Site-specific Srb10-dependent phosphorylation of the yeast subunit Med2 regulates expression from the 2-␮m plasmid

Magnus Hallberg*, Gennady V. Polozkov*, Guo-Zhen Hu†, Jenny Beve‡, Claes M. Gustafsson‡, Hans Ronne†, and Stefan Bjo¨ rklund*§

*Department of Medical Biochemistry and Biophysics, Umeå University, SE-901 87 Umeå, Sweden; †Department of Plant Biology and Forest Genetics, Swedish University of Agricultural Sciences, Box 7080, SE-75007 Uppsala, Sweden; and ‡Department of Medical Nutrition, Karolinska Institute, Novum, SE-141 86 Huddinge, Sweden

Communicated by Roger D. Kornberg, Stanford University School of Medicine, Stanford, CA, January 12, 2004 (received for review November 11, 2003) The yeast Mediator complex is required for transcriptional regu- holo-pol II to specific promoters, it is difficult to understand why lation both in vivo and in vitro, and its function is conserved in all recruitment in this case would lead to down-regulation of eukaryotes. Mediator interacts with both transcriptional activators transcription. Recruitment might therefore be required for and RNA polymerase II, but little is known about the mechanisms regulation of transcription, both activation and repression, but by which it operates at the molecular level. Here, we show that the yet unknown mechanisms may determine the effect of the cyclin-dependent kinase Srb10 interacts with, and phosphorylates, recruitment. Recent reports suggest that recruitment of holoen- the Med2 subunit of Mediator both in vivo and in vitro. A point zyme is not needed for each round of transcription. Rather, the mutation of the single phosphorylation site in Med2 results in a mediator-pol II interaction is dynamic, and both Mediator and strongly reduced expression of the REP1, REP2, FLP1, and RAF1 several general transcription factors remain at the promoter , which are all located on the endogenous 2-␮m plasmid. after release of pol II where they function as a scaffold for Combined with previous studies on the effects of SRB10͞SRB11 reinitiation by pol II. It is therefore of interest to study how deletions, our data suggest that posttranslational modifications of interactions between Mediator subunits, and interactions be- Mediator subunits are important for regulation of gene expression. tween Mediator, general transcription factors, and pol II can be regulated. transcriptional regulation ͉ Srb11 ͉ RNA polymerase II We here present evidence that posttranslational modifications may significantly modify the ability of Mediator to support he Mediator complex was originally identified in Saccharo- transcription of specific genes. We believe that this observation Tmyces cerevisiae as an activity required for transcriptional suggests a mechanism by which Mediator may switch between activation in an in vitro transcription system reconstituted from being a positive or a negative coregulator of transcription. highly purified RNA polymerase II (pol II) and general tran- Materials and Methods scription factors (1, 2). Mediator was later purified to homoge- neity and shown to be a complex composed of 20 subunits that Yeast Strains. All yeast strains except Rgr1-TAP (22) and its interacts with the C-terminal domain of the largest pol II subunit derivative MHY2 were congenic to W303-1A and thus carry the (3, 4). More recent work has described several subunit–subunit ade2-1 can1-100 his3-11,15 leu2-3,112 trp1-1 ura3-1 markers. Strains H707 (MATa -⌬2::HIS3), H844 (MATa interactions and subdomains within Mediator (5–8), and low- ⌬ ␣ ⌬ resolution structures of Mediator alone or in complex with pol srb10- 1::LEU2) and H906 (MAT med2- 1::HIS3) have been II have been determined by three-dimensional reconstruction described (8, 23). To create the site-directed serine-208 to from electron