Identification of a non-basic domain in the histone H4 N-terminus required for repression of the yeast silent mating loci.

We have shown previously that a stretch of four charged residues (16-19) at the histone H4 N-terminus is involved in repression of the yeast silent mating loci. One of these residues, Lys16, is a site for acetylation, which may prevent repression of the silent mating loci. In this paper we ask whether other sequences in histone H4, possibly in conjunction with H3 residues, are required for repression. We find that even in combination, the other seven acetylatable lysines in H3 and H4 do not function in repression. In contrast, we have found that an adjacent relatively uncharged domain (residues 21-29) is required for repression and that single amino acid insertions and deletions in this region are extremely detrimental. We propose that the basic and non-basic domains together form a DNA (or protein) induced amphipathic alpha-helix required in the formation of a repressive chromatin structure.     

Extremely conserved histone H4 N terminus is dispensable for growth but essential for repressing the silent mating loci in yeast

Yeast histone H4 function was probed in vivo by deleting segments of this extremely conserved 102 amino acid protein. Deletions in the hydrophobic core of H4 are lethal and block chromosomal segregation. In contrast, deletions at the hydrophilic N terminus (residues 4-28) and C terminus (residues 100-102) are viable. However, N-terminal deletion alters normal chromatin structure and lengthens the cell cycle, especially G2. Surprisingly, removal of the H4 N terminus also derepresses the silent mating type loci, HML alpha and HMRa, disrupting mating. This activation is specific since other regulated genes (GAL10, PHO5, CUP1) are repressed and induced normally in these cells. Deletions of the hydrophilic N termini of H2A or H2B do not show this effect on mating. These experiments allow us to define a unique H4 function that is not shared by other histones (H2A and H2B).     

Identification of a functional domain within the essential core of histone H3 that is required for telomeric and HM silencing in Saccharomyces cerevisiae

Fourteen novel single-amino-acid substitution mutations in histone H3 that disrupt telomeric silencing in Saccharomyces cerevisiae were identified, 10 of which are clustered within the alpha1 helix and L1 loop of the essential histone fold. Several of these mutations cause derepression of silent mating locus HML, and an additional subset cause partial loss of basal repression at the GAL1 promoter. Our results identify a new domain within the essential core of histone H3 that is required for heterochromatin-mediated silencing.     

Point mutations in the yeast histone H4 gene prevent silencing of the silent mating type locus HML.

The N-terminal serine and four conserved lysine residues near the N-terminus of yeast histone H4 are acetylated. We found that a mutation that changed the fourth lysine to alanine resulted in specific derepression of the silent mating type locus HML, while mutations that altered the N-terminal serine or the first three lysines had only minor phenotypic effects. Our results support an active role for histone H4 in the silencing of gene expression at this locus.     

Ubp8p, a histone deubiquitinase whose association with SAGA is mediated by Sgf11p, differentially regulates lysine 4 methylation of histone H3 in vivo

Despite recent advances in characterizing the regulation of histone H3 lysine 4 (H3-K4) methylation at the GAL1 gene by the H2B-K123-specific deubiquitinase activity of Saccharomyces cerevisiae SAGA (Spt-Ada-Gcn5-acetyltransferase)-associated Ubp8p, our knowledge on the general role of Ubp8p at the SAGA-dependent genes is lacking. For this study, using a formaldehyde-based in vivo cross-linking and chromatin immunoprecipitation (ChIP) assay, we have analyzed the role of Ubp8p in the regulation of H3-K4 methylation at three other SAGA-dependent yeast genes, namely, PHO84, ADH1, and CUP1. Like that at GAL1, H3-K4 methylation is increased at the PHO84 core promoter in the UBP8 deletion mutant. We also show that H3-K4 methylation remains invariant at the PHO84 open reading frame in the Deltaubp8 mutant, demonstrating a highly localized role of Upb8p in regulation of H3-K4 methylation at the promoter in vivo. However, unlike that at PHO84, H3-K4 methylation at the two other SAGA-dependent genes is not controlled by Ubp8p. Interestingly, Ubp8p and H3-K4 methylation are dispensable for preinitiation complex assembly at the core promoters of these genes. Our ChIP assay further demonstrates that the association of Ubp8p with SAGA is mediated by Sgf11p, consistent with recent biochemical data. Collectively, the data show that Ubp8p differentially controls H3-K4 methylation at the SAGA-dependent promoters, revealing a complex regulatory network of histone methylation in vivo.