1. Volume 22, Issue 2, Pages 179-192 (April 2006)
Genome-Wide Location of the Coactivator Mediator: Binding without Activation and Transient Cdk8 Interaction on DNA 
Jean-Christophe Andrau, Loes van de Pasch, Philip Lijnzaad, Theo Bijma, Marian Groot Koerkamp, Jeroen van de Peppel, Michel Werner, Frank C.P. Holstege 
Molecular Cell 
Volume 22, Issue 2, Pages (April 2006)
DOI: /j.molcel
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2. Figure 1
Similar Location of Individual Mediator Subunits on DNA Regardless of Transcription Activity
(A) Histograms of individual ChIPs all showing the same sites. These were selected for general Mediator binding (all subunits merged) and false discovery rate (FDR) < 5% and are depicted for BRs above 1.2 (left) or 1.5 (right). The bottom panel (no Tag) shows an experiment from a strain without a tag. The horizontal axis represents normalized BRs and the vertical axis the number of sites. The second panel from the bottom (Pol II SP) shows the BRs of Pol II in stationary phase (Radonjic et al., 2005) on these Mediator midlog phase binding sites.
(B) Mediator binding trend on GCRs and IGRs in relation to mRNA expression levels. All genomic elements are ranked according to average expression levels (Experimental Procedures and Supplemental Experimental Procedures). The binding trend is the average BR of a sliding window of 100 genes from low to high expression. For the IGR binding trend, only genes and IGRs are included whereby the IGR can be unambiguously associated with a single gene. This was achieved by excluding IGRs between converging or diverging genes.
(C) Pol II binding trends on GCRs or IGRs in relation to mRNA expression, as described in (B).
(D) Mediator BRs for all genomic elements, according to chromosomal position. Underenriched sequences are in dark blue (lower boundary BR less than 0.5) and enriched sequences in orange (upper boundary BR higher than 2.5).
(E) Pol II BRs as in (D), with lower boundary BR of 0.4 and upper boundary BR of 4.
(F) Histogram of Pol II BRs for all microarray elements located less than 20 kb from chromosomal ends. The horizontal axis represents the normalized BRs and the vertical axis the number of microarray elements.
(G) Histogram of Mediator BRs for all telomeric features as in (F).
Molecular Cell  , DOI: ( /j.molcel )
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3. Figure 2 Mediator Binding Upstream of Active Genes
(A) BRs of Med3, Med7, Med19, CycC, and Pol II on the PMA1 region from the genome-wide ChIP. The line labeled Mediator is the average of all Mediator subunit analyses (28 hybridizations). For the individual subunits, the average of four hybridizations is shown and for Pol II the average of 13 hybridizations. The position and length of the microarray PCR products are indicated as black bars below the horizontal axis. Areas shaded gray represent gene coding regions. Blue boxes are transcription factor binding sites. The black box in the left panel indicates the region analyzed.
(B) Higher resolution standard ChIP of Pol II and Med3 on PMA1 region. The left panel shows the gel image of the radioactively labeled PCR products used for quantification. PCR probes are represented as thick black lines and numbered according to the Supplemental Experimental Procedures. For Figures 2C, 3, and 5, the genome-wide ChIP results are presented in Figure S2.
(C) Standard ChIP for Pol II, Med3, and TBP on CCW12.
(D) Summary of Mediator and Pol II binding at various sites with expression levels of the corresponding genes. The left axis indicates BRs from standard ChIP on upstream regulatory sequences (URS) or GCRs, respectively. The right axis shows the expression levels (Experimental Procedures).
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4. Figure 3 Mediator Binding Upstream of Inactive Genes
(A) Standard ChIP of Pol II and Med3 on the ROX1 locus, as described for Figure 2B.
(B) Standard ChIP of Pol II, Med3, and Zap1 on the ZRT2 locus as described for Figure 2B. High- and low-affinity (LA) binding sites for Zap1 are represented as blue and red boxes, respectively.
