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

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

The ABCs of CENPs

Equal distribution of DNA in mitosis requires the assembly of a large proteinaceous ensemble onto the centromeric DNA, called the kinetochore. With few exceptions, kinetochore specification is independent of the DNA sequence and is determined epigenetically by deposition at the centromeric chromatin of special nucleosomes containing an H3-related histone, CENP-A. Onto centromeric CENP-A chromatin is assembled the so-called constitutive centromere-associated network (CCAN) of 16 proteins distributed in several functional groups as follows: CENP-C, CENP-H/CENP-I/CENP-K/, CENP-L/CENP-M/CENP-N, CENP-O/CENP-P/CENP-Q/CENP-R/CENP-U(50), CENP-T/CENP-W, and CENP-S/CENP-X. One role of the CCAN is to recruit outer kinetochore components further, such as KNL1, the Mis12 complex, and the Ndc80 complex (KMN network) to which attach the spindle microtubules with their structural and regulatory proteins. Among the CENPs in CCAN, CENP-C and CENP-T are required in parallel for operational kinetochore specification and spindle attachment. This review presents discussion of the latest structural and functional data on CENP-A and CENPs from the CCAN as well as their interaction with the KMN network.     

Premitotic assembly of human CENPs -T and -W switches centromeric chromatin to a mitotic state

Centromeres are differentiated chromatin domains, present once per chromosome, that direct segregation of the genome in mitosis and meiosis by specifying assembly of the kinetochore. They are distinct genetic loci in that their identity in most organisms is determined not by the DNA sequences they are associated with, but through specific chromatin composition and context. The core nucleosomal protein CENP-A/cenH3 plays a primary role in centromere determination in all species and directs assembly of a large complex of associated proteins in vertebrates. While CENP-A itself is stably transmitted from one generation to the next, the nature of the template for centromere replication and its relationship to kinetochore function are as yet poorly understood. Here, we investigate the assembly and inheritance of a histone fold complex of the centromere, the CENP-T/W complex, which is integrated with centromeric chromatin in association with canonical histone H3 nucleosomes. We have investigated the cell cycle regulation, timing of assembly, generational persistence, and requirement for function of CENPs -T and -W in the cell cycle in human cells. The CENP-T/W complex assembles through a dynamic exchange mechanism in late S-phase and G2, is required for mitosis in each cell cycle and does not persist across cell generations, properties reciprocal to those measured for CENP-A. We propose that the CENP-A and H3-CENP-T/W nucleosome components of the centromere are specialized for centromeric and kinetochore activities, respectively. Segregation of the assembly mechanisms for the two allows the cell to switch between chromatin configurations that reciprocally support the replication of the centromere and its conversion to a mitotic state on postreplicative chromatin.     

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.     

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

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

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

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

Cell-cycle-dependent structural transitions in the human CENP-A nucleosome in vivo

In eukaryotes, DNA is packaged into chromatin by canonical histone proteins. The specialized histone H3 variant CENP-A provides an epigenetic and structural basis for chromosome segregation by replacing H3 at centromeres. Unlike exclusively octameric canonical H3 nucleosomes, CENP-A nucleosomes have been shown to exist as octamers, hexamers, and tetramers. An intriguing possibility reconciling these observations is that CENP-A nucleosomes cycle between octamers and tetramers in?vivo. We tested this hypothesis by tracking CENP-A nucleosomal components, structure, chromatin folding, and covalent modifications across the human cell cycle. We report that CENP-A nucleosomes alter from tetramers to octamers before replication and revert to tetramers after replication. These structural transitions are accompanied by reversible chaperone binding, chromatin fiber folding changes, and previously undescribed modifications within the histone fold domains of CENP-A and H4. Our results reveal a cyclical nature to CENP-A nucleosome structure and have implications for the maintenance of epigenetic memory after centromere replication.     

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.     

Epigenetic specification of centromeres by CENP-A

Centromeres are the chromosomal loci that direct the formation of the kinetochores. These macromolecular assemblies mediate the interaction between chromosomes and spindle microtubules and thereby power chromosome movement during cell division. They are also the sites of extensive regulation of the chromosome segregation process. Except in the case of budding yeast, centromere identity does not rely on DNA sequence but on the presence of a special nucleosome that contains a histone H3 variant known as CenH3 or CENP-A (Centromere Protein A). It has been therefore proposed that CENP-A is the epigenetic mark of the centromere. Upon DNA replication the mark is diluted two-fold and must be replenished to maintain centromere identity. What distinguishes CENP-A nucleosomes from those containing histone H3, how CENP-A nucleosomes are incorporated specifically into centromeric chromatin, and how this incorporation is coordinated with other cell cycle events are key issues that have been the focus of intensive research over the last decade. Here we review some of the highlights of this research.     

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.     

