2009 Vienna - European Society of Human Genetics

Genomics, Genomic technology and Epigenetics
P11.028
Linking macro ncRNAs to human imprinted gene clusters and
cimP (cpG island methylator Phenotype) regions in normal and
cancer cells
I. M. Vlatkovic, R. Huang, F. M. Pauler, F. Santoro, D. P. Barlow;
Center for Molecular Medicine of the Austrian Academy of Science, Vienna,
Austria.
Genomic imprinting results in parental-specific gene expression
and offers one of the best examples of an epigenetic gene silencing
mechanism in mammals. The analysis of imprinted gene expression
in mouse models has identified two important but unexpected,
epigenetic mechanisms. First, that DNA methylation acts to silence
macro non-protein-coding RNAs. Second, that macro ncRNAs act
to silence flanking genes in cis. To date, two examples of imprinted
macro ncRNAs with a silencing function are known (Air and Kcnq1ot1,
reviewed in Pauler & Barlow 2006). In order to identify and characterize
new macro ncRNAs we selected imprinted regions from the human
genome and CIMP (CpG Island Methylator Phenotype) regions
to generate HIRTA (Human Imprinted Region Tiling Array) Chips. RNA
samples from different tissues, ES cells and from normal or tumor cell
lines are hybridized to these chips using the RETA (RNA Expression
Tiling Array) technique. In order to analyze the results from the Chips
we have developed the NORBERT (NOn- coding RNA identification
Based on Enrichment on RNA Tiling array) program. Our goals are to
test: (1) if macro ncRNAs are a common feature of imprinted regions
in the human genome, (2) if imprinted macro ncRNAs are deregulated
in cancer, and (3) if macro ncRNAs play a role in regulating non-imprinted
genes. We are particularly interested to test if macro ncRNAs
play a role in the gain DNA methylation of tumor suppressor genes
in tumors. Our preliminary data show that by this approach we can
identify 5 well-known imprinted ncRNAs and that we are able to detect
novel macro ncRNA candidates. Our immediate goal is to characterize
the transcriptional features of novel candidate macro ncRNAs.
P11.029
Assessing the levels of cREB1 after siRNA mediated knockdown
in K562 cells
Z. Deilami Khiabani 1 , M. Banan 2 , J. Gharesouran 3 , A. Asgharian 2 , M. Hoseini 2 ,
S. Farashi 2 , H. Najmabadi 2 ;
1 Isalamic Azad University, Zanjan Branch, Zanjan, Islamic Republic of Iran, 2 Genetics
Research Center, University of Social Welfare &Rehabilitation Sciences,
Tehran, Islamic Republic of Iran, 3 Department of Medical Genetics, Faculty of
Medicine, University of Medical Sciences, Tabriz, Islamic Republic of Iran.
CREB1 is an important downstream protein for many signaling pathways.
By designing efficient siRNAs against CREB1, it may be possible
to assess the role of molecules involved in signaling pathways
in different cell types. In this research the efficiency of CREB1 knockdown
by 2 different siRNAs in K562 cells have been studied. siRNAs
have been designed according to the criteria suggested by Reynolds
et al. K562 cells were transfected by siRNA using Lipofectamine 2000.
The efficiency of CREB1 knockdown has been assessed by quantitative
relative real time PCR. Our results have shown that only one of
the siRNAs has a high level of inhibitory effect on CREB1 gene expression.
The expression of CREB1 by this siRNA was knocked-down
79.5% in K562 cells. Reasons other than the aforementioned criteria
may be involved in effectiveness of siRNAs.
P11.030
Transcriptional profiling of mouse embryos with cardiac and
thymic defects induced by antagonist of retinoic acid and
recovered by supplementation with folic acid
L. Diano 1 , D. Cipollone 2 , S. Bueno 3 , L. Vecchione 1 , G. Prosperini 3 , A. Desideri 4 ,
G. Chillemi 3 , B. Marino 2 , G. Novelli 1 , F. Amati 1 ;
1 Dept. of Biopathology and Diagnostic Imagining,Tor Vergata, University, Roma,
Italy, 2 Dept. of Pediatrics, La Sapienza University, Roma, Italy, 3 CASPUR, Consortium
for Supercomputing Applications, Roma, Italy, 4 Dept. of Biology, Tor
Vergata University, Roma, Italy.
