Spt10 and Spt21 are required for transcriptional silencing in Saccharomyces cerevisiae

Abstract

In Saccharomyces cerevisiae, transcriptional silencing occurs at three classes of genomic regions: near the telomeres, at the silent mating type loci, and within the ribosomal DNA (rDNA) repeats. In all three cases, silencing depends upon several factors, including specific types of histone modifications. In this work we have investigated the roles in silencing for Spt10 and Spt21, two proteins previously shown to control transcription of particular histone genes. Building on a recent study showing that Spt10 is required for telomeric silencing, our results show that in both spt10 and spt21 mutants, silencing is reduced near telomeres and at HMLα, while it is increased at the rDNA. Both spt10 and spt21 mutations cause modest effects on Sir protein recruitment and histone modifications at telomeric regions, and they cause significant changes in chromatin structure, as judged by its accessibility to dam methylase. These silencing and chromatin changes are not seen upon deletion of HTA2-HTB2, the primary histone locus regulated by Spt10 and Spt21. These results suggest that Spt10 and Spt21 control silencing in S. cerevisiae by altering chromatin structure through roles beyond the control of histone gene expression.

Fig. 1.

Use of the GAL1pr-SPT10 allele to deplete Spt10. (A) Northern blot of wild type (WT) and GAL1pr-SPT10 strains grown on medium containing 2% glucose and no galactose. RNA was isolated from exponentially growing cultures and subjected to Northern blotting. ACT1 serves as a loading control. The slight migration difference for SPT10 in the GAL1pr-SPT10 lane is due to the replacement of the SPT10 5′ untranslated region (UTR) with the 5′ UTR of GAL1. (B) Generation times of wild-type, spt10Δ, GAL1pr-SPT10, and spt21Δ strains. Exponentially growing cultures were diluted to 100 cells/ml and then grown to a density of 1 × 10⁷ to 2 × 10⁷ cells/ml. The doubling time was calculated as time of incubation ÷ log₂(final density/initial density). Shown are the means ± standard errors of the means (SEM) for at least eight independent cultures. (C) Growth and Spt⁻ phenotypes of the GAL1pr-SPT10 mutant. Cultures were grown as described in Materials and Methods, subjected to 5-fold serial dilutions, and spotted onto the indicated media, which contain 2% glucose and no galactose. Plates were scanned after 2 days (SC) or 3 days (−Lys) of incubation at 30°C. Overall growth is shown on complete medium, and growth on SC without lysine (−Lys) indicates an Spt⁻ phenotype (suppression of lys2-128δ).

Fig. 2.

SPT10 and SPT21 are required for telomere position effect. All strains were grown in glucose, which represses expression of the GAL1pr-SPT10 allele. (A) Fivefold dilution spot assays with strains carrying the telV-R::URA3 reporter. Cultures were grown in YPD overnight to saturation, subjected to 5-fold serial dilutions, and spotted onto the indicated media. All strains have the telV-R::URA3 reporter, except for the wild-type URA3 strain, which has the wild-type URA3 gene at the endogenous locus, where it is not silenced. The mild growth defect of GAL1pr-SPT10 and spt21Δ mutants can be seen on the complete plate. (B) Real-time reverse transcription-PCR (RT-PCR) analysis of silencing of YFR057W. The measurement of YFR057W transcript levels was normalized to ACT1. Values are expressed relative to the wild-type level, which was assigned a value of 1. Shown are the means ± SEM for at least three independent experiments. The y axis was truncated to facilitate comparison; the value for sir2Δ is indicated above the corresponding bar.

Fig. 3.

GAL1pr-SPT10 and spt21Δ show mating type silencing defects in a MATa background. All strains were grown in glucose, which represses expression of the GAL1pr-SPT10 allele. (A) Quantitative mating assays with MATa and MATα haploid strains. Cells of each query strain were incubated for 5 h with a wild-type lawn of the opposite mating type, and the percentage of mated query cells was calculated (see Materials and Methods). Shown are the means ± standard deviations for at least three independent experiments. (B) Northern blot analysis of silencing at HMLα. The strains in lanes 1 to 11 are MATa, and the strain in lane 13 is MATα. The α1 and α2 transcripts are silenced in wild-type MATa haploids and expressed in wild-type MATα haploids.

Fig. 4.

