecular memory that enables 

the cell to have a stable phenotype. 

In Drosophila the Polycomb protein has been shown to be the anchor that recruits a whole 

complex of Polycomb Group proteins to the methylated lysine. In mammalian cells a gene 

expansion has produced five homologues of this anchor protein. Here we show that these 

structural homologues, termed CBX proteins in vertebrates, display a nuclear distribution 

pattern characteristic for each protein. This suggests that methylation of lysine 27 cannot 

be the only determinant of their binding to chromatin. Furthermore, we use Bimolecular 

Fluorescence Complementation (BiFC) to document that homologous domains have 

different roles in mediating Histone H3 binding of each CBX protein. For example, deletion 

of the chromodomain of CBX4 reduces interaction with H3 greatly, confirming in vitro 

studies. However, the homologous mutation in CBX6 and CBX7 does not affect H3 binding. 

Both, CBX6&7 are highly expressed in Embryonic Stem cells. These results are discussed 

in the context of the unusual distribution of H3K4 and H3K27 methylation in the genome of 

ES cells. We postulate that this atypical binding mode has evolved to accommodate the 

rapid genome wide changes in transcription that are induced upon differentiation in ES cells. 

167



Vikki Weake 

Abstract P118 

The SAGA histone acetyltransferase complex functions in the 

development of neuronal connectivity in the Drosophila 

compound eye 

Vikki M Weake, Kenneth K. Lee, Sebastian Guelman and Jerry L. Workman 

Stowers Institute for Medical Research, Kansas City, Missouri, U.S.A. 

The SAGA histone acetyltransferase complex is involved in transcriptional regulation via the 

covalent modification of histones. A proteomics approach is being used in our lab to identify 

and characterize the components of Drosophila SAGA. This approach has identified two 

novel components of the Drosophila SAGA complex: nonstop and CG13379. Nonstop is 

homologous to yeast Ubp8, which catalyzes removal of the ubiquitin group from histone 

H2B. A critical balance in the level of ubiquitylated H2B is required for correct transcription 

of SAGA regulated genes in yeast, and crosstalk between histone ubiquitylation and 

methylation has been observed in previous studies. CG13379 is homologous to yeast 

Sgf11, which together with Ubp8 constitutes a functional module within yeast SAGA. We 

have confirmed that nonstop and Sgf11 are stable components of Drosophila SAGA by coimmunoprecipitation, 

and a pupal-lethal mutation in sgf11 has been isolated. Furthermore, 

we show that nonstop can complement the ubp8 deletion strain in yeast and incorporates 

into yeast SAGA. The deubiquitiylation activity of nonstop is currently being characterized 

both in yeast and Drosophila. Independently of our studies, a mutation in nonstop was 

isolated during a screen for mutations disrupting neuronal connectivity in the Drosophila 

visual system. In order to determine whether the neuronal defect of the nonstop mutant is 

due to its role in SAGA, neuronal connectivity of other Drosophila SAGA mutants has been 

analyzed. Mutation of other components of SAGA results in an identical phenotype to that 

of nonstop, indicating that SAGA function is required for correct axon targeting in the 

developing fly eye. The eventual aim of this work is to identify the underlying genetic 

pathways regulated by SAGA that are required for the establishment of correct neuronal 

connectivity in the developing Drosophila eye. 

168



Stephanie Williams 

Abstract P119 

Mechanistic insights into promoter chromatin disassembly 

Stephanie Williams, Melissa Adkins and Jessica Tyler 

UCHSC, Dept. of Biochemistry, RC-1S, Rm 10403, 12801 E. 17th Ave., Aurora, CO 

80010, U.S.A. 

The disassembly of chromatin from promoter regions is a recently discovered mechanism 

for regulation of Eukaryotic gene expression. This process is best understood at the 

budding yeast PHO5 gene, where the histone H3/H4 chaperone Anti-silencing function 1 

(Asf1) is essential for removing 3-4 nucleosomes from the PHO5 promoter upon phosphate 

depletion - the signal for activation of the PHO5 gene (Adkins et al., Molecular Cell 2004). 

