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Direct Interaction of RNA Polymerase II and Mediator Required for Transcription in Vivo

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Science  18 Mar 2011:
Vol. 331, Issue 6023, pp. 1451-1454
DOI: 10.1126/science.1200188

Abstract

Gene transcription is highly regulated. Altered transcription can lead to cancer or developmental diseases. Mediator, a multisubunit complex conserved among eukaryotes, is generally required for RNA polymerase II (Pol II) transcription. An interaction between the two complexes is known, but its molecular nature and physiological role are unclear. We identify a direct physical interaction between the Rpb3 Pol II subunit of Saccharomyces cerevisiae and the essential Mediator subunit, Med17. Furthermore, we demonstrate a functional element in the Mediator–Pol II interface that is important for genome-wide Pol II recruitment in vivo. Our findings suggest that a direct interaction between Mediator and Pol II is generally required for transcription of class II genes in eukaryotes.

Mediator is a large multisubunit complex conserved in all eukaryotes (1). It acts as a link between specific protein regulators and the RNA polymerase II (Pol II) transcription machinery (2). Mediator is required at most Pol II–transcribed gene promoters for regulated gene expression (35). In Saccharomyces cerevisiae, Mediator is composed of 25 subunits and is organized in four structural modules: the tail, middle, head, and Cdk8 modules (6). A direct Mediator–Pol II interaction is indicated by previous copurification, coimmunoprecipitation (CoIP) experiments (79) and by in vivo formaldehyde cross-linking (10). A model of the Mediator–Pol II complex determined by electron microscopy (EM) at 35 Å resolution suggests that several Pol II subunits (Rpb1, 2, 3, 6, and 11) might contact the middle or the head of Mediator (11). It was recently suggested that Rpb4 and Rpb7 could also be implicated in interactions with Mediator (1214). However, the requirement of a direct interaction between Mediator and Pol II for transcription activation has not been demonstrated. Moreover, the identity of the Mediator subunits contacting Pol II is unknown because of the low resolution of the Mediator structure. As a consequence, the mechanism by which Mediator recruits Pol II is poorly understood.

To identify the subunit(s) of Mediator that directly contact Pol II and determine the role of these interactions in transcription regulation, we used an in vivo photo–cross-linking approach based on the incorporation by the cell-translation system of photo-activable analogs of methionine and leucine in proteins [see supporting online material (SOM) text and figs. S1 and S2] (15, 16).

Because EM results (11) suggested potential interactions of 16 Mediator subunits belonging to the head (Med6, 8, 11, 17, 18, 19, 20, 22) and middle (Med1, 4, 5, 7, 9, 10, 21, 31) modules with Rpb1, 2, 3, 6, or 11 Pol II subunits, we immunoprecipitated hemagglutinin (HA)–tagged proteins after in vivo cross-linking. Among the 80 pairwise contacts that we tested, only Myc-tagged Rpb3 and HA-tagged Med17 cross-linked (Fig. 1). These results demonstrate that the Rpb3 Pol II subunit directly interacts with the essential Med17 subunit of the Mediator head module, in line with the peripheral location of Rpb3 in the Pol II structure (17, 18) and with the general requirement for Med17 in transcription (4).

Fig. 1

Direct in vivo interaction of the Rpb3 Pol II subunit with the Med17 Mediator subunit. Rpb3-Myc Med17-HA or Rpb3-Myc Med11-HA strains grown in the presence or absence of photo-leucine plus photo-methionine (PLM) were ultraviolet (UV)–irradiated or not, as indicated. Proteins were immunoprecipitated (IP) with α-HA antibody from crude extracts (Input). Immunoprecipitated or coimmunoprecipitated (CoIP) proteins were analyzed by Western blotting (Wb) with α-HA or α-Myc antibodies, respectively. The cross-linked Rpb3-Med17 band is indicated. The position of unidentified cross-linked proteins with the tagged Mediator or Rpb3 subunits is indicated by a vertical bar. The position of the proteins and immunoglobulin G (IgG) heavy or light chains is indicated. An asterisk marks a nonspecific band. MW, molecular weight in kilodaltons.

