The NuRD (Nucleosome Remodelling and Deacetylation) complex is a highly conserved and abundant transcriptional repression complex. Embryonic Stem (ES) cells lacking a central structural component of the complex, Mbd3, proliferate slowly and are unable to differentiate in the absence of LIF. We show here that in addition they show significant demethylation of DNA at a number of repetitive sequences, such as the centromeric repeats and IAP elements, as well as some single-copy DMRs and CpG islands throughout the genome. This demethylation is associated with histone hyperacetylation and aberrant transcription of these sequences or, in the case of regulatory regions, genes controlled by them. Many of the sequences demethylated here are also demethylated in <i>Dnmt3a/3b<sup>-/-</sup></i> ES cells, and interestingly <i>Mbd3<sup>-/-</sup> </i>ES cells contain significantly reduced levels of the methyltransferase<i> Dnmt3b. </i>However <i>Dnmt3b<sup>-/-</sup> </i>ES cells are demethylated to a lesser extent than <i>Mbd3<sup>-/-</sup></i>cells, suggesting that a decrease in Dnmt3b levels is not the sole cause of the demethylation observed here. An interaction has been detected between Dnmt3b and the NuRD components Mta2 and Mbd3 in wild type cells, leading to speculation that they may co-operate to bring about a repressive chromatin structure at target sites, but chromatin immunoprecipitation of a tagged version of Mbd3 and another NuRD component, Mi-2β, does not reveal binding of the complex at affected sequences. However immunofluorescence of Mbd3, Mta2 and Mi-2β reveals localisation to centromeric regions in approximately 30% of wild type cells, suggesting localisation is cell-cycle dependent. The degree of centromeric localisation of Mta2 is significantly reduced in <i>Mbd3<sup>‑/-</sup></i> cells. We propose that NuRD binds to target sequences in a cell cycle dependent manner, where it participates with DNA methyltransferases in the formation and/or maintenance of a repressive chromatin structure.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:660715 |
| Date | January 2007 |
| Creators | Powell, Christine |
| Publisher | University of Edinburgh |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://hdl.handle.net/1842/12134 |
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