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. 2014 May 23;289(21):14981-95.
doi: 10.1074/jbc.M113.529354. Epub 2014 Apr 11.

Mediator, TATA-binding protein, and RNA polymerase II contribute to low histone occupancy at active gene promoters in yeast

Suraiya A Ansari  1 , Emily Paul  1 , Sebastian Sommer  2 , Corinna Lieleg  2 , Qiye He  3 , Alexandre Z Daly  1 , Kara A Rode  1 , Wesley T Barber  1 , Laura C Ellis  1 , Erika LaPorta  3 , Amanda M Orzechowski  1 , Emily Taylor  1 , Tanner Reeb  1 , Jason Wong  1 , Philipp Korber  2 , Randall H Morse  4
Affiliations

Mediator, TATA-binding protein, and RNA polymerase II contribute to low histone occupancy at active gene promoters in yeast

Suraiya A Ansari et al. J Biol Chem. .

Erratum in

Abstract

Transcription by RNA polymerase II (Pol II) in eukaryotes requires the Mediator complex, and often involves chromatin remodeling and histone eviction at active promoters. Here we address the role of Mediator in recruitment of the Swi/Snf chromatin remodeling complex and its role, along with components of the preinitiation complex (PIC), in histone eviction at inducible and constitutively active promoters in the budding yeast Saccharomyces cerevisiae. We show that recruitment of the Swi/Snf chromatin remodeling complex to the induced CHA1 promoter, as well as its association with several constitutively active promoters, depends on the Mediator complex but is independent of Mediator at the induced MET2 and MET6 genes. Although transcriptional activation and histone eviction at CHA1 depends on Swi/Snf, Swi/Snf recruitment is not sufficient for histone eviction at the induced CHA1 promoter. Loss of Swi/Snf activity does not affect histone occupancy of several constitutively active promoters; in contrast, higher histone occupancy is seen at these promoters in Mediator and PIC component mutants. We propose that an initial activator-dependent, nucleosome remodeling step allows PIC components to outcompete histones for occupancy of promoter sequences. We also observe reduced promoter association of Mediator and TATA-binding protein in a Pol II (rpb1-1) mutant, indicating mutually cooperative binding of these components of the transcription machinery and indicating that it is the PIC as a whole whose binding results in stable histone eviction.

Keywords: Chromatin Immunoprecipitation (ChiP); Chromatin Remodeling; Gene Transcription; Mediator; Nucleosome; Saccharomyces Cerevisiae; Swi/Snf Complex; Transcription; Transcription Coactivators; Yeast Genetics.

