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Status |
Public on Mar 01, 2024 |
Title |
Comparative cofactor screens reveal the influence of transactivation domains and core promoters on the mechanisms of transcription [ChIP-nexus] |
Organism |
Homo sapiens |
Experiment type |
Genome binding/occupancy profiling by high throughput sequencing
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Summary |
Eukaryotic transcription factors (TFs) activate gene expression by recruiting cofactors to promoters. However, the relationships between TFs, promoters and their associated cofactors remain poorly understood. Here, we combine GAL4-transactivation assays with comparative CRISPR-Cas9 screens to identify the cofactors required by nine different TFs in human cells. Using this dataset, we associate key TFs with their cofactors, classify cofactors as ubiquitous or specific, discover novel transcriptional co-dependencies and demonstrate that certain TFs use the tail 2 and kinase submodules of Mediator to potentiate transcriptional elongation. By employing a reductionistic and comparative approach, we demonstrate that TFs do not display discrete mechanisms of activation. Instead, each TF is dependent on a unique combination of cofactors, which influences distinct steps in the transcriptional process. We also extend our screens to nine different core promoters to explore how core promoter elements influence cofactor dependence. Our data suggest that different classes of promoter are constrained by either initiation or pause release, which influences their dynamic range of gene expression and compatibility with specific cofactors. Overall, our comparative cofactor screens uncover the interplay between TFs, cofactors and core promoters and reveal general principles by which they influence transcription.
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Overall design |
ChIP nexus for RNA-POL2 at a reporter activated with different TADs or different promoters. Cells were harvested 4hrs after addition of degron or relevant drug (CDK8i). For all ChIP experiments, at least 20 million cells were crosslinked for 15 mins with 1% formaldehyde. Crosslinked material was sonicated to approximately 200-1000bp using the Covaris Ultrasonicator e220. Sonicated material was incubated overnight with each antibody, then incubated for 3hrs with Protein A magnetic beads. Beads were washed with low and high salt wash buffers, LiCl buffer and TE, before being eluted and de-crosslinked overnight. DNA was purified using Qiagen Minelute columns. All ChIP antibodies were used at ~10ug per IP and are listed under the antibodies section. Sequencing libraries were prepared from eluted DNA using Rubicon ThruPLEX DNA-seq kit. Libraries were size selected between 200-500bps and sequenced on the NextSeq500 using the 75bp single-end chemistry.
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Contributor(s) |
Bell CC, Dawson MA, Talarmain L, Gillespie A |
Citation missing |
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Submission date |
Feb 20, 2024 |
Last update date |
Mar 02, 2024 |
Contact name |
Charles C Bell |
E-mail(s) |
charles.bell@petermac.org
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Organization name |
Peter MacCallum Cancer Centre
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Department |
Cancer Research
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Street address |
VCCC, Gate 1, 14 Flemington Road
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City |
Melbourne |
State/province |
VICTORIA |
ZIP/Postal code |
3000 |
Country |
Australia |
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Platforms (1) |
GPL18573 |
Illumina NextSeq 500 (Homo sapiens) |
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Samples (18)
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This SubSeries is part of SuperSeries: |
GSE198944 |
Comparative cofactor screens reveal the influence of transactivation domains and core promoters on the mechanisms of transcription. |
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Relations |
BioProject |
PRJNA1078371 |