(C) Standard ChIP of Pol II, Med3, and Zap1 on the ZRT1 locus as described for Figure 2B.
Molecular Cell  , DOI: ( /j.molcel )
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5. Figure 4
Genomic Location of Mediator during Exponential Growth Overlaps with Pol II Location in Quiescent Cells
(A) Venn diagram showing overlap between Mediator IGR binding sites during ML growth (this study, FDR < 5%, BR > 1.2, blue) and Pol II IGR binding sites in SP (Radonjic et al., 2005) (FDR < 5%, BR > 1.4, yellow). The p value of this overlap is 10−131.
(B) Functional categories enriched in the genes depicted in the overlap in (A). FDRs are indicated on the right.
(C–H) Examples of genomic regions showing both Mediator binding in ML and Pol II binding in SP at the same IGRs. BRs for Mediator and Pol II in ML are derived from the genome-wide ChIPs as described in Figure 2A and from four individual experiments of Pol II location in SP (Radonjic et al., 2005).
Molecular Cell  , DOI: ( /j.molcel )
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6. Figure 5 Mediator Binding to Gene Coding Regions
(A) Pol II binding trend on GCRs compared to Mediator binding on GCRs (blue) or Mediator binding to the corresponding IGRs (black, only for IGRs that can unambiguously be assigned to one gene), as described for Figure 2A.
(B) Pol II binding trend on IGRs compared to Mediator binding on GCRs or IGRs.
(C) Standard ChIP of Pol II, Med3, Med19, and TBP binding on the FBA1 locus as described for Figure 3B.
(D) Standard ChIP of Pol II, Med3, Med19, and TBP on the ILV5 locus as described for Figure 3B.
(E) Gene ontology categories enriched for genes with significant Mediator binding to GCRs (BR > 1.2, FDR < 5%), compared to highly transcribed genes (top 200 genes ranked for Pol II binding on GCRs with FDR < 5%). FDR is indicated on the right.
Molecular Cell  , DOI: ( /j.molcel )
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7. Figure 6
Transient Association of the Cdk Module with the Core Mediator Complex
(A) Cdk8 is accompanied by the Med15 core complex subunit on various Mediator binding sites. ReChIP with Cdk8-TAP followed by anti-Med15 (black) yields signals significantly higher than with TAP-Cdk8 (red) alone. In theory, a complete reChIP should yield signals in the range of the product of the single IPs. Mediator binding sites were selected from the examples used for verification of genome-wide results (Figures 2, 3, and 5). Note that the signals from the single ChIPs in these panels are slightly weaker than for other experiments for these regions (Figure S3 and Figures 2, 3, and 5), due to modification of the protocol for reChIP. Signals are calculated as ratios over input and POL1 control (Supplemental Experimental Procedures).
(B) Only a fraction of Med15 is accompanied by Cdk8 on DNA. Reversal of the order of the experiment depicted in (A) shows that reChIP with Med15 followed by Cdk8-TAP (black) yields signals that are a fraction of the signal with anti-Med15 alone (blue).
(C) Western blot analysis of Cdk8-TAP for the ChIP and reChIP experiments presented in (A) and (B).
(D) Anti-Med15 followed by anti-Med7-TAP reChIP.
(E) Western blot analysis of ChIP and reChIP presented in (D).
(F) ROX1 and ZRT2 mRNA level changes as a ratio of deletion mutant versus wild-type in CDK submodule mutants as determined previously by microarray analysis (van de Peppel et al., 2005). The average of four ratio measurements is plotted for each deletion mutant. Whereas the changes for ROX1 are all statistically significant (p < 0.05), ZRT2 mRNA levels show no statistically significant changes in the deletion mutants relative to wild-type.
(G) Models for the lower CDK module occupancy observed at core Mediator binding sites.
Molecular Cell  , DOI: ( /j.molcel )
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