Biotin-Tag Affinity Purification of a Centromeric Nucleosome Assembly Complex

Centromeres are chromosomal sites of microtubule binding that ensure correct mitotic segregation of chromosomes to daughter cells. This process is mediated by a special centromere-specific histone H3 variant (CenH3), which packages centromeric chromatin and epigenetically maintains the centromere at a distinct chromosomal location. However, CenH3 is present at low abundance relative to canonical histones, presenting a challenge for the isolation and characterization of the chaperone machinery that assembles CenH3 into nucleosomes at centromeres. To address this challenge, we used controlled overexpression of Drosophila CenH3 (CID) and an efficient biochemical purification strategy offered by in vivo biotinylation of CID to successfully purify and characterize the soluble CID nucleosome assembly complex. It consists of a singlechaperone protein, RbAp48, complexed with CID and histone H4. RbAp48 is also found in protein complexes that assemble canonical histone H3 and replacement histone H3.3. Here, we highlight the benefits of our improved biotin-mediated purification method, and address the question of how the simple CID/H4-RbAp48 chaperone complex can mediate nucleosome assembly specifically at centromeres.     

Conserved organization of centromeric chromatin in flies and humans

Recent studies have highlighted the importance of centromere-specific histone H3-like (CENP-A) proteins in centromere function. We show that Drosophila CID and human CENP-A appear at metaphase as a three-dimensional structure that lacks histone H3. However, blocks of CID/CENP-A and H3 nucleosomes are linearly interspersed on extended chromatin fibers, and CID is close to H3 nucleosomes in polynucleosomal preparations. When CID is depleted by RNAi, it is replaced by H3, demonstrating flexibility of centromeric chromatin organization. Finally, contrary to models proposing that H3 and CID/CENP-A nucleosomes are replicated at different times in S phase, we show that interspersed H3 and CID/CENP-A chromatin are replicated concurrently during S phase in humans and flies. We propose that the unique structural arrangement of CID/CENP-A and H3 nucleosomes presents centromeric chromatin to the poleward face of the condensing mitotic chromosome.     

Dimerization of the CENP‐A assembly factor HJURP is required for centromeric nucleosome deposition

The epigenetic mark of the centromere is thought to be a unique centromeric nucleosome that contains the histone H3 variant, centromere protein‐A (CENP‐A). The deposition of new centromeric nucleosomes requires the CENP‐A‐specific chromatin assembly factor HJURP (Holliday junction recognition protein). Crystallographic and biochemical data demonstrate that the Scm3‐like domain of HJURP binds a single CENP‐A–histone H4 heterodimer. However, several lines of evidence suggest that HJURP forms an octameric CENP‐A nucleosome. How an octameric CENP‐A nucleosome forms from individual CENP‐A/histone H4 heterodimers is unknown. Here, we show that HJURP forms a homodimer through its C‐terminal domain that includes the second HJURP_C domain. HJURP exists as a dimer in the soluble preassembly complex and at chromatin when new CENP‐A is deposited. Dimerization of HJURP is essential for the deposition of new CENP‐A nucleosomes. The recruitment of HJURP to centromeres occurs independent of dimerization and CENP‐A binding. These data provide a mechanism whereby the CENP‐A pre‐nucleosomal complex achieves assembly of the octameric CENP‐A nucleosome through the dimerization of the CENP‐A chaperone HJURP.     

Assembly of Drosophila centromeric chromatin proteins during mitosis

Semi-conservative segregation of nucleosomes to sister chromatids during DNA replication creates gaps that must be filled by new nucleosome assembly. We analyzed the cell-cycle timing of centromeric chromatin assembly in Drosophila, which contains the H3 variant CID (CENP-A in humans), as well as CENP-C and CAL1, which are required for CID localization. Pulse-chase experiments show that CID and CENP-C levels decrease by 50% at each cell division, as predicted for semi-conservative segregation and inheritance, whereas CAL1 displays higher turnover. Quench-chase-pulse experiments demonstrate that there is a significant lag between replication and replenishment of centromeric chromatin. Surprisingly, new CID is recruited to centromeres in metaphase, by a mechanism that does not require an intact mitotic spindle, but does require proteasome activity. Interestingly, new CAL1 is recruited to centromeres before CID in prophase. Furthermore, CAL1, but not CENP-C, is found in complex with pre-nucleosomal CID. Finally, CENP-C displays yet a different pattern of incorporation, during both interphase and mitosis. The unusual timing of CID recruitment and unique dynamics of CAL1 identify a distinct centromere assembly pathway in Drosophila and suggest that CAL1 is a key regulator of centromere propagation.     

