Role of the pre-initiation complex in Mediator recruitment and dynamics
- University at Albany, United States
- National Center for Biotechnology Information, National Library of Medicine, United States
- New York State Department of Health, United States
Figures
Figure 1 with 1 supplement
Mediator recruitment in med2∆ med3∆ med15∆ yeast.
(A) Spot dilutions (five-fold from left to right) of the indicated strains were made on YPD or CSM plates and allowed to grow at 30 ˚C for 3 days. The two med2∆ med3∆ med15∆ strains are independent …
Figure 1—figure supplement 1
ChIP signal metagene and peak analysis in kin28-AA yeast after rapamycin treatment.
(A) Occupancy in kin28-AA yeast after rapamycin treatment for Med17 (Srb4; head), Med18 (Srb5; head), Med15 (Gal11; tail) and for Rpb1, and signal for an untagged control (KHW127, the kin28-AA …
Figure 2
Decreased Pol II occupancy in med2∆ med3∆ med15∆ yeast.
(A) ChIP-seq of Rpb1 in wild type (BY4741) and med2∆ med3∆ med15∆ (∆tail) yeast. Left panel: normalized occupancy at S. cerevisiae UAS genes, defined as in Figure 1. Right panel: normalized …
Figure 3 with 4 supplements
Effect of depletion of TBP and Rpb3 on Mediator occupancy.
ChIP-seq was performed against (A) Med15 and (B) Med18 in tbp-AA, rpb3-AA, and taf1-AA yeast with (‘AAR’) and without (‘AA’) rapamycin treatment, and in kin28-AA yeast after rapamycin treatment. …
Figure 3—figure supplement 1
Loss of viability upon rapamycin treatment of anchor away strains used in this study.
(A) Ten-fold dilutions of indicated strains spotted onto YPD plates from left to right incubated 48 hr at 30 ˚C. (B) Growth in liquid YPD medium measured by OD600 at the indicated times after …
Figure 3—figure supplement 2
Control experiments for PIC component depletion.
(A) Time course of TBP occupancy at RPL12A and PDC1 after rapamycin treatment measured by ChIP followed by qPCR. ChIP signals were normalized to a mock (no antibody) control after first subtracting …
Figure 3—figure supplement 3
Effect of PIC component depletion on normalized occupancy (reads per million) by other PIC components, shown at UAS genes.
(A) TBP occupancy in tbp-AA yeast with and without rapamycin addition. (B) Pol II occupancy in rpb3-AA yeast with and without rapamycin addition. ChIP was performed against Rpb3 in Replicate one and …
Figure 3—figure supplement 4
Browser scans showing depletion of PIC components and differential ChIP signal for Mediator tail and head module subunits at proximal promoter and UAS regions.
(A) Browser scans showing depletion of PIC components by anchor away in replicate experiments. Scans are normalized for read number within each set of strains (e.g., all four tbp-AA TBP experiments …
Figure 4
Effect of depletion of TBP on Mediator occupancy at ‘non-UAS’ genes.
(A) Med15 occupancy measured by ChIP-seq at the 2000 most highly transcribed genes, after removal of the 498 genes exhibiting detectable Mediator ChIP signal at UAS regions in wild type yeast (Jeroni…
Figure 5 with 4 supplements
Effect of depletion of PIC components together with Kin28 on Mediator occupancy.
(A) Heat maps showing occupancy of Med15 after depletion of Kin28 alone, or in combination with Taf1, TBP, or Rpb3, at SAGA-dominated and TFIID-dominated genes. (B) Same as (A), for Med18 occupancy. …
Figure 5—figure supplement 1
Effect of depletion of PIC components together with Kin28 on Mediator occupancy at similarly expressed SAGA-dominated and TFIID-dominated genes.
Normalized Med15 (left) and Med18 (right) occupancy at similarly expressed (transcript levels between 5 and 10 (Nagalakshmi et al., 2008) SAGA-dominated and TFIID-dominated genes after depletion of …
Figure 5—figure supplement 2
Browser scans of normalized Mediator occupancy after simultaneous depletion of Kin28 and Rpb3 or Kin28 and Taf1.
Browser scans of two chromosomal regions showing greater reduction in normalized occupancy of Med15 (tail, red traces) and Med18 (head, green traces) at a TFIID-regulated gene (AFB1) and a …
Figure 5—figure supplement 3
Med15 occupancy at UAS regions after depletion of TBP, Kin28, or Kin28 and TBP together.
https://doi.org/10.7554/eLife.39633.014
Figure 5—figure supplement 4
Normalized occupancy of Med2 and Med17 at SAGA and TFIID genes after depletion of Kin28 and TBP together compared to Kin28 alone
(Med15 was used as a proxy for Med2 in kin28-AA yeast).
Figure 6
TBP depletion shifts Mediator occupancy from promoter to UAS sites.