We now show that Asf1-mediated promoter chromatin disassembly is also required for the 

activation of additional yeast genes. To understand the mechanism whereby promoter 

chromatin disassembly mediates transcriptional activation, we examined factor occupancy in 

vivo. Somewhat surprisingly, promoter chromatin disassembly is not required to enable 

access of the transcriptional activators to the promoter, but is required to enable access by 

the general transcription machinery, including TBP and RNA polymerase II. We will also 

present our recent analyses of the involvement of chromatin remodelers and histone 

acetyltransferases in promoter chromatin disassembly during transcriptional activation. 

169



Jon Wilson 

Abstract P120 

Structural studies of SET domain methyltransferases 

Jon Wilson 

Institute of Cancer Research, Section of Structural Biology, 237 Fulham Road, Chelsea, 

London, SW3 6JB, U.K. 

The methylation of lysine residues on histone tails is a potent signalling mechanism for 

defining patterns of gene expression via variation of chromatin organisation. Only a limited 

number of the lysines in histone tails are subject to modification but the possibility of the 

discreet addition of either one, two or three methyl groups allows for a high combinatorial 

potential. Correctly regulating the activity of histone methyltransferase enzymes is crucial as 

the consequence of mis-regulation can effect the expression of a large number of genes 

downstream. It is becoming increasingly apparent that epigenetic processes are involved in 

disease especially cancer. Whether lysine methylation leads to activation or repression is 

context specific. Disease can be caused by either activation of oncogenes or repression of 

tumour suppressors. 

Building upon previous analysis on Set7/9[1] and PR-Set7[2] we are establishing a better 

understanding of the molecular mechanisms involved in SET domain catalysed methyl 

transfer. The major area of interest is targeting of a particular lysine residue substrate and 

the determination of activity in terms of how many methyl groups a particular enzyme can 

add. This is a complex area given that some SET domain methyltransferase enzymes seem 

to have varying specificity depending on interactions within the multi-protein complexes of 

which they are components. 

Refs: 

[1] Xiao, B., Jing, C., Wilson, J.R., Walker, P.A., Vasisht, N., Kelly, G., Howell, S.A., Taylor, 

I.A., Blackburn, G.M. & Gamblin, S.J. (2003) Structure and catalytic mechanism of the 

human histone methyltransferase SET7/9. Nature 421, 652-656. 

[2] Xiao,B., Jing, C., Kelly, G., Walker, P.A., Muskett, F.W., Frenkiel, T.A., Martin, S.R., Sarma, 

K., Reinberg, D., Gamblin, S.J. and Wilson, J.R. (2005) Specificity and Mechanism of the 

Histone Methyltransferase Pr-Set7. Genes & Development 19, 1444-1454. 

170



Zhaodong Xu 

Abstract P121 

Remote elements critical for cytokine induced gene expression 

Zhaodong Xu, Zuyao Ni, Mohamed Abou El Hassan, Tao Yu, Monika 

Sangwan, Mohamad Ahmad and Rod Bremner 

Toronto Western Research Institute, Toronto Western Research Institute, 399 Bathurst 

Street, Mc6-424,Toronto, Ontario M5T 2S8, Canada 

The role played by long-range elements in the regulation of gene transcription is still 

underappreciated. Hunting for these elements is a daunting task and developing a rapid and 

easy procedure is essentially important. Towards this end, we utilized histone acetylation to 

precisely mark interferon gamma (IFNgamma) specific regulatory elements throughout 

megabase (Mb) regions around 56 known IFN-responsive genes. IFNgamma induced 

STAT1 and IRF1 binding specifically at promoter proximal and remote sites from gene starts. 

