Centromeric chromatin pliability and memory at a human neocentromere

We show that Trichostatin A (TSA)-induced partial histone hyperacetylation causes a unidirectional shift in the position of a previously defined binding domain for the centromere-specific histone H3 homologue CENP-A at a human neocentromere. The shift of approximately 320 kb is fully reversible when TSA is removed, but is accompanied by an apparent reduction in the density of CENP-A per unit length of genomic DNA at the neocentromere. TSA treatment also instigates a reversible abolition of a previously defined major domain of differentially delayed replication timing that was originally established at the neocentromeric site. None of these changes has any measurable deleterious effects on mitosis or neocentromere function. The data suggest pliability of centromeric chromatin in response to epigenetic triggers, and the non-essential nature of the regions of delayed replication for centromere function. Reversibility of the CENP-A-binding position and the predominant region of delayed replication timing following removal of TSA suggest strong memory at the original site of neocentromeric chromatin formation.     

Formation of novel CENP-A domains on tandem repetitive DNA and across chromosome breakpoints on human chromosome 8q21 neocentromeres

Endogenous human centromeres form on megabase-sized arrays of tandemly repeated alpha satellite DNA. Human neocentromeres form epigenetically at ectopic sites devoid of alpha satellite DNA and permit analysis of centromeric DNA and chromatin organization. In this study, we present molecular cytogenetic and CENP-A chromatin immunoprecipitation (ChIP) on CHIP analyses of two neocentromeres that have formed in chromosome band 8q21 each with a unique DNA and CENP-A chromatin configuration. The first neocentromere was found on a neodicentric chromosome 8 with an inactivated endogenous centromere, where the centromeric activity and CENP-A domain were repositioned to band 8q21 on a large tandemly repeated DNA. This is the first example of a neocentromere forming on repetitive DNA, as all other mapped neocentromeres have formed on single copy DNA. Quantitative fluorescent in situ hybridization (FISH) analysis showed a 60% reduction in the alpha satellite array size at the inactive centromere compared to the active centromere on the normal chromosome 8. This neodicentric chromosome may provide insight into centromere inactivation and the role of tandem DNA in centromere structure. The second neocentromere was found on a neocentric ring chromosome that contained the 8q21 tandemly repeated DNA, although the neocentromere was localized to a different genomic region. Interestingly, this neocentromere is composed of two distinct CENP-A domains in bands 8q21 and 8q24, which are brought into closer proximity on the ring chromosome. This neocentromere suggests that chromosomal rearrangement and DNA breakage may be involved in neocentromere formation. These novel examples provide insight into the formation and structure of human neocentromeres.     

Human centromere protein B induces translational positioning of nucleosomes on alpha-satellite sequences

The human centromere proteins A (CENP-A) and B (CENP-B) are the fundamental centromere components of chromosomes. CENP-A is the centromere-specific histone H3 variant, and CENP-B specifically binds a 17-base pair sequence (the CENP-B box), which appears within every other alpha-satellite DNA repeat. In the present study, we demonstrated centromere-specific nucleosome formation in vitro with recombinant proteins, including histones H2A, H2B, H4, CENP-A, and the DNA-binding domain of CENP-B. The CENP-A nucleosome wraps 147 base pairs of the alpha-satellite sequence within its nucleosome core particle, like the canonical H3 nucleosome. Surprisingly, CENP-B binds to nucleosomal DNA when the CENP-B box is wrapped within the nucleosome core particle and induces translational positioning of the nucleosome without affecting its rotational setting. This CENP-B-induced translational positioning only occurs when the CENP-B box sequence is settled in the proper rotational setting with respect to the histone octamer surface. Therefore, CENP-B may be a determinant for translational positioning of the centromere-specific nucleosomes through its binding to the nucleosomal CENP-B box.     

Stable complex formation of CENP-B with the CENP-A nucleosome

CENP-A and CENP-B are major components of centromeric chromatin. CENP-A is the histone H3 variant, which forms the centromere-specific nucleosome. CENP-B specifically binds to the CENP-B box DNA sequence on the centromere-specific repetitive DNA. In the present study, we found that the CENP-A nucleosome more stably retains human CENP-B than the H3.1 nucleosome in vitro. Specifically, CENP-B forms a stable complex with the CENP-A nucleosome, when the CENP-B box sequence is located at the proximal edge of the nucleosome. Surprisingly, the CENP-B binding was weaker when the CENP-B box sequence was located in the distal linker region of the nucleosome. This difference in CENP-B binding, depending on the CENP-B box location, was not observed with the H3.1 nucleosome. Consistently, we found that the DNA-binding domain of CENP-B specifically interacted with the CENP-A-H4 complex, but not with the H3.1-H4 complex, in vitro. These results suggested that CENP-B forms a more stable complex with the CENP-A nucleosome through specific interactions with CENP-A, if the CENP-B box is located proximal to the CENP-A nucleosome. Our in vivo assay also revealed that CENP-B binding in the vicinity of the CENP-A nucleosome substantially stabilizes the CENP-A nucleosome on alphoid DNA in human cells.     