micrographs of single particles (9, 10). These alanine mutation we used overlap extension PCR. The resulting structures indicate a division of Mediator into distinct domains. PCR fragment was cloned into a derivative of the pFL34 vector (24) in which the HindIII site had been destroyed. The resulting In parallel with the identification of yeast Mediator, a pol II plasmid was digested with HindIII before transformation of the holoenzyme that comprised both Mediator subunits [a subset of Rgr1 TAP-tagged strain and the W303-1A strain. Transformants general transcription factors and additional (i.e., Srb8- were selected on plates lacking uracil. Mutated revertants se- 11)] was purified (11). Nine SRB genes [SRB2,-4,-5,-6,-7, -8, lected on plates containing 5-fluoro-orotic acid were analyzed by -9, and -10 (encoding the cyclin C-dependent kinase), and -11 sequencing. (encoding cyclin C)] were identified in a genetic screen for suppressors of a cold-sensitive phenotype caused by a truncation Growth Media. Low-phosphate medium (LPM) contains 30 mg of of the C-terminal domain. Although Srb2, -4, -5, -6, and -7 are KH PO , 25 mg of MgSO ϫ 7HO, 100 mg of NaCl, 100 mg of well established as Mediator subunits, the Srb8, -9, -10, and -11 2 4 4 2 CaCl ϫ 2HO,2gofasparagine,1gofKCl, amino acids (SC proteins have not been identified in Mediator described by 2 2 media), and 20 g of glucose per liter. No-phosphate medium is Kornberg and coworkers (1–4). In contrast, homologues of identical to LPM without KH PO . Srb10 and Srb11 are present in some Mediator preparations 2 4 from human cells (12–16). Recent studies suggest that Mediator Expression and Purification of Proteins. Cloning, expression, and exists in two specific forms. Mediator containing the Srb8 to -11 purification of a 6ϫ histidine-tagged Med1 has been described subunits is always present in free form whereas Mediator lacking (8). The Med1 used in this study was further purified by this submodule may associate with pol II. The relative levels of chromatography on a UnoQ column (Bio-Rad). The coding Srb8–11 vary because this submodule is degraded when yeast is grown under conditions of nutrient limitation (17, 18), and the relative levels are also sensitive to the extraction procedure (19). Abbreviations: pol II, RNA polymerase II; PVDF, poly(vinylidene difluoride); CDK, cyclin- Activators contact individual Mediator subunits and recruit dependent kinase; TAP, tandem affinity purification. the Mediator complex to promoters during transcriptional acti- §To whom correspondence should be addressed. E-mail: stefan.bjorklund@medchem. vation. However, repressors have also been shown to interact umu.se. directly with Mediator subunits (20, 21). If repressors also recruit © 2004 by The National Academy of Sciences of the USA

3370–3375 ͉ PNAS ͉ March 9, 2004 ͉ vol. 101 ͉ no. 10 www.pnas.org͞cgi͞doi͞10.1073͞pnas.0400221101 Downloaded by guest on September 30, 2021 sequences for Srb10 and Srb11 were amplified from genomic package MICROARRAY SUITE 5.0. Data were used for all probe DNA by PCR and then cloned into the pGEX-6p-2 vector. We sets representing transcripts, called ‘‘present’’ by MICROARRAY expressed the GST-tagged Srb10 and Srb11 in Escherichia coli SUITE 5.0, in at least two arrays. The transcription analysis was BL21 cells and purified the recombinant proteins by using performed with RNA prepared from two WT and two med2- glutathione-Sepharose 4B beads according to the manufactures S208A cell cultures. Each of the two WT RNA preparations was recommendations (Amersham Pharmacia Biosciences). For compared with both the med2-S208A RNA preparations, gen- analysis of cyclin-dependent kinase (CDK)-phosphorylated pro- erating in total four comparisons (Exps. 1–4). The fold changes teins, Mediator