Proteolysis restricts localization of CID, the centromere-specific histone H3 variant of Drosophila, to centromeres

Centromere identity is determined by the formation of a specialized chromatin structure containing the centromere-specific histone H3 variant CENP-A. The precise molecular mechanism(s) accounting for the specific deposition of CENP-A at centromeres are still poorly understood. Centromeric deposition of CENP-A, which is independent of DNA replication, might involve specific chromatin assembly complexes and/or specific interactions with kinetochore components. However, transiently expressed CENP-A incorporates throughout chromatin indicating that CENP-A nucleosomes can also be promiscuously deposited during DNA replication. Therefore, additional mechanisms must exist to prevent deposition of CENP-A nucleosomes during replication and/or to remove them afterwards. Here, using transient expression experiments performed in Drosophila Kc cells, we show that proteasome-mediated degradation restricts localization of Drosophila CENP-A (CID) to centromeres by eliminating mislocalized CID as well as by regulating available CID levels. Regulating available CID levels appears essential to ensure centromeric deposition of transiently expressed CID as, when expression is increased in the presence of proteasome inhibitors, newly synthesized CID mislocalizes. Mislocalization of CID affects cell cycle progression as a high percentage of cells showing mislocalized CID are reactive against αPSer¹⁰H3 antibodies, enter mitosis at a very low frequency and show strong segregation defects. However, cells showing reduced amounts of mislocalized CID show normal cell cycle progression.     

Centromere identity in Drosophila is not determined in vivo by replication timing

Centromeric chromatin is uniquely marked by the centromere-specific histone CENP-A. For assembly of CENP-A into nucleosomes to occur without competition from H3 deposition, it was proposed that centromeres are among the first or last sequences to be replicated. In this study, centromere replication in Drosophila was studied in cell lines and in larval tissues that contain minichromosomes that have structurally defined centromeres. Two different nucleotide incorporation methods were used to evaluate replication timing of chromatin containing CID, a Drosophila homologue of CENP-A. Centromeres in Drosophila cell lines were replicated throughout S phase but primarily in mid S phase. However, endogenous centromeres and X-derived minichromosome centromeres in vivo were replicated asynchronously in mid to late S phase. Minichromosomes with structurally intact centromeres were replicated in late S phase, and those in which centric and surrounding heterochromatin were partially or fully deleted were replicated earlier in mid S phase. We provide the first in vivo evidence that centromeric chromatin is replicated at different times in S phase. These studies indicate that incorporation of CID/CENP-A into newly duplicated centromeres is independent of replication timing and argue against determination of centromere identity by temporal sequestration of centromeric chromatin replication relative to bulk genomic chromatin.     

着丝粒核小体结构研究进展

着丝粒是构成真核生物染色体的必需元件。在细胞有丝分裂或减数分裂时,微管通过动粒与染色体着丝粒连接,参与细胞分裂的染色体分离与分配过程,使染色体平均分配到子细胞中。构成着丝粒的基本单位是着丝粒特异的核小体,与常规核小体不同的是着丝粒核小体中的组蛋白H3被其变种——着丝粒组蛋白H3所替换。最近几年,着丝粒核小体的结构成为细胞生物学研究的热点之一。该文综述了最近在多种真核生物研究中,通过体外和体内实验,提出的着丝粒核小体结构的八聚体、六聚体、同型四聚体以及半八聚体模型,并对着丝粒核小体结构的动态模型与功能的关系进行了探讨。     

Scm3 is a centromeric nucleosome assembly factor

The Cse4 nucleosome at each budding yeast centromere must be faithfully assembled each cell cycle to specify the site of kinetochore assembly and microtubule attachment for chromosome segregation. Although Scm3 is required for the localization of the centromeric H3 histone variant Cse4 to centromeres, its role in nucleosome assembly has not been tested. We demonstrate that Scm3 is able to mediate the assembly of Cse4 nucleosomes in vitro, but not H3 nucleosomes, as measured by a supercoiling assay. Localization of Cse4 to centromeres and the assembly activity depend on an evolutionarily conserved core motif in Scm3, but localization of the CBF3 subunit Ndc10 to centromeres does not depend on this motif. The centromere targeting domain of Cse4 is sufficient for Scm3 nucleosome assembly activity. Assembly does not depend on centromeric sequence. We propose that Scm3 plays an active role in centromeric nucleosome assembly.     

