Centromere identity in Drosophila is not determined in vivo by replication timing

Centromeric chromatin is uniquely marked by the centromere-specific histone CENP-A. For assembly of CENP-A into nucleosomes to occur without competition from H3 deposition, it was proposed that centromeres are among the first or last sequences to be replicated. In this study, centromere replication in Drosophila was studied in cell lines and in larval tissues that contain minichromosomes that have structurally defined centromeres. Two different nucleotide incorporation methods were used to evaluate replication timing of chromatin containing CID, a Drosophila homologue of CENP-A. Centromeres in Drosophila cell lines were replicated throughout S phase but primarily in mid S phase. However, endogenous centromeres and X-derived minichromosome centromeres in vivo were replicated asynchronously in mid to late S phase. Minichromosomes with structurally intact centromeres were replicated in late S phase, and those in which centric and surrounding heterochromatin were partially or fully deleted were replicated earlier in mid S phase. We provide the first in vivo evidence that centromeric chromatin is replicated at different times in S phase. These studies indicate that incorporation of CID/CENP-A into newly duplicated centromeres is independent of replication timing and argue against determination of centromere identity by temporal sequestration of centromeric chromatin replication relative to bulk genomic chromatin.     

Mi-2/NuRD complex function is required for normal S phase progression and assembly of pericentric heterochromatin

During chromosome duplication, it is essential to replicate not only the DNA sequence, but also the complex nucleoprotein structures of chromatin. Pericentric heterochromatin is critical for silencing repetitive elements and plays an essential structural role during mitosis. However, relatively little is understood about its assembly and maintenance during replication. The Mi2/NuRD chromatin remodeling complex tightly associates with actively replicating pericentric heterochromatin, suggesting a role in its assembly. Here we demonstrate that depletion of the catalytic ATPase subunit CHD4/Mi-2β in cells with a dampened DNA damage response results in a slow-growth phenotype characterized by delayed progression through S phase. Furthermore, we observe defects in pericentric heterochromatin maintenance and assembly. Our data suggest that chromatin assembly defects are sensed by an ATM-dependent intra-S phase chromatin quality checkpoint, resulting in a temporal block to the transition from early to late S phase. These findings implicate Mi-2β in the maintenance of chromatin structure and proper cell cycle progression.     

The role of heterochromatin in centromere function

Chromatin at centromeres is distinct from the chromatin in which the remainder of the genome is assembled. Two features consistently distinguish centromeres: the presence of the histone H3 variant CENP-A and, in most organisms, the presence of heterochromatin. In fission yeast, domains of silent "heterochromatin" flank the CENP-A chromatin domain that forms a platform upon which the kinetochore is assembled. Thus, fission yeast centromeres resemble their metazoan counterparts where the kinetochore is embedded in centromeric heterochromatin. The centromeric outer repeat chromatin is underacetylated on histones H3 and H4, and methylated on lysine 9 of histone H3, which provides a binding site for the chromodomain protein Swi6 (orthologue of Heterochromatin Protein 1, HP1). The remarkable demonstration that the assembly of repressive heterochromatin is dependent on the RNA interference machinery provokes many questions about the mechanisms of this process that may be tractable in fission yeast. Heterochromatin ensures that a high density of cohesin is recruited to centromeric regions, but it could have additional roles in centromere architecture and the prevention of merotely, and it might also act as a trigger for kinetochore assembly. In addition, we discuss an epigenetic model for ensuring that CENP-A is targeted and replenished at the kinetochore domain.     