was purified from a strain expressing a tandem were calculated by using the average of the four comparisons. affinity purification (TAP)-tagged Rgr1 as described previously Expression of REP1, REP2, FLP1, RAF1, and MED1 mRNAs (25). For preparation of whole-cell extracts (WCE), S. cerevisiae was quantified by using the Titanium one-step RT-PCR Kit strains were grown to an OD600 of 1.2 in 100 ml of YNB (CLONTECH). In brief, 2 ng of total RNA from either the WT (W303-1A) or YNB lacking uracil and tryptophane [H905 or the med2-S208A strain were reverse transcribed at 50°Cfor1h (med2) and H844 (srb10)]. Isolation of WCE was performed as and then amplified by using 30 PCR cycles. RT-PCR products described (23). The Med2͞Pgd1 dimer purification has been were subsequently analyzed by agarose gel electrophoresis. described (J.B., and C.M.G., unpublished results). Results In Vivo Phosphorylation Assays. Yeast strains W303-1A, H707 Phosphorylation of Mediator Subunits in Vivo. We have previously (med1), and H844 (srb10) were grown at 30°C in low-phosphate reported that a LexA fusion to the Med1 may strongly medium to a concentration of 1 ϫ 107 cells per ml. The cells were activate transcription from promoters containing LexA binding harvested by centrifugation and resuspended at 5 ϫ 107 cells per sites and that this LexA-Med1 dependent activation is regulated 32 ͞ ͞ ml in no-phosphate medium. To these cells, 0.5 ml of PO4 (10 by the Srb10 Srb11 cdk cyclin complex (23). We therefore mCi͞ml)(1 Ci ϭ 37 GBq) was added, and the cultures were wanted to test whether Med1 was a target for the Srb10 kinase incubated at 30°C with shaking for 20 min. The cultures were activity. To this end, we made in vivo phosphorylation experi- then diluted 10 times with low-phosphate medium and incubated ments where Mediator was immunopurified by using Med1 with shaking at 30°C for 4–6 h. Mediator was immunoprecipi- antibodies, both from WT cells and from cells lacking either tated from the whole-cell extract by using anti-Med1 antibodies Srb10 or Med1 (negative control for immunoprecipitation) in 32 (8) coupled to Protein A agarose beads. the presence of PO4. We identified two labeled polypeptides of Ϸ40 and 60 kDa that were present in Mediator purified from WT In Vitro Phosphorylation Assays. Samples containing 0.5 ␮gof cells but absent in Mediator purified from cells lacking Srb10 substrate (Med1 or Med2͞Pgd1) were incubated at 30°C for 20 (Fig. 1a). We have previously reported that Med4 is phosphor- min with GST-Srb10 (0.25 ␮g) and GST-Srb11 (0.25 ␮g) in ylated in vivo (23). The labeled 40-kDa polypeptide could ͞ ͞ CDK-buffer (20 mM Hepes-KOH 10 mM MgCl2 1mM therefore correspond to Med4 because it has a predicted mo- EGTA͞1mMDTT͞2mg͞ml BSA and protease inhibitors), 100 lecular mass of 32 kDa but migrates as an Ϸ38-kDa protein. ␮M ATP, and 1 ␮l[␥-32P]ATP (3,000 Ci͞mmol) in a final However, we found no indication that Srb10 phosphorylates volume of 25 ␮l. Labeled proteins were visualized by separation Med4 in any of the other experiments presented in this article. on 10% SDS͞PAGE followed by autoradiography. As for the labeled 60-kDa polypeptide, the only Mediator subunits in that molecular mass range are Med1 and Med2. We Immunoprecipitation Assays. For studies of the in vivo substrate for therefore proceeded to investigate whether Srb10 can phosphor- Srb10-phosphorylation, whole-cell extracts (350 ␮g) from the ylate either of these two proteins. W303-1A, med2, and srb10 strains expressing a lexA-Srb7 fusion protein were isolated, and Mediator was immunopurified by Phosphorylation of Med2 by Srb10 in Vitro. We next investigated using monoclonal lexA antibodies coupled to protein A-agarose whether the Srb10 kinase could phosphorylate Med1 or Med2 in beads as