The WD40-repeat protein Pwp1p associates in vivo with 25S ribosomal chromatin in a histone H4 tail-dependent manner

The tails of core histones (H2A, H2B, H3 and H4) are critical for the regulation of chromatin dynamics. Each core histone tail is specifically recognized by various tail binding proteins. Here we screened for budding yeast histone H4-tail binding proteins in a protein differential display approach by two-dimensional gel electrophoresis (2DGE). To obtain highly enriched chromatin proteins, we used a Mg(2+)-dependent chromatin oligomerization technique. The Mg(2+)-dependent oligomerized chromatin from H4-tail deleted cells was compared with that from wild-type cells. We used mass spectrometry to identify 22 candidate proteins whose amounts were reduced in the oligomerized chromatin from the H4-tail deleted cells. A Saccharomyces Genome Database search revealed 10 protein complexes, each of which contained more than two candidate proteins. Interestingly, 7 out of the 10 complexes have the potential to associate with the H4-tail. We obtained in vivo evidence, by a chromatin immunoprecipitation assay, that one of the candidate proteins, Pwp1p, associates with the 25S ribosomal DNA (rDNA) chromatin in an H4-tail-dependent manner. We propose that the complex containing Pwp1p regulates the transcription of rDNA. Our results demonstrate that the protein differential display approach by 2DGE, using a histone-tail mutant, is a powerful method to identify histone-tail binding proteins

Centromere Silencing and Function in Fission Yeast Is Governed by the Amino Terminus of Histone H3

AbstractBackground: Centromeric domains often consist of repetitive elements that are assembled in specialized chromatin, characterized by hypoacetylation of histones H3 and H4 and methylation of lysine 9 of histone H3 (K9-MeH3). Perturbation of this underacetylated state by transient treatment with histone deacetylase inhibitors leads to defective centromere function, correlating with delocalization of the heterochromatin protein Swi6/HP1. Likewise, deletion of the K9-MeH3 methyltransferase Clr4/Suvar39 causes defective chromosome segregation. Here, we create fission yeast strains retaining one histone H3 and H4 gene; the creation of these strains allows mutation of specific N-terminal tail residues and their role in centromeric silencing and chromosome stability to be investigated.Results: Reduction of H3/H4 gene dosage to one-third does not affect cell viability or heterochromatin formation. Mutation of lysines 9 or 14 or serine 10 within the amino terminus of histone H3 impairs centromere function, leading to defective chromosome segregation and Swi6 delocalization. Surprisingly, silent centromeric chromatin does not require the conserved lysine 8 and 16 residues of histone H4.Conclusions: To date, mutation of conserved N-terminal residues in endogenous histone genes has only been performed in budding yeast, which lacks the Clr4/Suvar39 histone methyltransferase and Swi6/HP1. We demonstrate the importance of conserved residues within the histone H3 N terminus for the maintenance of centromeric heterochromatin in fission yeast. In sharp contrast, mutation of two conserved lysines within the histone H4 tail has no impact on the integrity of centromeric heterochromatin. Our data highlight the striking divergence between the histone tail requirements for the fission yeast and budding yeast silencing pathways

HDA2 and HDA3 are related proteins that interact with and are essential for the activity of the yeast histone deacetylase HDA1

Histone deacetylase HDA1, the prototype for the class II mammalian deacetylases, is likely the catalytic subunit of the HDA1-containing complex that is involved in TUP1-specific repression and global deacetylation in yeast. Although the class I RPD3-like enzymatic complexes have been well characterized, little is known about the identity and interactions of the factors that associate to form the HDA1 complex. In this paper, we identify related HDA2 and HDA3 proteins that are found in the HDA1 complex and show that HDA1 interacts with itself and with the HDA2-HDA3 subcomplex to form a likely tetramer. These interactions are necessary for catalytic activity because mutations in any of the three components disrupt activity both in vitro and in vivo. In this respect the HDA1 complex differs from yeast RPD3, which has components such as SIN3 that are not essential for activity in vitro, and yeast HOS3, which has intrinsic in vitro activity as a homodimer in the absence of other subunits