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Spt10 and Spt21 Are Required for Transcriptional Silencing in Saccharomyces cerevisiae

Jennifer S. Chang, Fred Winston
Jennifer S. Chang
Department of Genetics, Harvard Medical School, 77 Avenue Louis Pasteur, Boston, Massachusetts 02115
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Fred Winston
Department of Genetics, Harvard Medical School, 77 Avenue Louis Pasteur, Boston, Massachusetts 02115
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  • For correspondence: winston@genetics.med.harvard.edu
DOI: 10.1128/EC.00246-10
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  • Fig. 1.
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    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.
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    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.
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    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.
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    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.
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    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.
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    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.
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    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.
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    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.

Tables

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  • Additional Files
  • Table 1.

    S. cerevisiae strains used in this study

    StrainGenotype
    FY2200MATa lys2-128δ his3Δ200 ura3Δ0 leu2Δ0
    FY1856MATα lys2-128δ his3Δ200 ura3Δ0 leu2Δ0
    FY2813MATa lys2-128δ his3Δ200 ura3Δ0 leu2Δ0 GAL1pr-SPT10::KanMX
    FY2814MATα lys2-128δ his3Δ200 ura3Δ0 leu2Δ0 GAL1pr-SPT10::KanMX
    FY2815MATα his3Δ200 ura3Δ0 leu2Δ0
    FY2816MATa lys2-128δ his3Δ200 ura3Δ0 leu2Δ0 spt21Δ::HIS3
    FY2817MATα lys2-128δ his3Δ200 ura3Δ0 leu2Δ0 spt21Δ::HIS3
    FY2818MATa lys2-128δ his3Δ200 ura3Δ0 leu2Δ0 sir2Δ::NatMX
    FY2819MATα lys2-128δ his3Δ200 ura3Δ0 leu2Δ0 sir2Δ::NatMX
    FY2820MATa lys2-128δ his3Δ200 ura3Δ0 leu2Δ0 GAL1pr-SPT10::KanMX spt21Δ::HIS3
    FY2821MATα lys2-128δ his3Δ200 ura3Δ0 leu2Δ0 GAL1pr-SPT10::KanMX spt21Δ::HIS3
    FY605MATa ura3-52 his3Δ200 leu2Δ1 trp1Δ63(hta2-htb2)Δ::TRP1
    FY2822MATα lys2-128δ his3Δ200 ura3-52 (leu2Δ0 or leu2Δ1) trp1Δ63(hta2-htb2)Δ::TRP1
    FY2823MATa lys2-128δ his3Δ200 ura3Δ0 leu2Δ0 sir1Δ::NatMX
    FY2824MATα lys2-128δ his3Δ200 ura3Δ0 leu2Δ0 sir1Δ::NatMX
    FY2825MATa lys2-128δ his3Δ200 ura3Δ0 leu2Δ0 sir1Δ::NatMX GAL1pr-SPT10::KanMX
    FY2826MATα lys2-128δ his3Δ200 ura3Δ0 leu2Δ0 sir1Δ::NatMX GAL1pr-SPT10::KanMX
    FY2827MATa lys2-128δ his3Δ200 ura3Δ0 leu2Δ0 sir1Δ::NatMX spt21Δ::HIS3
    FY2828MATα lys2-128δ his3Δ200 ura3Δ0 leu2Δ0 sir1Δ::NatMX spt21Δ::HIS3
    FY2829MATa lys2-128δ his3Δ200 ura3Δ0 leu2Δ0 sir1Δ::NatMX GAL1pr-SPT10::KanMX spt21Δ::HIS3
    FY2830MATα lys2-128δ his3Δ200 ura3Δ0 leu2Δ0 sir1Δ::NatMX GAL1pr-SPT10::KanMX spt21Δ::HIS3
