Multiple bromodomain genes are involved in restricting the spread of heterochromatic silencing at the Saccharomyces cerevisiae HMR-tRNA boundary

Abstract

The transfer RNA gene downstream from the HMR locus in S. cerevisiae functions as part of a boundary (barrier) element that restricts the spread of heterochromatic gene silencing into the downstream region of chromosome III. A genetic screen for identifying additional genes that, when mutated, allow inappropriate spreading of silencing from HMR through the tRNA gene was performed. YTA7, a gene containing bromodomain and ATPase homologies, was identified multiple times. Previously, others had shown that the bromodomain protein Bdf1p functions to restrict silencing at yeast euchromatin-heterochromatin boundaries; therefore we deleted nonessential bromodomain-containing genes to test their effects on heterochromatin spreading. Deletion of RSC2, coding for a component of the RSC chromatin-remodeling complex, resulted in a significant spread of silencing at HMR. Since the bromodomain of YTA7 lacks a key tyrosine residue shown to be important for acetyllysine binding in other bromodomains, we confirmed that a GST-Yta7p bromodomain fusion was capable of binding to histones in vitro. Epistasis analysis suggests that YTA7 and the HMR-tRNA function independently to restrict the spread of silencing, while RSC2 may function through the tRNA element. Our results suggest that multiple bromodomain proteins are involved in restricting the propagation of heterochromatin at HMR.

Figure 1.

Mating assay to identify extragenic mutations that lead to spreading of silencing through the HMR right boundary. (Top) The 1.0-kb region downstream of HMR containing the boundary tRNA was cloned into the a2 gene, and the resulting construct was integrated back into chromosome III in a MATα strain. The boundary element blocks the spread of silencing into a1, resulting in a nonmating phenotype (DDY277). The mating assay was performed essentially as described previously (Donze et al. 1999). DDY277 was mutagenized by transformation with a yeast genomic DNA library containing random transposon-LEU2 insertions (Ross-Macdonald et al. 1999), and Leu+ recombinants were tested for mating. (Bottom) Thirteen independent isolates that showed increased silencing were confirmed, representing mutations in four different genes, YTA7, SAS5, SAS4, and RPD3. All strain genotypes are listed in Table 1.

Figure 2.

YTA7 encodes a histone-binding bromodomain protein. (A) Schematic of the predicted YTA7 open reading frame, showing the relative locations of the two AAA family ATPase domains (amino acids 449–580 and 771–957) and the single bromodomain (amino acids 1003–1091) as determined by query of the Conserved Domain Database (Marchler-Bauer et al. 2005). (B) Alignment of yeast bromodomain sequences reveals that YTA7 lacks a key conserved tyrosine residue (arrow, red) that is critical for histone binding in other bromodomains, but contains a tandem serine-threonine in the same position (green). (C) The GST-Yta7p pull-down assay reveals that the bromodomain of Yta7p specifically retains histones H3 and H4. Reactions and washes were carried out in 150 mm KCl (see materials and methods). Increasing KCl to 500 mm in binding and wash steps reduced the level of histone binding by ∼50% (data not shown).

Figure 3.

Mutation of multiple bromodomain-containing genes results in spreading of silencing through the HMR boundary. Specific deletions or insertions were created or crossed into DDY277 to test their effects on boundary function in the mating assay described in Figure 1. Null mutations in YTA7, BDF1, RSC2, and SAS2 led to a significant spread of silencing, while mutation of GCN5, RSC1, BDF1, and HTZ1 showed a weaker phenotype. Mutation of SPT7 or SNF2 showed no phenotype.

Figure 4.

YTA7 and the HMR-tRNA function through different pathways to restrict the spread of silencing at HMR. (A) The ADE2 gene was inserted into chromosome III downstream of HMR in S. cerevisiae (HMR-ADE2). Isogenic strains lacking the HMR-tRNA (HMR-trnaΔ-ADE2) or lacking HMR (hmrΔ-ADE2) were also constructed. (B) Each strain was crossed into rpd3, sas5, yta7, or rsc2 mutant backgrounds, and silencing was assessed by the degree of pigmentation exhibited by the presence or absence of ADE2 expression. The combination of yta7Δ trnaΔ leads to a more silenced phenotype (more and darker red colonies), suggesting that each functions independently. No difference in phenotype is seen between strains containing rsc2Δ and rsc2Δ trnaΔ. Strains depicted are: first row [wild type (wt)], DDY814, DDY811, and DDY2114; second row (rpd3), DDY2093, DDY2143, and DDY2128; third row (sas5), DDY2106, DDY2156, and DDY2142; fourth row (yta7), DDY2205, DDY2200, and DDY2198; and fifth row (rsc2), DDY2496, DDY2489, and DDY2450.