The glucanosyltransferase Gas1 functions in transcriptional silencing

Abstract

Transcriptional silencing is a crucial process that is mediated through chromatin structure. The histone deacetylase Sir2 silences genomic regions that include telomeres, ribosomal DNA (rDNA) and the cryptic mating-type loci. Here, we report an unsuspected role for the enzyme Gas1 in locus-specific transcriptional silencing. GAS1 encodes a beta-1,3-glucanosyltransferase previously characterized for its role in cell wall biogenesis. In gas1 mutants, telomeric silencing is defective and rDNA silencing is enhanced. We show that the catalytic activity of Gas1 is required for normal silencing, and that Gas1's role in silencing is distinct from its role in cell wall biogenesis. Established hallmarks of silent chromatin, such as Sir2 and Sir3 binding, H4K16 deacetylation, and H3K56 deacetylation, appear unaffected in gas1 mutants. Thus, another event required for telomeric silencing must be influenced by GAS1. Because the catalytic activity of Gas1 is required for telomeric silencing, Gas1 localizes to the nuclear periphery, and Gas1 and Sir2 physically interact, we propose a model in which carbohydrate modification of chromatin components provides a new regulatory element that may be critical for chromatin function but which is virtually unexplored in the current landscape of chromatin analysis.

Fig. 1.

GAS1 functions in transcriptional silencing. (A) Deletion of GAS1 does not affect HML silencing. All growth plates in A–D are synthetic complete (SC) medium. Wild-type (WT) (LPY13659), sir1Δ (LPY13660), and gas1Δ (LPY13661) strains with a HML::TRP1 reporter were plated on SC lacking tryptophan (SC-trp). Increased growth on SC-trp indicates defective silencing. (B) Deletion of GAS1 does not affect HMR silencing. WT (LPY4912), sir1Δ (LPY4958), sir2Δ (LPY4980), and gas1Δ (LPY13665) strains with an hmrΔE::TRP1 reporter were assayed as in A. Levels of WT silencing differ at these TRP1 HM loci reporters due to differences in the structure of the reporter. The HML::TRP1 reporter (47) contains a TRP1 reporter at HML whereas hmrΔE::TRP1 contains a mutated silencer (48). (C) Deletion of GAS1 causes increased silencing at the rDNA. WT (LPY2446), sir2Δ (LPY2447), gas1Δ (LPY10074), and gas1Δ sir2Δ (LPY10078) with an mURA3 NTS1 rDNA reporter were assayed for silencing on SC plates lacking uracil (SC-ura). Increased growth on SC-ura indicates defective silencing (see Fig. 3A for location of this reporter in the rDNA repeat). (D) Deletion of GAS1 causes a telomeric silencing defect. WT (LPY4916), sir2Δ (LPY10397), and gas1Δ (LPY10362) with a URA3 telomeric reporter on chromosome V-R, and a gas1Δ control strain (LPY10129) with no telomeric reporter (gas1Δ ura3–1), to monitor gas1Δ 5-FOA sensitivity, were plated on SC containing 5-FOA. Decreased growth on 5-FOA indicates defective silencing. (E) Expression of an endogenous telomeric gene, YFR057W, is increased in gas1Δ. cDNAs from WT (LPY1029), sir2Δ (LPY12660), and gas1Δ (LPY10358) strains were analyzed by quantitative PCR, with the bars representing YFR057W cDNA signal minus control reactions without reverse transcriptase, normalized to ACT1.

Fig. 2.

The telomeric silencing function of GAS1 is not shared with other cell wall genes. WT (LPY4916), sir2Δ (LPY10397), gas1Δ (LPY10362), bgl2Δ (LPY13094), gas3Δ (LPY12337), and gas5Δ (LPY12348) strains were assayed for telomeric silencing as in Fig. 1D.

Fig. 3.

