1. Volume 22, Issue 2, Pages 169-178 (April 2006)
Genome-Wide Occupancy Profile of Mediator and the Srb8-11 Module Reveals Interactions with Coding Regions 
Xuefeng Zhu, Marianna Wirén, Indranil Sinha, Nina N. Rasmussen, Tomas Linder, Steen Holmberg, Karl Ekwall, Claes M. Gustafsson 
Molecular Cell 
Volume 22, Issue 2, Pages (April 2006)
DOI: /j.molcel
Copyright © 2006 Elsevier Inc. Terms and Conditions

2. Figure 1 Mediator Associates with the Coding Region of Many Genes
(A) Mediator interactions with IGR and ORF regions are more prevalent on highly transcribed genes. The vertical axis shows the fraction of IGR and ORF fragments bound by Mediator. Genes were separated into three groups: the 1048 most transcribed genes (High), the 992 least transcribed genes (Low), and the 2984 genes with intermediate transcription levels (Medium). The cutoff used for Mediator binding was 2-fold enrichment in the immunoprecipitated fraction relative to a whole genome DNA sample.
(B) Mediator localizes to coding regions. Enrichment in the immunoprecipitated fraction relative to a whole genome DNA sample is shown along the left arm of chromosome 2 (positions 32–332 kbp). The data were normalized by using the 50th percentile. The y axis scale is log2, and each bar represents the average of 11 oligonucleotide probes within adjacent 250 bp windows. The horizontal axis shows kilobase units (kb). Noncoding regions (black), ORFs on the Watson strand (blue), and the Crick strand (pink) are indicated. “SPBC” has been omitted from the systematic gene names.
(C) Moving average plot analysis showed Mediator enrichment (y axis) versus levels of gene transcription (x axis). The window size was 150 and the step size 1.
Molecular Cell  , DOI: ( /j.molcel )
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3. Figure 2
Mediator Interactions with the Coding Region Display a Distinct Distribution
(A) Venn diagrams demonstrating a positive correlation between the presence of Mediator on longer genes (1000–2000 bp, left) and the absence of Mediator on shorter genes (<500 bp, right). P values indicate the statistical significance of the observed overlap.
(B) Mediator enrichment on the coding region varies with the ORF location as calculated from the ORF start site. Signals absent according to the Affymetrix detection algorithm were excluded from the data analysis, and the cutoff used for Mediator binding was 1.5-fold enrichment in the immunoprecipitated fraction relative to a whole genome DNA sample. The total number of genes analyzed for each position is indicated. A significantly (p < 0.05) lower value than expected is indicated with an asterisk (∗), whereas two asterisks (∗∗) indicate a significantly higher value than expected.
(C) The amount of Mediator on the promoter and in the coding region of SPCC645.06c was analyzed by ChIP. Precipitated DNA was quantified by real-time PCR with the appropriate primers. Error bars show standard deviations.
(D) The amount of Mediator on the promoter and in the coding region of SPBC was analyzed as described in (C).
Molecular Cell  , DOI: ( /j.molcel )
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4. Figure 3
Downregulation of Transcription in a med17ts Strain Correlates with Changes in Mediator-ORF Interactions
(A) Moving average plot of changes in gene transcription at the nonpermissive temperature in a med17ts strain (y axis) versus normal absolute levels of gene transcription (x axis).
(B) Venn diagrams demonstrating that no significant correlation could be observed between transcription downregulation at the nonpermissive temperature in the med17ts strain and increased (data not shown) or decreased Mediator-IGR interactions (left). In contrast, a positive correlation is observed between downregulation of transcription and increased Mediator-ORF interactions (right). The p value indicates the statistical significance of the observed overlap.
Molecular Cell  , DOI: ( /j.molcel )
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5. Figure 4
Gene Induction Leads to Increased Mediator Occupancy in Both the IGR and Coding Region
(A) The amount of Pol II, Mediator, TBP, TFIIE, and TFIIF on the promoter and in the coding region of the S. pombe nmt1 promoter was analyzed by ChIP. Precipitated DNA was quantified by real-time PCR with the appropriate primers. The nmt1 promoter was induced in the absence of thiamine. The vertical axis scale is log2, the level of transcription factor occupancy under uninduced conditions was set to 1, and the graph shows the fold of difference relative to this uninduced sample. Error bars indicate standard deviations. Below the graph is a schematic representation of the nmt1 gene. The thiamin-responsive element is denoted as a red box (−165 to −155, where A of the ATG is +1). An arrow denotes the transcription initiation site (−79). Regions amplified in the ChIP PCRs are noted below the gene.
(B) The amount of Pol II, Mediator, TBP, TFIIE, and TFIIF on the promoter and in the coding region of the S. cerevisiae GAL1 promoter was analyzed by ChIP as described in (A). Error bars indicate standard deviations. The vertical axis indicates the increase in Mediator occupancy after induction of the GAL1 gene in the presence of galactose. Below the graph is a schematic representation of the GAL1 gene. The Gal4 binding sites are represented as a red box (−460 to −330, where A of the ATG is +1). An arrow denotes the transcription initiation site (−60). Regions amplified in the ChIP PCRs are noted below the gene.
Molecular Cell  , DOI: ( /j.molcel )
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6. Figure 5
Mediator and Pol II Mutations Negatively Affect Both IGR and Coding Region Binding
(A) The Pol II mutant strain rpb1-1 was used at the permissive and nonpermissive temperatures to measure Mediator enrichment after galactose induction on the promoter and in the coding region of the S. cerevisiae GAL1 gene. The ChIP analysis was as described in Figure 4A. The vertical axis scale is log2, the level of Mediator occupancy under uninduced conditions was set to 1, and the graph shows the fold difference relative to the uninduced sample. Error bars indicate standard deviations. Below the graph is a schematic representation of the GAL1 gene. The Gal4 binding sites are represented as a red box (−460 to −330, where A of the ATG is +1). An arrow denotes the transcription initiation site (−60). Regions amplified in the ChIP PCRs are noted below the gene.
(B) The amount of med7ts (srb4-138) Mediator on the promoter and in the coding region of the S. cerevisiae GAL1 promoter was analyzed at the permissive and nonpermissive temperature. The ChIP analysis was as described in Figure 4A. The vertical axis scale is log2, the level of Mediator occupancy under uninduced conditions was set to 1, and the graph shows fold difference relative the uninduced sample. Error bars indicate standard deviations.
Molecular Cell  , DOI: ( /j.molcel )
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7. Figure 6
The Srb8-11 Module and Core Mediator Associate with the Same Genomic Regions
(A) Correlation between core Mediator (TAP-Med7) and Srb8-11 module (TAP-Med13) binding in the IGR region.
(B) Correlation between core Mediator (TAP-Med7) and Srb8-11 module (TAP-Med13) binding in the ORF region.
(C) Moving average plot of Srb8-11 module enrichment (y axis) versus levels of gene transcription (x axis).
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2006 Elsevier Inc. Terms and Conditions