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.     

Mutational analysis of the central centromere targeting domain of human centromere protein C, (CENP-C)

Human centromere protein C (CENP-C) is an essential component of the inner kinetochore plate. A central region of CENP-C can bind DNA in vitro and is sufficient for targeting the protein to centromeres in vivo, raising the possibility that this domain mediates centromere localization via direct DNA binding. We performed a detailed molecular dissection of this domain to understand the mechanism by which CENP-C assembles at centromeres. By a combination of PCR mutagenesis and transient expression of GFP-tagged proteins in HeLa cells, we identified mutations that disrupt centromere localization of CENP-C in vivo. These cluster in a 12 amino acid region adjacent to the core domain required for in vitro DNA binding. This region is conserved between human and mouse, but is divergent or absent in invertebrate and plant CENP-C homologues. We suggest that these 12 amino acids are essential to confer specificity to DNA binding by CENP-C in vivo, or to mediate interaction with another as yet unidentified centromere component. A differential yeast two-hybrid screen failed to identify interactions specific to this sequence, but nonetheless identified 14 candidate proteins that interact with the central region of CENP-C. This collection of mutations and interacting proteins comprise a useful resource for further elucidating centromere assembly.     

Dissection of CENP-C–directed Centromere and Kinetochore Assembly

Eukaryotic cells ensure accurate chromosome segregation in mitosis by assembling a microtubule-binding site on each chromosome called the kinetochore that attaches to the mitotic spindle. The kinetochore is assembled specifically during mitosis on a specialized region of each chromosome called the centromere, which is constitutively bound by >15 centromere-specific proteins. These proteins, including centromere proteins A and C (CENP-A and -C), are essential for kinetochore assembly and proper chromosome segregation. How the centromere is assembled and how the centromere promotes mitotic kinetochore formation are poorly understood. We have used Xenopus egg extracts as an in vitro system to study the role of CENP-C in centromere and kinetochore assembly. We show that, unlike the histone variant CENP-A, CENP-C is not maintained at centromeres through spermatogenesis but is assembled at the sperm centromere from the egg cytoplasm. Immunodepletion of CENP-C from metaphase egg extract prevents kinetochore formation on sperm chromatin, and depleted extracts can be complemented with in vitro–translated CENP-C. Using this complementation assay, we have identified CENP-C mutants that localized to centromeres but failed to support kinetochore assembly. We find that the amino terminus of CENP-C promotes kinetochore assembly by ensuring proper targeting of the Mis12/MIND complex and CENP-K.     

CENP-B interacts with CENP-C domains containing Mif2 regions responsible for centromere localization

Recently, human artificial chromosomes featuring functional centromeres have been generated efficiently from naked synthetic alphoid DNA containing CENP-B boxes as a de novo mechanism in a human cultured cell line, but not from the synthetic alphoid DNA only containing mutations within CENP-B boxes, indicating that CENP-B has some functions in assembling centromere/kinetochore components on alphoid DNA. To investigate whether any interactions exist between CENP-B and the other centromere proteins, we screened a cDNA library by yeast two-hybrid analysis. An interaction between CENP-B and CENP-C was detected, and the CENP-C domains required were determined to overlap with three Mif2 homologous regions, which were also revealed to be involved in the CENP-C assembly of centromeres by expression of truncated polypeptides in cultured cells. Overproduction of truncated CENP-B containing no CENP-C interaction domains caused abnormal duplication of CENP-C domains at G2 and cell cycle delay at metaphase. These results suggest that the interaction between CENP-B and CENP-C may be involved in the correct assembly of CENP-C on alphoid DNA. In other words, a possible molecular linkage may exist between one of the kinetochore components and human centromere DNA through CENP-B/CENP-B box interaction.     

