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dc.contributor.advisorHill, Robert
dc.contributor.authorReddington, James Peter
dc.date.accessioned2013-11-14T15:31:04Z
dc.date.available2013-11-14T15:31:04Z
dc.date.issued2012-11-30
dc.identifier.urihttp://hdl.handle.net/1842/8138
dc.description.abstractChromatin 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.en_US
dc.contributor.sponsorMedical Research Council (MRC)en_US
dc.contributor.sponsorCancer Research UKen_US
dc.language.isoenen_US
dc.publisherThe University of Edinburghen_US
dc.relation.hasversionNestor, C. E., R. Ottaviano, J. Reddington, D. Sproul, D. Reinhardt, D. Dunican, E. Katz, J. M. Dixon, D. J. Harrison and R. Meehan (2011). Tissue-type is a major modifier of the 5- hydroxymethylcytosine content of human genes. Genome Res.en_US
dc.relation.hasversionRuzov, A., E. Savitskaya, J. A. Hackett, J. P. Reddington, A. Prokhortchouk, M. J. Madej, N. Chekanov, M. Li, D. S. Dunican, E. Prokhortchouk, S. Pennings and R. R. Meehan (2009). The non-methylated DNA-binding function of Kaiso is not required in early Xenopus laevis development. Development. 136(5): 729-738.en_US
dc.subjectDNA methylationen_US
dc.subjectpolycomben_US
dc.subjectgene regulationen_US
dc.subjectchromatinen_US
dc.titleRole for the DNA methylation system in polycomb proteinmediated gene regulationen_US
dc.typeThesis or Dissertationen_US
dc.type.qualificationlevelDoctoralen_US
dc.type.qualificationnamePhD Doctor of Philosophyen_US


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