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.     

SGT1-HSP90 complex is required for CENP-A deposition at centromeres

The centromere plays an essential role in accurate chromosome segregation, and defects in its function lead to aneuploidy and thus cancer. The centromere-specific histone H3 variant CENP-A is proposed to be the epigenetic mark of the centromere, as active centromeres require CENP-A–containing nucleosomes to direct the recruitment of multiple kinetochore proteins. CENP-A K124 ubiquitylation, mediated by CUL4A-RBX1-COPS8 E3 ligase activity, is required for CENP-A deposition at the centromere. However, the mechanism that controls the E3 ligase activity of the CUL4A-RBX1-COPS8 complex remains obscure. We have discovered that the SGT1-HSP90 complex is required for recognition of CENP-A by COPS8. Thus, the SGT1-HSP90 complex contributes to the E3 ligase activity of the CUL4A complex that is necessary for CENP-A ubiquitylation and CENP-A deposition at the centromere.     

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.     

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.     

Incorporation of Drosophila CID/CENP-A and CENP-C into centromeres during early embryonic anaphase

The centromere/kinetochore complex is indispensable for accurate segregation of chromosomes during cell divisions when it serves as the attachment site for spindle microtubules. Centromere identity in metazoans is believed to be governed by epigenetic mechanisms, because the highly repetitive centromeric DNA is neither sufficient nor required for specifying the assembly site of the kinetochore. A candidate for an epigenetic mark is the centromere-specific histone H3 variant CENP-A that replaces H3 in alternating blocks of chromatin exclusively in active centromeres. CENP-A acts as an initiator of kinetochore assembly, but the detailed dynamics of the deposition of metazoan CENP-A and of other constitutive kinetochore components are largely unknown. Here we show by quantitative fluorescence measurements in living early embryos that functional fluorescent fusion proteins of the Drosophila CENP-A and CENP-C homologs are rapidly incorporated into centromeres during anaphase. This incorporation is independent of ongoing DNA synthesis and pulling forces generated by the mitotic spindle, but strictly coupled to mitotic progression. Thus, our findings uncover a strikingly dynamic behavior of centromere components in anaphase.     

Biphasic incorporation of centromeric histone CENP-A in fission yeast

CENP-A is a centromere-specific histone H3 variant that is essential for kinetochore formation. Here, we report that the fission yeast Schizosaccharomyces pombe has at least two distinct CENP-A deposition phases across the cell cycle: S and G2. The S phase deposition requires Ams2 GATA factor, which promotes histone gene activation. In Deltaams2, CENP-A fails to retain during S, but it reaccumulates onto centromeres via the G2 deposition pathway, which is down-regulated by Hip1, a homologue of HIRA histone chaperon. Reducing the length of G2 in Deltaams2 results in failure of CENP-A accumulation, leading to chromosome missegregation. N-terminal green fluorescent protein-tagging reduces the centromeric association of CENP-A, causing cell death in Deltaams2 but not in wild-type cells, suggesting that the N-terminal tail of CENP-A may play a pivotal role in the formation of centromeric nucleosomes at G2. These observations imply that CENP-A is normally localized to centromeres in S phase in an Ams2-dependent manner and that the G2 pathway may salvage CENP-A assembly to promote genome stability. The flexibility of CENP-A incorporation during the cell cycle may account for the plasticity of kinetochore formation when the authentic centromere is damaged.     

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.     

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.     

A cell cycle-regulated GATA factor promotes centromeric localization of CENP-A in fission yeast

CENP-A, the centromere-specific histone H3 variant, plays a crucial role in organizing kinetochore chromatin for precise chromosome segregation. We have isolated Ams2, a Daxx-like motif-containing GATA factor, and histone H4, as multicopy suppressors of cnp1-1, an S. pombe CENP-A mutant. While depletion of Ams2 results in the reduction of CENP-A binding to the centromere and chromosome missegregation, increasing its dosage restores association of a CENP-A mutant protein with centromeres. Conversely, overexpression of CENP-A or histone H4 suppresses an ams2 disruptant. The intracellular amount of Ams2 thus affects centromeric nucleosomal constituents. Ams2 is abundant in S phase and associates with chromatin, including the central centromeres through binding to GATA-core sequences. Ams2 is thus a cell cycle-regulated GATA factor that is required for centromere function.     

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.     

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.     

A tale of two centromeres—diversity of structure but conservation of function in plants and animals

The structural and functional aspects of two specific centromeres, one drawn from the animal kingdom (Drosophila) and the other from the plant kingdom (maize), are compared. Both cases illustrate an epigenetic component to centromere specification. The observations of neocentromeres in Drosophila and inactive centromeres in maize constitute one line of evidence for this hypothesis. Another common feature is the divisibility of centromere function with reduced stability as the size decreases. The systems differ in that Drosophila has no common sequence repeat at all centromeres, whereas maize has a 150-bp unit present in tandem arrays together with a centromere-specific transposon, centromere retrotransposon maize, present at all primary constrictions. Aspects of centromere structure known only from one or the other system might be common to both, namely, the presence of centromere RNAs in the kinetochore as found in maize and the organization of the centromeric histone 3 in tetrameric nucleosomes.     

Centromeric chromatin exhibits a histone modification pattern that is distinct from both euchromatin and heterochromatin

Post-translational histone modifications regulate epigenetic switching between different chromatin states. Distinct histone modifications, such as acetylation, methylation and phosphorylation, define different functional chromatin domains, and often do so in a combinatorial fashion. The centromere is a unique chromosomal locus that mediates multiple segregation functions, including kinetochore formation, spindle-mediated movements, sister cohesion and a mitotic checkpoint. Centromeric (CEN) chromatin is embedded in heterochromatin and contains blocks of histone H3 nucleosomes interspersed with blocks of CENP-A nucleosomes, the histone H3 variant that provides a structural and functional foundation for the kinetochore. Here, we demonstrate that the spectrum of histone modifications present in human and Drosophila melanogaster CEN chromatin is distinct from that of both euchromatin and flanking heterochromatin. We speculate that this distinct modification pattern contributes to the unique domain organization and three-dimensional structure of centromeric regions, and/or to the epigenetic information that determines centromere identity.     

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.