Remarkably the vast majority of IFNgamma-induced STAT1 and IRF1 binding at promoters 

as well as remote sites was flagged by acetylated histones, verifying the role of acetylated 

chromatin in the prediction of promoters and most importantly of long-range elements. The 

activity at novel long-range elements, residing at as far as 70 kb of gene starts, were 

confirmed at the CIITA and SOCS1 loci using ChIP, reporter and looping assays. Thus, longrange 

elements are a novel aspect of IFNgamma-mediated gene induction and mapping 

acetylated chromatin is an excellent tool to find them, implying the specificity of the present 

method in finding remote regulatory elements in large genomic scales. 

171



Xiaofang Yang 

Abstract P122 

Dissecting SWI/SNF ATP-dependent chromatin remodeling 

complex in Saccharomyces cerevisiae 

Xiaofang Yang 1 , Roser Zaurin 2 , Sharmistha Kundu 1 , Miguel Beato 2 and 

Craig Peterson 1 

1 

Program in Molecular Medicine, University of Massachusetts Medical School, Worcester, 

MA 01605. 2 Centre de Regulacio Genomica, Universitat Pompeu Fabra, Passeig Maritim 37- 

49, E-08003 Barcelona, Spain 

Yeast SWI/SNF is the founding member of an ATP-dependent chromatin remodeling 

superfamily involved in transcriptional regulation for a subset of genes. Most SWI/SNF-like 

chromatin remodeling enzymes have been purified as multiprotein complexes that contain 

an ATPase catalytic subunit highly homologous to yeast Swi2/Snf2. The fact that ATPase 

subunit like human Brg1 alone is active for chromatin remodeling raised the question on the 

role of other subunits for chromatin remodeling. Interestingly, mutations of core subunits of 

hSWI/SNF such as hSwi2/BRG1, hSnf5/INI1, or hSWI3 are found in many types of cancers. 

In this study, we investigated the role of different subunits for yeast SWI/SNF function 

through a partial deletion of the SANT domain of Swi3. Genome-wide transcriptional profile 

suggested that SWI3deltaSANT was basically a weak null allele of SWI3. SWI3deltaSANT 

crippled the recruitment of SWI/SNF to target promoter of CDC6, SIC1 and HO. In the 

absence of an intact SANT domain, tethering SWI/SNF via LexA-Swi2 was insufficient for 

transcriptional activation of a LexAop-GAL1TATA-LacZ reporter in vivo. Surprisingly, 

SWI3deltaSANT, SWI3R564E or swi3 caused dissociation of SWI/SNF into at least 4 stable 

subcomplexes, indicating that the SANT domain of Swi3 is critical for SWI/SNF assembly. 

Intriguingly, a triplex of Swi2/Arp7/Arp9, the minimal complex of SWI/SNF, was completely 

competent for ATP hydrolysis, generating superhelical torsion, inducing restriction enzyme 

accessibility and catalyzing nucleosome mobility change in vitro. However, the minimal 

complex was defective to catalyze histone H2A/H2B dimer loss from MMTV 

mononucleosomes. Finally, we found that the minimal SWI/SNF was functional to regulate 

SRG1 transcription in vivo, based on Northern blot and ChIP. We propose that SWI/SNF is 

an integration of at least 4 functional modules that are responsible for multi-step chromatin 

remodeling process. Our data help to understand both structural and functional organization 

of SWI/SNF ATP-dependent chromatin remodeling complex. 

172



Juan I. Young 

Post-transcriptional functions of MeCP2 

Abstract P123 

Mauricio A. Saez, Matias Alvarez-Saavedra, Huda Y. Zoghbi and 

Juan I. Young 

Centro de Estudios Cientificos and Universidad Austral de Chile, Valdivia, 905-9100, Chile. 

Department of Molecular and Human Genetics, Department of Neuroscience and Howard 

Hughes Medical Institute, Baylor College of Medicine, Houston, TX 77030, U.S.A. 

