The C-terminal domain of CENP-C displays multiple and critical functions for mammalian centromere formation.
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.
Trazzi
Stefania
S
Department of Biology, University of Bologna, Bologna, Italy.
Perini
Giovanni
G
Bernardoni
Roberto
R
Zoli
Monica
M
Reese
Joseph C
JC
Musacchio
Andrea
A
Della Valle
Giuliano
G
D001324:Autoantigens / Q000737:chemistry; D002460:Cell Line; D002503:Centromere / Q000648:ultrastructure*; D000076247:Centromere Protein A; D002868:Chromosomal Proteins, Non-Histone / Q000737:chemistry*; D004247:DNA / Q000737:chemistry; D004268:DNA-Binding Proteins / Q000737:chemistry; D019281:Dimerization; D000939:Epitopes / Q000737:chemistry; D005456:Fluorescent Dyes / Q000494:pharmacology; D005976:Glutaral / Q000737:chemistry; D006657:Histones / Q000737:chemistry; D006801:Humans; D018386:Kinetochores / Q000737:chemistry; D017434:Protein Structure, Tertiary; D012441:Saccharomyces cerevisiae / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry
D001324:Autoantigens; C000619700:CENPA protein, human; D000076247:Centromere Protein A; D002868:Chromosomal Proteins, Non-Histone; D004268:DNA-Binding Proteins; D000939:Epitopes; D005456:Fluorescent Dyes; D006657:Histones; C083768:MIF2 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; C060905:centromere protein C; D004247:DNA; D005976:Glutaral