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. 2015 Oct 30;43(19):9214-31.
doi: 10.1093/nar/gkv782. Epub 2015 Aug 3.

Mediator independently orchestrates multiple steps of preinitiation complex assembly in vivo

Fanny Eyboulet  1 , Sandra Wydau-Dematteis  1 , Thomas Eychenne  1 , Olivier Alibert  2 , Helen Neil  1 , Claire Boschiero  1 , Marie-Claire Nevers  3 , Hervé Volland  3 , David Cornu  4 , Virginie Redeker  4 , Michel Werner  1 , Julie Soutourina  5
Affiliations

Mediator independently orchestrates multiple steps of preinitiation complex assembly in vivo

Fanny Eyboulet et al. Nucleic Acids Res. .

Abstract

Mediator is a large multiprotein complex conserved in all eukaryotes, which has a crucial coregulator function in transcription by RNA polymerase II (Pol II). However, the molecular mechanisms of its action in vivo remain to be understood. Med17 is an essential and central component of the Mediator head module. In this work, we utilised our large collection of conditional temperature-sensitive med17 mutants to investigate Mediator's role in coordinating preinitiation complex (PIC) formation in vivo at the genome level after a transfer to a non-permissive temperature for 45 minutes. The effect of a yeast mutation proposed to be equivalent to the human Med17-L371P responsible for infantile cerebral atrophy was also analyzed. The ChIP-seq results demonstrate that med17 mutations differentially affected the global presence of several PIC components including Mediator, TBP, TFIIH modules and Pol II. Our data show that Mediator stabilizes TFIIK kinase and TFIIH core modules independently, suggesting that the recruitment or the stability of TFIIH modules is regulated independently on yeast genome. We demonstrate that Mediator selectively contributes to TBP recruitment or stabilization to chromatin. This study provides an extensive genome-wide view of Mediator's role in PIC formation, suggesting that Mediator coordinates multiple steps of a PIC assembly pathway.