described (23). For studies of interaction between Med2 recombinant form (Fig. 1b). Expression and purification of 6 ϫ and Srb10͞Srb11, we used the following: (i)20␮g of GST- histidine-tagged Med1 has been described (8). Med2 is insoluble protein, (ii)25␮g of GST-Srb10, (iii)25␮g of GST-Srb11, and when expressed alone, but soluble untagged Med2 protein was (iv)25␮g of GST-Srb10 plus 25 ␮g of GST-Srb11. Proteins were obtained by coexpression with the Mediator subunit Pgd1 in a then immunoprecipitated by using anti-GST monoclonal anti- baculovirus expression system. The Med2͞Pgd1 dimer was then bodies (Amersham Pharmacia Biotech) coupled to protein purified to near homogeneity. Finally, the Srb10 and Srb11 G-agarose with 25 ␮g of Med2͞Pgd1 protein being added to each proteins were expressed as GST-fusion proteins in E. coli and tube. After 2 h incubation by rotation at 4°C, the beads were purified as described in Materials and Methods. ϫ ͞

washed three times with buffer W (1 PBS 0.8 M potassium Initially, we performed in vitro phosphorylation experiments BIOCHEMISTRY acetate͞0.5% Nonidet P-40) and once with 1ϫ PBS. The pre- where Srb10͞Srb11 was incubated with [32P]ATP alone or with cipitated proteins were analyzed by Western blotting by using either Med1 or Med2͞Pgd1. We could not observe any phos- antibodies specific for Med2. phorylation of Med1 or Srb10. Thus, the phosphorylated 60-kDa polypeptide identified in Fig. 1a was not due to autophosphor- Affymetrix GeneChip Probe Array Analyses and RT-PCR. Total yeast ylation of Srb10͞Srb11 or to phosphorylation of Med1. We did, RNA was isolated by using a hot acid phenol extraction protocol however, observe a phosphorylated polypeptide of Ϸ60 kDa (26). Poly(A)ϩ RNA was prepared from total RNA by using a when Srb10͞Srb11 was incubated with Med2͞Pgd1 (Fig. 1c). Qiagen (Valencia, CA) Oligotex Midi Kit. The cDNA synthesis, This observation suggested to us that Med2 was phosphorylated, cRNA synthesis, and labeling, as well as array hybridization to but we could not rule out Pgd1 because a phosphorylation could Affymetrix yeast S98 arrays, were performed at the Karolinska change the protein’s behavior during SDS͞PAGE. Institute Affymetrix core facility as described in the Affymetrix To identify which protein of Med2 and Pgd1 that is phosphor- users’ manual [Affymetrix GeneChip Expression Analysis Tech- ylated by Srb10, we performed in vitro phosphorylation exper- nical Manual (2000), Affymetrix, Santa Clara, CA]. Arrays were iments with purified, recombinant Srb10, Srb11, Med2͞Pgd1, washed and stained by using the GeneChip Fluidics Station 400 and unlabeled ATP. After incubation, the proteins were sepa- and scanned in an Affymetrix GeneArray Scanner. Acquisition rated by SDS͞PAGE, transferred to poly(vinylidene difluoride) and quantification of array images as well as primary data (PVDF) membranes, and blotted with Med2 antibodies. Fig. 2a analysis were performed by using the Affymetrix software shows that a fraction of Med2 migrated faster when incubated

Hallberg et al. PNAS ͉ March 9, 2004 ͉ vol. 101 ͉ no. 10 ͉ 3371 Downloaded by guest on September 30, 2021 Fig. 2. Med2 is phosphorylated by and interacts with the Srb10 kinase in vitro.