1. Mediator, Not Holoenzyme, Is Directly Recruited to the Heat Shock Promoter by HSF upon Heat Shock 
Jin Mo Park, Janis Werner, Jung Mo Kim, John T Lis, Young-Joon Kim 
Molecular Cell 
Volume 8, Issue 1, Pages 9-19 (July 2001)
DOI: /S (01)

2. Figure 1
Mediator Localization Relative to Pol IIa or Pol IIo on Polytene Chromosomes
Antibodies to Mediator (dTRAP80), Pol IIa (8WG16; [A] and [C]), and Pol IIo (a mix of H5 and H14 antibodies; [B] and [D]) were used to stain polytene chromosomes from CXM40 larvae without ([A] and [B]) or with ([C] and [D]) heat shock (HS).
(A) Clusters of lines mark a series of sites labeled comparably by antibodies to dTRAP80 and Pol IIa. Arrows show sites of strong Pol IIa labeling.
(B) Symbols are as in (A), except arrows show sites of labeling that are stronger for Pol IIo than for dTRAP80.
(C and D) Sets of lines mark major heat shock loci at 87A and 87C. Arrows mark transgenic sites of insertion of HSE polymers at 30A and 43E
Molecular Cell 2001 8, 9-19DOI: ( /S (01) )

3. Figure 2
Mediator or TFIID Localization Relative to HSF during Heat Shock on the HSE Polymer Transgenic Line
Lines mark the heat shock loci at 87A and 87C that contain native hsp70 genes, and arrows mark the synthetic HSE polymer sites at 30A and 43E in the CXM40 transgenic line.
(A) HSF and Mediator colocalization on chromosomes from a heat-shocked larva. Antibodies to HSF and the Mediator subunit dTRAP80 were used to stain polytene chromosomes from the transgenic line CXM40 (Shopland and Lis, 1996).
(B) TFIID and HSF localization on chromosomes from a heat-shocked larva. Costaining was with antibody to the TAF250 subunit of TFIID and with antibody to HSF.
(C) Various Mediator subunits concentrate on HSF binding sites. Polyclonal antibodies to the indicated subunits of Mediator were each used to stain chromosomes from the CXM40 line
Molecular Cell 2001 8, 9-19DOI: ( /S (01) )

4. Figure 3
Time Course of Recruitment of Mediator and HSF to Native Heat Shock Loci and Synthetic HSE Polymers and the Dependence on Functional HSF
(A) The recruitment of HSF to native heat shock loci and the transgenic loci containing synthetic HSE polymers are shown in the minutes following heat shock activation.
(B) The recruitment of Mediator to these same loci. The HSF antibody and Mediator antibody (dp34) were used as described in Figure 2. The CXM40 transgenic line used here contains the polymeric HSEs at cytological positions 30A and 43F. Shown are images focused on the native heat shock loci at 87A and 87C that contain two and four copies of the hsp70 gene, respectively, and the loci containing synthetic HSE polymers at 30A and 43F. The images in each column are collected from a nucleus in which the staining is representative of nuclei from several salivary gland squashes prepared at that time point. Time points following a heat shock treatment at which squashes were prepared are indicated at the top of the columns of images, and NHS represents animals not submitted to heat shock, or effectively, time 0 min. The left-most column is a DNA stain of the corresponding region mapped under non-heat shock conditions (0 min heat shock) and is used to identify the heat shock loci at high resolution.
(C) Mediator distribution in an hsf4 ts mutant stock. Following a 20 min heat shock of larvae from this line, nuclei were spread and stained with Mediator antibody (anti-dSrb7) as described in Figure 2. Shown are the major heat shock loci 87A and 87C and a control segment of chromosome 2 covering regions 21 and 22. Adjacent to these short segments of antibody-stained chromosome 3R is the same region stained for DNA with Hoechst. The control segment does not contain heat shock genes but shows faint bands stained with anti-dSrb7
Molecular Cell 2001 8, 9-19DOI: ( /S (01) )