Plasticity and Epigenetic Inheritance of Centromere-specific Histone H3 (CENP-A)-containing Nucleosome Positioning in the Fission Yeast

Nucleosomes containing the specific histone H3 variant CENP-A mark the centromere locus on each chromatin and initiate kinetochore assembly. For the common type of regional centromeres, little is known in molecular detail of centromeric chromatin organization, its propagation through cell division, and how distinct organization patterns may facilitate kinetochore assembly. Here, we show that in the fission yeast S. pombe, a relatively small number of CENP-A/Cnp1 nucleosomes are found within the centromeric core and that their positioning relative to underlying DNA varies among genetically homogenous cells. Consistent with the flexible positioning of Cnp1 nucleosomes, a large portion of the endogenous centromere is dispensable for its essential activity in mediating chromosome segregation. We present biochemical evidence that Cnp1 occupancy directly correlates with silencing of the underlying reporter genes. Furthermore, using a newly developed pedigree analysis assay, we demonstrated the epigenetic inheritance of Cnp1 positioning and quantified the rate of occasional repositioning of Cnp1 nucleosomes throughout cell generations. Together, our results reveal the plasticity and the epigenetically inheritable nature of centromeric chromatin organization.     

How two become one: HJURP dimerization drives CENP‐A assembly

EMBO J 32 15, 2113–2124 doi:10.1038/emboj.2013.142; published online June142013Curr Biol 23 9, 764–769 doi:10.1016/j.cub.2013.03.037; published online May062013Curr Biol 23 9, 770–774 doi:10.1016/j.cub.2013.03.042; published online May062013CENP-A containing nucleosomes epigenetically specify centromere position on chromosomes. Deposition of CENP-A into chromatin is mediated by HJURP, a specific CENP-A chaperone. Paradoxically, HJURP binding sterically prevents dimerization of CENP-A, which is critical to form functional centromeric nucleosomes. A recent publication in The EMBO Journal (Zasadzińska et al, 2013) demonstrates that HJURP itself dimerizes through a C-terminal repeat region, which is essential for centromeric assembly of nascent CENP-A.CENP-A containing nucleosomes have a well-established role in the epigenetic specification of centromere position. However, the composition of the CENP-A nucleosome has been the subject of intense investigation and debate (as has been extensively reviewed, e.g., in Black and Cleveland, 2011). X-ray crystallography data, biochemical interaction experiments and in vivo mutational analysis provide strong evidence that CENP-A nucleosomes are octameric (CENP-A/H4/H2A/H2B)₂, analogous to their histone H3-containing counterparts (Tachiwana et al, 2011; Bassett et al, 2012). Alternatively, based primarily on AFM data and nucleosome crosslinking assays, a tetrameric CENP-A/H4/H2A/H2B ‘hemisome'' has been proposed to be present at centromeres, at least during part of the cell cycle (Dalal et al, 2007; Bui et al, 2012). Whether both nucleosome types exist under specific conditions remains an unresolved question. However, recent studies by the Maddox and Black labs have reported single-molecule fluorescence measurements of CENP-A nucleosomes and high-resolution DNA protection assays of centromeric chromatin, respectively, both of which indicate that octamers are the predominant species of CENP-A in vivo (Hasson et al, 2013; Padeganeh et al, 2013).HJURP is the centromeric histone chaperone that is responsible for timely assembly of CENP-A nucleosomes. HJURP binds to soluble CENP-A and is recruited to centromeric chromatin in early G1 phase, concurrently with nascent CENP-A (Stellfox et al, 2013). Importantly, HJURP facilitates CENP-A nucleosome formation in vitro and its transient targeting to non-centromeric chromatin is sufficient to stably deposit CENP-A at these sites in vivo (Barnhart et al, 2011). Together, these observations identify HJURP as a bona fide centromeric CENP-A histone assembly factor.However, there is an apparent discrepancy between the role of HJURP in CENP-A assembly and the octameric nature of CENP-A nucleosomes. The crystal structure of the human prenucleosomal complex clearly shows that HJURP binds to CENP-A/H4 dimers in a manner that precludes CENP-A/H4 hetero-tetramerization (Hu et al, 2011). Interestingly, however, mutational analysis of CENP-A has shown that tetramerization is crucial