(a) Purified, recombinant Med2͞Pgd1 was incubated alone, with calf intestine alkaline phosphatase (CIAP), or with Srb10͞Srb11 and unlabeled ATP. Proteins were separated on a 10% SDS͞PAGE gel, transferred to PVDF membranes, and blotted with antibodies specific for the Med2 protein. The same batch of prestained molecular mass markers was used to align this Western blot with the autoradiograph presented in Fig. 1c. The asterisk next to the plus sign above the rightmost lane in the figure indicates that [32P]ATP was added in this experiment. Arrows to the left indicate the positions of Med2 and phosphorylated Med2 (Med2-P). (b) Copurified, recombinant un- tagged Med2 and Pgd1 were subjected to coimmunoprecipitation with re- combinant GST protein (lane 1), GST-tagged Srb10 alone (lane 2), GST-tagged Srb11 alone (lane 3), or with GST-Srb10 and GST-Srb11 together (lane 4), by using monoclonal anti-GST antibodies. Precipitated proteins from each reac- tion were separated on a 10% SDS͞PAGE gel and immunoblotted with anti- Fig. 1. Phosphorylation of a Mediator subunit by Srb10 and Srb11. (a) WT, bodies specific for the Med2 protein. Arrows to the right indicate the positions 32 of prestained molecular mass markers. med1⌬, and srb10⌬ yeast strains were labeled in vivo with PO4. Mediator was immunoprecipitated from each strain by using Med1 antibodies coupled to Protein A agarose beads, and bound proteins were separated on a 10% SDS͞PAGE gel. Immunoprecipitation from extracts isolated from cells lacking vious in vitro phosphorylation experiment showed that the faster Med1 was made as a control for nonspecific immunoprecipitation. The aster- migrating Med2 species migrated at the same position as the isks indicate the position of the 60-kDa protein, which is specifically labeled labeled 60-kDa protein (the rightmost lane in Fig. 1c is aligned only in the WT strain. (b) Coomassie-stained protein gels of the peak fractions from the final purification steps of Srb10, Srb11, Med1, and Med2͞Pgd1. with lane 3 in Fig. 2a). We conclude that the phosphorylated Arrows indicate the position of each protein as indicated above each lane. The protein was Med2 and that the phosphorylated form of Med2 identity of Med1, Srb10, and Srb11 was confirmed by N-terminal amino acid migrated slightly faster than the unphosphorylated form. Finally, sequencing. (c) Purified, recombinant Srb10͞Srb11 was incubated with it should be noted that only a fraction of Med2 was phosphor- [32P]ATP alone, together with purified recombinant Med1, or with purified ylated under the conditions used here. recombinant Med2͞Pgd1 as indicated in the figure. Labeled proteins were separated on a 10% SDS͞PAGE gel and subjected to autoradiography. Arrows Med2 Interacts with Srb10 in Vitro. Phosphorylation of Med2 by the to the left (in a and c) indicate the positions of prestained molecular mass ͞ markers. Srb10 Srb11 cyclin-kinase suggested a direct physical interaction between Med2 and Srb10 or Srb11. To test whether such an interaction could be detected in vitro, we analyzed whether Med2 with Srb10, Srb11, and ATP. A similar increase in the migration could be coimmunoprecipitated with either GST-tagged Srb10 rate after phosphorylation has been reported previously, i.e., alone, GST-Srb11 alone, or with GST-Srb10 and GST-Srb11 when Cdk2 is phosphorylated by CDK-activating kinase (CAK) together. Fig. 2b shows that Med2 is specifically precipitated only 1, another cyclin-dependent kinase (27). Alignment of the filter when GST-Srb10 is prebound to the beads, i.e., lane 2 (GST- from this Western blot with the autoradiograph from the pre- Srb10 alone) and lane 4 (GST-Srb10 and GST-Srb11). In con-

3372 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0400221101 Hallberg et al. Downloaded by guest on September 30, 2021 Fig. 4. Mutation of the Med2 serine-208 to alanine abolishes the Srb10- dependent phosphorylation of Med2. (a) The predicted amino acid sequence of Med2. The positions of the two potential CDK-dependent phosphorylation sites are indicated. (b) Mediator was purified from a WT (Wt) strain and from the med2-S208A strain. Both strains also expressed TAP-tagged Rgr1, which was used for affinity purification. Purified Mediator from each strain was immunoblotted with anti-SerP-Pro͞Lys, anti-Med2, anti-Med4, and anti- Med1 antibodies as indicated.

