Volume 31, Issue 3, Pages 337-346 (August 2008) Mediator-Dependent Recruitment of TFIIH Modules in Preinitiation Complex  Cyril Esnault, Yad Ghavi-Helm, Sylvain Brun, Julie Soutourina, Nynke Van Berkum, Claire Boschiero, Frank Holstege, Michel Werner  Molecular Cell  Volume 31, Issue 3, Pages 337-346 (August 2008) DOI: 10.1016/j.molcel.2008.06.021 Copyright © 2008 Elsevier Inc. Terms and Conditions

Figure 1 Med11 Mediator Subunit Interacts with Rad3 Helicase of TFIIH (A) Two-hybrid interaction between Med11 and Rad3. Two-hybrid assays were performed in Y190 strains transformed by pGBT9, expressing Gal4 DNA-binding domain (GBD) or pGBT9-Med11 (GBD-Med11) and pACTII, expressing the Gal4 activating domain (GAD) or pACTII-Rad3-[72–256] (GAD-Rad3-[72–256]). Interaction activated a lacZ reporter gene that was revealed by a blue color in an X-gal overlay assay (Werner et al., 1993). (B) Rad3 conserved domain organization according to PFAM database (www.sanger.ac.uk/Software/Pfam/). A line indicates the Rad3 interaction domain with Med11. (C) Coimmunoprecipitation of Med11 and Rad3. Proteins from strains overexpressing TAP-Med11, Rad3-3HA, or both proteins were immunoprecipitated with magnetic beads coupled to IgG. The IPs were performed in buffers containing 100 to 400 mM NaCl. After immunoprecipitation, the proteins were revealed by western blotting using 12CA5 or PAP antibodies, binding to HA or TAP tag, respectively. Molecular Cell 2008 31, 337-346DOI: (10.1016/j.molcel.2008.06.021) Copyright © 2008 Elsevier Inc. Terms and Conditions

Figure 2 Med11 Mutations Affect the Interactions with Its Partners (A) Effect of the mutations on two-hybrid interactions between Med11 and its partners. Two-hybrid assays were performed in Y190 strains transformed by pGBT9 or pGBT9-Med11 mutants (GBD or GBD-Med11), pACTII, pACTII-Med17, pACTII-Med22, or pACTII-Rad3-[72–256] (GAD, GAD-Med17, GAD-Med22 or GAD-Rad3-[72–256]). The X-gal agarose assay was performed as in Figure 1. An interaction between Med11 or its mutants and Med17, Med22, or Rad3-[72–256] is indicated by the blue color. A paler shade of blue compared to wild-type indicates a decrease in the strength of interaction between a Med11 mutant protein and its partner. (B) Med11-T47A mutation decreases Rad3 coimmunoprecipitation with Mediator. Strains expressing TAP, TAP-Med11 or TAP-Med11-T47A, and Rad3-3HA were grown to exponential phase at 30°C. Protein extracts of these strains were immunoprecipitated in a buffer containing 400 mM NaCl with magnetic beads coupled to mouse IgG (Invitrogen). After immunoprecipitation, the proteins were revealed by western blotting using 12CA5 or PAP antibodies. (C) Mediator integrity revealed by CoIP in mutant strains. Wild-type and mutant strains were grown 45 min at 37°C. Med5-3HA was immunoprecipitated from cell extracts using magnetic beads coupled to anti-HA antibodies. Coimmunoprecipitations of Mediator subunits were revealed by western blotting using anti-HA, -Med6, -Med8, -Med14, -Med17, -Med18, or -Med20 antibodies. (D) Mediator and Pol II coimmunoprecipitated in Med11 mutant strains. Wild-type and mutant strains were grown 45 min at 37°C. Pol II was immunoprecipitated from cell extracts using magnetic beads coupled to an anti-Rpb1 antibody (8WG16). Negative controls were magnetic beads without antibody. After immunoprecipitation, the coimmunoprecipitated proteins were revealed by western blotting using anti-Rpb1 or anti-HA (12CA5) antibodies. Molecular Cell 2008 31, 337-346DOI: (10.1016/j.molcel.2008.06.021) Copyright © 2008 Elsevier Inc. Terms and Conditions

