Abstract
CENP-C is a fundamental component of functional centromeres. The elucidation of its structure-function relationship with centromeric DNA and other kinetochore proteins is critical to the understanding of centromere assembly. CENP-C carries two regions, the central and the C-terminal domains, both of which are important for the ability of CENP-C to associate with the centromeric DNA. However, while the central region is largely divergent in CENP-C homologues, the C-terminal moiety contains two regions that are highly conserved from yeast to humans, named Mif2p homology domains (blocks II and III). The activity of these two domains in human CENP-C is not well defined. In this study we performed a functional dissection of C-terminal CENP-C region analyzing the role of single Mif2p homology domains through in vivo and in vitro assays. By immunofluorescence and Chromatin immunoprecipitation assay (ChIP) we were able to elucidate the ability of the Mif2p homology domain II to target centromere and contact alpha satellite DNA. We also investigate the interactions with other conserved inner kinetochore proteins by means of coimmunoprecipitation and bimolecular fluorescence complementation on cell nuclei. We found that the C-terminal region of CENP-C (Mif2p homology domain III) displays multiple activities ranging from the ability to form higher order structures like homo-dimers and homo-oligomers, to mediate interaction with CENP-A and histone H3. Overall, our findings support a model in which the Mif2p homology domains of CENP-C, by virtue of their ability to establish multiple contacts with DNA and centromere proteins, play a critical role in the structuring of kinethocore chromatin.
Highlights
Proper chromosome segregation during cell divisions depends on a specialized chromosomal site, the centromere
Our results show that the Mif2p homology domain III contained in the HA::760/943 CENP-C construct is the only one able to interact both with CENP-A and H3
We have extended our previous results by further dissecting the C-terminal region of CENP-C and defining specific functions for the highly conserved Mif2p homology domains
Summary
Proper chromosome segregation during cell divisions depends on a specialized chromosomal site, the centromere. Centromeres are constituted by large arrays of repeats known as satellite sequences (alpha satellite in humans) These DNA sequences direct the assembly of kinetochore proteins and are strikingly divergent between even closely related species. Numerous kinetochore proteins have been identified in both human and model organisms and found to be very conserved during evolution [2,3,4,5] Understanding how these highly conserved proteins assemble onto divergent satellite DNA to form functional centromeres remains one of the key problems in chromosome biology. Mammalian centromeres contain mega bases of repetitive satellite DNA This is organized into specialized chromatin consisting of nucleosomes in which histone H3 is replaced by CENP-A, an H3-like variant. Since CENP-A depletion reduces fidelity of chromosome segregation and causes mislocalization of various kinetochore proteins [7,8], it is believed that this protein may hierarchically recruit other centromere and kinetochore components to generate a high-order chromatin structure required for the formation of the inner kinetochore surface (for recent reviews see: [9,10])
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