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.     

The Oligomerization Landscape of Histones

In eukaryotes, DNA is packaged within nucleosomes. The DNA of each nucleosome is typically centered around an octameric histone protein core: one central tetramer plus two separate dimers. Studying the assembly mechanisms of histones is essential for understanding the dynamics of entire nucleosomes and higher-order DNA packaging. Here, we investigate canonical histone assembly and that of the centromere-specific histone variant, centromere protein A (CENP-A), using molecular dynamics simulations. We quantitatively characterize their thermodynamical and dynamical features, showing that two H3/H4 dimers form a structurally floppy, weakly bound complex, the latter exhibiting large instability around the central interface manifested via a swiveling motion of two halves. This finding is consistent with the recently observed DNA handedness flipping of the tetrasome. In contrast, the variant CENP-A encodes distinctive stability to its tetramer with a rigid but twisted interface compared to the crystal structure, implying diverse structural possibilities of the histone variant. Interestingly, the observed tetramer dynamics alter significantly and appear to reach a new balance when H2A/H2B dimers are present. Furthermore, we found that the preferred structure for the (CENP-A/H4)₂ tetramer is incongruent with the octameric structure, explaining many of the unusual dynamical behaviors of the CENP-A nucleosome. In all, these data reveal key mechanistic insights and structural details for the assembly of canonical and variant histone tetramers and octamers, providing theoretical quantifications and physical interpretations for longstanding and recent experimental observations. Based on these findings, we propose different chaperone-assisted binding and nucleosome assembly mechanisms for the canonical and CENP-A histone oligomers.     

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.     

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.     

A neocentromere in the DAZ region of the human Y chromosome

We describe a novel rearranged human Y chromosome consisting of an inverted duplication of the long arm heterochromatin and a small amount of euchromatin: rea(Y)(qter-q11.2::q11.2-qter). The normal centromere has been deleted and a neocentromere containing CENP-A, -C, -E and Mad2 but not CENP-B has formed close to the breakpoint. A 2.7 Mb yeast artificial chromosome contig spanning the breakpoint was constructed and the breakpoint was localised to a region of <120 kb close to the DAZ gene cluster. Combined immunofluorescence and fluorescence in situ hybridisation showed that the centromeric protein-binding domain of the neocentromere was located near the breakpoint and within the DAZ cluster.     

Analysis of Primary Structural Determinants That Distinguish the Centromere-Specific Function of Histone Variant Cse4p from Histone H3

Cse4p is a variant of histone H3 that has an essential role in chromosome segregation and centromere chromatin structure in budding yeast. Cse4p has a unique 135-amino-acid N terminus and a C-terminal histone-fold domain that is more than 60% identical to histone H3 and the mammalian centromere protein CENP-A. Cse4p and CENP-A have biochemical properties similar to H3 and probably replace H3 in centromere-specific nucleosomes in yeasts and mammals, respectively. In order to identify regions of Cse4p that distinguish it from H3 and confer centromere function, a systematic site-directed mutational analysis was performed. Nested deletions of the Cse4p N terminus showed that this region of the protein contains at least one essential domain. The C-terminal histone-fold domain of Cse4p was analyzed by changing Cse4p amino acids that differ between Cse4p and H3 to the analogous H3 residues. Extensive substitution of contiguous Cse4p residues with H3 counterparts resulted in cell lethality. However, all large lethal substitution alleles could be subdivided into smaller viable alleles, many of which caused elevated rates of mitotic chromosome loss. The results indicate that residues critical for wild-type Cse4p function and high-fidelity chromosome transmission are distributed across the entire histone-fold domain. Our findings are discussed in the context of the known structure of H3 within the nucleosome and compared with previous results reported for CENP-A.     

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.     

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.     

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.     

Formation of a centromere-specific chromatin structure

The kinetochore is formed on centromeric DNA as a key interface with microtubules from the mitotic spindle to achieve accurate chromosome segregation during mitosis. However, in contrast to other regions of the chromosome, the position of the kinetochore is specified by sequence-independent epigenetic mechanisms. Most recent work on kinetochore specification has focused on the centromere-specific histone H3-variant CENP-A. Whereas CENP-A is an important epigenetic marker for the kinetochore specification, it is unclear how centromeric chromatin structure is organized. To understand centromeric chromatin structure, we focused on additional centromere proteins that have an intrinsic DNA binding activity and identified the DNA binding CENP-T-W-S-X complex. Tetramer formation of CENP-T-W-S-X is essential for functional kinetochore assembly in vertebrate cells. Our structural and biochemical analysis reveals that the CENP-T-W-S-X complex is composed of four histone-fold domains with structural similarity to nucleosomes and displays DNA supercoiling activity. These results suggest that the CENP-T-W-S-X complex forms a unique nucleosome-like structure at centromeric chromatin. In addition, CENP-S and CENP-X function at non-centromeric sites. The intriguing histone-like properties of these proteins suggest that they may form nucleosome-like structures at various genome loci, extending the chromatin code beyond classical histone variants.