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Figures

FIGURE 1.
FIGURE 1.
CHA1 induction depends on the Swi/Snf complex. A, schematic diagram of the CHA1 promoter. The dashed ellipse represents the nucleosome that occludes the TATA element in the uninduced promoter and is remodeled upon activation. B, CHA1 expression in CSM containing 1 mg/ml of serine was measured by Northern analysis and normalized to PYK1 mRNA in wild type (BY4741) and deletion mutants as indicated. Error bars represent S.D. (n = 4). C, CHA1 expression in CSM containing 1 mg/ml of serine was measured by Northern analysis and normalized to 26 S rRNA in wild type (BY4741) and snf/swi deletion mutants as indicated. Error bars represent S.D. (n = 4). D, delayed induction kinetics of CHA1 in snf5Δ and swi3Δ yeast. Transcription was halted by addition of 10 mm sodium azide at the indicated times following addition of 1 mg/ml of serine to yeast cultures grown in CSM, and RNA analyzed by Northern blotting. Error bars represent S.D. (n = 2–4; note n = 4 for t = 0, 5, 15, 30, and 60 min (left panel)). For the kinetic analyses in D, data were normalized to allow easier visual comparison (see “Experimental Procedures”); this erases the difference in transcription level between wild type and mutant, which was otherwise consistent with that in C.
FIGURE 2.
FIGURE 2.
Dependence on Mediator of Swi/Snf and SAGA association with active gene promoters. A, Snf2 and Snf5 association with the CHA1 promoter in wild type (WT; BY4742 for Snf5 and Research Genetics Snf2-TAP strain for Snf2) and gal11/med15Δ med3Δ (LS10 for Snf5 and RMY525 for Snf2) yeast grown in CSM without (uninduced) or with 1 mg/ml of serine (induced) was measured by ChIP. B, Snf2 association with the MET2 and MET6 promoters in WT (Snf2-TAP strain, Research Genetics) and gal11/med15Δ med3Δ (RMY525) yeast grown in YPD without (uninduced) or with (induced) 0.5 mm CdCl2. C, Snf5 association with the indicated promoters was measured in WT (RMY521) and srb4/med17 ts (RMY522) yeast grown in YPD after 1 h at 37 °C. D, Snf5 association with promoters whose transcription is dependent or independent of the Mediator tail module, as indicated, was measured in WT (Snf2-TAP strain, Research Genetics) and gal11/med15Δ med3Δ (RMY525) yeast grown in YPD. E, effect of Mediator tail mutation on SAGA recruitment. Left panel: Spt3 association with the CHA1 promoter was measured by ChIP in wild type (Snf2-TAP strain, Research Genetics) and gal11/med15Δ med3Δ yeast (RMY525) grown in CSM without (uninduced) or with (induced) 1 mg/ml of serine, and association with the MET2 and MET6 promoters was measured in wild type (RMY521) and gal11/med15Δ med3Δ yeast (RMY415) grown in YPD without (uninduced) or with (induced) 0.5 mm CdCl2. Right panel, Ada2 and Gcn5 association at the uninduced and induced MET2 and MET6 promoters were measured by ChIP, and the log2 ratios of the association at the induced/uninduced promoters in wild type and gal11/med15Δ med3Δ yeast are shown, as indicated. Relative association in all cases was determined by quantitative PCR analysis of input and immunoprecipitated (IP) samples, and normalized to a non-transcribed region of ChrV (25). Error bars represent S.D. (n = 3–4).
FIGURE 3.
FIGURE 3.
Chromatin remodeling of the induced CHA1 promoter occurs normally in yeast harboring Mediator tail mutants that are defective for CHA1 transcriptional activation. MNase cleavage sites were mapped relative to the BamHI site at +602 in the CHA1 ORF in chromatin prepared from yeast grown in CSM without or with 1 mg/ml of serine (30 min induction). Increasing amounts of MNase were used for each sample as indicated by the triangles at the bottom; the lowest level in each group corresponds to no MNase addition, and the highest to 50 units/ml. Marker lanes (M) contain 100-bp DNA ladders. The small arrow in each panel indicates the strong cleavage induced by serine addition in the vicinity of the CHA1 TATA element.
FIGURE 4.
FIGURE 4.
Histone H3 association with active gene promoters is increased in Mediator mutants. A, histone H3 association with the CHA1 promoter was measured by ChIP using wild type (BY4742) and gal11/med15Δ med3Δ (LS10) yeast grown in CSM without (uninduced) or with (induced) 1 mg/ml of serine. B, histone H3 association with the indicated promoters was measured in wild type (RMY521) and srb4/med17 ts (RMY522) yeast grown in YPD after 1 h growth at 37 °C. C, histone H3 association with promoters whose transcription is dependent or independent of the Mediator tail module, as indicated, was measured in wild type (BY4742) and gal11/med15Δ med3Δ (LS10) yeast grown in YPD. Relative association in all cases was determined by quantitative PCR analysis of input and immunoprecipitated (IP) samples, and normalized to a non-transcribed region of ChrV (25). Error bars represent S.D. (n = 3).
FIGURE 5.
FIGURE 5.
