The centromere is a specialized chromatin region that is essential for proper segregation of mitotic chromosomes by forming a kinetochore. Centromere dysfunction results in aneuploidy or loss of chromosomes and is associated with cancer and miscarriage. In most eukaryotes centromeres are not encoded by DNA, but instead epigenetically defined by the histone H3-variant CENP-AcenH3 (dCENP-A/CID in flies). Although significant progress has been made to catalogue the protein composition of centromeres, the mechanisms by which CENP-A is deposited and how centromere identity is propagated through many cell divisions are poorly understood. We address this question by analysis of the less complex centromere in Drosophila by dissecting the function of dCENP-A in combination with the two additional proteins required for centromere maintenance, namely CAL1 and dCENP-C.
In this project, we propose to reconstitute the dCENP-A deposition machinery (CAL1 and dCENP-C) bound to centromeric dCENP-A nucleosomes in vitro using recombinant proteins from E.Coli/baculovirus. We aim to investigate the specifics of their binding and and subunit stoichiometry using biochemical/biophysical analysis. Further insight into the structure of the dCENP-A machinery will be gained using X-ray crystallography and cryo electron microscopy in collaboration with the Arulanandam lab.
Structurally predicted key protein binding domains/interactions will be validated by testing specific mutants in a variety of different in vivo assays using Drosophila tissue culture cells. These include co-immunoprecipitations and the biosynthetic LacO/LacI system combined with immunofluorescence microscopy. Together, the results obtained during this Ph.D. project will represent a significant step towards understanding the molecular underpinnings of epigenetic propagation of centromere identity.
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