for centromere assembly (Bassett et al, 2012). Thus, a mechanism must exist to allow for two trimeric HJURP/CENP-A/H4 complexes to coordinately assemble a tetrameric (CENP-A/H4)₂ particle.In this issue, a study by the Foltz lab sheds light on these paradoxical observations (Zasadzińska et al, 2013). Human HJURP contains two C-terminal repeat regions (HJURP C-terminal domains; HCTD). Expression of short fragments of HJURP containing either of these was sufficient to allow for centromere targeting. However, depletion of endogenous HJURP abolished centromere targeting of the C-terminally located HCTD2 fragment, without affecting the localization of the fragment containing HCTD1. These observations suggest that HCTD1 is required for centromere targeting, while HCTD2 allows for HJURP dimerization. Indeed, the authors go on to show that the latter fragment is both necessary and sufficient to form functional dimers of HJURP. RNAi replacement experiments show that HJURP lacking the HCTD2 dimerization domain is incapable of loading nascent CENP-A into centromeres. Importantly, Zasadzińska et al (2013) demonstrate that the defect in CENP-A loading can be directly attributed to a lack of HJURP dimerization. In an elegant experiment where the HCTD2 containing domain is replaced by an unrelated dimerization domain (that of bacterial LacI), CENP-A assembly is rescued to wild-type levels (Figure 1). This indicates that dimerization of HJURP is an essential step in centromeric chromatin assembly and provides a potential mechanism for the assembly of tetrameric (CENP-A/H4)₂ structures into chromatin as precursors to octameric nucleosomes.Open in a separate windowFigure 1Human HJURP contains separate protein domains that are responsible for CENP-A/H4 binding (blue), centromere targeting (brown) and dimerization (red). Full-length HJURP containing all these domains is capable of assembling CENP-A nucleosomes at centromeres (left). Zasadzińska et al (2013) now show that HJURP lacking the dimerization domain is still able to localize to centromeres, but is unable to assemble CENP-A nucleosomes (middle). However, replacement of the HJURP dimerization domain by an exogenous dimerization domain fully rescues the capability to form CENP-A nucleosomes at centromeres (right). These findings show that HJURP dimerization is an essential feature in the process of nucleosome formation, and explain how (CENP-A/H4)₂ tetramers can be formed by a chaperone that exclusively binds to CENP-A/H4 dimers.While the composition of the HJURP complex suggests a likely mechanism for the formation of octameric nucleosomes, this poses a new challenge to the field. Future studies will be needed to dissect how the shielded HJURP-bound state of CENP-A/H4 can transition to a tetramer on DNA. Interestingly, HJURP is not the only histone chaperone that exclusively binds to histone dimers. Crystal structures of trimeric complexes of both Asf1a/H3.1/H4 (English et al, 2006) as well as DAXX/H3.3/H4 (Elsässer et al, 2012) clearly show sterical incompatibility between chaperone binding and histone tetramerization. It follows that efficient chromatin assembly requires a mode for two histone chaperones to deposit histone dimers in a coordinated fashion, e.g., through dimerization as has been shown for Nap1 (McBryant and Peersen, 2004) and now for HJURP. However, dimerization does not appear to be a universal feature for histone chaperones, as a single CAF1 chaperone is able to bind two H3/H4 dimers as well as (H3/H4)₂ tetramers (Winkler et al, 2012). Thus, while deposition of H3.1/H4 at the replication fork may be driven by the high density of pre-assembly complexes, assembly of nucleosomes containing the replacement variant H3.3, H3.1 nucleosomes at DNA damage sites, and CENP-A at the centromere would require a more active form of coordination. Histone chaperone dimerization may therefore be a common feature in the pipeline to chromatin formation.In summary, Zasadzińska et al (2013) propose a solution to a paradox in the assembly pathway of CENP-A. They show that while each HJURP molecule can exclusively bind a single CENP-A/H4 dimer, HJURP itself dimerizes, ultimately allowing for the formation of tetrameric (CENP-A/H4)₂ structures in chromatin. Interestingly, exclusive dimer binding has been observed for a number of histone chaperones, suggesting that chaperone dimerization may be a more general process in the nucleosome assembly pathway.     

