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Epigenetic mechanism of CENP-A loading to centromeres

Abstract : Centromere is a specialized chromosomal locus, where kinetochore assembles, which is required for correct chromosome segregation during cell division. In higher eukaryotes, centromere specification is independent of the DNA sequence and is determined epigenetically by the presence of a unique nucleosome that contains a histone H3 variant, called CENP-A. A fundamental question in centromere biology is that how CENP-A is specifically delivered to and maintained on centromeres. The aim of my thesis was to identify specific chaperone in human, responsible for CENP-A loading to centromeres, by using biochemical and proteomic strategies. To identify CENP-A deposition machinery, I purified the prenucleosomal CENP-A complex from HeLa cells stably expressing epitope tagged CENP-A. By mass spectrometry analysis of proteins present in CENP-A and H3.1 complex, I found HJURP uniquely in CENP-A prenucleosomal complex. Down regulation of HJURP by specific siRNA strongly diminished centromeric localization of CENP-A. Bacteriallyexpressed HJURP specifically binds to the CATD domain of CENP-A, via a highly conserved Nterminal domain, called CBD. Finally, I showed that HJURP is able to facilitate the efficient deposition of CENP-A/H4 tetramer on naked DNA. Taken together, my data demonstrate that HJURP is a key chaperone responsible for the targeting and deposition of newly synthesized CENPA at centromeres.
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Muhammad Shuaib. Epigenetic mechanism of CENP-A loading to centromeres. Biochemistry, Molecular Biology. Université de Strasbourg, 2012. English. ⟨NNT : 2012STRAJ028⟩. ⟨tel-00845987⟩

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