Evidence for Multiple Mediator Complexes in Yeast Independently Recruited by Activated Heat Shock Factor

Abstract

Mediator is an evolutionarily conserved coactivator complex essential for RNA polymerase II transcription. Although it has been generally assumed that in Saccharomyces cerevisiae, Mediator is a stable trimodular complex, its structural state in vivo remains unclear. Using the "anchor away" (AA) technique to conditionally deplete select subunits within Mediator and its reversibly associated Cdk8 kinase module (CKM), we provide evidence that Mediator's tail module is highly dynamic and that a subcomplex consisting of Med2, Med3, and Med15 can be independently recruited to the regulatory regions of heat shock factor 1 (Hsf1)-activated genes. Fluorescence microscopy of a scaffold subunit (Med14)-anchored strain confirmed parallel cytoplasmic sequestration of core subunits located outside the tail triad. In addition, and contrary to current models, we provide evidence that Hsf1 can recruit the CKM independently of core Mediator and that core Mediator has a role in regulating postinitiation events. Collectively, our results suggest that yeast Mediator is not monolithic but potentially has a dynamic complexity heretofore unappreciated. Multiple species, including CKM-Mediator, the 21-subunit core complex, the Med2-Med3-Med15 tail triad, and the four-subunit CKM, can be independently recruited by activated Hsf1 to its target genes in AA strains.

FIG 1

Yeast Mediator and its location within HSP genes following acute heat shock. (A) Yeast Mediator subunit arrangement and modular structure, canonical view. The schematic illustrates a current model of S. cerevisiae core Mediator and the reversibly associated Cdk8-kinase module. The model is based on both structural analyses and protein-protein interaction assays (20, 22, 72, 73). (Adapted from reference and used with permission.) (B) ChIP analysis of Pol II and representative head (Med17), tail (Med15), and scaffold (Med14) subunits of core Mediator at four Hsf1-regulated genes. Yeast cells were subjected to a 5-min, 39°C heat shock prior to fixation with 1% formaldehyde. Chromatin was isolated and sonicated as described in Materials and Methods. Antisera raised against recombinant proteins were used to detect Mediator subunits; antiserum raised against the CTD of Rpb1 was used to detect Pol II. Shown is the occupancy of each factor normalized to input. The data are shown as means and standard deviations (SD) of 2 or 3 independent biological replicates (n = 4 for Pol II). The midpoint coordinates of qPCR amplicons used in this analysis, presented relative to the ATG start codon (+1), are as follows: SSA4 UAS (−333), Prom (promoter) (−128), 5′ open reading frame (ORF) (+142), mid-ORF (+881), and 3′ untranslated region (UTR) (+1959); HSP82 UAS (−283), Prom (−123), 5′ ORF (+658), mid-ORF (+1346), and 3′ UTR (+2181); HSP104 UAS (−232), Prom (−111), ORF (+1723), and 3′ UTR (+2665); and ZPR1 UAS (−258), Prom (−103), ORF (+765), and 3′ UTR (+1492).

FIG 2

Heat shock-activated UAS/promoter regions in cells exposed to rapamycin are efficiently depleted of Med14-FRB, and the abundance of most, but not all, core subunits is reduced in parallel. (A) Anchor away technique (52). See the text for details. (B) Spot dilution analysis of parental (Med14⁺) and Med14-FRB AA strains (HHY212 and YM101, respectively). Depicted are 5-fold serial dilutions spotted onto YPDA medium supplemented with drug as indicated. The plates were incubated at 30° or 37°C for 2 to 3 days and at 15°C for 7 days. (C) ChIP analysis of Med14-FRB at SSA4, HSP82, and ZPR1 UAS/promoter regions in YM101 cells subjected to treatment with 1 μg/ml rapamycin for the indicated times, followed by a 5-min heat shock. ChIP was performed as in Fig. 1B. Med14 was detected with anti-Rgr1/Med14 antiserum, and its abundance is presented relative to that seen in nontreated cells similarly subjected to a 5-min heat shock (0′). Depicted are means and SD; n = 2. (D) ChIP analysis of representative Mediator subunits conducted and quantified as in panel C. Antibodies raised against recombinant proteins were used to detect Mediator subunits lacking a C-terminal Myc tag; those containing one were detected with an anti-Myc monoclonal antibody. For both panels C and D, mock IP signal (beads alone for Med14, Med15, Med17, and Med21; anti-Myc IP of chromatin isolated from the corresponding parental strain for the Myc-tagged subunits) was subtracted from each ChIP signal prior to normalization. Depicted are means and SD; n = 2 or 3. The strains used were YM103, YM105, YM106, YM112, YM113, and YM123.

FIG 3

Cytoplasmic anchoring of scaffold subunit Med14 leads to a strong reduction in Med5 and Med16 occupancy at activated HSP genes but only a mild reduction in the occupancy of either Med2 or Med3. (A) ChIP was conducted and quantified as in Fig. 2. Cells were exposed to 1 μg/ml rapamycin for 120 min (or not), followed by a 5-min heat shock. Med14 was detected using anti-FRB Ab; Med17 using anti-Srb4/Med17 antiserum; and Med2, Med3, and Med5 using anti-Myc Ab. Background was determined as described in Fig. 2 and subtracted from each ChIP signal. Means and SD are depicted; n = 2 in each case except Med14-FRB (n = 6). The strains used were AJ101, AJ102, and AJ103. (B) ChIP analysis was performed and quantified as described for panel A, except that rapamycin pretreatment was for 90 min and Med16 was detected using an anti-Myc Ab. The strain used was YM107.

