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Title: Biochemical and functional analysis of the chromatin remodeller Mi-2β and the associated NuRD complex in pluripotent cells
Author: Costello, Ita
Awarding Body: University of Edinburgh
Current Institution: University of Edinburgh
Date of Award: 2007
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Mi-2β, encoded by the Chd4 gene, is an ATP-dependent chromatin remodelling protein belonging to the CHD protein family. Structurally, Mi-2β contains two PHD finger domains, two chromodomains and an ATPase helicase domain. It is a key component of the NuRD (nucleosome remodelling and histone deacetylase) complex. Due to its deacetylase activities, the NuRD complex is considered to be a transcriptional repressor complex. In my thesis, I biochemically and functionally characterised Mi-2β and the associated NuRD complex. I used a gene-trap strategy to analyse the role of Mi-2β in development. Chd4 gene-trap homozygous embryos were not evident in post-implantation development, indicating that Mi-2β is essential in early embryonic development. Ex vivo analysis revealed a critical role of Mi-2β in the viability of early embryos, highlighting its importance in development. Additionally, I investigated the biochemical function of the PHD fingers and chromodomains. The chromodomains of Mi-2β do not appear to bind di-methylated lysine 4 of histone H3 or unmodified histone H3. Additionally, the PHD fingers of Mi-2β may be important in binding histone tails, irrespective of the modification state of these tails. I also employed a biotinylation tagging strategy to biochemically characterise the NuRD complex in embryonic stem (ES) cells. This method identified the subunits of the complex in ES cells and revealed that the complex was a heterogeneous complex, potentially explaining the differences in previously reported purifications. Additionally, the biotinylation purification allowed the identification of interacting transcriptional regulators in ES cells, which included the pluripotency-associated protein Sall4. In addition to the identification of previously known interactors, the purification implied roles for the NuRD complex not previously acknowledged, including a potential role in transcription elongation.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available