5. Figure 4
Heat Shock-Induced Promoter Localization of Mediator at hsp70 in Larval Salivary Glands
(A) Schematic representation of hsp70. The diagram shows the PCR-amplified fragments (thick lines), the position of the oligonucleotide used in primer extension (arrow), and the location of the TATA element (closed rectangle), HSEs (open rectangles), and GAGA elements (gray triangles).
(B) Primer extension analysis of the heat shock response in cultured salivary glands. The reverse transcriptase-extended products for hsp70 and EF-1α transcripts were visualized. NHS, non-heat shock; HS, heat shock.
(C) ChIP of cultured salivary gland cells. Crosslinked chromatin from salivary glands subjected to heat shock (HS) or not (NHS) were immunoprecipitated with (α-dSOH1) or without (Mock) anti-dSOH1 antibody. Immunoprecipitated chromatin DNAs and 1% of the crosslinked chromatin used in the immunoprecipitation (Input) were amplified with primers specific to the promoter or coding sequence (CDS) as shown in (A)
Molecular Cell 2001 8, 9-19DOI: ( /S (01) )

6. Figure 5
Heat Shock-Induced Localization of Mediator at Heat Shock Promoters in Schneider Cells
(A) ChIP of SL2 cells. Crosslinked chromatins from cultured cells subjected to heat shock (HS) or not (NHS) were immunoprecipitated with antibodies specific to HSF (α-HSF) or dSOH1 (α-dSOH1). Immunoprecipitated chromatin DNAs and 1% of the crosslinked chromatin used in immunoprecipitation (Input) were amplified with primers specific to the promoter regions of the genes indicated at left.
(B) ChIP of SL2 cells stably expressing epitope-tagged proteins. SL2 cells stably expressing the HA or FLAG epitope-tagged proteins indicated at the top were used in crosslinking IP analysis as in (A). Monoclonal antibodies specific for each epitope were used in the immunoprecipitation step.
(C) Quantitative analysis of protein occupancy at hsp70 and hsp26. After crosslinking IP experiments were carried out in triplicate as in (A) and (B), the occupancy of proteins listed on the abscissa was calculated from the amount of immunoprecipitated DNA as a percentage of total input DNA. Values on the ordinate represent the fold increase of the protein occupancy under heat shock conditions compared to that under non-heat shock conditions. Standard deviations are in proportion to the length of error bars
Molecular Cell 2001 8, 9-19DOI: ( /S (01) )

7. Figure 6 Recruitment of Mediator, Not Holoenzyme, by HSF
SL2 cells were pretreated with α-amanitin at the indicated concentration for 1 hr, then analyzed by crosslinking IP with (HS) or without (NHS) heat shock.
(A) Inhibition of Pol II binding at hsp70 by α-amanitin. The presence of Pol II at the hsp70 promoter and coding regions was analyzed in SL2 cells containing a FLAG-tagged Pol II (F:dRPB9) with anti-FLAG antibodies (α-FLAG). The PCR products analyzed for the promoter and coding (CDS) regions are as shown in Figure 4A.
(B) ChIP analysis of Mediator (α-HA) and Pol II (α-CTD) on the hsp70 promoter. Cells containing HA-tagged MED6 (HA2:dMED6) were analyzed with the antibodies indicated above. A minimal α-amanitin concentration (100 μg/ml), which gave a near complete inhibition of Pol II elongation under the assay condition, was used.
(C) Quantitative analysis of protein occupancy at hsp70 promoter region. After crosslinking IP experiments were carried out in triplicate as in (B), the fold increases of the promoter-bound proteins by heat shock in the presence (+) or absence (−) of α-amanitin (100 μg/ml) pretreatment are shown. Standard deviations are in proportion to the length of error bars
Molecular Cell 2001 8, 9-19DOI: ( /S (01) )

8. Figure 7 Direct Interaction of HSF with dTRAP80 of Mediator
(A) Transcription assay of Mediator activity. G4-HSF protein and SNFs immunodepleted with either anti-β-galactosidase (SNFmock) or anti-Trfp (SNFαTrfp) were used in transcription reactions. The immunopurified Mediator using anti-dSOH1 antibody resin was added to the reactions (+).The transcripts from the E4 templates, which contain five copies of the Gal4 DNA binding site (G5-E4), are indicated by arrows at the left of the panel.
(B) Physical interaction of Mediator with the HSF activation domain. SNF was incubated with beads that contained GST or GST derivatives containing GST fused to either full-length HSF (amino acids 1–691) or the carboxy-terminal HSF activation domain (amino acids 582–691). Input SNF (lane 1) and bead-bound proteins (lanes 2–4) were separated by SDS-PAGE and then immunoblotted with antibodies specific for the proteins indicated at the left.
(C) GST pull-down assay of individual Mediator proteins. Mediator proteins (indicated at the left) were synthesized separately and 35S labeled in vitro, then incubated with GST beads and analyzed as in (B)
Molecular Cell 2001 8, 9-19DOI: ( /S (01) )