A thermosensitive rpb3-2 mutant defective in activator-dependent transcription in vitro and in vivo was described previously by Tan et al. (19). The mutation contains two amino acid substitutions [Cys92→Arg92 (C92R) and Ala159→Gly159 (A159G)] (20) that have a strong synergistic effect on the thermosensitive phenotype. We mapped the mutated amino acid residues of rpb3-2 on the crystallographic structure of Pol II. C92 is located on the subunit surface, and A159 is close (~7 Å) to C92 (Fig. 2A). We tested whether the Med17-Rpb3 contact is modified in the rpb3-2 mutant. The in vivo photo–cross-linking approach could not be used in the rpb3-2 mutant, because the incorporation of the photo–amino acids was strongly diminished due to the mutant slow growth. We turned to a formaldehyde cross-linking strategy and detected Rpb3-Med17 cross-linking in the wild-type (WT) strain (Fig. 2B). This contact was strongly reduced in the rpb3-2 mutant. CoIP experiments with chromatin and soluble extracts further confirmed that Mediator–Pol II association was impaired in the rpb3-2 mutant (see SOM text and figs. S3 and S4).

Fig. 2

The rpb3-2 mutant is defective in Mediator-Pol II association and Pol II occupancy. (A) Positions of the residues (A159, C92) mutated in rpb3-2. A ribbon model of the yeast Pol II structure (Protein Data Bank code 1WCM) is presented. Pol II subunits are shown in gray, except for Rpb3, which is shown in orange. The blue spheres mark A159 and C92. A box zooms in on the surface of Rpb3 with C92 in blue. A159 is located beneath the subunit surface and is thus invisible on the enlargement. To focus on the Rpb3 surface, the structure in the box was rotated. (B) Med17-Rpb3 contact is affected in the rpb3-2 mutant. Rpb3-Myc Med17-HA or rpb3-2-Myc Med17-HA strains were cross-linked (or not) with formaldehyde (FA), as indicated. Med17-HA was immunoprecipitated with α-HA antibody from crude extracts (Input) and analyzed by Western blotting with α-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. An asterisk marks a nonspecific band. (C) Pol II and Mediator occupancy on the GAL1 gene upon galactose induction. The rpb3-2 and WT strains were grown in a raffinose-supplemented medium, then galactose was added for 45 or 120 min. ChIP was performed with a α-Rpb1 antibody (Pol II) and with a α-HA antibody against Med17-HA (Mediator). Mean values and SDs (indicated by error bars) of three independent experiments are shown. The location of the polymerase chain reaction (PCR) fragments is indicated (P1, O1, O2). A control corresponds to a nontranscribed region on chromosome V. IP/IN, ratio of immunoprecipitated DNA (IP) over input DNA (IN). (D) Genome-wide Pol II enrichment in rpb3-2 was compared with that of the wild type. A linear regression (red line) and its equation are indicated. The dotted line corresponds to y = x. Four biological replicates were performed for each experiment. R2, correlation coefficient.

Previous results have shown that the mRNA level of the GAL1-inducible gene was decreased in the rpb3-2 mutant (19). We therefore examined Pol II and Mediator occupancy on the GAL1 gene by chromatin immunoprecipitation (ChIP) experiments after galactose induction. Pol II occupancy of the GAL1 gene was significantly reduced in the rpb3-2 mutant compared with the wild type (Fig. 2C). In contrast, Mediator occupancy of the GAL1 promoter in the rpb3-2 mutant was similar to the WT level with only a marginal reduction after a 45-min induction. As for the GAL1 gene, a clear decrease of Pol II occupancy can be observed in the rpb3-2 mutant, but not for Mediator association to the ADH1 and PYK1 genes that are constitutively expressed (fig. S5). Our results suggest that the rpb3-2 mutant is affected in the Mediator–Pol II interaction leading to defective Pol II recruitment, even though other factors could also contribute to this defect. To investigate whether the decrease of Pol II occupancy in the rpb3-2 mutant affects transcription in general, we analyzed the genome-wide Pol II occupancy by ChIP followed by DNA hybridization on microarrays. Regression analysis of Pol II binding in the WT versus rpb3-2 mutant revealed a global 1.5-fold decrease in the mutant with a high correlation coefficient (Fig. 2D), suggesting that Med17-Rpb3 interaction is required for the recruitment of Pol II on most active genes.