Overlapping kinetochore targets of CK2 and Aurora B kinases in mitotic regulation

Protein kinase CK2 is one of the most conserved kinases in eukaryotic cells and plays essential roles in diverse processes. While we know that CK2 plays a role(s) in cell division, our understanding of how CK2 regulates cell cycle progression is limited. In this study, we revealed a regulatory role for CK2 in kinetochore function. The kinetochore is a multi-protein complex that assembles on the centromere of a chromosome and functions to attach chromosomes to spindle microtubules. To faithfully segregate chromosomes and maintain genomic integrity, the kinetochore is tightly regulated by multiple mechanisms, including phosphorylation by Aurora B kinase. We found that a loss of CK2 kinase activity inhibits anaphase spindle elongation and results in chromosome missegregation. Moreover, a lack of CK2 activates the spindle assembly checkpoint. We demonstrate that CK2 associates with Mif2, the Saccharomyces cerevisiae homologue of human CENP-C, which serves as an important link between the inner and outer kinetochore. Furthermore, we show Mif2 and the inner kinetochore protein Ndc10 are phosphorylated by CK2, and this phosphorylation plays antagonistic and synergistic roles with Aurora B phosphorylation of these targets, respectively.     

CENP-C recruits M18BP1 to centromeres to promote CENP-A chromatin assembly

Eukaryotic chromosomes segregate by attaching to microtubules of the mitotic spindle through a chromosomal microtubule binding site called the kinetochore. Kinetochores assemble on a specialized chromosomal locus termed the centromere, which is characterized by the replacement of histone H3 in centromeric nucleosomes with the essential histone H3 variant CENP-A (centromere protein A). Understanding how CENP-A chromatin is assembled and maintained is central to understanding chromosome segregation mechanisms. CENP-A nucleosome assembly requires the Mis18 complex and the CENP-A chaperone HJURP. These factors localize to centromeres in telophase/G1, when new CENP-A chromatin is assembled. The mechanisms that control their targeting are unknown. In this paper, we identify a mechanism for recruiting the Mis18 complex protein M18BP1 to centromeres. We show that depletion of CENP-C prevents M18BP1 targeting to metaphase centromeres and inhibits CENP-A chromatin assembly. We find that M18BP1 directly binds CENP-C through conserved domains in the CENP-C protein. Thus, CENP-C provides a link between existing CENP-A chromatin and the proteins required for new CENP-A nucleosome assembly.     

The Elusive Structure of Centro-Chromatin: Molecular Order or Dynamic Heterogenetity?

The centromere is an essential chromatin domain required for kinetochore recruitment and chromosome segregation in eukaryotes. To perform this role, centro-chromatin adopts a unique structure that provides access to kinetochore proteins and maintains stability under tension during mitosis. This is achieved by the presence of nucleosomes containing the H3 variant CENP-A, which also acts as the epigenetic mark defining the centromere. In this review, we discuss the role of CENP-A on the structure and dynamics of centromeric chromatin. We further discuss the impact of the CENP-A binding proteins CENP-C, CENP-N, and CENP-B on modulating centro-chromatin structure. Based on these findings we provide an overview of the higher order structure of the centromere.     

A cooperative mechanism drives budding yeast kinetochore assembly downstream of CENP-A

Kinetochores are megadalton-sized protein complexes that mediate chromosome–microtubule interactions in eukaryotes. How kinetochore assembly is triggered specifically on centromeric chromatin is poorly understood. Here we use biochemical reconstitution experiments alongside genetic and structural analysis to delineate the contributions of centromere-associated proteins to kinetochore assembly in yeast. We show that the conserved kinetochore subunits Ame1^CENP-U and Okp1^CENP-Q form a DNA-binding complex that associates with the microtubule-binding KMN network via a short Mtw1 recruitment motif in the N terminus of Ame1. Point mutations in the Ame1 motif disrupt kinetochore function by preventing KMN assembly on chromatin. Ame1–Okp1 directly associates with the centromere protein C (CENP-C) homologue Mif2 to form a cooperative binding platform for outer kinetochore assembly. Our results indicate that the key assembly steps, CENP-A recognition and outer kinetochore recruitment, are executed through different yeast constitutive centromere-associated network subunits. This two-step mechanism may protect against inappropriate kinetochore assembly similar to rate-limiting nucleation steps used by cytoskeletal polymers.     

