Histone H3 lysine 36 methylation antagonizes silencing in Saccharomyces cerevisiae independently of the Rpd3S histone deacetylase complex

Abstract

In yeast, methylation of histone H3 on lysine 36 (H3-K36) is catalyzed by the NSD1 leukemia oncoprotein homolog Set2. The histone deacetylase complex Rpd3S is recruited to chromatin via binding of the chromodomain protein Eaf3 to methylated H3-K36 to prevent erroneous transcription initiation. Here we identify a distinct function for H3-K36 methylation. We used random mutagenesis of histones H3 and H4 followed by a reporter-based screen to identify residues necessary to prevent the ectopic spread of silencing from the silent mating-type locus HMRa into flanking euchromatin. Mutations in H3-K36 or deletion of SET2 caused ectopic silencing of a heterochromatin-adjacent reporter. Transcriptional profiling revealed that telomere-proximal genes are enriched for those that display decreased expression in a set2Delta strain. Deletion of SIR4 rescued the expression defect of 26 of 37 telomere-proximal genes with reduced expression in set2Delta cells, implying that H3-K36 methylation prevents the spread of telomeric silencing. Indeed, Sir3 spreads from heterochromatin into neighboring euchromatin in set2Delta cells. Furthermore, genetic experiments demonstrated that cells lacking the Rpd3S-specific subunits Eaf3 or Rco1 did not display the anti-silencing phenotype of mutations in SET2 or H3-K36. Thus, antagonism of silencing is independent of the only known effector of this conserved histone modification.

Figure 1.—

A genetic screen identifies several mutations in H3 and H4 that affect anti-silencing adjacent to HMRa. (a) Schematic of the HMRa locus and the surrounding region, indicating the site of integration of the Candida albicans URA3 reporter gene. Ty1 long terminal repeats (LTRs) are shown in light purple, the Ty5 LTR is shown in dark purple, and the tRNA-Thr gene is shown in green. The right boundary element has been defined as the tRNA gene and flanking sequences (Donze and Kamakaka 2001). BE, boundary element. Not to scale. (b) Phenotypes of histone point mutants. To determine the extent of loss of expression from URA3, strains were grown to saturation and diluted in 10-fold steps and plated on media containing or lacking 5′-FOA. Examples of increased plating efficiency on 5′-FOA conferred by several mutations in H3 and H4 and suppression of this effect by deletion of SIR2 are shown. The reporter strain also contains ADE2 integrated near TELV, which confers a pink or red coloring when silenced and white when expressed. All histone mutants that display increased growth on 5′-FOA also show ectopic telomeric silencing as assayed by darker pink coloration. (c) Comparison of phenotypes of strains with mutations in H3-K36 or SET2. Plating of WT and mutant strains on 5′-FOA as described in b is shown.

Figure 2.—

Set2 prevents ectopic silencing of regions bordering heterochromatin genomewide. (a) Histogram of Set2-dependent genes plotted as a function of distance from telomere. Whole-genome transcriptional profiling of wild-type and set2Δ strains was performed as described (Meneghini et al. 2003), using four replicate experiments from separate cultures. Genes with significant changes in expression between wild type and set2Δ were determined using SAM (Tusher et al. 2001). (b) Suppression of the subtelomeric gene expression defect by deletion of SIR4. Whole-genome transcriptional profiling was performed as in a with WT and set2Δsir4Δ strains. Genes from the plot in a were divided into two classes, those that decreased in both set2Δ and set2Δsir4Δ strains (Sir independent) and those that decreased in a set2Δ strain but not in set2Δsir4Δ strains (Sir dependent). (c) Venn diagram showing overlap between genes that decrease in set2Δ cells and genes that decrease in htz1Δ cells (Meneghini et al. 2003). Subtelomeric: genes within 30 kb of telomeres with decreased expression in htz1Δ or set2Δ. Whole genome: all genes with decreased expression in htz1Δ or set2Δ. Purple numbers indicate overlap between genes with decreased expression in each strain.

Figure 3.—

Dot1 does not cause spread of silencing to the right of HMRa. Reporter gene assays are shown. SET2, DOT1, or both were deleted in the reporter strain used for the anti-silencing screen, and strains were plated on media containing or lacking 5′-FOA as described in the Figure 1 legend.

Figure 4.—

(a) Schematic of the right telomeres and neighboring regions of chromosomes III and XIV. Locations of primer sets used for ChIP in c are denoted by A–I and Z. Arrows indicate genes that decrease significantly in expression in set2Δ cells as assayed by expression microarray. Genes that are derepressed in set2Δsir4Δ cells are shown in boldface type. This is adapted from Figure 3A in Meneghini et al. (2003). (b) Effect of deletion of SET2 or SET2 and SIR4 on the expression of HMR- and telomere-proximal genes. Shown are the average expression ratios derived from microarray hybridization relative to wild type of the indicated genotypes. Plotted are means and standard error of the mean for four independent experiments performed on each genotype. (c) ChIP using antibodies specific for Sir3. Quantitative PCR was performed using primers as indicated in A. Immunoprecipitation (IP) values were normalized to input values. Plotted are means and standard error of the mean for three independent experiments. (d) Relative enrichments of H3-3meK36 in regions of chromosomes III and XIV studied. Graphs represent data from experiments in Pokholok et al. (2005), where chromatin IPs against H3 or H3-3meK36 were performed and competitively hybridized to whole-genome microarrays.

Figure 5.—

Set2 antagonizes spread of silencing independently of Rpd3S. (a) Reporter gene assays. SET2, RCO1, or EAF3 were deleted in the reporter strain and plated as in Figure 1. (b) A genetic model that proposes distinct pathways that mediate the roles of H3-K36 methylation in anti-silencing and repression of ectopic initiation sites.