RNA polymerase II (RNAPII)-mediated gene expression is spatially and temporally regulated by auxiliary protein factors to ensure that transcription occurs at the right place and time on the human genome. Two cyclin-dependent kinases, CDK7 and CDK9, play a major role in this regulation at all three phases of transcription: initiation, elongation, and termination. This regulation is in part achieved by CDK7 and CDK9’s phosphorylation of the RNAPII C-terminal domain (CTD), at serines 2 (CDK9), 5 (CDK7), and 7 (CDK7) of the CTD’s 52 repeats with the consensus heptad sequence YS2PTS5PS7. This CTD phosphorylation “code” is thought to play a major role in recruiting protein factors to RNAPII that help regulate the different phases of transcription, effectively ensuring that the process proceeds from one checkpoint to the next. However, these kinases’ role in RNAPII transcription remains poorly understood due to historically limited technologies with which to inhibit and study CDKs. In this thesis, specific, next-generation, small molecule inhibitors SY-5609 and KB-0742 were used to inhibit CDK7 and CDK9 respectively in IFN-γ stimulated HCT116 cells. ChIP-Seq experiments with antibodies for pSer2, 5, 7, and total RNAPII were then performed to determine the genomic locations of differentially phosphorylated forms of RNAPII when CDK7 and CDK9 were inhibited.

This work showed that CDK9 inhibition results in an accumulation of RNAPII at gene 5’ ends, consistent with previous understandings of this kinase’s role in RNAPII promoter-proximal pause release. This work also revealed that pSer2 marks are found at the 5’ ends of many human genes, a distribution which is distinct from yeast and has been identified but ignored in previous literature and about which little is known. Additionally, pSer7 is more negatively impacted than pSer5 by CDK7 inhibition. These results suggest that CDK7 and CDK9 may be subject to levels of compensation by other kinases, and that inhibition of CDK7 and CDK9 may result in phosphatase dysregulation that impacts CTD phosphorylation patterns. While preliminary, these results raise new questions about the distribution of RNAPII CTD phosphomarks on human genes and the regulatory roles of the kinases that deposit them.