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Figures

Figure 1.
Figure 1.
med17 thermosensitive mutants. (A) Med17 mutant phenotypes. Cultures of WT and mutant med17 yeast strains were serially diluted, spotted on YPD agar plates and incubated for 3 days at permissive (30°C) or non-permissive (37°C) temperatures. (B) Mass spectrometry analysis of Mediator integrity in med17 mutants. Mediator subunits identified by mass spectrometry analysis in WT and med17 mutants are indicated. (Immunoprecipitation) Mediator was immunoprecipitated through Med5-HA from crude extracts using magnetic protein G beads coupled to anti-HA antibodies. The MED17 strain carrying a non-tagged Mediator subunit was used as a negative control (C for Control IP). (Mediator purification) Core Mediator complex containing head, middle and tail modules was purified from med17 mutants and a wild-type strain. An asterisk marks Med21 subunit in med17-98 mutant that was detected in a second data acquisition identifying two unique peptides and also by western blotting with anti-Med21 antibody (Supplementary Figure S3B). (C) Silver-stain SDS-PAGE analysis of purified Mediator complex from the WT strain and med17 mutants. (D) Interaction between Pol II and Mediator in med17-504 mutant. Rpb3-Myc Med17-EGFP strains with WT MED17 or a med17 mutation were grown at 30°C in YPD medium and cross-linked or not with formaldehyde (FA), as indicated. Med17-EGFP was immunoprecipitated (IP) with anti-EGFP antibody from crude extracts (Input) and analyzed by western blotting with anti-Myc antibody (CoIP) against Rpb3. The cross-linked Rpb3-Med17 band is indicated in red. The position of unidentified cross-linked proteins with the tagged Med17 or Rpb3 subunits is indicated by a vertical bar.
Figure 2.
Figure 2.
Effect of med17 mutations on Mediator, Pol II, TFIIH and TBP occupancy. Cells were grown to exponential phase at 30°C on YPD medium and then transferred for 45 min to 37°C. Quantitative ChIP experiments were performed using anti-HA antibody (12CA5) against Mediator subunits Med6-HA (A), Med15-HA (B) or Med5-HA (C), Rad3-HA (E), Kin28-HA (F), TBP-HA (G) or anti-Rpb1 Pol II antibody (8WG16) (D). Immunoprecipitated DNA was amplified with primers corresponding to ADH1, PMA1 and PYK1 ORF (O) or promoters (P). P1 primers listed in Supplementary Table S3 located close to upstream regulatory regions were used for Mediator ChIP experiments and P2 primers located close to core promoters were used for Pol II, Rad3, Kin28 and TBP ChIP experiments. GAL1 ORF was used as a negative control. The mean values and standard deviation (indicated by error bars) of three independent experiments are shown.
Figure 3.
Figure 3.
Kinetics of galactose induction in med17-444, -504 and WT strains. Yeast strains were grown in raffinose-supplemented medium at 30°C, then galactose was added, cultures were transferred at the same time to 37°C and samples were collected for ChIP experiments at indicated time points upon galactose induction (T0, T20, T40 and T60 min). Quantitative ChIP experiments were performed using anti-HA antibody (12CA5) against Mediator subunit Med5-HA (A), TBP-HA (C) or anti-Rpb1 Pol II antibody (8WG16) (B). The mean values and standard deviation (indicated by error bars) of three independent experiments are shown. Immunoprecipitated fragments from ChIP experiments were amplified with primers corresponding to the GAL1 gene promoter (GAL1 P) or ORF (GAL1 O). A nontranscribed region on chromosome V was used as a negative control.
Figure 4.
Figure 4.
Enrichment profiles of Mediator, Pol II, TFIIH and TBP on yeast genome. Cells were grown at 30°C in YPD medium and then transferred for 45 minutes to 37°C. (A) Examples of Mediator, Pol II, TBP, and TFIIH ChIP-seq enrichment profiles on selected class II genes. Densities of sequence tags were assessed from ChIP-seq experiments performed with Med15-HA (Mediator), TBP-HA, Rad3-HA (TFIIH core), and Kin28-HA (TFIIK) strains using anti-HA antibody. Pol II was immunoprecipitated using anti-Rpb1 antibody. ChIP-seq density profiles are displayed using the IGB yeast genome browser. Input DNA and DNA from ChIP with an untagged strain were used as negative controls. Densities of sequence tags were displayed after subtraction of the normalized control of an untagged strain. The scales in kiloreads are indicated for each profile. (B) Distribution of Mediator, TBP, TFIIH and Pol II ChIP-seq densities around TSS. Intergenic regions encompassing Pol III-transcribed genes and divergent genes were excluded. The tag density was determined for each protein in a 1600-bp window centred on the TSS. The TSS positions were provided from previous studies (4,5). Mean tag density for each nucleotide position was then calculated and plotted over the window.
Figure 5.
Figure 5.
Global destabilization of PIC assembly in med17-444 and -504 mutant strains. The density of sequence tags in Med15, Rad3, Kin28 and TBP ChIP-seq experiments was calculated for promoter regions of Pol II-transcribed genes. Cells were grown at 30°C in YPD medium and then transferred for 45 minutes to 37°C. The density of sequence tags in the Pol II ChIP-seq experiments was calculated for the Pol II-transcribed genes. In all, 3852 Pol II-enriched genes were used for these analyses. Tag densities were normalized relative to qPCR data on a set of selected genes. Each point on the plot corresponds to one promoter region or one ORF. Promoter regions correspond to intergenic regions in tandem or in divergent orientation, excluding intergenic regions encompassing Pol III-transcribed genes. A linear regression (red line) for ChIP-seq density in mutant versus ChIP-seq density in WT and an R2 correlation coefficient are indicated. (A) Med15, Pol II, Rad3, Kin28 and TBP ChIP-seq density in med17-444 versus ChIP-seq density in WT on class II promoter regions or ORFs (for Pol II). (B) Med15, Pol II, Rad3, Kin28 and TBP ChIPseq density in med17-504 versus ChIP-seq density in WT on class II promoter regions or ORFs (for Pol II).
Figure 5.
Figure 5.
Global destabilization of PIC assembly in med17-444 and -504 mutant strains. The density of sequence tags in Med15, Rad3, Kin28 and TBP ChIP-seq experiments was calculated for promoter regions of Pol II-transcribed genes. Cells were grown at 30°C in YPD medium and then transferred for 45 minutes to 37°C. The density of sequence tags in the Pol II ChIP-seq experiments was calculated for the Pol II-transcribed genes. In all, 3852 Pol II-enriched genes were used for these analyses. Tag densities were normalized relative to qPCR data on a set of selected genes. Each point on the plot corresponds to one promoter region or one ORF. Promoter regions correspond to intergenic regions in tandem or in divergent orientation, excluding intergenic regions encompassing Pol III-transcribed genes. A linear regression (red line) for ChIP-seq density in mutant versus ChIP-seq density in WT and an R2 correlation coefficient are indicated. (A) Med15, Pol II, Rad3, Kin28 and TBP ChIP-seq density in med17-444 versus ChIP-seq density in WT on class II promoter regions or ORFs (for Pol II). (B) Med15, Pol II, Rad3, Kin28 and TBP ChIPseq density in med17-504 versus ChIP-seq density in WT on class II promoter regions or ORFs (for Pol II).
Figure 6.
Figure 6.
Global decrease of TFIIH core association independent of TFIIK in med17-670 and -98 mutant strains. The density of sequence tags in the Med15, Pol II, Rad3, Kin28 and TBP ChIP-seq experiments was calculated as in Figure 5. (A) Med15, Pol II, Rad3, Kin28 and TBP ChIP-seq density in med17-670 versus ChIP-seq density in WT on class II promoter regions or ORFs (for Pol II). (B) Med15, Pol II, Rad3, Kin28 and TBP ChIPseq density in med17-98 versus ChIP-seq density in WT on class II promoter regions or ORFs (for Pol II).
Figure 6.
Figure 6.
Global decrease of TFIIH core association independent of TFIIK in med17-670 and -98 mutant strains. The density of sequence tags in the Med15, Pol II, Rad3, Kin28 and TBP ChIP-seq experiments was calculated as in Figure 5. (A) Med15, Pol II, Rad3, Kin28 and TBP ChIP-seq density in med17-670 versus ChIP-seq density in WT on class II promoter regions or ORFs (for Pol II). (B) Med15, Pol II, Rad3, Kin28 and TBP ChIPseq density in med17-98 versus ChIP-seq density in WT on class II promoter regions or ORFs (for Pol II).
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