 |
 |
GEO help: Mouse over screen elements for information. |
|
| Status |
Public on May 28, 2020 |
| Title |
TFIID enables RNA polymerase II promoter-proximal pausing |
| Organisms |
Drosophila melanogaster; Homo sapiens |
| Experiment type |
Other
|
| Summary |
RNA polymerase II (pol II) transcribes all protein-coding and many non-coding RNAs in the human genome. Pol II transcription initiation is governed by the Pre-Initiation Complex (PIC), which contains TFIIA, TFIIB, TFIID, TFIIE, TFIIF, TFIIH, pol II, and Mediator. After initiation, pol II enzymes typically pause after transcribing less than 100 bases, and paused polymerases represent a common regulatory intermediate. Accordingly, paused pol II has been implicated in enhancer function, development and homeostasis, and diseases ranging from cancer to viral pathogenesis. Precisely how pol II promoter-proximal pausing is enforced and regulated remains unclear; however, protein complexes such as NELF and DSIF increase pausing whereas the activity of CDK9 (P-TEFb complex) correlates with pause release. To address specific mechanistic questions about pol II pausing and its regulation, we reconstituted human pol II promoter-proximal pausing in vitro, entirely with purified factors (no extracts). As expected, NELF and DSIF increased pol II pausing in vitro, whereas P-TEFb promoted pause release. Unexpectedly, the PIC alone was sufficient to reconstitute pol II pausing, suggesting that pausing is an inherent property of the PIC. In agreement, pol II pausing was lost upon replacement of the TFIID complex with TATA-binding protein (TBP); moreover, pausing was dependent upon TFIID subunits TAF1 and TAF2. TAF1/2 bind genomic DNA downstream of the pol II initiation site, invoking a “complex interaction” model for pausing. Consistent with this model, PRO-Seq experiments revealed increased transcription upon acute depletion (t=60 min) of TAF1 and TAF2 in human cells, and pol II pausing was disrupted at thousands of genes. Similar results were obtained in TAF1-depleted Drosophila S2 cells. Collectively, these data establish the general transcription factor TFIID as a genome-wide regulator of pol II promoter-proximal pausing.
|
| |
|
| Overall design |
Examination of pol II pausing by persicion run-on nucelar sequencing (PROseq) in HCT116 and S2 cells where TAF1 is knocked down vs. control.
|
| |
|
| Contributor(s) |
Fant C, Levandowski C, Maas Z, Moir J |
| Citation(s) |
32229306 |
| |
| Submission date |
Jun 14, 2019 |
| Last update date |
May 28, 2020 |
| Contact name |
Dylan J Taatjes |
| E-mail(s) |
[email protected]
|
| Phone |
303 492-6929
|
| Organization name |
University of Colorado Boulder
|
| Department |
Biochemistry
|
| Lab |
Taatjes Lab
|
| Street address |
Campus Box 596
|
| City |
Boulder |
| State/province |
CO |
| ZIP/Postal code |
80303-0596 |
| Country |
USA |
| |
|
| Platforms (2) |
| GPL18573 |
Illumina NextSeq 500 (Homo sapiens) |
| GPL19132 |
Illumina NextSeq 500 (Drosophila melanogaster) |
|
| Samples (10)
|
| GSM3891565 |
wild-type HCT116, TAF1 Trim-Away, Replicate 1 |
| GSM3891566 |
wild-type HCT116, TAF1 Trim-Away, Replicate 2 |
| GSM3891567 |
wild-type HCT116, control, Replicate 1 |
| GSM3891568 |
wild-type HCT116, control, Replicate 2 |
| GSM3891569 |
wild type S2 cells, LacI dsRNA control, Replicate 1 |
| GSM3891570 |
wild type S2 cells, LacI dsRNA control, Replicate 2 |
| GSM3891571 |
wild type S2 cells, LacI dsRNA control, Replicate 3 |
| GSM3891572 |
wild-type S2 cells, TAF1 dsRNA knockdown, Replicate 1 |
| GSM3891573 |
wild-type S2 cells, TAF1 dsRNA knockdown, Replicate 2 |
| GSM3891574 |
wild-type S2 cells, TAF1 dsRNA knockdown, Replicate 3 |
|
| Relations |
| BioProject |
PRJNA548938 |
| SRA |
SRP201480 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSE132764_RAW.tar |
2.1 Gb |
(http)(custom) |
TAR (of BEDGRAPH) |
| GSE132764_counts_hct116.txt.gz |
456.8 Kb |
(ftp)(http) |
TXT |
| GSE132764_counts_s2.txt.gz |
320.5 Kb |
(ftp)(http) |
TXT |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data are available on Series record |
| Processed data provided as supplementary file |
|
|
|
|
 |
Less...
More...