Functional genomics identifies a Myb domain-containing protein family required for assembly of CENP-A chromatin

Nucleosomes containing the centromere-specific histone H3 variant centromere protein A (CENP-A) create the chromatin foundation for kinetochore assembly. To understand the mechanisms that selectively target CENP-A to centromeres, we took a functional genomics approach in the nematode Caenorhabditis elegans, in which failure to load CENP-A results in a signature kinetochore-null (KNL) phenotype. We identified a single protein, KNL-2, that is specifically required for CENP-A incorporation into chromatin. KNL-2 and CENP-A localize to centromeres throughout the cell cycle in an interdependent manner and coordinately direct chromosome condensation, kinetochore assembly, and chromosome segregation. The isolation of KNL-2-associated chromatin coenriched CENP-A, indicating their close proximity on DNA. KNL-2 defines a new conserved family of Myb DNA-binding domain-containing proteins. The human homologue of KNL-2 is also specifically required for CENP-A loading and kinetochore assembly but is only transiently present at centromeres after mitotic exit. These results implicate a new protein class in the assembly of centromeric chromatin and suggest that holocentric and monocentric chromosomes share a common mechanism for CENP-A loading.     

Transgenerational inheritance of centromere identity requires the CENP-A N-terminal tail in the C. elegans maternal germ line

Centromere protein A (CENP-A) is a histone H3 variant that defines centromeric chromatin and is essential for centromere function. In most eukaryotes, CENP-A-containing chromatin is epigenetically maintained, and centromere identity is inherited from one cell cycle to the next. In the germ line of the holocentric nematode Caenorhabditis elegans, this inheritance cycle is disrupted. CENP-A is removed at the mitosis-to-meiosis transition and is reestablished on chromatin during diplotene of meiosis I. Here, we show that the N-terminal tail of CENP-A is required for the de novo establishment of centromeres, but then its presence becomes dispensable for centromere maintenance during development. Worms homozygous for a CENP-A tail deletion maintain functional centromeres during development but give rise to inviable offspring because they fail to reestablish centromeres in the maternal germ line. We identify the N-terminal tail of CENP-A as a critical domain for the interaction with the conserved kinetochore protein KNL-2 and argue that this interaction plays an important role in setting centromere identity in the germ line. We conclude that centromere establishment and maintenance are functionally distinct in C. elegans.

This study of the nematode Caenorhabditis elegans shows that centromere identity is set in the maternal germ line and passed on to the progeny via an epigenetic mechanism that requires the N-terminal tail of the centromeric histone H3 variant CENP-A.     

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.     

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.     

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.     

Misregulation of Scm3p/HJURP causes chromosome instability in Saccharomyces cerevisiae and human cells

The kinetochore (centromeric DNA and associated proteins) is a key determinant for high fidelity chromosome transmission. Evolutionarily conserved Scm3p is an essential component of centromeric chromatin and is required for assembly and function of kinetochores in humans, fission yeast, and budding yeast. Overexpression of HJURP, the mammalian homolog of budding yeast Scm3p, has been observed in lung and breast cancers and is associated with poor prognosis; however, the physiological relevance of these observations is not well understood. We overexpressed SCM3 and HJURP in Saccharomyces cerevisiae and HJURP in human cells and defined domains within Scm3p that mediate its chromosome loss phenotype. Our results showed that the overexpression of SCM3 (GALSCM3) or HJURP (GALHJURP) caused chromosome loss in a wild-type yeast strain, and overexpression of HJURP led to mitotic defects in human cells. GALSCM3 resulted in reduced viability in kinetochore mutants, premature separation of sister chromatids, and reduction in Cse4p and histone H4 at centromeres. Overexpression of CSE4 or histone H4 suppressed chromosome loss and restored levels of Cse4p at centromeres in GALSCM3 strains. Using mutant alleles of scm3, we identified a domain in the N-terminus of Scm3p that mediates its interaction with CEN DNA and determined that the chromosome loss phenotype of GALSCM3 is due to centromeric association of Scm3p devoid of Cse4p/H4. Furthermore, we determined that similar to other systems the centromeric association of Scm3p is cell cycle regulated. Our results show that altered stoichiometry of Scm3p/HJURP, Cse4p, and histone H4 lead to defects in chromosome segregation. We conclude that stringent regulation of HJURP and SCM3 expression are critical for genome stability.     

The CENP-A chaperone Scm3 becomes enriched at kinetochores in anaphase independently of CENP-A incorporation

Centromeres are specialized chromatin domains where kinetochores assemble. Centromeres contain as a conserved feature nucleosomes that are composed of the canonical histones H2A, H2B and H4 and a centromere-specific histone H3 variant, known as CENP-A in humans and Cse4 in budding yeast. The incorporation of CENP-A homologs into centromeric chromatin is cell cycle regulated and is assisted by related assembly factors named Scm3 in yeast and HJURP in human cells. Here, we describe that the budding yeast Scm3 binds weakly to centromeres during interphase including S phase when Cse4 assembles into centromeres. In anaphase Scm3 then becomes 2.5-fold enriched at kinetochores where it is dynamic with a half recovery time t_½ of 36 sec. In contrast, Cse4 is stably integrated into kinetochores. In addition, ten Scm3 molecules bind to a cluster of 16 kinetochores with 32 Cse4 molecules suggesting a 1:3 ratio at kinetochores between the two proteins. Analysis of conditional lethal scm3–1 mutant cells indicated that Scm3 participates in maintaining Cse4 at centromeres in anaphase. Thus, Scm3 interacts transiently with kinetochores in anaphase where it safeguards Cse4 even after its S phase incorporation into 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.     