Levels of rDNA silencing are increased in GAL1pr-SPT10 and spt21Δ mutants. All strains were grown in glucose, which represses expression of the GAL1pr-SPT10 allele. Fivefold dilution spot tests were done on the indicated strains, all of which carry an mURA3 reporter in a single copy within the rDNA. Growth on medium without uracil (−Ura) assesses the degree of reporter silencing, and complete medium controls for growth. Papillation reflects the stochastic nature of silencing changes.

Fig. 5.

(A) A high-copy-number plasmid encoding all four histones can partially suppress spt21Δ and GAL1pr-SPT10 silencing phenotypes. All strains were grown in glucose, which represses expression of the GAL1pr-SPT10 allele. The indicated strains carry either an empty vector (2μ LEU2) or a high-copy-number plasmid encoding the first copy of each histone protein (2μ LEU2 HTA1-HTB1 HHT1-HHF1). Fivefold dilution spot tests were grown on either medium without leucine (−Leu), which selects for plasmid maintenance, or on −Leu plus 5-FOA. Cells are able to grow in the presence of 5-FOA only when the telomere position effect is intact, and they are able to silence URA3 expression. (B) A high-copy-number plasmid with SIR3 can partially suppress the silencing defects of GAL1pr-SPT10 and spt21Δ. Fivefold dilution spot tests of telomere position effect were performed on the indicated strains carrying either an empty vector (2μ-LEU2) or a high-copy-number plasmid with SIR3 (2μ-LEU2-SIR3). All strains have the telV-R::URA3 reporter.

Fig. 6.

Measurement of Sir protein levels and Sir protein association with silenced chromatin in GAL1pr-SPT10 and spt21Δ mutants. All strains were grown in glucose, which represses expression of the GAL1pr-SPT10 allele. (A) Measurement of Sir protein levels. Total protein was isolated from the indicated strains and analyzed by Western analysis. Pgk1 is a glycolytic enzyme and serves as a loading control. (B) Chromatin immunoprecipitation (ChIP) of Sir2 on chromosome VI. Antibodies specific to Sir2 were used to immunoprecipitate the proteins in sonicated chromatin extracts from wild-type, GAL1pr-SPT10, and spt21Δ strains. Following reversal of cross-linking, the amount of immunoprecipitated DNA was quantitated using real-time PCR with primers specific to regions 0.6 kb and 2.8 kb from the right telomere of chromosome VI. Values were calculated relative to binding at the ACT1 gene, which is not subjected to silencing, and normalized to input DNA. Shown is the relative enrichment of Sir2 binding at the two subtelomeric locations. Plotted are the means ± SEM for four independent experiments. (C) ChIP of Sir3 was performed as described for Sir2, using an anti-Sir3 antiserum. (D) ChIP of histone H3 was conducted as described for Sir2, using an antiserum specific to histone H3.

Fig. 7.

Measurement of histone H3 K79 dimethylation and histone H4 acetylation levels at telomere VI-R in GAL1pr-SPT10 and spt21Δ mutants. All strains were grown in glucose, which represses expression of the GAL1pr-SPT10 allele. (A) ChIP of histone H3 K79 dimethylation was performed using the same strains, primers, and calculations as for Fig. 6, using antibodies specific to dimethyl histone H3 K79. Values are shown relative to total histone H3 ChIP. (B) ChIP of acetylated histone H4. (C) Western blot to measure total cellular levels of dimethylated histone H3 K79, acetylated histone H4, total histone H3, and Pgk1 as a loading control.

Fig. 8.

Dam methylation assays to probe chromatin structure in vivo. (A) A restriction map of relevant sites within the regions probed. The region 1.3 kb from the right telomere of chromosome VI is subject to silencing, and the telomere-proximal end lies 200 bp from the sequences probed in ChIP and YFR057W RT-PCR (Fig. 2B, 6, and 7). The region approximately 20 kb from the same telomere is at a distance not expected to be subject to telomere position effect (23). In both cases, multiple dam methylation sites occur between NdeI digest sites, resulting in multiple bands. For the 1.3-kb region, a dam methylation site is present 16 bp from the NdeI site, and digestion does not result in a significant mobility shift (band A). A region within MSN5, where no dam methylation sites occur between two NdeI sites, serves as a loading control. (B) Southern blot analysis of genomic DNA digested with NdeI and the indicated methylation-specific enzymes, separated on an agarose gel and probed for the regions indicated in panel A. The same blot was probed in all three panels. All strains were grown in glucose, which represses expression of the GAL1pr-SPT10 allele.