Assembly in G1 phase and long-term stability are unique intrinsic features of CENP-A nucleosomes

Centromeres are the site of kinetochore formation during mitosis. Centromere protein A (CENP-A), the centromere-specific histone H3 variant, is essential for the epigenetic maintenance of centromere position. Previously we showed that newly synthesized CENP-A is targeted to centromeres exclusively during early G1 phase and is subsequently maintained across mitotic divisions. Using SNAP-based fluorescent pulse labeling, we now demonstrate that cell cycle–restricted chromatin assembly at centromeres is unique to CENP-A nucleosomes and does not involve assembly of other H3 variants. Strikingly, stable retention is restricted to the CENP-A/H4 core of the nucleosome, which we find to outlast general chromatin across several cell divisions. We further show that cell cycle timing of CENP-A assembly is independent of centromeric DNA sequences and instead is mediated by the CENP-A targeting domain. Unexpectedly, this domain also induces stable transmission of centromeric nucleosomes, independent of the CENP-A deposition factor HJURP. This demonstrates that intrinsic properties of the CENP-A protein direct its cell cycle–restricted assembly and induces quantitative mitotic transmission of the CENP-A/H4 nucleosome core, ensuring long-term stability and epigenetic maintenance of centromere position.     

Centromeres are specialized replication domains in heterochromatin

The properties that define centromeres in complex eukaryotes are poorly understood because the underlying DNA is normally repetitive and indistinguishable from surrounding noncentromeric sequences. However, centromeric chromatin contains variant H3-like histones that may specify centromeric regions. Nucleosomes are normally assembled during DNA replication; therefore, we examined replication and chromatin assembly at centromeres in Drosophila cells. DNA in pericentric heterochromatin replicates late in S phase, and so centromeres are also thought to replicate late. In contrast to expectation, we show that centromeres replicate as isolated domains early in S phase. These domains do not appear to assemble conventional H3-containing nucleosomes, and deposition of the Cid centromeric H3-like variant proceeds by a replication-independent pathway. We suggest that late-replicating pericentric heterochromatin helps to maintain embedded centromeres by blocking conventional nucleosome assembly early in S phase, thereby allowing the deposition of centromeric histones.     

Tetrameric structure of centromeric nucleosomes in interphase Drosophila cells

Centromeres, the specialized chromatin structures that are responsible for equal segregation of chromosomes at mitosis, are epigenetically maintained by a centromere-specific histone H3 variant (CenH3). However, the mechanistic basis for centromere maintenance is unknown. We investigated biochemical properties of CenH3 nucleosomes from Drosophila melanogaster cells. Cross-linking of CenH3 nucleosomes identifies heterotypic tetramers containing one copy of CenH3, H2A, H2B, and H4 each. Interphase CenH3 particles display a stable association of approximately 120 DNA base pairs. Purified centromeric nucleosomal arrays have typical “beads-on-a-string” appearance by electron microscopy but appear to resist condensation under physiological conditions. Atomic force microscopy reveals that native CenH3-containing nucleosomes are only half as high as canonical octameric nucleosomes are, confirming that the tetrameric structure detected by cross-linking comprises the entire interphase nucleosome particle. This demonstration of stable half-nucleosomes in vivo provides a possible basis for the instability of centromeric nucleosomes that are deposited in euchromatic regions, which might help maintain centromere identity.     

Tetrameric Structure of Centromeric Nucleosomes in Interphase Drosophila Cells

Centromeres, the specialized chromatin structures that are responsible for equal segregation of chromosomes at mitosis, are epigenetically maintained by a centromere-specific histone H3 variant (CenH3). However, the mechanistic basis for centromere maintenance is unknown. We investigated biochemical properties of CenH3 nucleosomes from Drosophila melanogaster cells. Cross-linking of CenH3 nucleosomes identifies heterotypic tetramers containing one copy of CenH3, H2A, H2B, and H4 each. Interphase CenH3 particles display a stable association of approximately 120 DNA base pairs. Purified centromeric nucleosomal arrays have typical “beads-on-a-string” appearance by electron microscopy but appear to resist condensation under physiological conditions. Atomic force microscopy reveals that native CenH3-containing nucleosomes are only half as high as canonical octameric nucleosomes are, confirming that the tetrameric structure detected by cross-linking comprises the entire interphase nucleosome particle. This demonstration of stable half-nucleosomes in vivo provides a possible basis for the instability of centromeric nucleosomes that are deposited in euchromatic regions, which might help maintain centromere identity.     