To investigate the in vivo role of Med17 in Pol II recruitment, we selected 29 med17 thermosensitive mutants and showed that 5 of them (med17-68, -158, -208, -257, and -327) were colethal with the rpb3-2 mutation (Fig. 3A and fig. S6A). In a RPB3 WT background, these five med17 mutants had diverse slow-growth phenotypes at 30°C (fig. S6B). The allele-specific lethality between the rpb3-2 and med17 mutations is consistent with a physiological role for the Med17-Rpb3 interaction.

Fig. 3

Specific med17 thermosensitive mutations are colethal with rpb3-2 and show severe Pol II occupancy defects. (A) med17 thermosensitive mutations are colethal with rpb3-2. Cells were serially diluted, spotted on yeast extract, peptone, and dextrose (YPD)– or 5-FOA–containing agar plates (to counterselect the WT MED17-bearing plasmid), and incubated at 30°C for 7 days. (B) Cells were grown at 30°C. ChIP was performed with a α-Rpb1 antibody for Pol II. Mean values and SDs (error bars) of three independent experiments are shown. The GAL1 open reading frame (ORF) was used as a control.

To analyze the molecular nature of the med17 thermosensitive mutations showing specific synthetic phenotypes in rpb3-2 background, we determined the Pol II and the Mediator occupancy in the med17-68, -208, and -257 mutants by ChIP experiments in a RPB3 WT background. A strong decrease in Pol II association with the ADH1 and PYK1 genes was observed in these mutants compared with the wild type (Fig. 3B). In contrast, the Mediator association (measured by the Med5 middle, Med8 head, or Med15 tail subunit ChIP) to the ADH1 and PYK1 gene promoters was similar to the WT level (fig. S6, C, D, and E). The integrity of Mediator in med17 mutants has been verified by CoIP experiments (fig. S7).

Using random mutagenesis of MED17, we looked for med17 mutants that would suppress the growth defects of rpb3-2 (C92R A159G) at 37°C. No suppressor was obtained, but we isolated several med17 mutants that suppressed the mild thermosensitive defect of rpb3-A159G single mutant (Fig. 4A). One representative suppressor mutant, med17-sup1 (I128V R582G N595D), was chosen for further analysis. We determined the effect of med17-sup1 mutation on Pol II and Mediator occupancy in the rpb3-A159G background. Figure 4B and fig. S8 show that the association of Pol II to the ADH1, PYK1, and PHO84 genes is slightly but significantly decreased in the rpb3-A159G mutant and is restored to WT levels in the rpb3-A159G med17-sup1 strain. The Mediator association to the ADH1, PYK1, and PHO84 promoters remained at WT levels (fig. S8B). As for Pol II, the association of the general transcription factor TFIIH was also diminished in the rpb3-A159G mutant, and the med17 suppressor restored near WT TFIIH occupancy (fig. S8C).

Fig. 4

The med17 mutation suppresses the rpb3-A159G growth phenotype and Pol II–association defects. (A) The med17-sup1 mutation suppresses the rpb3-A159G growth phenotype. Cells were grown in a liquid YPD medium at 30°C and then shifted to 37°C at time 0 min. The optical density at 600 nm (OD600) is indicated on the y axis. The growth curves for WT strain, rpb3-A159G, and rpb3-A159G med17-sup1 mutants are shown in black, green, and blue, respectively. (B) Cells were grown at 30°C and then shifted to 37°C for 5 hours. ChIP was performed with a α-Rpb1 antibody for Pol II. Mean values and SDs (error bars) of three independent experiments are shown. The GAL1 ORF was used as a control. The location of the PCR fragments is indicated (P1, P2, O1, O2, O3).