    FY2831MATa lys2-128δ his3Δ200 (ura3Δ0 or ura3-52) (leu2Δ0 or leu2Δ1) trp1Δ63 sir1Δ::NatMX (hta2-htb2)Δ::TRP1
    FY2832MATα lys2-128δ his3Δ200 (ura3Δ0 or ura3-52) (leu2Δ0 or leu2Δ1) trp1Δ63 sir1Δ::NatMX (hta2-htb2)Δ::TRP1
    FY2833MATa lys2-128δ his3Δ200 ura3Δ0 leu2Δ0 SPT10-13×MYC:KanMX
    FY2834MATα lys2-128δ his3Δ200 ura3Δ0 leu2Δ0 SPT10-13×MYC:KanMX
    FY2835MATa lys2-128δ his3Δ200 ura3Δ0 leu2Δ0 SPT21-13×MYC:KanMX
    FY2836MATα lys2-128δ his3Δ200 ura3Δ0 leu2Δ0 SPT21-13×MYC:KanMX
    FY2837MATa lys2-128δ his3Δ200 ura3Δ0 leu2Δ0 sir3Δ::KanMX
    FY2838MATa lys2-128δ his3Δ200 ura3Δ0 leu2Δ0 sir4Δ::KanMX
    FY636MATα his4-912δ ura3-52 leu2Δ1 lys2Δ202::LYS2-dam
    L1134MATα lys2-128δ his3Δ200 ura3Δ0 leu2Δ0 telV-R::URA3
    L1135MATα lys2-128δ his3Δ200 (ura3Δ0 or ura3-167) (leu2Δ0 or leu2Δ1) telV-R::URA3 sir2Δ::NatMX
    L1136MATα ura3Δ0 leu2Δ0 telV-R::URA3 GAL1pr-SPT10::KanMX
    L1137MATα (ura3Δ0 or ura3-167) (leu2Δ0 or leu2Δ1) telV-R::URA3 spt21Δ::HIS3
    L1138MATα lys2-128δ his3Δ200 (ura3-52 or ura3Δ0) (leu2Δ1 or leu2Δ0) trp1Δ63 telV-R::URA3 (hta2-htb2)Δ::TRP1
    L1139MATa lys2-128Δ his3Δ200 (ura3Δ0 or ura3-167) (leu2Δ0 or leu2Δ1) rDNA::mURA3-LEU2
    L1140MATα lys2-128Δ his3Δ200 (ura3Δ0 or ura3-167) (leu2Δ0 or leu2Δ1) rDNA::mURA3-LEU2 sir2Δ::NatMX
    L1141MATa lys2-128Δ his3Δ200 (ura3Δ0 or ura3-167) (leu2Δ0 or leu2Δ1) rDNA::mURA3-LEU2 sir4Δ::NatMX
    L1142MATa lys2-128Δ his3Δ200 (ura3Δ0 or ura3-167) (leu2Δ0or leu2Δ1) rDNA::mURA3-LEU2 GAL1pr-SPT10:KanMX
    L1143MATa lys2-128Δ his3Δ200 (ura3Δ0 or ura3-167) (leu2Δ0 or leu2Δ1) rDNA::mURA3-LEU2 spt21Δ::HIS3
    L1144MATa lys2-128Δ his3Δ200 (ura3Δ0 or ura3-167) (leu2Δ0 or leu2Δ1) trp1Δ63 rDNA::mURA3-LEU2 (hta2-htb2)Δ::TRP1
    L1145MATa lys2-128Δ his3Δ200 ura3Δ0 leu2Δ0 telV-R::URA3
    L1146MATa his3Δ200 ura3Δ0 leu2Δ0 telV-R::URA3 GAL1pr-SPT10:KanMX
    L1147MATa lys2-128δ his3Δ200 ura3Δ0 leu2Δ0 telV-R::URA3 spt21Δ::HIS3
    L1148MATa (his3Δ200 or his4-912δ) (ura3Δ0 or ura3-52) (leu2Δ0 or leu2Δ1) lys2Δ202::LYS2-dam
    L1149MAT? (his3Δ200 or HIS3) (his4-912δ or HIS4) (ura3Δ0 or ura3-52) (leu2Δ0 or leu2Δ1) lys2Δ202::LYS2-dam sir2Δ::NatMX
    L1150MATa (ura3Δ0 or ura3-52) (leu2Δ0 or leu2Δ1) lys2Δ202::LYS2-dam GAL1pr-SPT10:KanMX
    L1151MATα (his3Δ200 or HIS3) (ura3Δ0 or ura3-52) (leu2Δ0 or leu2Δ1) lys2Δ202::LYS2-dam spt21Δ::HIS3

Additional Files

  • Figures
  • Tables
  • Supplemental material

    Files in this Data Supplement:

    • Supplemental file 1 - Supplemental table listing primers used in this work.
      PDF file, 59K.
    • Supplemental file 2 - Supplemental figure showing that Spt10 and Spt21 are not detectably associated with a silenced telomeric region on chromosome VI.
      PDF file, 212K.
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Spt10 and Spt21 Are Required for Transcriptional Silencing in Saccharomyces cerevisiae
Jennifer S. Chang, Fred Winston
Eukaryotic Cell Jan 2011, 10 (1) 118-129; DOI: 10.1128/EC.00246-10

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Spt10 and Spt21 Are Required for Transcriptional Silencing in Saccharomyces cerevisiae
Jennifer S. Chang, Fred Winston
Eukaryotic Cell Jan 2011, 10 (1) 118-129; DOI: 10.1128/EC.00246-10
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