A region of the nucleosome required for multiple types of transcriptional silencing in Saccharomyces cerevisiae

Abstract

Extended heterochromatin domains, which are repressive to transcription and help define centromeres and telomeres, are formed through specific interactions between silencing proteins and nucleosomes. This study reveals that in Saccharomyces cerevisiae, the same nucleosomal surface is critical for the formation of multiple types of heterochromatin, but not for local repression mediated by a related transcriptional repressor. Thus, this region of the nucleosome may be generally important to long-range silencing. In S. cerevisiae, the Sir proteins perform long-range silencing, whereas the Sum1 complex acts locally to repress specific genes. A mutant form of Sum1p, Sum1-1p, achieves silencing in the absence of Sir proteins. A genetic screen identified mutations in histones H3 and H4 that disrupt Sum1-1 silencing and fall in regions of the nucleosome previously known to disrupt Sir silencing and rDNA silencing. In contrast, no mutations were identified that disrupt wild-type Sum1 repression. Mutations that disrupt silencing fall in two regions of the nucleosome, the tip of the H3 tail and a surface of the nucleosomal core (LRS domain) and the adjacent base of the H4 tail. The LRS/H4 tail region interacts with the Sir3p bromo-adjacent homology (BAH) domain to facilitate Sir silencing. By analogy, this study is consistent with the LRS/H4 tail region interacting with Orc1p, a paralog of Sir3p, to facilitate Sum1-1 silencing. Thus, the LRS/H4 tail region of the nucleosome may be relatively accessible and facilitate interactions between silencing proteins and nucleosomes to stabilize long-range silencing.

Figure 1.—

Genetic screens for mutations in histones H3 and H4 that disrupt Sum1-1 silencing or Sum1 repression. (A) Reporter yeast strains were transformed with a library of mutated histone genes generated by homologous recombination between a PCR product, generated under error-prone conditions, and a linearized vector (pDM18), the ends of which were homologous to the PCR product. Shaded bars indicate the digestion sites. The reporter strains had their only copies of the H3 and H4 genes on a URA3-containing plasmid (pDM9), which was subsequently shuffled out by selecting for 5-FOA–resistant cells. To identify mutations that disrupted Sum1-1 silencing, a MATα sir2Δ SUM1-1 strain (LRY1450) was used, and colonies that failed to mate were identified. To identify mutations that disrupted Sum1 repression, the starting strain (LRY1849) had two reporters in which the promoters of the Sum1p-regulated genes PES4 and YGL138C drive the expression of HIS3 and LEU2. Colonies that grew in the absence of histidine and leucine were identified. (B) Mutations were assessed in semiquantitative assays. Sum1-1 silencing of HMRa was assessed using a mating assay, shown for representative histone mutations. Sum1 repression of the pPES4-HIS3 and pYGL138C-LEU2 reporters was assessed by growth on medium lacking histidine and leucine, shown for yeast strains SUM1 HST1 (LRY1849), sum1Δ HST1 (LRY1854), SUM1 hst1Δ (LRY1853), and sum1Δ hst1Δ (LRY1855). For both assays, 10-fold serial dilutions were plated on selective medium. (C) The numbers of colonies screened, plasmids identified, and disruptive mutations identified in each screen are indicated.

Figure 2.—

Histone mutations that disrupt Sum1-1 silencing cluster in discrete regions of the nucleosome. (A) Positions of histone substitutions tested (x-axis) and the number of substitutions tested at each position (y-axis). Mutations that did not have a silencing defect are in blue for H3 and green for H4. Disruptive mutations (>10-fold decrease in mating) are in red. Fifty-seven H3 and 31 H4 mutations were tested, and 22 mutations caused a silencing defect. (B) The 22 histone mutations that caused silencing defects map to three distinct regions: the H3 tail, the H3 LRS region, and the H4 tail. Mutations in white were identified in the genetic screen, mutations in light gray were identified by testing previously described LRS mutations, and the mutation in dark gray was identified through alanine scanning of modifiable residues. (C) The nucleosome structure, displaying H2A (yellow), H2B (orange), H3 (blue), and H4 (green) from PDB1ID3 (White et al. 2001) in Pymol. The positions of disruptive mutations are shown in red. A dashed line indicates where the H3 tails exit the nucleosome.