Key features of silent chromatin are unaltered in gas1Δ mutants. (A) Map of primer sites used for ChIP. Chromosome VI-R primers amplify regions 0.2 kb and 1 kb from the end of the telomere. Chromosome XII primers amplify regions near the 25S rRNA and 5S rRNA genes. Also shown are ADE2-CAN1 and URA3 reporter locations for rDNA silencing assays. (B) Sir2 occupancy in gas1Δ is increased slightly at the telomere and 5S rDNA. ChIP of Sir2 was done in WT (LPY5), sir2Δ (LPY11), and gas1Δ (LPY10129) strains. Input and IP DNA were analyzed with primers shown in A and the nonspecific locus ACT1. Sir2 enrichment at the telomere and rDNA was normalized to ACT1. (C) H4K16 is deacetylated at the telomere and rDNA in gas1Δ. ChIP of acetylated H4K16 (AcH4K16) was done in the same strains as (B). AcH4K16 enrichment at the telomere and rDNA was normalized to the Chr. V intergenic region. (D) H3K56 is deacetylated at the telomere in gas1Δ. ChIP of acetylated H3K56 (AcH3K56) was done in the same strains as B, and as a negative control, hht2-K56Q (LPY13166). AcH3K56 enrichment at the telomere was normalized to the intergenic region.

Fig. 4.

Sir2 interacts with Gas1 by two-hybrid and GST pull-down. (A) GBD-core Sir2 interacts with GAD-Gas1. The constructs GBD vector (pLP956), GBD-core Sir2 (pLP1073), and GBD-Sir2 (pLP1074) were expressed from 2μ TRP1 plasmids. GAD-Gas1 (pLP1205) was expressed from a 2μ LEU2 plasmid. The two-hybrid strain (LPY3374) was transformed with pairs of these plasmids to form LPY7251 (with pLP956, pLP1205), LPY7251 (with pLP1073, pLP1205), and LPY7253 (with pLP1074, 1205). The growth control plate is SC-leu-trp medium. The interaction plate is this medium also lacking histidine and adenine, to simultaneously monitor for GAL1-HIS3 and GAL2-ADE2 reporter activation. Growth on this plate indicates a physical interaction between GBD-core Sir2 and GAD-Gas1. (B) GST-Sir2 physically interacts with Gas1. GST (pLP1302) and GST-Sir2 (pLP1275) were purified and incubated with whole-cell extracts from strains used in Fig. 3B. Bound protein was analyzed by immunobloting for Gas1 (125 kDa).

Fig. 5.

Gas1 enzymatic activity is required for transcriptional silencing and is linked to Sir2. (A) gas1 enzymatically inactive point mutants are defective in telomeric silencing. The diagram of Gas1 shows the location of the E161Q and E262Q mutations in the catalytic domain. Dark bar at the C terminus of Gas1 indicates GPI anchor position. WT (LPY4916) strains were transformed with the 2μ HIS3 plasmids: vector (pLP359), GAS1 (pLP2091), and gas1-E161Q, E262Q (pLP2117). Transformed WT strains are LPY13554, LPY13559, and LPY13562. The gas1Δ strain (LPY10362) was transformed with 2μ HIS3 plasmids: pLP359, pLP2091, pLP2093 (gas1-E161Q), pLP2094 (gas1-E262Q), and pLP2117. Transformed gas1Δ strains are LPY13563 and LPY13568-LPY13571. Growth was examined on SC plates lacking histidine (SC-his). Silencing was examined on SC-his containing 5-FOA (SC-his 5-FOA). Growth at elevated temperature was examined at 37 °C. (B) Anti-β-1,3-glucan immunoprecipitates Sir2. β-1–3-glucan immunoprecipitations were performed in extracts from wild-type (LPY5), gas1Δ (LPY10129), and gas1Δ gas3Δ gas5Δ (LPY13543) strains overexpressing SIR2 (pLP349) and from sir2Δ (LPY11) expressing a vector construct (pLP135). Transformed strains are LPY13545, LPY13549, LPY13553, and LPY13546, respectively. Immunoprecipitated material was analyzed by immunoblot for Sir2 (65 kDa).