Mitotic Regulator Mis18β Interacts with and Specifies the Centromeric Assembly of Molecular Chaperone Holliday Junction Recognition Protein (HJURP)

The centromere is essential for precise and equal segregation of the parental genome into two daughter cells during mitosis. CENP-A is a unique histone H3 variant conserved in eukaryotic centromeres. The assembly of CENP-A to the centromere is mediated by Holliday junction recognition protein (HJURP) in early G₁ phase. However, it remains elusive how HJURP governs CENP-A incorporation into the centromere. Here we show that human HJURP directly binds to Mis18β, a component of the Mis18 complex conserved in the eukaryotic kingdom. A minimal region of HJURP for Mis18β binding was mapped to residues 437–460. Depletion of Mis18β by RNA interference dramatically impaired HJURP recruitment to the centromere, indicating the importance of Mis18β in HJURP loading. Interestingly, phosphorylation of HJURP by CDK1 weakens its interaction with Mis18β, consistent with the notion that assembly of CENP-A to the centromere is achieved after mitosis. Taken together, these data define a novel molecular mechanism underlying the temporal regulation of CENP-A incorporation into the centromere by accurate Mis18β-HJURP interaction.     

The N-terminal tail of C. elegans CENP-A interacts with KNL-2 and is essential for centromeric chromatin assembly

Centromeres are epigenetically defined by the centromere-specific histone H3 variant CENP-A. Specialized loading machinery, including the histone chaperone HJURP/Scm3, participates in CENP-A nucleosome assembly. However, Scm3/HJURP is missing from multiple lineages, including nematodes, with CENP-A-dependent centromeres. Here, we show that the extended N-terminal tail of Caenorhabditis elegans CENP-A contains a predicted structured region that is essential for centromeric chromatin assembly; removal of this region prevents CENP-A loading, resulting in failure of kinetochore assembly and defective chromosome condensation. By contrast, the N-tail mutant CENP-A localizes normally in the presence of endogenous CENP-A. The portion of the N-tail containing the predicted structured region binds to KNL-2, a conserved SANTA domain and Myb domain-containing protein (referred to as M18BP1 in vertebrates) specifically involved in CENP-A chromatin assembly. This direct interaction is conserved in the related nematode Caenorhabditis briggsae, despite divergence of the N-tail and KNL-2 primary sequences. Thus, the extended N-tail of CENP-A is essential for CENP-A chromatin assembly in C. elegans and partially substitutes for the function of Scm3/HJURP, in that it mediates a direct interaction between CENP-A and KNL-2. These results highlight an evolutionary variation on centromeric chromatin assembly in the absence of a dedicated CENP-A–specific chaperone/targeting factor of the Scm3/HJURP family.     

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.     

HJURP is a CENP-A chromatin assembly factor sufficient to form a functional de novo kinetochore

Centromeres of higher eukaryotes are epigenetically marked by the centromere-specific CENP-A nucleosome. New CENP-A recruitment requires the CENP-A histone chaperone HJURP. In this paper, we show that a LacI (Lac repressor) fusion of HJURP drove the stable recruitment of CENP-A to a LacO (Lac operon) array at a noncentromeric locus. Ectopically targeted CENP-A chromatin at the LacO array was sufficient to direct the assembly of a functional centromere as indicated by the recruitment of the constitutive centromere-associated network proteins, the microtubule-binding protein NDC80, and the formation of stable kinetochore–microtubule attachments. An amino-terminal fragment of HJURP was able to assemble CENP-A nucleosomes in vitro, demonstrating that HJURP is a chromatin assembly factor. Furthermore, HJURP recruitment to endogenous centromeres required the Mis18 complex. Together, these data suggest that the role of the Mis18 complex in CENP-A deposition is to recruit HJURP and that the CENP-A nucleosome assembly activity of HJURP is responsible for centromeric chromatin assembly to maintain the epigenetic mark.     