Congenital heart diseases (CHDs) account for 25% of all human congenital
abnormalities and affect 1-2% of newborn children. Specific
malformations of the outflow portions of the heart are termed conotruncal
malformations (CTHM; OMIM 217095) and account for a fourth to a
third of all nonsyndromic congenital heart defects.
By induction of a retinoic acid competitive antagonist (BMS-189453)
we developed a mouse model of CTHMs (81.3%), thymic abnormalities
(98.4%) and neural tube defects (NTD, 20.3%). A nutritive therapy
based on folic acid (FA) administered to mouse embryos previously
treated with BMS-189453, resulted in a reduction of CTHM (64.8%),
thymic abnormalities (27.8%) and NTD (3.7%)
We performed a global transcription analysis by microarray to identify
genes or molecular pathways affected in both the experimental models.
A total of 447 genes were differentially expressed (FC= ± 1,5) in BMStreated
mouse; while a total of 239 genes were differentially expressed
in BMS+FA-treated embryos. A comparative analysis of these gene
expression patterns revealed 140 common genes; 70 of them includes
genes that were down or up regulated in BMS-treated embryos, but
returned to a “wild-type” level in BMS + FA-treated embryos. These
genes were mainly involved in protein metabolism (14.8%), transport
(10.2%), signal transduction (13%), cell cycle (7.4%) and transcription
(6.5%).
QRT-PCR assay performed on a selected group of commonly regulated
genes confirmed the microarray data.
The discrete number of genes which resulted from our data might be
considered as candidate genes for conotruncal heart and thymic malformations
in humans.
P11.031
the cYLD tumor suppressor sensitizes cells to microtubule
destabilization
S. Krauss 1 , J. So 1 , M. Huber 2 , A. Koehler 3 , R. Schneider 3 , S. Schweiger 4 ;
1 Max-Planck Institute for Molecular Genetics, Berlin, Germany, 2 Department of
Dermatology, Lausanne, Switzerland, 3 Department of Biochemistry, Innsbruck,
Austria, 4 University of Dundee, Dundee, United Kingdom.
Mutations in the CYLD tumor suppressor have been identified in patients
with familial cylindromatosis and familial trichoepithelioma, which
are both autosomal dominant genetic predispositions to multiple tumors
of the skin appendages. CYLD has been shown to deubiquitinate
TRAF proteins and Bcl-3, both leading to inhibition of NF-κB activation.
We have now found that CYLD is a microtubule-associated protein that
accelerates microtubule destruction in cells treated with the microtubule-depolymerizing
agent nocodazole. CYLD protein carrying a point
mutation that truncates the protein at a.a. 485 and thereby deletes
the C-terminus, including the majority of the third predicted CAP-GLY
domain, still associates to microtubules, but has no influence on microtubule
stability. Accordingly, specific knockdown of CYLD results in an
increase of microtubule stability and faster recovery after nocodazole
withdrawal. Our data strongly suggest that, in addition to upregulation
of NF-κB signalling, microtubule dynamics plays an important role in
the development of skin cancer induced by mutations in the tumor suppressor
CYLD.
P11.032
the integration of approaches for the study of biological
networks in the inner ear
T. Elkan 1 , R. Hertzano 1 , I. Ulitsky 2 , R. Elkon 1 , M. Irmler 3 , R. Shamir 2 , J. Beckers
3 , K. B. Avraham 1 ;
1 Dept. of Human Molecular Genetics & Biochemistry, Tel Aviv University, Sackler
Faculty of Medicine, Tel Aviv, Israel, 2 Blavatnik School of Computer Science,
Tel Aviv University, Tel Aviv, Israel, 3 Institute of Experimental Genetics, Neuherberg,
Germany.
Systems biology involves studying the interaction and interplay of
many levels of biological regulation. We combined comparative transcriptomic
and proteomic analyses of early post-natal cochlear and
vestibular sensory epithelia to identify networks of genes and proteins
essential for the development and function of these inner ear organs.
Expression profiling of vestibular and cochlear sensory epithelia was
performed using Affymetrix microarrays. Proteomics analysis was
performed using the Q-TOF mass spectrometer with ITRAQ labeling
(Smoler Proteomics Center, Technion). Integration of the transcriptome
and proteome data led to the identification of genes/proteins that
may play an important role in the inner ear. These genes/proteins are
being examined in further detail.
In addition, we identified microRNAs (miRNAs) that are expressed in
these sensory epithelia using the miRCURY LNA array system. We
integrated the transcriptome, proteome and miRNA levels to efficiently
predict targets of miRNAs in the inner ear using newly developed algo-