Dual recognition of CENP-A nucleosomes is required for centromere assembly

Centromeres contain specialized nucleosomes in which histone H3 is replaced by the histone variant centromere protein A (CENP-A). CENP-A nucleosomes are thought to act as an epigenetic mark that specifies centromere identity. We previously identified CENP-N as a CENP-A nucleosome-specific binding protein. Here, we show that CENP-C also binds directly and specifically to CENP-A nucleosomes. Nucleosome binding by CENP-C required the extreme C terminus of CENP-A and did not compete with CENP-N binding, which suggests that CENP-C and CENP-N recognize distinct structural elements of CENP-A nucleosomes. A mutation that disrupted CENP-C binding to CENP-A nucleosomes in vitro caused defects in CENP-C targeting to centromeres. Moreover, depletion of CENP-C with siRNA resulted in the mislocalization of all other nonhistone CENPs examined, including CENP-K, CENP-H, CENP-I, and CENP-T, and led to a partial reduction in centromeric CENP-A. We propose that CENP-C binds directly to CENP-A chromatin and, together with CENP-N, provides the foundation upon which other centromere and kinetochore proteins are assembled.     

Building the centromere: from foundation proteins to 3D organization

At each mitosis, accurate segregation of every chromosome is ensured by the assembly of a kinetochore at each centromeric locus. Six foundation kinetochore proteins that assemble hierarchically and co-dependently have been identified in vertebrates. CENP-A, Mis12, CENP-C, CENP-H and CENP-I localize to a core domain of centromeric chromatin. The sixth protein, CENP-B, although not essential in higher eukaryotes, has homologues in fission yeast that bind pericentric DNA and are essential for heterochromatin formation. Foundation kinetochore proteins have various roles and mutual interactions, and their associations with centromeric DNA and heterochromatin create structural domains that support the different functions of the centromere. Advances in molecular and microscopic techniques, coupled with rare centromere variants, have enabled us to gain fresh insights into the linear and 3D organization of centromeric chromatin.     

<Emphasis Type="Italic">Drosophila</Emphasis> CENP-C is essential for centromere identity

Centromeres are specialized chromosomal domains that direct mitotic kinetochore assembly and are defined by the presence of CENP-A (CID in Drosophila) and CENP-C. While the role of CENP-A appears to be highly conserved, functional studies in different organisms suggest that the precise role of CENP-C in kinetochore assembly is still under debate. Previous studies in vertebrate cells have shown that CENP-C inactivation causes mitotic delay, chromosome missegregation, and apoptosis; however, in Drosophila, the role of CENP-C is not well-defined. We have used RNA interference depletion in S2 cells to address this question and we find that depletion of CENP-C causes a kinetochore null phenotype, and consequently, the spindle checkpoint, kinetochore–microtubule interactions, and spindle size are severely misregulated. Importantly, we show that CENP-C is required for centromere identity as CID, MEI-S332, and chromosomal passenger proteins fail to localize in CENP-C depleted cells, suggesting a tight communication between the inner kinetochore proteins and centromeres. We suggest that CENP-C might fulfill the structural roles of the human centromere-associated proteins not identified in Drosophila.     

Induced ectopic kinetochore assembly bypasses the requirement for CENP-A nucleosomes

Accurate chromosome segregation requires assembly of the multiprotein kinetochore complex at centromeres. Although prior work identified the centromeric histone H3-variant CENP-A as the important upstream factor necessary for centromere specification, in human cells CENP-A is not sufficient for kinetochore assembly. Here, we demonstrate that two constitutive DNA-binding kinetochore components, CENP-C and CENP-T, function to direct kinetochore formation. Replacing the DNA-binding regions of CENP-C and CENP-T with alternate chromosome-targeting domains recruits these proteins to ectopic loci, resulting in CENP-A-independent kinetochore assembly. These ectopic kinetochore-like foci are functional based on the stoichiometric assembly of multiple kinetochore components, including the microtubule-binding KMN network, the presence of microtubule attachments, the microtubule-sensitive recruitment of the spindle checkpoint protein Mad2, and the segregation behavior of foci-containing chromosomes. We additionally find that CENP-T phosphorylation regulates the mitotic assembly of both endogenous and ectopic kinetochores. Thus, CENP-C and CENP-T form a critical regulated platform for vertebrate kinetochore assembly.     

