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Takehito Furuyama

Publications and source records attributed to Takehito Furuyama.

5 recordsLinked to original sources
PubMed2006

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.

Animals↗
PubMed2006

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.

Animals↗
PubMed2004

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.

Amino Acid Sequence↗
PubMed2003

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.

Animals↗