Acidic Nucleoplasmic DNA-binding Protein (And-1) Controls Chromosome Congression by Regulating the Assembly of Centromere Protein A (CENP-A) at Centromeres

The centromere is an epigenetically designated chromatin domain that is essential for the accurate segregation of chromosomes during mitosis. The incorporation of centromere protein A (CENP-A) into chromatin is fundamental in defining the centromeric loci. Newly synthesized CENP-A is loaded at centromeres in early G₁ phase by the CENP-A-specific histone chaperone Holliday junction recognition protein (HJURP) coupled with other chromatin assembly factors. However, it is unknown whether there are additional HJURP-interacting factor(s) involving in this process. Here we identify acidic nucleoplasmic DNA-binding protein 1 (And-1) as a new factor that is required for the assembly of CENP-A nucleosomes. And-1 interacts with both CENP-A and HJURP in a prenucleosomal complex, and the association of And-1 with CENP-A is increased during the cell cycle transition from mitosis to G₁ phase. And-1 down-regulation significantly compromises chromosome congression and the deposition of HJURP-CENP-A complexes at centromeres. Consistently, overexpression of And-1 enhances the assembly of CENP-A at centromeres. We conclude that And-1 is an important factor that functions together with HJURP to facilitate the cell cycle-specific recruitment of CENP-A to centromeres.     

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.     

HJURP uses distinct CENP-A surfaces to recognize and to stabilize CENP-A/histone H4 for centromere assembly

Centromeres are defined by the presence of chromatin containing the histone H3 variant, CENP-A, whose assembly into nucleosomes requires the chromatin assembly factor HJURP. We find that whereas surface-exposed residues in the CENP-A targeting domain (CATD) are the primary sequence determinants for HJURP recognition, buried CATD residues that generate rigidity with H4 are also required for efficient incorporation into centromeres. HJURP contact points adjacent to the CATD on the CENP-A surface are not used for binding specificity but rather to transmit stability broadly throughout the histone fold domains of both CENP-A and H4. Furthermore, an intact CENP-A/CENP-A interface is a requirement for stable chromatin incorporation immediately upon HJURP-mediated assembly. These data offer insight into the mechanism by which HJURP discriminates CENP-A from bulk histone complexes and chaperones CENP-A/H4 for a substantial portion of the cell cycle prior to mediating chromatin assembly at the centromere.     

Diaphanous formin mDia2 regulates CENP-A levels at centromeres

Centromeres of higher eukaryotes are epigenetically defined by centromere protein A (CENP-A), a centromere-specific histone H3 variant. The incorporation of new CENP-A into centromeres to maintain the epigenetic marker after genome replication in S phase occurs in G1 phase; however, how new CENP-A is loaded and stabilized remains poorly understood. Here, we identify the formin mDia2 as essential for stable replenishment of new CENP-A at centromeres. Quantitative imaging, pulse-chase analysis, and high-resolution ratiometric live-cell studies demonstrate that mDia2 and its nuclear localization are required to maintain CENP-A levels at centromeres. Depletion of mDia2 results in a prolonged centromere association of holiday junction recognition protein (HJURP), the chaperone required for CENP-A loading. A constitutively active form of mDia2 rescues the defect in new CENP-A loading caused by depletion of male germ cell Rac GTPase-activating protein (MgcRacGAP), a component of the small GTPase pathway essential for CENP-A maintenance. Thus, the formin mDia2 functions downstream of the MgcRacGAP-dependent pathway in regulating assembly of new CENP-A containing nucleosomes at centromeres.     

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.     

CAL1 is the Drosophila CENP-A assembly factor

Centromeres are specified epigenetically by the incorporation of the histone H3 variant CENP-A. In humans, amphibians, and fungi, CENP-A is deposited at centromeres by the HJURP/Scm3 family of assembly factors, but homologues of these chaperones are absent from a number of major eukaryotic lineages such as insects, fish, nematodes, and plants. In Drosophila, centromeric deposition of CENP-A requires the fly-specific protein CAL1. Here, we show that targeting CAL1 to noncentromeric DNA in Drosophila cells is sufficient to heritably recruit CENP-A, kinetochore proteins, and microtubule attachments. CAL1 selectively interacts with CENP-A and is sufficient to assemble CENP-A nucleosomes that display properties consistent with left-handed octamers. The CENP-A assembly activity of CAL1 resides within an N-terminal domain, whereas the C terminus mediates centromere recognition through an interaction with CENP-C. Collectively, this work identifies the “missing” CENP-A chaperone in flies, revealing fundamental conservation between insect and vertebrate centromere-specification mechanisms.     