Med2 and Pgd1 usually are present in substoichiometric amounts (4). The purified fraction was then analyzed by Western blotting by using either Med2 antibodies (Fig. 3b Left), or anti-SerP- Pro͞Lys antibodies (monoclonal antibody PSER-16B4, nano- Tools Antiko¨rpertechnik, Teningen, Germany; Fig. 3b Right) specific for proteins containing a phosphorylated CDK phos- phorylation site (a phosphorylated serine or threonine followed by proline). In agreement with the in vivo labeling result (Fig. 1a), the Med2 antibodies detected one protein of Ϸ60 kDa in the purified Mediator, which migrated at the same position as the Fig. 3. Med2 is phosphorylated by Srb10 in vivo.(a) A silver-stained gel from protein detected with the anti-SerP-Pro͞Lys antibody. This the peak-fraction of TAP-purified Mediator. Arrows to the right indicate the molecular mass of protein standards. (b) The Mediator fraction shown in a was result corroborates the in vitro phosphorylation results presented transferred to PVDF membranes and blotted with anti-Med2 antibodies (left in Fig. 2a. The absence of the slower migrating species seen in filter) and anti-SerP-Pro͞Lys antibodies (right filter). The two filters were Fig. 2a further suggests that only the phosphorylated, fast aligned in silico by using the prestained protein marker proteins as reference. migrating form of Med2 is present in Mediator. (c) Mediator was immunoprecipitated with anti-lexA antibodies from whole- To further establish that Med2 is an in vivo substrate for the cell extracts isolated from WT, med2⌬, and srb10⌬ strains. Each strain also Srb10 kinase, Mediator was immunoprecipitated with anti-lexA expressed a lexA-Srb7 fusion protein from a low copy number CEN plasmid antibodies from yeast strains expressing the lexA-Srb7 fusion BIOCHEMISTRY (23). The same amount of protein from each strain was separated on a 10% SDS͞PAGE gel, transferred to PVDF membranes, and blotted with either protein in different genetic backgrounds. Mediator isolated from anti-Med1 antibodies (Lower) or anti-SerP-Pro͞Lys antibodies (Upper). WT, med2, and srb10 strains was first blotted with anti-Med1 antibodies to confirm that equal amounts of Mediator was loaded to each lane (Fig. 3c Lower). The same filter was then trast, Med2 did not interact with GST-Srb11 alone (lane 3) or blotted with the anti-SerP-Pro͞Lys antibodies (Fig. 3c Upper). A with recombinant GST (lane 1). phosphorylated protein of Ϸ60 kDa was detected only in Me- diator isolated from the WT strain but absent in Mediator Phosphorylation of Med2 by Srb10 in Vivo. We conclude from our isolated from strains lacking either Med2 or Srb10. We conclude experiments that Srb10 can phosphorylate Med2 in vitro, but it that the 60-kDa polypeptide reflects Srb10-dependent phos- was still unclear whether the 60-kDa phosphoprotein identified phorylation of Med2 in vivo. in the in vivo phosphorylation experiments (Fig. 1a) was Med2. We therefore isolated Mediator from a yeast strain expressing a Identification of the Phosphorylation Site in Med2. The predicted TAP-tagged Rgr1 Mediator subunit (Fig. 3a). The Med2 protein amino acid sequence of Med2 contained only two potential appeared as a weak, fuzzy band in the purified Mediator, and the CDK-phosphorylation sites, at positions 6 and 208 (Fig. 4a). For Pgd1 subunit was nearly undetectable. The observed pattern is technical reasons, we first chose to mutate serine-208 to alanine consistent with earlier reports of purified Mediator where both (see Materials and Methods). Mediator was then affinity purified

Hallberg et al. PNAS ͉ March 9, 2004 ͉ vol. 101 ͉ no. 10 ͉ 3373 Downloaded by guest on September 30, 2021 from a med2-S208A strain expressing a TAP-tagged Rgr1, and proteins were transferred to PVDF membranes. Immunoblotting with anti-Med2, anti-Med4, and anti-Med1 antibodies (Fig. 4b) showed that Med2 was precipitated equally efficiently from extracts isolated from both the WT strain and the med2-S208A mutant. We also blotted the same filter with Pgd1 antibodies to study whether the Med2-S208A mutation had any effect on the interaction between Med2 and Pgd1. However, the anti-Pgd1 antibodies we had available showed crossreactivity to several proteins, which made it impossible to interpret the results (data not shown). Reblotting of the same membrane using the anti- SerP-Pro͞Lys antibodies showed that a phosphorylated polypep- tide with the same molecular mass as Med2 is present in the WT, but absent in the med2-S208A mutant. This finding demon- strated that the serine-208 to alanine mutation abolished the Srb10-dependent phosphorylation of Med2. Finally, the equally efficient precipitation of Med1, Med2, and Med4 from both extracts showed that phosphorylation of Med2 has no major effect on the subunit composition of Mediator.