A cell-free CENP-A assembly system defines the chromatin requirements for centromere maintenance

Centromeres are defined by the presence of CENP-A nucleosomes in chromatin and are essential for accurate chromosome segregation. Centromeric chromatin epigenetically seeds new CENP-A nucleosome formation, thereby maintaining functional centromeres as cells divide. The features within centromeric chromatin that direct new CENP-A assembly remain unclear. Here, we developed a cell-free CENP-A assembly system that enabled the study of chromatin-bound CENP-A and soluble CENP-A separately. We show that two distinct domains of CENP-A within existing CENP-A nucleosomes are required for new CENP-A assembly and that CENP-A nucleosomes recruit the CENP-A assembly factors CENP-C and M18BP1 independently. Furthermore, we demonstrate that the mechanism of CENP-C recruitment to centromeres is dependent on the density of underlying CENP-A nucleosomes.     

Centromere regulation: new players, new rules, new questions

Centromeres support the assembly of the kinetochore on every chromosome and are therefore essential for the proper segregation of sister chromatids during cell division. Centromere identity is regulated epigenetically through the presence of the histone H3 variant CENP-A. CENP-A regulation and incorporation specifically into centromeric nucleosomes are the matter of intensive studies in many different model organisms. Here we briefly review the current knowledge in centromere biology with a focus on Drosophila melanogaster and how these insights lead to new rules and challenges.     

A simple model explains the cell cycle-dependent assembly of centromeric nucleosomes in holocentric species

Centromeres are essential for chromosome movement. In independent taxa, species with holocentric chromosomes exist. In contrast to monocentric species, where no obvious dispersion of centromeres occurs during interphase, the organization of holocentromeres differs between condensed and decondensed chromosomes. During interphase, centromeres are dispersed into a large number of CENH3-positive nucleosome clusters in a number of holocentric species. With the onset of chromosome condensation, the centromeric nucleosomes join and form line-like holocentromeres. Using polymer simulations, we propose a mechanism relying on the interaction between centromeric nucleosomes and structural maintenance of chromosomes (SMC) proteins. Different sets of molecular dynamic simulations were evaluated by testing four parameters: (i) the concentration of Loop Extruders (LEs) corresponding to SMCs, (ii) the distribution and number of centromeric nucleosomes, (iii) the effect of centromeric nucleosomes on interacting LEs and (iv) the assembly of kinetochores bound to centromeric nucleosomes. We observed the formation of a line-like holocentromere, due to the aggregation of the centromeric nucleosomes when the chromosome was compacted into loops. A groove-like holocentromere structure formed after a kinetochore complex was simulated along the centromeric line. Similar mechanisms may also organize a monocentric chromosome constriction, and its regulation may cause different centromere types during evolution.     

Deposition,turnover, and release of CENH3 at <Emphasis Type="Italic">Arabidopsis</Emphasis> centromeres

The kinetochore is a complex multiprotein structure located at centromeres and required for the proper segregation of chromosomes during mitosis and meiosis. An important role in kinetochore assembly and function plays the centromeric histone H3 variant (CENH3). Cell cycle stage of CENH3 deposition to centromeres varies between different organisms. We confirmed by in vivo studies that deposition of Arabidopsis CENH3 takes place at centromeres during G2 and demonstrated that additionally a low turnover of CENH3 occurs along the cell cycle, apparently for replacement of damaged protein. Furthermore, enhanced yellow fluorescent protein (EYFP)-CENH3 of photobleached chromocenters is not replaced by EYFP-CENH3 molecules from unbleached centromeres of the same nucleus, indicating a stable incorporation of CENH3 into centromeric nucleosomes. In differentiated endopolyploid nuclei however, the amount of CENH3 at centromeres declines with age.     

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.