Regulation of RNA polymerase II promoter-proximal pausing by DSIF
Final Report Abstract
RNA Polymerase II (Pol II) of higher eukaryotes commonly pauses downstream of the transcription start site of most genes, in the so-called promoter-proximal region. Primarily based on indirect observations using cell-based assays, it appears that Pol II pausing is induced by the factors DSIF and NELF. Dynamic phosphorylation of these factors triggers the release of Pol II into productive elongation. Here, I assessed DSIF (subunits Spt4 and Spt5) function by performing in vitro transcription reactions using purified factors. To study the role of high confidence phosphorylation sites within Spt5, those residues were mutated and the mutant Spt5 was compared to wildtype Spt5 in transcription assays. The assays revealed that DSIF promotes overall RNA synthesis by RNA Polymerase II in a phosphorylation-dependent manner. Additionally, phosphorylation of Spt5 was found to regulate pause duration and/or transcriptional turnover. In summary, these findings highlight that DSIF contributes to Pol II processivity and regulation of promoter-proximal pausing, with contribution of phosphorylation of Spt5. To study the function of DSIF in human cells, a HCT116 cell line was engineered, in which Spt5 is tagged with the degradation domain FKBP12F36V that can be facilitated to recruit a ubiquitin ligase upon addition of a small dTAG molecule, triggering the degradation of Spt5. I obtained clonal cell lines with either N-terminal or C-terminal FKBP12F36V as confirmed by PCR and Sanger sequencing. Treatment of engineered cells with the small molecule dTAGV-1 resulted in rapid depletion of Spt5 at nanomolar concentration within 30 minutes. In studies these cells can be utilized in functional cellular assays including transcriptome sequencing strategies such as precision nuclear run-on sequencing (PRO-seq).
Publications
- (2019) Pol II phosphorylation regulates a switch between transcriptional and splicing condensates. Nature 572: 543–548
Guo YE, Manteiga JC, Henninger JE, Sabari BR, Dall’Agnese A, Hannett NM, Spille J-H, Afeyan LK, Zamudio A V., Shrinivas K, Abraham BJ, Boija A, Decker T-M, Rimel JK, Fant CB, Lee TI, Cisse II, Sharp PA, Taatjes DJ & Young RA
(See online at https://doi.org/10.1038/s41586-019-1464-0) - (2020) Partitioning of cancer therapeutics in nuclear condensates. Science. 368: 1386–1392
Klein IA, Boija A, Afeyan LK, Hawken SW, Fan M, Dall’Agnese A, Oksuz O, Henninger JE, Shrinivas K, Sabari BR, Sagi I, Clark VE, Platt JM, Kar M, McCall PM, Zamudio A V., Manteiga JC, Coffey EL, Li CH, Hannett NM, Guo YE, Decker T-M. et al.
(See online at https://doi.org/10.1126/science.aaz4427) - (2020) Selective inhibition of CDK7 reveals high-confidence targets and new models for TFIIH function in transcription. Genes Dev. 34: 1452-1473
Rimel JK, Poss ZC, Erickson B, Maas ZL, Ebmeier CC, Johnson JL, Decker T-M, Yaron TM, Bradley MJ, Hamman KB, Hu S, Malojcic G, Marineau JJ, White PW, Brault M, Tao L, DeRoy P, Clavette C, Nayak S, Damon LJ, et al.
(See online at https://doi.org/10.1101/gad.341545.120)