FIG 4

Med18 and Med16 efficiently relocate from nucleus to cytoplasm following addition of rapamycin to Med14 AA cells, while Med15 persists in the nucleus. Fluorescence microscopy analysis of early-log-phase cells expressing the indicated C-terminally tagged proteins was conducted following addition of rapamycin (1 μg/ml) for the indicated times. The cells were maintained at 25 to 30°C throughout. See Materials and Methods for details. (A) Strain AJ204 (MED14-FRB-GFP, MED18-mCherry). (B) Strain AJ203 (MED14-FRB-GFP, MED16-mCherry). (C) Strain AJ202 (MED14-FRB-GFP, MED15-mCherry).

FIG 5

Anchoring either Med7 or Med16 results in parallel depletion of Med17 but only partial loss of Med15. (A) ChIP analysis of the indicated Mediator subunits was conducted using strain ASK202 and quantified as in Fig. 2. (B) As in panel A, except strain ASK203 was used. For both panels, means and SD are depicted; n = 2. (C) As in panel B, except antibodies raised against recombinant proteins were used to detect nontagged proteins; anti-FRB Ab was used to detect Med16. Shown are means and SD; n = 2. The strain used was YM124. (B and C) Similar results were obtained with (B) or without (C) C-terminal tagging of Med15.

FIG 6

Anchoring Med15 obviates core Mediator recruitment but not that of the CKM, while anchoring Hsf1 obviates both. (A) ChIP analysis of Med15 AA strain YM125 conducted and quantified as in Fig. 2. Untagged subunits were detected using antibodies raised against the recombinant proteins; Med16-Myc9 was detected using Myc Ab. Means and SD are depicted; n = 2 or 3. (B) As in panel A, except Med15 AA strains AJ126, AJ127, AJ128, and YM119 were evaluated. Subunits were detected using antibodies directed against their C-terminal tags; Med12 was detected using anti-Srb8/Med12 antiserum. n = 2 for all data except Med15-FRB (n = 8). (C) As in panel A, except Hsf1 AA strain BY4742-Hsf1-AA was used. Cells were exposed to rapamycin for 90 min prior to subjecting them to a 5-min heat shock. All proteins were detected using antibodies raised against their recombinant counterparts. n = 2 or 3, except in the case of Hsf1 (n = 5).

FIG 7

Anchoring Cdk8 leads to parallel depletion of CKM subunit Med12 but has no effect on Med17 recruitment. ChIP analysis of Cdk8 AA strain YM115. Cells were pretreated with 1 μg/ml rapamycin at 30°C for the times indicated, followed by a 5-min heat shock at 39°C. Depicted are means and SD; n = 2. This experiment demonstrates that recruitment of core Mediator (as signified by Med17) to heat shock-induced HSP genes is unaffected by prior exposure of cells to rapamycin.

FIG 8

Anchoring Med14 strongly reduces Pol II promoter occupancy and HSP gene expression during heat shock. (A) Pol II and Med17 ChIP analysis of strain YM103 pretreated with rapamycin for the indicated times, followed by 5-min heat shock. Depicted is Pol II occupancy at HSP gene promoters and Med17 occupancy at HSP UAS regions. Shown are means and SD; n = 3 or 4. (B) Same as panel A, except Pol II occupancy within HSP coding regions is shown. (C) RT-qPCR analysis of HSP mRNA levels in YM103 cells either pretreated or not with rapamycin for 90 min (red and blue bars, respectively), followed by heat shock for the indicated times. Shown are means and SD; n ≥ 2.

FIG 9

Anchoring of either tail subunit, Med15 or Med16, strongly reduces HSP gene transcription whereas anchoring of Cdk8 has no effect. (A) RT-qPCR analysis of Med16-FRB strain YM124 subjected to a heat shock time course following pretreatment or not with rapamycin as in Fig. 8C. Shown are means and SD; n ≥ 2. Blue bars, without rapamycin; red bars, with rapamycin. (B) As for panel A, except Med15-FRB strain YM117 was analyzed. (C) As for panel A, except Cdk8-FRB strain YM115 was analyzed.

FIG 10

Nuclear depletion of SAGA subunit Spt20 has minimal effect on CKM-Mediator recruitment to heat shock-induced HSP genes. (A) ChIP analysis of SAGA subunit Spt20-FRB, core Mediator subunit Med17, and CKM subunits Med12 and Cdk8 in strain YM120 exposed to rapamycin for the indicated times, followed by a 5-min heat shock. Shown are means and SD; n = 3. The asterisk indicates that occupancy significantly differs from that of the control (0′) condition (P < 0.05; two-tailed t test; equal variance). (B) As in panel A, except Med14-FRB strain YM108 was analyzed, and occupancy of Med17 and SAGA subunit Spt3 was evaluated.

FIG 11

Yeast Mediator complexes suggested by this study. Evidence obtained from both ChIP and fluorescence microscopy analyses of AA strains suggests the existence of multiple Mediator complexes in vivo. Those marked with asterisks were detected at the regulatory regions of heat shock-induced HSP genes. Note that the relative abundances of the Mediator species depicted may differ in wild-type strains versus the AA strains examined here. In particular, dissociation of intact core Mediator into tail triad and 18-mer subcomplexes, suggested by the ChIP data (Fig. 2, 3, 5, and 6), may be more pronounced in AA strains. H, head; M, middle; T, tail; S, scaffold; K, kinase.