15th International Conference on Arabidopsis Research - TAIR

T09-027
Histone methylation and heterochromatin assembly
in Arabidopsis thaliana
Jörg Fuchs(1), Zuzana Jasencakova(1, 2), Armin Meister(1), Steve Jacobsen(3),
Ingo Schubert(1)
1-Institute of Plant Genetics and Crop Plant Research (IPK), Corrensstr. 3, D-06466 Gatersleben,
Germany
2-present address: Institute of Genetics and Biophysics (IGB), 80131 Naples, Italy
3-Department of Molecular Cell and Developmental Biology, University of California, Los Angeles,
CA90095, USA
The N-terminal residues of nucleosomal core histones are subjected to a
variety of post-translational modifications such as acetylation, phosphorylation,
methylation and ubiquitination. The acetylation and in particular the
methylation of lysine residues of histones H3 and H4 concerted with the
cytosine methylation of DNA seem to play a crucial role in heterochromatin
formation in a variety of organisms. Each methylatable histone residue can
be either mono-, di- or trimethylated (Paik and Kim 1971). Using specific
antibodies against the three different methylation states of histone H3 at
lysine K9 and K27 we performed immunostaining experiments on interphase
nuclei of Arabidopsis thaliana to analyse the distribution of these isoforms
between heterochromatic and euchromatic subdomains. The heterochromatin
of A. thaliana, mainly composed of tandem and dispersed repeats, is located
around centromeres (and NORs) and identifiable as distinct densely stained
chromocenters in interphase nuclei (Fransz et al. 2002). Immunostaining
revealed mono- and dimethylated H3-K9 predominantly at chromocenters
(Jackson et al. 2004). The occurrence of trimethylated H3-K9 in plants is still
a matter of discussion. For H3-K27 methylation all three methylation states
were found dispersed over the interphase chromatin, but the mono- and
dimethylated H3-K27 were enriched at the chromocenters. Together with
previous findings, these data suggest that wildtype heterochromatin in
Arabidopsis is characterized by high levels of DNA methylation, mono- and
dimethylated H3-K9 and H3-K27 and low levels of histone H3 and H4 acetylation
(except for H3K18 and H4K16 which show an increased acetylation
at chromocenters during and after replication) and dimethylated H3-K4.
The transcriptionally permissive euchromatin in contrast shows high levels
of acetylation of histones H3 and H4, dimethylation of H3-K4 and moderate
methylation of H3-K27 (mono-, di- and tri-).
In order to identify genes responsible for the distinct types of histone methylation
and to characterize the interdependence of the different chromatin
modifications putative chromatin mutants of Arabidopsis are being tested.
Similarities and differences as to heterochromatin assembly between Arabidopsis
and other organisms are discussed.
Jackson et al. (2004) Chromosoma 112:308-15
Fransz et al. (2002) PNAS 99:14584-9
Paik and Kim (1971) Science 174:114-9
T09 Genetic Mechanisms (Transcriptional and Chromatin Regulation)
T09-028
Contribution of target transgene position and
structure to RNA-directed promoter methylation and
TGS
Ute Fischer(1), Renate Schmidt(2), M. Florian Mette(1)
1-Institute of Plant Genetics and Crop Plant Research, Corrensstraße 3, 06466 Gatersleben,
Germany
2-Max-Plank-Institute of Molecular Plant Physiology, Am Mühlenberg 1, 14476 Golm, Germany
RNA-directed transcriptional gene silencing (TGS), the specific repression of
transcription of a gene in correlation with extensive promoter DNA methylation
in the presence of double stranded (ds) RNA with homology to the
respective promoter, is an epigenetic mechanism so far only known from
plants. For its study, transgene systems employing the nopaline synthase
promoter (NOSpro), a moderately strong constitutive promoter widely used in
T-DNA constructs, have proven very useful [1]. In tobacco and Arabidopsis
thaliana, transcription of dsRNA from a silencer transgene with a NOSpro
inverted repeat (IR) can trigger TGS of unlinked homologous target promoters
in trans. Similar to other silencing mechanisms involving dsRNA, the
NOSpro dsRNA is processed to short interfering RNAs predominantly 21-24
nucleotides in length. Both, TGS and DNA methylation at the target NOSpro
are dependent on the NOSpro dsRNA, upon its removal, NOSpro-driven
reporter-gene expression is reactivated and the NOSpro DNA methylation is
lost. But reactivation and release from DNA methylation are not immediate,
indicating that there is some level of maintenance of the silenced state in the
absence of the inducing RNA signal. Testing Arabidopsis thaliana mutants
known to affect TGS in other systems identified the DNA methyltransferases
DRM1/DRM2 and MET1 as well as the putative SWI2/SNF2 chromatin
remodelling factor DDM1 as essential for RNA-directed transcriptional gene
silencing. New genetic screens yielded additional mutations, of which one
was mapped to histone deacetylase HDA6, a candidate enzyme for histone
modification. Interestingly, not all target transgenes containing NOSpro-driven
reporter genes show the same susceptibility to RNA-directed TGS, indicating
that the chromosomal location of target transgenes and / or the particular
arrangement of NOSpro copies in the target transgenes might contribute to
the silencing process. To approach this problem in a systematic way, collections
of well characterized transgenes with the same structure integrated at
different chromosomal positions [2] or of transgenes with differing structures
integrated at the same chromosomal positions are being challenged by a
silencer transgene providing NOSpro dsRNA. Initial results of the analysis of
the levels of induced transcriptional repression and NOSpro DNA methylation
will be presented. The work is supported by DFG grant ME 2122/1-1.
[1] Matzke et al. (2004) Biochim Biophys Acta 1677:129-141
[2] Forsbach et al. (2003) Plant Mol Biol 52:161-176
15 th <strong>International</strong> <strong>Conference</strong> on Arabidopsis Research 2004 · Berlin