Reconstitution of hemisomes on budding yeast centromeric DNA.

The structure of nucleosomes that contain the cenH3 histone variant has been controversial. In budding yeast, a single right-handed cenH3/H4/H2A/H2B tetramer wraps the ∼80-bp Centromere DNA Element II (CDE II) sequence of each centromere into a 'hemisome'. However, attempts to reconstitute cenH3 particles in vitro have yielded exclusively 'octasomes', which are observed in vivo on chromosome arms only when Cse4 (yeast cenH3) is overproduced. Here, we show that Cse4 octamers remain intact under conditions of low salt and urea that dissociate H3 octamers. However, particles consisting of two DNA duplexes wrapped around a Cse4 octamer and separated by a gap efficiently split into hemisomes. Hemisome dimensions were confirmed using a calibrated gel-shift assay and atomic force microscopy, and their identity as tightly wrapped particles was demonstrated by gelFRET. Surprisingly, Cse4 hemisomes were stable in 4 M urea. Stable Cse4 hemisomes could be reconstituted using either full-length or tailless histones and with a 78-bp CDEII segment, which is predicted to be exceptionally stiff. We propose that CDEII DNA stiffness evolved to favor Cse4 hemisome over octasome formation. The precise correspondence between Cse4 hemisomes resident on CDEII in vivo and reconstituted on CDEII in vitro without any other factors implies that CDEII is sufficient for hemisome assembly.

The unconventional structure of centromeric nucleosomes.

The centromere is a defining feature of the eukaryotic chromosome, required for attachment to spindle microtubules and segregation to the poles at both mitosis and meiosis. The fundamental unit of centromere identity is the centromere-specific nucleosome, in which the centromeric histone 3 (cenH3) variant takes the place of H3. The structure of the cenH3 nucleosome has been the subject of controversy, as mutually exclusive models have been proposed, including conventional and unconventional left-handed octamers (octasomes), hexamers with non-histone protein constituents, and right-handed heterotypic tetramers (hemisomes). Hemisomes have been isolated from native centromeric chromatin, but traditional nucleosome assembly protocols have generally yielded partially unwrapped left-handed octameric nucleosomes. In budding yeast, topology analysis and high-resolution mapping has revealed that a single right-handed cenH3 hemisome occupies the ~80-bp Centromere DNA Element II (CDEII) of each chromosome. Overproduction of cenH3 leads to promiscuous low-level incorporation of octasome-sized particles throughout the yeast genome. We propose that the right-handed cenH3 hemisome is the universal unit of centromeric chromatin, and that the inherent instability of partially unwrapped left-handed cenH3 octamers is an adaptation to prevent formation of neocentromeres on chromosome arms.

Centromeric nucleosomes induce positive DNA supercoils.

Centromeres of higher eukaryotes are epigenetically maintained; however, the mechanism that underlies centromere inheritance is unknown. Centromere identity and inheritance require the assembly of nucleosomes containing the CenH3 histone variant in place of canonical H3. Although H3 nucleosomes wrap DNA in a left-handed manner and induce negative supercoils, we show here that CenH3 nucleosomes reconstituted from Drosophila histones induce positive supercoils. Furthermore, we show that CenH3 likewise induces positive supercoils in functional centromeres in vivo, using a budding yeast minichromosome system and temperature-sensitive mutations in kinetochore proteins. The right-handed wrapping of DNA around the histone core implied by positive supercoiling indicates that centromere nucleosomes are unlikely to be octameric and that the exposed surfaces holding the nucleosome together would be available for kinetochore protein recruitment. The mutual incompatibility of nucleosomes with opposite topologies could explain how centromeres are efficiently maintained as unique loci on chromosomes.

Structure, dynamics, and evolution of centromeric nucleosomes.

Centromeres are defining features of eukaryotic chromosomes, providing sites of attachment for segregation during mitosis and meiosis. The fundamental unit of centromere structure is the centromeric nucleosome, which differs from the conventional nucleosome by the presence of a centromere-specific histone variant (CenH3) in place of canonical H3. We have shown that the CenH3 nucleosome core found in interphase Drosophila cells is a heterotypic tetramer, a "hemisome" consisting of one molecule each of CenH3, H4, H2A, and H2B, rather than the octamer of canonical histones that is found in bulk nucleosomes. The surprising discovery of hemisomes at centromeres calls for a reevaluation of evidence that has long been interpreted in terms of a more conventional nucleosome. We describe how the hemisome structure of centromeric nucleosomes can account for enigmatic properties of centromeres, including kinetochore accessibility, epigenetic inheritance, rapid turnover of misincorporated CenH3, and transcriptional quiescence of pericentric heterochromatin. Structural differences mediated by loop 1 are proposed to account for the formation of stable tetramers containing CenH3 rather than stable octamers containing H3. Asymmetric CenH3 hemisomes might interrupt the global condensation of octameric H3 arrays and present an asymmetric surface for kinetochore formation. We suggest that this simple mechanism for differentiation between centromeric and packaging nucleosomes evolved from an archaea-like ancestor at the dawn of eukaryotic evolution.

Biotin-tag affinity purification of a centromeric nucleosome assembly complex.

