In mammals, epigenetic modifications and trans-acting effectors coordinate gene expression during development and impose transcriptional memories that define specific cell lineages and cell-types. Methylation at CpG dinucleotides is an epigenetic mechanism through which transcriptional silencing is established and heritably maintained through development. Functionally, DNA methylation regulates key biological processes such as X-chromosome inactivation, transposon repression and genomic imprinting. However, the extent to which DNA methylation is the primary regulator of single-copy gene expression and the precise mechanism of methylation-dependent silencing remain undetermined. Here, I identify a novel set of germline-specific candidate genes putatively regulated by DNA methylation. Analysis of one candidate gene, Tex19, demonstrates that promoter CpG methylation is the primary and exclusive mechanism for regulating developmental silencing in somatic lineages. Genetic or pharmacological removal of CpG methylation triggers robust de-repression of Tex19 and loss of transcriptional memory. Moreover, Tex19 critically relies on de novo methylation, mediated by Dnmt3b, to impose silencing in differentiating ES cells and somatic cells in vivo from embryonic day (E)7.5. Reporter gene and ChIP analysis demonstrate that Tex19 is strongly activated by general transcription factors and is not marked by repressive histone modifications in somatic lineages, consistent with differential DNA methylation per se being the primary mechanism of regulating expression. Full transcriptional silencing of Tex19 is critically dependent on the methyl-binding protein (MBP) Kaiso, which is only recruited to methylated Tex19 promoter. The reliance on DNA methylation and Kaiso for silencing in somatic cells establishes an epigenetic memory responsible for maintaining expression in germline and pluripotent cell types through successive developmental cycles. This thesis represents the first causal report of lineagespecific promoter DNA methylation directing silencing of an in vivo gene through recruitment of an MBP.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:563205 |
| Date | January 2010 |
| Creators | Hackett, Jamie Alexander |
| Contributors | Meehan, Richard. ; Van Heyningen, Veronica |
| Publisher | University of Edinburgh |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://hdl.handle.net/1842/5931 |
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