The med2-S208A Mutation Strongly Reduces Expression of Genes Encoded on the Endogenous 2-␮m Plasmid. To investigate whether the Med2-S208A mutation has a detectable phenotype, we compared its growth to that of the W303-1A parental strain under a number of different growth conditions, for example, growth on galactose or at 37°C. However, the med2-S208A mutant behaved similarly to the WT strain under all conditions tested. To search for more specific differences between the WT strain and the med2-S208A mutant, whole-genome expression profiles were determined with DNA chip technology (Affymetrix). Dif- ferences in specific transcript levels were determined under rich conditions [yeast extract͞peptone͞dextrose (YPD)]. In agree- ment with the mild phenotypes observed, the transcription of only a subset of 19 genes was changed Ͼ1.4-fold in the med2- S208A mutant (Fig. 5a). Most of these genes were only mildly affected, but the transcription of four genes was dramatically decreased in the med2-S208A mutant strain. The REP1, REP2, FLP1, and RAF1 genes are all encoded for on the endogenous 2-␮m plasmid, and the transcription of these genes was high in the WT strain, but essentially quenched in the med2-S208A mutant strain. These results were confirmed by quantitative RT-PCR assays (Fig. 5b). As a control, we also determined the relative levels of the 2-␮m plasmid in both strains by using Fig. 5. Mutation of the Med2 serine-208 to alanine strongly reduces expres- quantitative PCR. Fig. 5c shows that the levels of the 2-␮m sion of REP1, REP2, FLP1, and RAF1.(a) List of genes whose expression is most plasmid are comparable in the two strains. Apparently, a single affected by the Med2 serine-208 to alanine mutation. Only genes that are amino acid substitution at position 208 of Med2 inhibits gene down-regulated at least 1.4-fold (green) or up-regulated at least 1.4-fold (red) ␮ have been included. The expression analysis was made with the Affymetrix expression from the 2- m plasmid. yeast S98 arrays as described in Materials and Methods.(b) Quantification of the FLP1, REP1, REP2, and RAF1 mRNAs in the WT and med2-S208A strains by Discussion using RT-PCR. (c) Quantification of the 2-␮m plasmid in the WT and med2- Srb10 and Srb11 have been shown to have a negative effect on S208A strains. An equal amount of genomic DNA isolated from the two strains transcription of a limited set of genes, and it was suggested that was used as templates for PCRs with primers specific for the 2-␮m plasmid and this effect operates through phosphorylation of the C-terminal the SRB8 cDNA. Samples were removed from the PCRs after 17 (lanes 1 and 2), domain before formation of the initiation complex on promoter 22 (lanes 3 and 4), 27 (lanes 5 and 6), 32 (lanes 7 and 8), and 37 (lanes 9 and 10) DNA (17, 28). The Srb10͞Srb11 complex has also been shown cycles as indicated. to operate through mechanisms acting directly on DNA-bound transcriptional activators. Thus, phosphorylation of Gal4, Ste12, lexA-Med1 (8). In both cases, the ability of lexA-Med1 to Sip4, Gcn4, and Msn2 by Srb10 suggests that modulation of activate transcription depended on Med2. Furthermore, Medi- specific transactivators in response to physiological signals is ator purified from yeast mutants lacking Med1 also lacks Med2. involved in coordinating inducible transcription with the cellular ͞ environment (18, 29–31). Taken together, our data demonstrated that the Srb10 Srb11 In previous experiments, we used fusion proteins where the complex functionally interacts with Med1 and Med2 within the lexA DNA-binding domain was fused to different Mediator Mediator complex. subunits (8, 23). We found that lexA-Med1 was unable to We here provide yet another example of a functional inter- activate transcription in WT cells although the fusion protein was action between the Srb10͞Srb11 complex and Med2. Our results functionally incorporated into the Mediator complex (23). How- show that Med2 is a target for the Srb10 kinase activity both in ever, lexA-Med1 was a potent activator (400-fold) in cells lacking vivo and in vitro. It is interesting to note that Med2 physically components of the Srb8-11 complex or in cells overexpressing interacts with Srb10, and not with Srb11, because target speci-