Architecture of the budding yeast kinetochore reveals a conserved molecular core

How kinetochore proteins are organized to connect chromosomes to spindle microtubules, and whether any structural and organizational themes are common to kinetochores from distantly related organisms, are key unanswered questions. Here, we used affinity chromatography and mass spectrometry to generate a map of kinetochore protein interactions. The budding yeast CENP-C homologue Mif2p specifically copurified with histones H2A, H2B, and H4, and with the histone H3-like CENP-A homologue Cse4p, strongly suggesting that Cse4p replaces histone H3 in a specialized centromeric nucleosome. A novel four-protein Mtw1 complex, the Nnf1p subunit of which has homology to the vertebrate kinetochore protein CENP-H, also copurified with Mif2p and a variety of central kinetochore proteins. We show that Mif2 is a critical in vivo target of the Aurora kinase Ipl1p. Chromatin immunoprecipitation studies demonstrated the biological relevance of these associations. We propose that a molecular core consisting of CENP-A, -C, -H, and Ndc80/HEC has been conserved from yeast to humans to link centromeres to spindle microtubules.     

Human centromere chromatin protein hMis12, essential for equal segregation,is independent of CENP-A loading pathway

Kinetochores are the chromosomal sites for spindle interaction and play a vital role for chromosome segregation. The composition of kinetochore proteins and their cellular roles are, however, poorly understood in higher eukaryotes. We identified a novel kinetochore protein family conserved from yeast to human that is essential for equal chromosome segregation. The human homologue hMis12 of yeast spMis12/scMtw1 retains conserved sequence features and locates at the kinetochore region indistinguishable from CENP-A, a centromeric histone variant. RNA interference (RNAi) analysis of HeLa cells shows that the reduced hMis12 results in misaligned metaphase chromosomes, lagging anaphase chromosomes, and interphase micronuclei without mitotic delay, while CENP-A is located at kinetochores. Further, the metaphase spindle length is abnormally extended. Spindle checkpoint protein hMad2 temporally localizes at kinetochores at early mitotic stages after RNAi. The RNAi deficiency of CENP-A leads to a similar mitotic phenotype, but the kinetochore signals of other kinetochore proteins, hMis6 and CENP-C, are greatly diminished. RNAi for hMis6, like that of a kinetochore kinesin CENP-E, induces mitotic arrest. Kinetochore localization of hMis12 is unaffected by CENP-A RNAi, demonstrating an independent pathway of CENP-A in human kinetochores.     

Repurposing of synaptonemal complex proteins for kinetochores in Kinetoplastida

Chromosome segregation in eukaryotes is driven by the kinetochore, a macromolecular complex that connects centromeric DNA to microtubules of the spindle apparatus. Kinetochores in well-studied model eukaryotes consist of a core set of proteins that are broadly conserved among distant eukaryotic phyla. By contrast, unicellular flagellates of the class Kinetoplastida have a unique set of 36 kinetochore components. The evolutionary origin and history of these kinetochores remain unknown. Here, we report evidence of homology between axial element components of the synaptonemal complex and three kinetoplastid kinetochore proteins KKT16-18. The synaptonemal complex is a zipper-like structure that assembles between homologous chromosomes during meiosis to promote recombination. By using sensitive homology detection protocols, we identify divergent orthologues of KKT16-18 in most eukaryotic supergroups, including experimentally established chromosomal axis components, such as Red1 and Rec10 in budding and fission yeast, ASY3-4 in plants and SYCP2-3 in vertebrates. Furthermore, we found 12 recurrent duplications within this ancient eukaryotic SYCP^2–3 gene family, providing opportunities for new functional complexes to arise, including KKT16-18 in the kinetoplastid parasite Trypanosoma brucei. We propose the kinetoplastid kinetochore system evolved by repurposing meiotic components of the chromosome synapsis and homologous recombination machinery that were already present in early eukaryotes.     