Molecular underpinnings of centromere identity and maintenance

Centromeres direct faithful chromosome inheritance at cell division but are not defined by a conserved DNA sequence. Instead, a specialized form of chromatin containing the histone H3 variant, CENP-A, epigenetically specifies centromere location. We discuss current models where CENP-A serves as the marker for the centromere during the entire cell cycle in addition to generating the foundational chromatin for the kinetochore in mitosis. Recent elegant experiments have indicated that engineered arrays of CENP-A-containing nucleosomes are sufficient to serve as the site of kinetochore formation and for seeding centromeric chromatin that self-propagates through cell generations. Finally, recent structural and dynamic studies of CENP-A-containing histone complexes - before and after assembly into nucleosomes - provide models to explain underlying molecular mechanisms at the centromere.     

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.     

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.     

Active establishment of centromeric CENP-A chromatin by RSF complex

Centromeres are chromosomal structures required for equal DNA segregation to daughter cells, comprising specialized nucleosomes containing centromere protein A (CENP-A) histone, which provide the basis for centromeric chromatin assembly. Discovery of centromere protein components is progressing, but knowledge related to their establishment and maintenance remains limited. Previously, using anti-CENP-A native chromatin immunoprecipitation, we isolated the interphase–centromere complex (ICEN). Among ICEN components, subunits of the remodeling and spacing factor (RSF) complex, Rsf-1 and SNF2h proteins, were found. This paper describes the relationship of the RSF complex to centromere structure and function, demonstrating its requirement for maintenance of CENP-A at the centromeric core chromatin in HeLa cells. The RSF complex interacted with CENP-A chromatin in mid-G1. Rsf-1 depletion induced loss of centromeric CENP-A, and purified RSF complex reconstituted and spaced CENP-A nucleosomes in vitro. From these data, we propose the RSF complex as a new factor actively supporting the assembly of CENP-A chromatin.     

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.     

How to build a centromere: from centromeric and pericentromeric chromatin to kinetochore assembly.

The assembly of the centromere, a specialized region of DNA along with a constitutive protein complex which resides at the primary constriction and is the site of kinetochore formation, has been puzzling biologists for many years. Recent advances in the fields of chromatin, microscopy, and proteomics have shed a new light on this complex and essential process. Here we review recently discovered mechanisms and proteins involved in determining mammalian centromere location and assembly. The centromeric core protein CENP-A, a histone H3 variant, is hypothesized to designate centromere localization by incorporation into centromere-specific nucleosomes and is essential for the formation of a functional kinetochore. It has been found that centromere localization of centromere protein A (CENP-A), and therefore centromere determination, requires proteins involved in histone deacetylation, as well as base excision DNA repair pathways and proteolysis. In addition to the incorporation of CENP-A at the centromere, the formation of heterochromatin through histone methylation and RNA interference is also crucial for centromere formation. The assembly of the centromere and kinetochore is complex and interdependent, involving epigenetics and hierarchical protein-protein interactions.     

At the right place at the right time: novel CENP-A binding proteins shed light on centromere assembly

Centromeres, the chromosomal loci that form the sites of attachment for spindle microtubules during mitosis, are identified by a unique chromatin structure generated by nucleosomes containing the histone H3 variant CENP-A. The apparent epigenetic mode of centromere inheritance across mitotic and meiotic divisions has generated much interest in how CENP-A assembly occurs and how structurally divergent centromeric nucleosomes can specify the centromere complex. Although a substantial number of proteins have been implicated in centromere assembly, factors that can bind CENP-A specifically and deliver nascent protein to the centromere were, thus far, lacking. Several recent reports on experiments in fission yeast and human cells have now shown significant progress on this problem. Here, we discuss these new developments and their implications for epigenetic centromere inheritance.     

Structure of centromere chromatin: from nucleosome to chromosomal architecture

The centromere is essential for the segregation of chromosomes, as it serves as attachment site for microtubules to mediate chromosome segregation during mitosis and meiosis. In most organisms, the centromere is restricted to one chromosomal region that appears as primary constriction on the condensed chromosome and is partitioned into two chromatin domains: The centromere core is characterized by the centromere-specific histone H3 variant CENP-A (also called cenH3) and is required for specifying the centromere and for building the kinetochore complex during mitosis. This core region is generally flanked by pericentric heterochromatin, characterized by nucleosomes containing H3 methylated on lysine 9 (H3K9me) that are bound by heterochromatin proteins. During mitosis, these two domains together form a three-dimensional structure that exposes CENP-A-containing chromatin to the surface for interaction with the kinetochore and microtubules. At the same time, this structure supports the tension generated during the segregation of sister chromatids to opposite poles. In this review, we discuss recent insight into the characteristics of the centromere, from the specialized chromatin structures at the centromere core and the pericentromere to the three-dimensional organization of these regions that make up the functional centromere.     

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.