MED17 was initially isolated as SRB4, in reference to mutations that suppressed the growth phenotype of truncations of the Pol II Rpb1 C-terminal repeat domain (CTD) (9). The med17 suppressors were unable to compensate the conditional-growth phenotypes of the rpb1-Δ104 CTD-truncation mutant (fig. S9), indicating that the suppression is specific for rpb3-A159G.

A functional link between Mediator and Pol II CTD was proposed after the identification of suppressors of CTD-truncation mutants affecting the Mediator subunits (9, 21), because Mediator plays a stimulatory role in CTD phosphorylation by TFIIH (7, 22) and binds to glutathione S-transferase–CTD (23). However, a low-resolution model of the Mediator–Pol II complex suggests that, beyond its presumed interaction with CTD, Mediator might engage in multiple contacts with the enzyme (11). Here, we have shown that Med17 interacts with Rpb3, and we selected med17 suppressors specific for rpb3-A159G that had no SRB phenotype. Further, we observed allele-specific lethality between rpb3-2 and med17 thermosensitive mutations. The cross-linking approach that we used probably revealed only a subset of in vivo interactions. Indeed, we did not test all potential contacts between subunits of Mediator and Pol II, and we used only two photo-activable amino acids. Thus, it is possible that other contacts exist between the Mediator complex and Pol II.

We showed that the Rpb3 subunit plays an essential role in Pol II recruitment to most class II genes and that the Med17 Mediator subunit function is central to preinitiation complex formation. A direct contact between Med17 and Rpb3 that is structurally homologous to the bacterial α subunit implicated in transcription activation (19, 24) further supports the apparent conservation of the RNA polymerase surface involved in transcription regulation from prokaryotes to eukaryotes, even though the molecular mechanisms and interacting partners differ.

Our results support the notion that the direct interaction of the Rpb3 Pol II subunit with the Med17 Mediator subunit is essential for global Pol II recruitment in vivo. Therefore, Mediator might be considered a general transcription factor (25). In addition, with the conservation of Pol II (26) and Mediator (1), the mechanism of transcription activation by Pol II recruitment through the contact between Rpb3 and Med17 may extend to all eukaryotes.

Supporting Online Material

www.sciencemag.org/cgi/content/full/331/6023/1451/DC1

Materials and Methods

SOM Text

Figs. S1 to S9

Tables S1 to S3

References

References and Notes

  1. Materials and methods are available as supporting material on Science Online.
  2. Single-letter abbreviations for the amino acid residues are as follows: A, Ala; C, Cys; G, Gly; and R, Arg.
  3. We thank N. Woychik, P. Thuriaux, O. Gadal, and B. Guglielmi for strains or plasmids; S. Hahn and M. Ptashne for antibodies; the Service de Pharmacologie et d’Immuno Analyse (CEA/Saclay) for monoclonal antibodies; A. Sentenac, A. W. Rutherford, and P. Thuriaux for discussions and critical reading of the manuscript; P. Thuriaux for help with crystal-structures visualization; Y. Ghavi-Helm and M. Pinskaya for help with ChIP-chip data analyses; and B. Guglielmi for initial interest in Mediator–Pol II interaction. This work was supported by the Agence Nationale de la Recherche (ANR-08-BLAN-0229) and Association pour la Recherche sur le Cancer (subvention fixe 5118). J.S. and S.W. performed the experimental work and analyzed the results, Y.A. participated in photo–cross-linking experiments and C.B. in yeast-strain constructions, and J.S. and M.W. designed the research and wrote the manuscript. Raw ChIP-chip data are available at Array Express under accession number E-MEXP-3043 (www.ebi.ac.uk/arrayexpress).
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