Priming of centromere for CENP-A recruitment by human hMis18alpha, hMis18beta, and M18BP1

The centromere is the chromosomal site that joins to microtubules during mitosis for proper segregation. Determining the location of a centromere-specific histone H3 called CENP-A at the centromere is vital for understanding centromere structure and function. Here, we report the identification of three human proteins essential for centromere/kinetochore structure and function, hMis18alpha, hMis18beta, and M18BP1, the complex of which is accumulated specifically at the telophase-G1 centromere. We provide evidence that such centromeric localization of hMis18 is essential for the subsequent recruitment of de novo-synthesized CENP-A. If any of the three is knocked down by RNAi, centromere recruitment of newly synthesized CENP-A is rapidly abolished, followed by defects such as misaligned chromosomes, anaphase missegregation, and interphase micronuclei. Tricostatin A, an inhibitor to histone deacetylase, suppresses the loss of CENP-A recruitment to centromeres in hMis18alpha RNAi cells. Telophase centromere chromatin may be primed or licensed by the hMis18 complex and RbAp46/48 to recruit CENP-A through regulating the acetylation status in the centromere.     

Both tails and the centromere targeting domain of CENP-A are required for centromere establishment

The centromere—defined by the presence of nucleosomes containing the histone H3 variant, CENP-A—is the chromosomal locus required for the accurate segregation of chromosomes during cell division. Although the sequence determinants of human CENP-A required to maintain a centromere were reported, those that are required for early steps in establishing a new centromere are unknown. In this paper, we used gain-of-function histone H3 chimeras containing various regions unique to CENP-A to investigate early events in centromere establishment. We targeted histone H3 chimeras to chromosomally integrated Lac operator sequences by fusing each of the chimeras to the Lac repressor. Using this approach, we found surprising contributions from a small portion of the N-terminal tail and the CENP-A targeting domain in the initial recruitment of two essential constitutive centromere proteins, CENP-C and CENP-T. Our results indicate that the regions of CENP-A required for early events in centromere establishment differ from those that are required for maintaining centromere identity.     

CENP-A overexpression promotes aneuploidy with karyotypic heterogeneity

Chromosomal instability (CIN) is a hallmark of many cancers. Restricting the localization of centromeric histone H3 variant CENP-A to centromeres prevents CIN. CENP-A overexpression (OE) and mislocalization have been observed in cancers and correlate with poor prognosis; however, the molecular consequences of CENP-A OE on CIN and aneuploidy have not been defined. Here, we show that CENP-A OE leads to its mislocalization and CIN with lagging chromosomes and micronuclei in pseudodiploid DLD1 cells and xenograft mouse model. CIN is due to reduced localization of proteins to the kinetochore, resulting in defects in kinetochore integrity and unstable kinetochore–microtubule attachments. CENP-A OE contributes to reduced expression of cell adhesion genes and higher invasion of DLD1 cells. We show that CENP-A OE contributes to aneuploidy with karyotypic heterogeneity in human cells and xenograft mouse model. In summary, our results provide a molecular link between CENP-A OE and aneuploidy, and suggest that karyotypic heterogeneity may contribute to the aggressive phenotype of CENP-A–overexpressing cancers.     

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.     

Critical histone post-translational modifications for centromere function and propagation

The centromere is a critical genomic region that enables faithful chromosome segregation during mitosis, and must be distinguishable from other genomic regions to facilitate establishment of the kinetochore. The centromere-specific histone H3-variant CENP-A forms a special nucleosome that functions as a marker for centromere specification. In addition to the CENP-A nucleosomes, there are additional H3 nucleosomes that have been identified in centromeres, both of which are predicted to exhibit specific features. It is likely that the composite organization of CENP-A and H3 nucleosomes contributes to the formation of centromere-specific chromatin, termed ‘centrochromatin’. Recent studies suggest that centrochromatin has specific histone modifications that mediate centromere specification and kinetochore assembly. We use chicken non-repetitive centromeres as a model of centromeric activities to characterize functional features of centrochromatin. This review discusses our recent progress, and that of various other research groups, in elucidating the functional roles of histone modifications in centrochromatin.     