Dependence on Mediator for transcription, chromatin opening, and histone eviction of the PHO5v33 promoter. A, β-galactosidase activity (Miller units) was measured for wild type (BY4742) and gal11/med15Δ med3Δ (LS10) yeast harboring the plasmid pPpho5v33-lacZ and grown in raffinose-containing medium and induced by addition of galactose for the indicated times. Error bars represent S.D. (n = 3). B, ClaI accessibility of the PHO5v33 promoter was determined in nuclei prepared from strains and using growth conditions as in A. Biological replicates for the 30-min induction time point of wild type and mutant gave 88 and 94% ClaI accessibility, respectively. C, histone H3 association at the PHO5v33 promoter was measured by ChIP using strains and growth conditions as in A. Error bars represent the variation between two biological replicates.
FIGURE 6.
FIGURE 6.
Dependence of histone H3 association on the Swi/Snf complex. A, association of histone H3 with the CHA1 and SPO20 promoters was measured by ChIP in wild type (BY4741) or snf5Δ (yeast deletion collection) yeast grown in CSM under non-inducing conditions (no serine) or after induction for 15 or 30 min by addition of 1 mg/ml of serine. B, histone H3 association at the indicated promoters was measured using the samples from A obtained from yeast grown under non-inducing conditions for 30 min. Relative association in all cases was determined by quantitative PCR analysis of input and immunoprecipitated (IP) samples, and normalized to a non-transcribed region of ChrV (25). Error bars represent S.D. (n = 3).
FIGURE 7.
FIGURE 7.
Dependence of histone H3 association on Pol II and TBP. A, association of histone H3 with the CHA1 promoter was measured by ChIP in yeast grown in CSM without (uninduced) or with (induced) 1 mg/ml of serine and shifted to 37 °C for 1 h. Strains used were RMY521 (wild type) and Z111-Srb5-Myc (left panel), and BYΔ2 (wild type) and BYΔ2-ts1 (tbp ts-1). B, association of histone H3 with the indicated promoters was measured by ChIP for yeast grown in YPD after a 1-h shift to 37 °C in wild type (RMY521) and rpb1-1 ts (Z111-Srb5-Myc) yeast. CHA1 is partially induced in YPD medium. C, association of histone H3 with the indicated promoters was measured by ChIP in wild type (BYΔ2) and tbp ts-1 (BYΔ2-ts1) yeast grown in CSM (left panel) or YPD (right panel) medium after a 1-h shift to 37 °C. D, association of Rpb4 with the core promoter regions of the indicated genes was measured by ChIP in wild type (RMY521) and rpb1-1 ts (Z111-Srb5-Myc) yeast grown in YPD after 1 h at 37 °C. E, association of TBP with core promoter regions of the indicated genes was measured by ChIP in wild type (BYΔ2) and tbp ts-1 (BYΔ2 ts-1) yeast grown in CSM after 1 h at 37 °C. Relative association in all cases was determined by quantitative PCR analysis of input and immunoprecipitated (IP) samples, and normalized to a non-transcribed region of ChrV (25). All error bars represent S.D. (n = 3 except for C, left panel, and E, PDC1, for which n = 2); asterisk in A indicates p < 0.05 and the double asterisk indicates p < 0.01.
FIGURE 8.
FIGURE 8.
Mutations that affect DNA looping or Ser-5 phosphorylation of the Pol II carboxyl-terminal domain do not alter histone H3 occupancy at active promoters. A, core promoter occupancy by histone H3, Rpb1, and TBP was measured by ChIP at the CHA1 promoter in wild type and sua7-1 yeast grown in CSM without (uninduced) and with (induced) 1 mg/ml of serine. B, core promoter occupancy by histone H3, Rpb1, and TBP was measured by ChIP at the indicated promoters in wild type and sua7-1 yeast grown in CSM. C, core promoter occupancy by histone H3 was measured by ChIP at the indicated promoters in wild type and bur2Δ kin28-as yeast grown in CSM-ura supplemented with 1 mg/ml of serine 60 min after addition of 1 mm NA-PP1. Relative association in all cases was determined by quantitative PCR analysis of input and immunoprecipitated (IP) samples, and normalized to a non-transcribed region of ChrV (25). Error bars represent S.D. (n = 3), and p values shown in B are from a paired t test.
FIGURE 9.
FIGURE 9.
Dependence of TBP and Srb5/Med18 binding on Pol II. TBP (A) and Srb5/Med18 (B) association with the indicated core promoters was measured by ChIP in wild type (RMY521) and rpb1-1 ts (Z111-Srb5-Myc) yeast grown in YPD after 1 h at 37 °C. Relative association in all cases was determined by quantitative PCR analysis of input and immunoprecipitated (IP) samples, and normalized to a non-transcribed region of ChrV (25). Error bars represent S.D. (n = 3).
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References

    1. Ansari S. A., Morse R. H. (2013) Mechanisms of Mediator complex action in transcriptional activation. Cell Mol. Life Sci. 70, 2743–2756 - PubMed
    1. Larivière L., Seizl M., Cramer P. (2012) A structural perspective on Mediator function. Curr. Opin. Cell Biol. 24, 305–313 - PubMed
    1. Ansari S. A., He Q., Morse R. H. (2009) Mediator complex association with constitutively transcribed genes in yeast. Proc. Natl. Acad. Sci. U.S.A. 106, 16734–16739 - PMC - PubMed
    1. Thompson C. M., Young R. A. (1995) General requirement for RNA polymerase II holoenzymes in vivo. Proc. Natl. Acad. Sci. U.S.A. 92, 4587–4590 - PMC - PubMed
    1. Lacombe T., Poh S. L., Barbey R., Kuras L. (2013) Mediator is an intrinsic component of the basal RNA polymerase II machinery in vivo. Nucleic Acids Res. 41, 9651–9662 - PMC - PubMed

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