Chromatin assembly at kinetochores is uncoupled from DNA replication

The specification of metazoan centromeres does not depend strictly on centromeric DNA sequences, but also requires epigenetic factors. The mechanistic basis for establishing a centromeric "state" on the DNA remains unclear. In this work, we have directly examined replication timing of the prekinetochore domain of human chromosomes. Kinetochores were labeled by expression of epitope-tagged CENP-A, which stably marks prekinetochore domains in human cells. By immunoprecipitating CENP-A mononucleosomes from synchronized cells pulsed with [(3)H]thymidine we demonstrate that CENP-A-associated DNA is replicated in mid-to-late S phase. Cytological analysis of DNA replication further demonstrated that centromeres replicate asynchronously in parallel with numerous other genomic regions. In contrast, quantitative Western blot analysis demonstrates that CENP-A protein synthesis occurs later, in G2. Quantitative fluorescence microscopy and transient transfection in the presence of aphidicolin, an inhibitor of DNA replication, show that CENP-A can assemble into centromeres in the absence of DNA replication. Thus, unlike most genomic chromatin, histone synthesis and assembly are uncoupled from DNA replication at the kinetochore. Uncoupling DNA replication from CENP-A synthesis suggests that regulated chromatin assembly or remodeling could play a role in epigenetic centromere propagation.     

In and out: histone variant exchange in chromatin

Alterations in nucleosome structure affect the accessibility of the DNA and can generate specialized domains of chromatin in the genome. Such changes can be introduced by posttranslational modifications of histones, by chromatin remodeling, or by the incorporation of variants of H2A and H3 into nucleosomes. In contrast to the canonical histones, which are deposited behind the replication fork during S phase, histone variants are incorporated in a process that is independent of DNA replication. Recent studies have shown that distinct multiprotein complexes are responsible for the targeted deposition of histone variants at active genes, centromeres and silent loci. The incorporation of histone variants most probably has epigenetic consequences and contributes to architectural changes in chromosomes.     

A CAF-1 dependent pool of HP1 during heterochromatin duplication

To investigate how the complex organization of heterochromatin is reproduced at each replication cycle, we examined the fate of HP1-rich pericentric domains in mouse cells. We find that replication occurs mainly at the surface of these domains where both PCNA and chromatin assembly factor 1 (CAF-1) are located. Pulse-chase experiments combined with high-resolution analysis and 3D modeling show that within 90 min newly replicated DNA become internalized inside the domain. Remarkably, during this time period, a specific subset of HP1 molecules (alpha and gamma) coinciding with CAF-1 and replicative sites is resistant to RNase treatment. Furthermore, these replication-associated HP1 molecules are detected in Suv39 knockout cells, which otherwise lack stable HP1 staining at pericentric heterochromatin. This replicative pool of HP1 molecules disappears completely following p150CAF-1 siRNA treatment. We conclude that during replication, the interaction of HP1 with p150CAF-1 is essential to promote delivery of HP1 molecules to heterochromatic sites, where they are subsequently retained by further interactions with methylated H3-K9 and RNA.     

Epigenetics regulate centromere formation and kinetochore function

The eukaryote centromere was initially defined cytologically as the primary constriction on vertebrate chromosomes and functionally as a chromosomal feature with a relatively low recombination frequency. Structurally, the centromere is the foundation for sister chromatid cohesion and kinetochore formation. Together these provide the basis for interaction between chromosomes and the mitotic spindle, allowing the efficient segregation of sister chromatids during cell division. Although centromeric (CEN) DNA is highly variable between species, in all cases the functional centromere forms in a chromatin domain defined by the substitution of histone H3 with the centromere specific H3 variant centromere protein A (CENP-A), also known as CENH3. Kinetochore formation and function are dependent on a variety of regional epigenetic modifications that appear to result in a loop chromatin conformation providing exterior CENH3 domains for kinetochore construction, and interior heterochromatin domains essential for sister chromatid cohesion. In addition pericentric heterochromatin provides a structural element required for spindle assembly checkpoint function. Advances in our understanding of CENH3 biology have resulted in a model where kinetochore location is specified by the epigenetic mark left after dilution of CENH3 to daughter DNA strands during S phase. This results in a self-renewing and self-reinforcing epigenetic state favorable to reliably mark centromere location, as well as to provide the optimal chromatin configuration for kinetochore formation and function.     