Figure 3 Effect of med11 Mutations on Pol II, TFIIE, cTFIIH, and TFIIK Occupancy Immunoprecipitated DNA from ChIP experiments was amplified with primers as indicated in the Supplemental Experimental Procedures. GAL1 open reading frame (ORF) was used as a negative control since its transcription is repressed in YPD. Values represent the average of three independent experiments. Error bars indicate the standard deviation and an asterisk indicates a significant difference between the wild-type and the mutant at p value <0.05 in a Student's t test. (A) Pol II occupancy. Cells were grown on YPD to OD0.6 at 600 nm, then transferred for 45 min to 37°C. Standard ChIP assays were performed on chromatin prepared from WT, med11-T47A, med11-L82P, and med11G108S strains using an antibody against Rpb1 CTD (8WG16). (B) cTFIIH and TFIIK subunits occupancy. Strains expressing Rad3-3HA, Ssl1-3HA, Kin28-3HA, or Ccl1-3HA in wild-type or mutant backgrounds were grown for 45 min at 37°C. Standard ChIP assays were performed using anti-HA antibody (12CA5). (C) TFIIE occupancy. Strains expressing Tfa1-3HA in wild-type or mutant backgrounds were grown for 45 min at 37°C. Standard ChIP assays were performed using anti-HA antibody (12CA5). Molecular Cell 2008 31, 337-346DOI: (10.1016/j.molcel.2008.06.021) Copyright © 2008 Elsevier Inc. Terms and Conditions

Figure 4 Genome-wide Analysis Reveals that the med11-T47A Mutation Specifically Affects TFIIK Occupancy (A) Genome-wide analysis of TFIIH modules occupancy. ChIP-chip analysis was performed on Kin28-3HA (TFIIK) and Rad3-3HA (cTFIIH) in med11-T47A and wild-type strains with 12CA5 antibody. Wild-type or mutant cells were grown as in Figure 3. The graphic represents the correlation of Kin28-3HA and Rad3-3HA in med11-T47A and the wild-type strains. The red dots correspond to the yeast genome part that presents a decrease superior to 2-fold of TFIIK occupancy. A linear regression and its equation are indicated. (B) Genome-wide analysis of Mediator occupancy. ChIP-chip analysis was performed on Med5-3HA in med11-T47A and wild-type strains with 12CA5 antibody. Wild-type or mutant cells were grown as in Figure 3. The graphic represents the correlation of Med5-3HA binding in med11-T47A and the wild-type strains. A linear regression and its equation are indicated. Molecular Cell 2008 31, 337-346DOI: (10.1016/j.molcel.2008.06.021) Copyright © 2008 Elsevier Inc. Terms and Conditions

Figure 5 med11-T47A Mutation Affects Pol II Phosphorylation (A) ChIP-chip analysis of total Pol II and S5P Pol II in med11-T47A and wild-type strains. Wild-type or mutant cells were grown as described in Figure 3. ChIP-chip assays were performed using an antibody against total CTD (8WG16) or against CTD phosphorylated on serine 5 (H14). The graphics represent the correlation between Pol II and S5P Pol II occupancy in med11-T47A and the wild-type strains. A linear regression was performed, and its equation is indicated. (B) Profiles of total and S5P Pol II on HST2, ADH1, and PYK1 in wild-type and med11-T47A strains. On the left, schematic promoter and coding region organization of HST2, ADH1, and PYK1 are presented. The location of the PCR fragments amplified in ChIP analysis is indicated. The open reading frame (ORF) is indicated as an open box. The vertical line indicates the position of the transcription initiation site. Wild-type or mutant cells were grown as in Figure 3. Standard ChIP assays were performed using an antibody against total (8WG16) or S5P Pol II (H14). Values represent the average of three independent experiments. Error bars indicate the standard deviation. Molecular Cell 2008 31, 337-346DOI: (10.1016/j.molcel.2008.06.021) Copyright © 2008 Elsevier Inc. Terms and Conditions

Figure 6 Mediator, TFIIH, and Pol II Enrichment over Selected Genes in Wild-Type Strain Examples of genomic regions bound by TFIIH and Mediator but not Pol II. ChIP-chip assays were performed using antibodies against Rpb1 (8WG16) or against the HA tag (12CA5) in wild-type strain grown as in Figure 3. The graphics present binding ratios of Med5-3HA (Mediator), Kin28-3HA (TFIIK), Rad3-3HA (cTFIIH), and Rpb1 (Pol II). Molecular Cell 2008 31, 337-346DOI: (10.1016/j.molcel.2008.06.021) Copyright © 2008 Elsevier Inc. Terms and Conditions

Figure 7 Branched Pathway Model of PIC Formation (A–E) The steps leading to PIC formation are depicted in the cartoon. Arrows connect the preinitiation intermediates in a branched pathway leading to PIC formation and Pol II phosphorylation. The intermediates that have been observed are indicated by solid colors. The intermediates that were inferred are indicated by the translucent colors. The color code used is as follows: red, essential Mediator subunits; yellow, dispensable Mediator subunits; dark green, cTFIIH; light green, TFIIK; blue, TFIIE; violet, Pol II. Molecular Cell 2008 31, 337-346DOI: (10.1016/j.molcel.2008.06.021) Copyright © 2008 Elsevier Inc. Terms and Conditions