Role for the DNA methylation system in polycomb proteinmediated gene regulation
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Date
30/11/2012Author
Reddington, James Peter
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Abstract
Chromatin structure and epigenetic mechanisms play an important role in initiating and
maintaining the intricate patterns of gene expression required for embryonic development.
One such mechanism, DNA methylation (5mC), involves the chemical modification of
cytosine bases in DNA and is implicated in maintaining patterns of transcription. However,
many fundamental aspects of DNA methylation are not fully understood, including the
mechanisms by which it influences transcriptional states. Recent data suggest functional
links between DNA methylation and a second epigenetic mechanism that has important roles
in transcriptional repression, the polycomb group (PcG) repressor system.
Here, I suggest
that an intact DNA methylation system is required for the repression of many PcG target
genes by influencing the genomic targeting of the polycomb repressor 2 complex (PRC2)
and its signature histone modification, H3K27me3 (K27me3). I demonstrate differential
genomic localisation of K27me3 at gene promoter regions in hypomethylated mouse
embryonic fibroblast (MEF) cells deficient for the major maintenance DNA
methyltransferase, Dnmt1. Globally, Dnmt1-/- MEFs have a higher level of the K27me3
mark than controls, as assessed by western blot and immunofluorescence. I observe
increased K27me3 at a relatively small number of gene promoters in Dnmt1-/- MEFs that
often are associated with high levels of DNA methylation in wildtype MEFs, consistent with
the notion that DNA methylation is capable of antagonising PRC2 binding at certain loci.
Conversely, I show that a large number of developmentally important genes that are
normally repressed and highly bound by K27me3, including classic polycomb targets, the
Hox genes, display dramatically reduced association with K27me3 in Dnmt1-/- MEFs. Many
of these genes, but not all, show reciprocal increases in promoter H3K4me3 modification
and are transcriptionally de-repressed in Dnmt1-/- MEFs. I suggest that these genes are
mostly associated with CpG-rich promoters with low levels of DNA methylation in wildtype
cells, implying that their silencing is not dependent on the canonical role of DNA
methylation. Consistent with the findings of recently published work, I suggest a working
model where PRC2 binding in wildtype cells is restricted by CpG methylation. According to
this model, the differential genomic location of K27me3 in hypomethylated Dnmt1-/- MEFs
is explained by a redistribution of PRC2 to normally DNA methylated, unbound loci,
resulting in a titration effect and coincident loss of K27me3 from normal targets. It was also
apparent that certain PRC2-target genes, including the developmentally important Hox gene
clusters, are strongly affected in Dnmt1-/- MEFs, displaying striking loss of K27me3. As
intergenic transcription has been implicated in relief from polycomb silencing and abundant
intergenic transcription has been reported within Hox clusters, I measured RNA expression
at Hox clusters and a small number of other PcG target genes in Dnmt1-/- MEFs using highdensity
tiling arrays. In Dnmt1-deficient MEFs, widespread increases in intergenic
transcription were observed within Hox clusters. In addition, mapping of the elongatingpolymerase-
associated H3K36me3 histone modification showed widespread increases in this
mark at intergenic and promoter regions in Dnmt1-/- MEFs. Increased local intergenic RNA
and H3K36me3 were found to correlate with K27me3 loss for this cohort of genes. I suggest
a working model where increased intergenic transcription and H3K36me3 in Dnmt1-/- MEFs
leads to accelerated loss of K27me3 at certain loci, including Hox clusters. Taken together
with recently published data, this work suggests that a major role of DNA methylation is in
shaping the PRC2/K27me3 landscape. The potential implications of this putative role for
DNA methylation are widespread, including our knowledge of how DNA methylation
influences transcriptional regulation, and the consequence of rearranged DNA methylation
patterns that are observed in many diseases including cancers.