Epigenetic modifications of DNA provide an extra level of genetic information. The main 

epigenetic modification of mammalian genomes is cytosine methylation, commonly 

associated with gene silencing. The importance of this process for chromatin function has 

been underscored by the discovery that Rett syndrome, a disabling neurodevelopmental 

disease, is caused by mutations in MeCP2. MeCP2 (methyl-CpG-binding protein 2) is a 

methylation-dependent transcriptional repressor. We found that MeCP2 is a multifunctionalmultilevel 

regulator of gene expression; interacts with proteins involved in RNA processing 

and regulates alternative splicing in addition to its role as a transcriptional repressor. 

173



Veronica Yu 

Abstract P124 

Over-expression of Cks proteins causes gene derepression 

in Saccharomyces cerevisiae 

Roman Holic and Veronica Yu 

MRC Clinical Sciences Centre, Imperial College Hammersmith Campus, Du Cane Road, 

London, W12 0NN, U.K. 

Cks (cyclin-dependent kinase interacting) proteins are evolutionarily conserved and are 

frequently over-expressed in multiple cancers of high grade and stage. 

It has previously been demonstrated that the Saccharomyces cerevisiae cell cycle 

regulatory cyclin-dependent kinase interacting protein, Cks1 and its kinase partner, Cdc28 

(Cdk1); are involved in the control of transcription at multiple gene loci. This transcriptional 

role is mediated through a requirement of Cdc28/Cks1 for recruiting proteasomes to coding 

regions. However, it is independent of the protein kinase activity of Cdc28. 

We over-expressed yeast and mammalian Cks proteins in S. cerevisiae in order to mimic 

the protein over-expression pattern in tumour cells. We found that over-expression of Cks 

proteins caused derepression of a wide-range of genes under repressive conditions. 

Possible mechanisms and implications for this observation will be discussed. 

174



Rebekah Zinn 

Abstract P125 

hTERT is expressed in cancer despite promoter DNA 

methylation by preservation of unmethylated DNA and active 

chromatin around the transcription start site 

Rebekah L. Zinn 1,2 , Kevin Pruitt 1 , Sayaka Eguchi 1 , Stephen B. Baylin 1,2 and 

James G. Herman 1,2 

1 

The Sidney Kimmel Comprehensive Cancer Center at Johns Hopkins, Baltimore, Maryland 

21231 2 The Graduate Program in Cellular and Molecular Medicine, The Johns Hopkins 

University School of Medicine, Baltimore, Maryland 21231 

hTERT, which encodes the catalytic subunit of telomerase and is expressed in most 

immortalized and cancer cells, has been reported to have increased DNA methylation in its 

promoter region in many cancers. This pattern is inconsistent with observations that DNA 

methylation of promoter CpG islands is typically associated with gene silencing. Here we 

provide a comprehensive analysis of promoter DNA methylation, chromatin patterns, and 

expression of hTERT in cancer and immortalized cells. Methylation specific PCR (MSP) and 

bisulfite sequencing of the hTERT promoter in breast, lung, and colon cancer cells shows 

that all cancer cell lines retain alleles with little or no methylation around the transcription 

start site, despite being densely methylated in a region 600 bp upstream of the transcription 

start site. By real-time RT-PCR, all cancer cell lines express hTERT. Chromatin 

immunoprecipitation (ChIP) analysis reveals that both active (acetyl-H3K9, dimethyl-H3K4) 

and inactive (trimethyl-H3K9, trimethyl-H3K27) chromatin marks are present across the 

hTERT promoter. However, using a novel approach combining methylation analysis of ChIP 

DNA, we show that active chromatin marks are associated with unmethylated DNA, while 

inactive marks of chromatin are associated with methylated DNA in the region around the 

transcription start site. These results demonstrate that DNA methylation patterns of the 

hTERT promoter (-150 to +150 around the transcription start) are not inconsistent with the 

usual dynamics of gene expression in that the absence of methylation in this region and the 

association with active chromatin marks allow for the continued expression of hTERT. 