Figure 3.—

The BAH domain of Orc1p acts in Sum1-1 silencing by binding nucleosomes. (A) The BAH domain of Orc1p was not required for the interaction of Sum1-1p with ORC. MATa sum1Δ yeast strains with wild-type ORC1 (LRY1729) or orc1Δbah (LRY1835) were transformed with plasmids expressing LexA-Orc5 (pTT93) and the Gal4 activation domain (pGAD424), the Gal4 activation domain fused to Sum1p775-1062 (pRH01), or the Gal4 activation domain fused to Sum1-1p775-1062 (pRH02). Two-hybrid interactions between LexA-Orc5p and GAD-Sum1-1p775-1062 resulted in activation of a LacZ reporter gene. (B) Mutations in the BAH domain of Orc1p that disrupt nucleosome binding decreased mating. Semiquantitative mating assays of MATα SUM1-1 sir2Δ sir3Δ yeast with ORC1 (LRY2762), ORC1-HA (LRY2763), orc1E84K-HA (LRY2764), orc1P179L-HA (LRY2765), and orc1Δbah-HA (LRY2766) are shown.

Figure 4.—

The BAH domain of Orc1p is required for Sum1-1 silencing at a step other than recruitment. (A) ORC-binding sites at HMRa were replaced with Gal4-binding sites to create HMRa-Gal4. (B) Semiquantitative mating assay of MATα sir2Δ sir3Δ hmrΔ yeast containing SUM1-1 and ORC1-HA (LRY2804) or Gal4DBD-myc-SUM1-1 with ORC1 (LRY2752), ORC1-HA (LRY2753), orc1-E84K-HA (LRY2754), orc1-P179L-HA (LRY2755), and orc1Δbah-HA (LRY2756). These strains were transformed with pLR805 HMRa-Gal4 and tested for mating. (C and D) ChIP of Orc1p-HA (C) and Gal4-Sum1-1p (D) at HMRa-Gal4 in strains LRY2752, LRY2753, and LRY2804 described in B. Primers are spaced ∼1 kb apart, and enrichment values are relative to PHO5, which is not associated with Sum1-1p. Significance values (P < 0.0002) were calculated relative to SUM1-1 strain LRY2804. (E and F) ChIP of Orc1p-HA (E) and Gal4-Sum1-1p (F) at HMRa-Gal4 in strains LRY2752, LRY2753, LRY2754, LRY2755, and LRY2756 described in B. Significance values (P < 0.0002) were calculated relative to ORC1-HA strain LRY2753.

Figure 5.—

The BAH domain of Orc1p was not required for Sum1-1p self-association. (A) Co-immunoprecipitation of HA-Sum1p with myc-Sum1p. Yeast of the genotype sum1Δ (LRY144) or sum1Δ orc1Δbah (LRY1879) were transformed with plasmids expressing HA- and myc-tagged Sum1p (pLR052 and pLR021) or Sum1-1p (pLR047 and pJR2291). The samples were analyzed by immunoblotting with antibodies against the HA or myc tags. (B) Co-immunoprecipitation of myc-Sum1p with HA-Sum1p. HA-Sum1-1p or HA-Sum1p was immunoprecipitated from the same strains described in A.

Figure 6.—

Mutations in the N terminus of H3 disrupt Sum1-1 silencing at a step subsequent to recruitment of Sum1-1p. (A) Deletion of Set1p disrupts Sum1-1 silencing. A mating assay of MATα myc-SUM1-1 strains with SIR2 (LRY529), sir2Δ (LRY459), sir2Δ dot1Δ (LRY1364), and sir2Δ set1Δ (LRY1384) is shown. (B) Sum1-1 silencing blocks H3-K4 methylation at HMRa. ChIP is shown of di- and trimethylated H3-K4 at HMR in MATα strains with SUM1 SIR3 (LRY1007), SUM1 sir3Δ (LRY341), and SUM1-1 sir3Δ (LRY344). Enrichment values are relative to downstream ATG1, shown to be devoid of methylation (Pokholok et al. 2005). (C) H3-K4I did not disrupt expression of the Sum1 complex. RT–PCR analysis is shown of SUM1, HST1, RFM1, and ORC1 in MATα sir2Δ myc-SUM1-1 strains with SET1 (LRY459) or set1Δ (LRY1384) or a h3Δ h4Δ strain (LRY1450) transformed with a plasmid expressing wild-type histones (pDM18) or H3 K4I (pLR619). mRNA amounts were quantified relative to the control mRNA NTG1 and normalized to LRY459, containing wild-type genomic histones. (D) H3-K4I did not disrupt Sum1-1p enrichment at HMRa. Chromatin IP is shown of myc-Sum1-1p at HMRa in a MATα sir2Δ myc-SUM1-1 h3Δ h4Δ strain (LRY1450) transformed with plasmids expressing wild-type histones (pDM18), H3-K4I (pLR619), H3-E73G (pLR615), or H4-I21V (pLR640). Enrichment values are relative to PHO5, which is not associated with Sum1-1p.