Uncoupling global and fine-tuning replication timing determinants for mouse pericentric heterochromatin

Mouse chromocenters are clusters of late-replicating pericentric heterochromatin containing HP1 bound to trimethylated lysine 9 of histone H3 (Me3K9H3). Using a cell-free system to initiate replication within G1-phase nuclei, we demonstrate that chromocenters acquire the property of late replication coincident with their reorganization after mitosis and the establishment of a global replication timing program. HP1 dissociated during mitosis but rebound before the establishment of late replication, and removing HP1 from chromocenters by competition with Me3K9H3 peptides did not result in early replication, demonstrating that this interaction is neither necessary nor sufficient for late replication. However, in cells lacking the Suv39h1,2 methyltransferases responsible for K9H3 trimethylation and HP1 binding at chromocenters, replication of chromocenter DNA was advanced by 10-15% of the length of S phase. Reintroduction of Suv39h1 activity restored the later replication time. We conclude that Suv39 activity is required for the fine-tuning of pericentric heterochromatin replication relative to other late-replicating domains, whereas separate factors establish a global replication timing program during early G1 phase.     

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.     

Plasticity of fission yeast CENP-A chromatin driven by relative levels of histone H3 and H4

The histone H3 variant CENP-A assembles into chromatin exclusively at centromeres. The process of CENP-A chromatin assembly is epigenetically regulated. Fission yeast centromeres are composed of a central kinetochore domain on which CENP-A chromatin is assembled, and this is flanked by heterochromatin. Marker genes are silenced when placed within kinetochore or heterochromatin domains. It is not known if fission yeast CENP-A^Cnp1 chromatin is confined to specific sequences or whether histone H3 is actively excluded. Here, we show that fission yeast CENP-A^Cnp1 can assemble on noncentromeric DNA when it is inserted within the central kinetochore domain, suggesting that in fission yeast CENP-A^Cnp1 chromatin assembly is driven by the context of a sequence rather than the underlying DNA sequence itself. Silencing in the central domain is correlated with the amount of CENP-A^Cnp1 associated with the marker gene and is also affected by the relative level of histone H3. Our analyses indicate that kinetochore integrity is dependent on maintaining the normal ratio of H3 and H4. Excess H3 competes with CENP-A^Cnp1 for assembly into central domain chromatin, resulting in less CENP-A^Cnp1 and other kinetochore proteins at centromeres causing defective kinetochore function, which is manifest as aberrant mitotic chromosome segregation. Alterations in the levels of H3 relative to H4 and CENP-A^Cnp1 influence the extent of DNA at centromeres that is packaged in CENP-A^Cnp1 chromatin and the composition of this chromatin. Thus, CENP-A^Cnp1 chromatin assembly in fission yeast exhibits plasticity with respect to the underlying sequences and is sensitive to the levels of CENP-A^Cnp1 and other core histones.     

cis-acting DNA from fission yeast centromeres mediates histone H3 methylation and recruitment of silencing factors and cohesin to an ectopic site

BACKGROUND: Metazoan centromeres are generally composed of large repetitive DNA structures packaged in heterochromatin. Similarly, fission yeast centromeres contain large inverted repeats and two distinct silenced domains that are both required for centromere function. The central domain is flanked by outer repetitive elements coated in histone H3 methylated on lysine 9 and bound by conserved heterochromatin proteins. This centromeric heterochromatin is required for cohesion between sister centromeres. Defective heterochromatin causes premature sister chromatid separation and chromosome missegregation. The role of cis-acting DNA sequences in the formation of centromeric heterochromatin has not been established. RESULTS: A deletion strategy was used to identify centromeric sequences that allow heterochromatin formation in fission yeast. Fragments from the outer repeats are sufficient to cause silencing of an adjacent gene when inserted at a euchromatic chromosomal locus. This silencing is accompanied by the local de novo methylation of histone H3 on lysine 9, recruitment of known heterochromatin components, Swi6 and Chp1, and the provision of a new strong cohesin binding site. In addition, we demonstrate that the chromodomain of Chp1 binds to MeK9-H3 and that Chp1 itself is required for methylation of histone H3 on lysine 9. CONCLUSIONS: A short sequence, reiterated at fission yeast centromeres, can direct silent chromatin assembly and cohesin recruitment in a dominant manner. The heterochromatin formed at the euchromatic locus is indistinguishable from that found at endogenous centromeres. Recruitment of Rad21-cohesin underscores the link between heterochromatin and chromatid cohesion and indicates that these centromeric elements act independently of kinetochore activity to recruit cohesin.