175



Additional poster submissions 

Yoshimitsu Takahashi 

Abstract P126 

Degree of SUMO modification as a differential tag for targeting 

to specific chromosomal domains 

Yoshimitsu Takahashi and Alexander Strunnikov 

NIH, NICHD, LGRD, Bethesda, MD, U.S.A. 

SUMO is a ubiquitin-like modifier that regulates many proteins by direct conjugation. E1, E2 

and E3 enzymes are needed to transfer SUMO to a specific target. Recently, numerous 

potential SUMO substrates were identified by proteomic approaches. However, functional 

roles of individual modification are obscure, as only a small fraction of a given protein is 

sumoylated. In order to overcome this technical difficulty, we designed a novel technique 

Constitutive SUMO Modification (CSM), which allows to track only the modified form of the 

protein. We validated this method using Top2p as a model substrate. The advantages of 

Top2p for application of this technique are: (1) sumoylation sites are in a non-essential C- 

terminal domain. (2) Top2p expression is abundant. (3) Top2 modification is relatively strong 

both in vivo and in vitro. We have evidence that sumoylation of Top2p to different degrees 

changes intranuclear targeting of this essential protein. 

176



Marna S. Costanzo 

Abstract P127 

The evolutionary conservation of chromatin modifying 

proteins in malaria 

Marna S. Costanzo 1 , Szymon Kaczanowski 2 , Thanat Chookajorn 3 , and 

Daniel L. Hartl 1 

1 

Department of Organismic and Evolutionary Biology, Harvard University, Cambridge, MA 

02138, USA; 2 Institute of Biochemistry and Biophysics, Polish Academy of Sciences, 02- 

106 Warsaw, Poland; 3 Department of Biochemistry, Faculty of Science, Mahidol University, 

Bangkok 10400, Thailand 

The malaria parasite is responsible for most of the deaths related to infectious disease in 

the world today. This single cell eukaryote has a complex life-cycle consisting of two hosts 

and many morphologically different forms. The majority of genes expressed during the 

pathogenic bloodstages correlate to the cell cycle. Transcription of these genes is achieved 

monocistronically despite the apparent lack of specific transcription factors and covalent 

histone modification provides a possible mechanism of expression control. We investigate 

the apparent conservation of histone lysine methyltransferase function in malaria, despite 

an evolutionary history that is far diverged from most eukaryotes. The implications of these 

findings are discussed in context of the parasite biology and evolutionary implications. 

177



Philippe Prochasson 

Abstract P128 

Functional characterization of the HIR corepressor complex 

Philippe Prochasson, Laurence Florens and Jerry L. Workman 

The Stowers Institute for Medical Research, Kansas City, Missouri 64110, U.S.A. 

The histone regulatory (HIR) and histone promoter control (HPC) repressor proteins 

regulate three of the four histone gene loci during the Saccharomyces cerevisiae cell cycle. 

Previously, we showed that Hir1, Hir2, Hir3, and Hpc2 proteins form a stable HIR 

corepressor complex. The HIR complex promotes histone deposition onto DNA in vitro and 

constitutes a novel nucleosome assembly complex. The HIR complex stably binds to DNA 

and nucleosomes. Furthermore, we demonstrated that the HIR complex binding to 

nucleosomes forms a distinct protein/DNA complex resistant to remodeling by SWI/SNF. 

Thus, the HIR complex is a novel nucleosome assembly complex which functions with 

SWI/SNF to regulate transcription. 

We are now pursuing the functional characterization of the HIR complex to better understand 

its role in gene regulation and its interplay with the SWI/SNF chromatin remodeling complex. 

We will present data showing that the HIR complex is directly involved in the transcriptional 

regulation of the SUC2 gene and that the presence of the HIR proteins at the SUC2 

promoter renders its transcriptional activation swi2/snf2 dependent. In a second part, we will 

focus on the identification of post-translational modifications (PTMs) of the HIR proteins 

during the cell cycle and their role on the histone genes transcriptional regulation. 

178


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