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.     

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.     

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.     

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.     

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.     

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.     

Centromere identity maintained by nucleosomes assembled with histone H3 containing the CENP-A targeting domain

Active centromeres are marked by nucleosomes assembled with CENP-A, a centromere-specific histone H3 variant. The CENP-A centromere targeting domain (CATD), comprised of loop 1 and the alpha2 helix within the histone fold, is sufficient to target histone H3 to centromeres and to generate the same conformational rigidity to the initial subnucleosomal heterotetramer with histone H4 as does CENP-A. We now show in human cells and in yeast that depletion of CENP-A is lethal, but recruitment of normal levels of kinetochore proteins, centromere-generated mitotic checkpoint signaling, chromosome segregation, and viability can be rescued by histone H3 carrying the CATD. These data offer direct support for centromere identity maintained by a unique nucleosome that serves to distinguish the centromere from the rest of the chromosome.     

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.     

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.     

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.     

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.     

Posttranslational modifications of CENP-A: marks of distinction

Centromeres are specialized chromosome domain that serve as the site for kinetochore assembly and microtubule attachment during cell division, to ensure proper segregation of chromosomes. In higher eukaryotes, the identity of active centromeres is marked by the presence of CENP-A (centromeric protein-A), a histone H3 variant. CENP-A forms a centromere-specific nucleosome that acts as a foundation for centromere assembly and function. The posttranslational modification (PTM) of histone proteins is a major mechanism regulating the function of chromatin. While a few CENP-A site-specific modifications are shared with histone H3, the majority are specific to CENP-A-containing nucleosomes, indicating that modification of these residues contribute to centromere-specific function. CENP-A undergoes posttranslational modifications including phosphorylation, acetylation, methylation, and ubiquitylation. Work from many laboratories have uncovered the importance of these CENP-A modifications in its deposition at centromeres, protein stability, and recruitment of the CCAN (constitutive centromere-associated network). Here, we discuss the PTMs of CENP-A and their biological relevance.     

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.     

Human CENP-A contains a histone H3 related histone fold domain that is required for targeting to the centromere

Centromeres are the differentiated chromosomal domains that specify the mitotic behavior of chromosomes. To examine the molecular basis for the specification of centromeric chromatin, we have cloned a human cDNA that encodes the 17-kD histone-like centromere antigen, CENP-A. Two domains are evident in the 140 aa CENP-A polypeptide: a unique NH2- terminal domain and a 93-amino acid COOH-terminal domain that shares 62% identity with nucleosomal core protein, histone H3. An epitope tagged derivative of CENP-A was faithfully targeted to centromeres when expressed in a variety of animal cells and this targeting activity was shown to reside in the histone-like COOH-terminal domain of CENP-A. These data clearly indicate that the assembly of centromeres is driven, at least in part, by the incorporation of a novel core histone into centromeric chromatin.     

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.     

Xenopus CENP-A assembly into chromatin requires base excision repair proteins

CENP-A is an essential histone H3 variant found in all eukaryotes examined to date. To begin to determine how CENP-A is assembled into chromatin, we developed a binding assay using sperm chromatin in cell-free extract derived from Xenopus eggs. Our data suggest that the catalytic activities of an unidentified deoxycytidine deaminase and UNG2, a uracil DNA glycosylase, are involved in CENP-A assembly. In support of this model, inhibiting deoxycytidine deaminase with zebularine, or uracil DNA glycosylase with Ugi, uracil or UTP results in a lack of detectable CENP-A on sperm DNA. Conversely, inducing DNA damage increases the level of CENP-A detected on sperm chromatin. Our data suggest that base excision repair may be involved in assembly of this histone H3 variant.     

A minimal CENP-A core is required for nucleation and maintenance of a functional human centromere

Chromatin clusters containing CENP-A, a histone H3 variant, are found in centromeres of multicellular eukaryotes. This study examines the ability of alpha-satellite (alphoid) DNA arrays in different lengths to nucleate CENP-A chromatin and form functional kinetochores de novo. Kinetochore assembly was followed by measuring human artificial chromosome formation in cultured human cells and by chromatin immunoprecipitation analysis. The results showed that both the length of alphoid DNA arrays and the density of CENP-B boxes had a strong impact on nucleation, spreading and/or maintenance of CENP-A chromatin, and formation of functional kinetochores. These effects are attributed to a change in the dynamic balance between assembly of chromatin containing trimethyl histone H3-K9 and chromatin containing CENP-A/C. The data presented here suggest that a functional minimum core stably maintained on 30-70 kb alphoid DNA arrays represents an epigenetic memory of centromeric chromatin.     

Heterochromatin tells CENP-A where to go

The centromere is the region of the chromosome where the kinetochore forms. Kinetochores are the attachment sites for spindle microtubules that separate duplicated chromosomes in mitosis and meiosis. Kinetochore formation depends on a special chromatin structure containing the histone H3 variant CENP-A. The epigenetic mechanisms that maintain CENP-A chromatin throughout the cell cycle have been studied extensively but little is known about the mechanism that targets CENP-A to naked centromeric DNA templates. In a recent report published in Science, such de novo centromere assembly of CENP-A is shown to be dependent on heterochromatin and the RNA interference pathway.