Alpha Satellite Insertion Close to an Ancestral Centromeric Region

Human centromeres are mainly composed of alpha satellite DNA hierarchically organized as higher-order repeats (HORs). Alpha satellite dynamics is shown by sequence homogenization in centromeric arrays and by its transfer to other centromeric locations, for example, during the maturation of new centromeres. We identified during prenatal aneuploidy diagnosis by fluorescent in situ hybridization a de novo insertion of alpha satellite DNA from the centromere of chromosome 18 (D18Z1) into cytoband 15q26. Although bound by CENP-B, this locus did not acquire centromeric functionality as demonstrated by the lack of constriction and the absence of CENP-A binding. The insertion was associated with a 2.8-kbp deletion and likely occurred in the paternal germline. The site was enriched in long terminal repeats and located ∼10 Mbp from the location where a centromere was ancestrally seeded and became inactive in the common ancestor of humans and apes 20–25 million years ago. Long-read mapping to the T2T-CHM13 human genome assembly revealed that the insertion derives from a specific region of chromosome 18 centromeric 12-mer HOR array in which the monomer size follows a regular pattern. The rearrangement did not directly disrupt any gene or predicted regulatory element and did not alter the methylation status of the surrounding region, consistent with the absence of phenotypic consequences in the carrier. This case demonstrates a likely rare but new class of structural variation that we name “alpha satellite insertion.” It also expands our knowledge on alphoid DNA dynamics and conveys the possibility that alphoid arrays can relocate near vestigial centromeric sites.     

Xenopus HJURP and condensin II are required for CENP-A assembly

Centromeric protein A (CENP-A) is the epigenetic mark of centromeres. CENP-A replenishment is necessary in each cell cycle to compensate for the dilution associated to DNA replication, but how this is achieved mechanistically is largely unknown. We have developed an assay using Xenopus egg extracts that can recapitulate the spatial and temporal specificity of CENP-A deposition observed in human cells, providing us with a robust in vitro system amenable to molecular dissection. Here we show that this deposition depends on Xenopus Holliday junction-recognizing protein (xHJURP), a member of the HJURP/Scm3 family recently identified in yeast and human cells, further supporting the essential role of these chaperones in CENP-A loading. Despite little sequence homology, human HJURP can substitute for xHJURP. We also report that condensin II, but not condensin I, is required for CENP-A assembly and contributes to retention of centromeric CENP-A nucleosomes both in mitosis and interphase. We propose that the chromatin structure imposed by condensin II at centromeres enables CENP-A incorporation initiated by xHJURP.     

Three-dimensional localization of CENP-A suggests a complex higher order structure of centromeric chromatin

The histone H3 variant centromere protein A (CENP-A) is central to centromere formation throughout eukaryotes. A long-standing question in centromere biology has been the organization of CENP-A at the centromere and its implications for the structure of centromeric chromatin. In this study, we describe the three-dimensional localization of CENP-A at the inner kinetochore plate through serial-section transmission electron microscopy of human mitotic chromosomes. At the kinetochores of normal centromeres and at a neocentromere, CENP-A occupies a compact domain at the inner kinetochore plate, stretching across two thirds of the length of the constriction but encompassing only one third of the constriction width and height. Within this domain, evidence of substructure is apparent. Combined with previous chromatin immunoprecipitation results (Saffery, R., H. Sumer, S. Hassan, L.H. Wong, J.M. Craig, K. Todokoro, M. Anderson, A. Stafford, and K.H.A. Choo. 2003. Mol. Cell. 12:509–516; Chueh, A.C., L.H. Wong, N. Wong, and K.H.A. Choo. 2005. Hum. Mol. Genet. 14:85–93), our data suggest that centromeric chromatin is arranged in a coiled 30-nm fiber that is itself coiled or folded to form a higher order structure.     

The CCAN recruits CENP-A to the centromere and forms the structural core for kinetochore assembly

CENP-A acts as an important epigenetic marker for kinetochore specification. However, the mechanisms by which CENP-A is incorporated into centromeres and the structural basis for kinetochore formation downstream of CENP-A remain unclear. Here, we used a unique chromosome-engineering system in which kinetochore proteins are targeted to a noncentromeric site after the endogenous centromere is conditionally removed. Using this system, we created two distinct types of engineered kinetochores, both of which were stably maintained in chicken DT40 cells. Ectopic targeting of full-length HJURP, CENP-C, CENP-I, or the CENP-C C terminus generated engineered kinetochores containing major kinetochore components, including CENP-A. In contrast, ectopic targeting of the CENP-T or CENP-C N terminus generated functional kinetochores that recruit the microtubule-binding Ndc80 complex and chromosome passenger complex (CPC), but lack CENP-A and most constitutive centromere-associated network (CCAN) proteins. Based on the analysis of these different engineered kinetochores, we conclude that the CCAN has two distinct roles: recruiting CENP-A to establish the kinetochore and serving as a structural core to directly recruit kinetochore proteins.