3374 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0400221101 Hallberg et al. Downloaded by guest on September 30, 2021 ficity in CDK phosphorylation usually is mediated through expression, it does not affect the 2-␮m plasmid copy number. interaction between the cyclin and the substrate (32, 33). How- The reduced FLP1 expression in the med2-S208A mutant was ever, it has recently been shown that the activator protein Gal4, unexpected because Srb10 plays a negative role in regulating the which is phosphorylated by Srb10, also physically interacts with expression of many genes (36). One might therefore expect that the catalytic subunit of the kinase, i.e. Srb10 itself (34). loss of an Srb10-dependent phosphorylation site should have We performed several unsuccessful experiments searching for positive effects on gene expression. However, it is not clear a specific growth phenotype of the med2-S208A mutant strain whether any of the known negative effects of Srb10 on gene under different conditions. We proceeded with a gene expres- expression is mediated by its phosphorylation of Med2. Inter- sion study where we examined the expression profiles of the WT estingly, it was recently reported that deletion of Hst3, a Sir2 and med2-S208A strains by using Affymetrix microarrays. In histone deacetylase homolog, leads to an up-regulation of FLP1 total, we found 19 genes that were affected Ͼ1.4-fold. Most of gene expression (37). Similar to our results presented here, they these genes encoded proteins with diverse functions, and we found that the altered expression of FLP1 was not a result of found no correlation to previous microarray results obtained differences in copy number of the 2-␮m plasmid between the with cells lacking Med2 (7). However, those experiments de- WT and mutant strains. scribed the effects of a deletion of Med2 only when cells were Based on the data presented here, we suggest that posttrans- grown in galactose medium and after heat shock but not under lational modifications of Mediator subunits represent a so far normal conditions in rich glucose medium (YPD) as we use here. uncharacterized mechanism for regulation of gene expression Surprisingly, the four most strongly affected genes (FLP1, RAF1, that can function as a molecular switch that controls the ability REP1, and REP2) are all located on the endogenous 2-␮m of Mediator to act as either a or a in plasmid. These four genes are highly expressed in the WT strain, transcription. but their expression is strongly reduced in the mutant strain. Increased amounts of Flp1 protein have previously been shown We thank Roger Kornberg and Tilman Borggrefe for generous gifts of to induce 2-␮m recombination and thus increase the 2-␮m copy strains and antibodies. This work was supported by grants from the Swedish Research Council (to S.B., H.R., and C.M.G.), the Swedish number (35). However, PCR analysis showed that the copy ␮ Cancer Society, the Swedish Foundation for Strategic Research, and the number of the 2- m plasmid in med2-S208A mutant yeast strains Human Frontier Science Program (to S.B and C.M.G.), and by the Erik was indistinguishable from that of congenic WT strains. Thus, and Mai Pehrsson Foundation (to H.R.), and the Kempe Foundation (to although the phosphorylation status of Med2 regulates FLP1 M.H.).

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