PROJECT SUMMARY RNA Polymerase II (Pol II) transcription is regulated through the concerted action of a variety of factors including the 10-subunit complex transcription factor IIH (TFIIH). TFIIH contains a kinase subunit, CDK7, which has gained attention as a therapeutic target in aggressive, metastatic cancers, including glioblastoma multiforme and triple negative breast cancer. CDK7 expression is upregulated in these cancers, and they are vulnerable to CDK7 inhibition. A thorough understanding of CDK7 function is needed to gain further insight into its roles in cancer and its prospects as an anti-cancer therapeutic target. CDK7 phosphorylates the C-terminal domain (CTD) of Pol II, facilitating the transition from transcription initiation to elongation in ways that remain poorly understood. Pol II CTD phosphorylation is also vital for proper recruitment of RNA processing factors. Unpublished work in our lab indicates that CDK7 may also control Pol II promoter-proximal pausing and splicing. Promoter-proximal Pol II pausing is a major regulatory checkpoint involving the pausing factors NELF, DSIF, and P-TEFb. RNA splicing is a highly regulated process that requires SF3B1, a component of the U2 snRNP complex, and the associated factor U2AF2 for proper branch point selection. Regulation of these two transcriptional processes (Pol II pausing and splicing) is commonly disrupted in cancer. Here, I propose to gain mechanistic insight into 1.) how CDK7 regulates Pol II promoter-proximal pausing and 2.) how splicing is regulated by CDK7 using a combination of in vitro and cell-based techniques. My first aim will examine how CDK7 phosphorylation regulates Pol II promoter-proximal pausing through phosphorylation of the pausing factors NELF, DSIF, and P- TEFb. I will test a model, supported by preliminary data, in which CDK7 i) releases Pol II pausing through direct phosphorylation of NELF and DSIF and ii) activates P-TEFb to phosphorylate NELF and DSIF. I will utilize reconstituted in vitro transcription assays (with purified human factors, no extracts) to probe how CDK7 kinase activity regulates Pol II promoter-proximal pausing and/or promoter escape through phosphorylation of pausing factors and the Pol II CTD. These in vitro findings will be probed further with PRO-Seq experiments in the context of p53 response in human cells. My second aim will investigate how CDK7 may regulate RNA splicing through phosphorylation of the U2 snRNP splicing factor SF3B1 and its associated factor U2AF2. SILAC-MS experiments (unpublished) identified SF3B1 and U2AF2 as high-confidence CDK7 targets, which was further confirmed by in vitro kinase assays. Using RNA-Seq and ChIP-Seq following p53 induction, I will test a model in which CDK7 may be enriched at actively spliced loci and may regulate splicing through phosphorylation of SF3B1 and U2AF2. Furthermore, in vitro microscale thermophoresis (MST) binding assays will provide mechanistic insight into the potential role of C...