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 thus impairs genome stability. 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 in Drosophila, namely CAL1 and CENP-C.
In this project, we propose to make use of a heterologous biosynthetic system in which Drosophila centromere factors are expressed in human cells. Using the LacI/LacO targeting system we aim to re-constitute a synthetic centromere outside the endogenous centromere on integrated LacO arrays. Preliminary evidence suggests that we are able to reproduce know interactions between CENP-A and its binding partners CAL1 and CENP-C. Importantly, we find that centromere proteins of human and Drosophila cells don't interfere with each other, allowing us to dissect the molecular underpinnings of Drosophila centromere inheritance in a very controlled way. In a second complementary approach, we are using recombinant Drosophila proteins to reconstitute centromeric nucleosomes and binding partners in vitro to gain further understanding regarding direct interactions, size and stoichiometry of the relevant protein complexes. Further insight into the structure might be gained upon successful crystallization and diffraction of the generated protein complexes in collaboration with the Arulanandam lab. Together, the results obtained during this Ph.D. project will represent a significant step towards closing the epigenetic loop of centromere inheritance through cell generations.