HCP-4/CENP-C promotes the prophase timing of centromere resolution by enabling the centromere association of HCP-6 in Caenorhabditis elegans

Prior to microtubule capture, sister centromeres resolve from one another, coming to rest on opposite surfaces of the condensing chromosome. Subsequent assembly of sister kinetochores at each sister centromere generates a geometry favorable for equal levels of segregation of chromatids. The holocentric chromosomes of Caenorhabditis elegans are uniquely suited for the study of centromere resolution and subsequent kinetochore assembly. In C. elegans, only two proteins have been identified as being necessary for centromere resolution, the kinase AIR-2 (prophase only) and the centromere protein HCP-4/CENP-C. Here we found that the loss of proteins involved in chromosome cohesion bypassed the requirement for HCP-4/CENP-C but not for AIR-2. Interestingly, the loss of cohesin proteins also restored the localization of HCP-6 to the kinetochore. The loss of the condensin II protein HCP-6 or MIX-1/SMC2 impaired centromere resolution. Furthermore, the loss of HCP-6 or MIX-1/SMC2 resulted in no centromere resolution when either nocodazole or RNA interference (RNAi) of the kinetochore protein KNL-1 perturbed spindle-kinetochore interactions. This result suggests that normal prophase centromere resolution is mediated by condensin II proteins, which are actively recruited to sister centromeres to mediate the process of resolution.     

In vivo functional dissection of human inner kinetochore protein CENP-C

CENP-C is a fundamental component of the inner kinetochore plate and contributes to the formation of functional centromeres in eukaryotic organisms. Recruitment of CENP-C to kinetochore requires other centromere proteins, particularly CENP-A, CENP-H, and CENP-I. However, how CENP-C is correctly localized at the kinetochore is not clearly determined, mainly due to the functional variety of its domains, which hints at a complex recruitment mechanism. Here, by both immunofluorescent labeling and chromatin/immunoprecipitation we could show that human CENP-C contains two distinct domains, one in the central region, between amino acids 426 and 537, and the second one in the carboxyl terminal region, between amino acids 638 and 943, which are both capable of localizing at centromeres and binding alpha-satellite DNA. The presence of two domains that iterate the same function despite being significantly different in their amino acid sequence and structure suggests that CENP-C may target the centromere by establishing multiple contacts with both the DNA and protein constituents of the kinetochore.     

Identification of overlapping DNA-binding and centromere-targeting domains in the human kinetochore protein CENP-C.

The kinetochore in eukaryotes serves as the chromosomal site of attachment for microtubules of the mitotic spindle and directs the movements necessary for proper chromosome segregation. In mammalian cells, the kinetochore is a highly differentiated trilaminar structure situated at the surface of the centromeric heterochromatin. CENP-C is a basic, DNA-binding protein that localizes to the inner kinetochore plate, the region that abuts the heterochromatin. Microinjection experiments using antibodies specific for CENP-C have demonstrated that this protein is required for the assembly and/or stability of the kinetochore as well as for a timely transition through mitosis. From these observations, it has been suggested that CENP-C is a structural protein that is involved in the organization or the kinetochore. In this report, we wished to identify and map the functional domains of CENP-C. Analysis of CENP-C truncation mutants expressed in vivo demonstrated that CENP-C possesses an autonomous centromere-targeting domain situated at the central region of the CENP-C polypeptide. Similarly, in vitro assays revealed that a region of CENP-C with the ability to bind DNA is also located at the center of the CENP-C molecule, where it overlaps the centromere-targeting domain.     

Centromere Epigenetics in Plants

The centromere is an essential chromosome site at which the kinetochore forms and loads proteins needed for faithful segregation during the cell cycle and meiosis(Houben et al., 1999;Cleveland et al.,2003;Ma et al.,2007;Birchler and Han,2009).Centromere specific sequences such as tandem repeats or transposable elements evolve quickly both within and between the species but have conserved kinetochore proteins(Henikoff and Furuyama,2010).The universal feature