Centromeres are chromosomal sites of microtubule binding that ensure correct mitotic segregation of chromosomes to daughter cells. This process is mediated by a special centromere-specific histone H3 variant (CenH3), which packages centromeric chromatin and epigenetically maintains the centromere at a distinct chromosomal location. However, CenH3 is present at low abundance relative to canonical histones, presenting a challenge for the isolation and characterization of the chaperone machinery that assembles CenH3 into nucleosomes at centromeres. To address this challenge, we used controlled overexpression of Drosophila CenH3 (CID) and an efficient biochemical purification strategy offered by in vivo biotinylation of CID to successfully purify and characterize the soluble CID nucleosome assembly complex. It consists of a single chaperone protein, RbAp48, complexed with CID and histone H4. RbAp48 is also found in protein complexes that assemble canonical histone H3 and replacement histone H3.3. Here, we highlight the benefits of our improved biotin-mediated purification method, and address the question of how the simple CID/H4-RbAp48 chaperone complex can mediate nucleosome assembly specifically at centromeres.

Chaperone-mediated assembly of centromeric chromatin in vitro.

Every eukaryotic chromosome requires a centromere for attachment to spindle microtubules for chromosome segregation. Although centromeric DNA sequences vary greatly among species, centromeres are universally marked by the presence of a centromeric histone variant, centromeric histone 3 (CenH3), which replaces canonical histone H3 in centromeric nucleosomes. Conventional chromatin is maintained in part by histone chaperone complexes, which deposit the S phase-limited (H3) and constitutive (H3.3) forms of histone 3. However, the mechanism that deposits CenH3 specifically at centromeres and faithfully maintains its chromosome location through mitosis and meiosis is unknown. To address this problem, we have biochemically purified a soluble assembly complex that targets tagged CenH3 to centromeres in Drosophila cells. Two different affinity procedures led to purification of the same complex, which consists of CenH3, histone H4, and a single protein chaperone, RbAp48, a highly abundant component of various chromatin assembly, remodeling, and modification complexes. The corresponding CenH3 assembly complex reconstituted in vitro is sufficient for chromatin assembly activity, without requiring additional components. The simple CenH3 assembly complex is in contrast to the multisubunit complexes previously described for H3 and H3.3, suggesting that centromeres are maintained by a passive mechanism that involves exclusion of the complexes that deposit canonical H3s during replication and transcription.

SIR2 is required for polycomb silencing and is associated with an E(Z) histone methyltransferase complex.

BACKGROUND: SIR2 was originally identified in S. cerevisiae for its role in epigenetic silencing through the creation of specialized chromatin domains. It is the most evolutionarily conserved protein deacetylase, with homologs in all kingdoms. SIR2 orthologs in multicellular eukaryotes have been implicated in lifespan determination and regulation of the activities of transcription factors and other proteins. Although SIR2 has not been widely implicated in epigenetic silencing outside yeast, Drosophila SIR2 mutations were recently shown to perturb position effect variegation, suggesting that the role of SIR2 in epigenetic silencing may not be restricted to yeast. RESULTS: Evidence is presented that Drosophila SIR2 is also involved in epigenetic silencing by the Polycomb group proteins. Sir2 mutations enhance the phenotypes of Polycomb group mutants and disrupt silencing of a mini-white reporter transgene mediated by a Polycomb response element. Consistent with this, SIR2 is physically associated with components of an E(Z) histone methyltransferase complex. SIR2 binds to many euchromatic sites on polytene chromosomes and colocalizes with E(Z) at most sites. CONCLUSIONS: SIR2 is involved in the epigenetic inheritance of silent chromatin states mediated by the Drosophila Polycomb group proteins and is physically associated with a complex containing the E(Z) histone methyltransferase.

Polycomb group proteins ESC and E(Z) are present in multiple distinct complexes that undergo dynamic changes during development.

The Polycomb Group proteins are required for stable long-term maintenance of transcriptionally repressed states. Two distinct Polycomb Group complexes have been identified, a 2-MDa PRC1 complex and a 600-kDa complex containing the ESC and E(Z) proteins together with the histone deacetylase RPD3 and the histone-binding protein p55. We report here that there are at least two embryonic ESC/E(Z) complexes that undergo dynamic changes during development and a third larval E(Z) complex that forms after disappearance of ESC. We have identified a larger embryonic ESC complex containing RPD3 and p55, along with E(Z), that is present only until mid-embryogenesis, while the previously identified 600-kDa ESC/E(Z) complex persists until the end of embryogenesis. Constitutive overexpression of ESC does not promote abnormal persistence of the larger or smaller embryonic complexes and does not delay a dissociation of E(Z) from the smaller ESC complex or delay appearance of the larval E(Z) complex, indicating that these changes are developmentally programmed and not regulated by the temporal profile of ESC itself. Genetic removal of ESC prevents appearance of E(Z) in the smaller embryonic complex, but does not appear to affect formation of the large embryonic ESC complex or the PRC1 complex. We also show that the ESC complex is already bound to chromosomes in preblastoderm embryos and present genetic evidence that ESC is required during this very early period.