Browser scans showing normalized Med15 and Med18 occupancy in kin28-AA and kin28-tbp-AA yeast treated with rapamycin, as indicated. (A) Upstream shift of Mediator at a SAGA-dominated gene and loss …
Figure 7
Normalized PIC occupancy at UAS genes after PIC component depletion.
(A) Rbp1 occupancy after depletion of Kin28 (black), Kin28 and TBP (red), and Kin28 and Taf1 (green). (B) Taf1 occupancy before (black) and after (red) depletion of TBP (top graph) or TBP and Kin28 …
Figure 8
Pathways of Mediator recruitment.
(A) (i) Mediator recruitment via interactions between activator proteins bound to UASs and the Med2-Med3-Med15 triad. Contacts between Mediator and components of the general transcription machinery …
Author response image 1
Replicates used for graphs and heat maps of Figure 3.
Replicate data was combined except for taf1-AA and taf1-AAR for Med15, as the second ChIP experiment failed.
Author response image 2
Replicate data for Figure 5.
https://doi.org/10.7554/eLife.39633.025Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Strain, strain background (S. cerevisiae) | yFR1321 | PMID: 24704787 | Francois Robert (University of Montreal) | |
| Strain, strain background (S. cerevisiae) | KHW127 | PMID: 24746699 | Kevin Struhl (Harvard Medical School) | |
| Strain, strain background (S. cerevisiae) | LS01 | PMID: 15254252 | Alan Hinnebusch (National Institutes of Health) | |
| Strain, strain background (S. cerevisiae) | LS10 | PMID: 15254252 | Alan Hinnebusch (National Institutes of Health) | |
| Strain, strain background (S. pombe) | FWP510 | PMID: 24100010 | Fred Winston (Harvard Medical School) | |
| Antibody | 8WG16 (Pol II) (mouse monoclonal) | Covance | Cat# 664906 | ChIP, 2 μg (1:150) |
| Antibody | Rpb3 (mouse monoclonal) | Neoclone | Cat# W0012 | ChIP, 1 μg (1:300) |
| Antibody | 9E10 (Myc) (mouse monoclonal) | Roche | Cat# 11667141001 | ChIP, 2 μg (1:150) |
| Antibody | 9E10 (Myc) (mouse monoclonal) | Sigma | Cat# 11667149001 | ChIP, 5 μg (1:300) |
| Antibody | anti-protein A (rabbit polyclonal) | Sigma | Cat# P3775 | ChIP, 2.5 μg (1:250) |
| Antibody | TBP (affinity-purified rabbit polyclonal) | Tony Weil, Vanderbilt University | NA | ChIP, 2.5 μg (1:150) |
| Antibody | 58C9 (TBP) (mouse monoclonal) | Abcam | Cat# ab61411 | ChIP, 5 μg (1:300) |
| Antibody | Taf1 antisera (rabbit polyclonal) | Joseph Reese and Song Tan, Pennsylvania State University | NA | ChIP, 2 μL (1:250) |
| Recombinant DNA reagent | pFA6a-FRB-kanMX6 | Euroscarf | P30578 | |
| Recombinant DNA reagent | pFA6a-2-FKB12-His3MX6 | Euroscarf | P30583 | |
| Recombinant DNA reagent | pSC11 | Burke et al., 2000 | ||
| Commercial assay or kit | Magnetic Bead Isolation Kit | New EnglandBiolabs | Cat# S1550S | |
| Commercial assay or kit | NEB Next Ultra RNA Library Prep Kit | New England Biolabs | Cat# E7530S | |
| Commercial assay or kit | NEBNext Ultra II Library Prep Kit | New England Biolabs | Cat# E7645S | |
| Commercial assay or kit | NEBNext Multiplex Oligos for Illumina | New England Biolabs | Cat# E7710 | |
| Commercial assay or kit | NEXTflex barcodes | BIOO Scientific | Cat# 514122 | |
| Chemical compound, drug | Rapamycin | LC Laboratories | Cat# R-5000 | |
| Software, algorithm | bwa | PMID: 10571001 | RRID: SCR_010910 | |
| Software, algorithm | SICER | PMID: 19505939 | RRID: SCR_010843 | |
| Software, algorithm | BioConductor | PMID: 15461798 | RRID:SCR_006442 | |
| Software, algorithm | Integrative Genomics Viewer | PMID: 21221095 | RRID: SCR_011793 | |
| Software, algorithm | tophat2 | PMID: 23618408 | RRID: SCR_013035 (Tophat) |
Additional files
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Supplementary file 1
Yeast strains used in this study.
- https://doi.org/10.7554/eLife.39633.019
-
Supplementary file 2
Primers used in this study.
- https://doi.org/10.7554/eLife.39633.020
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Supplementary file 3
Summary of RNA-seq and ChIP-seq experiments.
- https://doi.org/10.7554/eLife.39633.021
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Transparent reporting form
- https://doi.org/10.7554/eLife.39633.022