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.     

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.     

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.     

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.     

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.     

A network of players in H3 histone variant deposition and maintenance at centromeres

Centromeres are key chromosomal landmarks important for chromosome segregation and are characterized by distinct chromatin features. The centromeric histone H3 variant, referred to as CENP-A or CenH3^CENP-A in mammals, has emerged as a key determinant for centromeric structure, function and epigenetic inheritance. To regulate the correct incorporation and maintenance of histones at this locus, the cell employs an intricate network of molecular players, among which histone chaperones and chromatin remodelling factors have been identified over the past years. The mammalian centromere-specific chaperone HJURP represents an interesting paradigm to understand the functioning of this network. This review highlights and discusses the latest findings on centromeric histone H3 variant deposition and regulation to delineate the current view on centromere establishment, maintenance and propagation throughout the cell cycle. This article is part of a Special Issue entitled: Chromatin and epigenetic regulation of animal development.     

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 is involved in the expansion of centromeric chromatin

The CENP-A–specific chaperone HJURP mediates CENP-A deposition at centromeres. The N-terminal region of HJURP is responsible for binding to soluble CENP-A. However, it is unclear whether other regions of HJURP have additional functions for centromere formation and maintenance. In this study, we generated chicken DT40 knockout cell lines and gene replacement constructs for HJURP to assess the additional functions of HJURP in vivo. Our analysis revealed that the middle region of HJURP associates with the Mis18 complex protein M18BP1/KNL2 and that the HJURP-M18BP1 association is required for HJURP function. In addition, on the basis of the analysis of artificial centromeres induced by ectopic HJURP localization, we demonstrate that HJURP exhibits a centromere expansion activity that is separable from its CENP-A–binding activity. We also observed centromere expansion surrounding natural centromeres after HJURP overexpression. We propose that this centromere expansion activity reflects the functional properties of HJURP, which uses this activity to contribute to the plastic establishment of a centromeric chromatin structure.     

Breaking the HAC Barrier: histone H3K9 acetyl/methyl balance regulates CENP-A assembly

The kinetochore is responsible for accurate chromosome segregation. However, the mechanism by which kinetochores assemble and are maintained remains unclear. Here we report that de novo CENP-A assembly and kinetochore formation on human centromeric alphoid DNA arrays is regulated by a histone H3K9 acetyl/methyl balance. Tethering of histone acetyltransferases (HATs) to alphoid DNA arrays breaks a cell type-specific barrier for de novo stable CENP-A assembly and induces assembly of other kinetochore proteins at the ectopic alphoid site. Similar results are obtained following tethering of CENP-A deposition factors hMis18α or HJURP. HAT tethering bypasses the need for hMis18α, but HJURP is still required for de novo kinetochore assembly. In contrast, H3K9 methylation following tethering of H3K9 tri-methylase (Suv39h1) to the array prevents de novo CENP-A assembly and kinetochore formation. CENP-A arrays assembled de novo by this mechanism can form human artificial chromosomes (HACs) that are propagated indefinitely in human cells.     

Centromeric chromatin gets loaded

Centromeric nucleosomes contain a histone H3 variant called centromere protein A (CENP-A) that is required for kinetochore assembly and chromosome segregation. Two new studies, Jansen et al. (see p. 795 of this issue) and Maddox et al. (see p. 757 of this issue), address when CENP-A is deposited at centromeres during the cell division cycle and identify an evolutionally conserved protein required for CENP-A deposition. Together, these studies advance our understanding of centromeric chromatin assembly and provide a framework for investigating the molecular mechanisms that underlie the centromere-specific loading of CENP-A.     

The outer kinetochore protein KNL-1 contains a defined oligomerization domain in nematodes

The kinetochore is a large, macromolecular assembly that is essential for connecting chromosomes to microtubules during mitosis. Despite the recent identification of multiple kinetochore components, the nature and organization of the higher-order kinetochore structure remain unknown. The outer kinetochore KNL-1/Mis12 complex/Ndc80 complex (KMN) network plays a key role in generating and sensing microtubule attachments. Here we demonstrate that Caenorhabditis elegans KNL-1 exists as an oligomer, and we identify a specific domain in KNL-1 responsible for this activity. An N-terminal KNL-1 domain from both C. elegans and the related nematode Caenorhabditis remanei oligomerizes into a decameric assembly that appears roughly circular when visualized by electron microscopy. On the basis of sequence and mutational analysis, we identify a small hydrophobic region as responsible for this oligomerization activity. However, mutants that precisely disrupt KNL-1 oligomerization did not alter KNL-1 localization or result in the loss of embryonic viability based on gene replacements in C. elegans. In C. elegans, KNL-1 oligomerization may coordinate with other kinetochore activities to ensure the proper organization, function, and sensory capabilities of the kinetochore–microtubule attachment.