TFIIH-Associated Cdk7 Kinase Functions in Phosphorylation of C-Terminal Domain Ser7 Residues, Promoter-Proximal Pausing, and Termination by RNA Polymerase II

Dynamic modification of the RNA polymerase II (Pol II) C-terminal domain (CTD) by phosphorylation and dephosphorylation plays important roles in controlling both transcription and cotranscriptional RNA processing (9, 31). There are 52 heptad repeats with the consensus sequence Y1S2P3T4S5P6S7 in the human CTD that can be phosphorylated cotranscriptionally on serines 2, 5, and 7 (S2, S5, and S7, respectively) (6, 31). S5 phosphorylation normally occurs early in the transcription cycle coincident with initiation, whereas S2 phosphorylation predominates later, during elongation and termination (13, 20). The complex pattern of heptad repeat phosphorylation serves in part to control binding of partner proteins, including elongation factors, RNA processing factors (31), and chromatin modifiers (25, 39). Ser5 phosphorylation enhances cotranscriptional mRNA capping (17), and Ser2 facilitates 3′-end formation by cleavage/polyadenylation (1, 4, 29). Ser7 phosphorylation has been specifically implicated in formation of U snRNA 3′ ends but is also found on mRNA coding genes (10) (6). Exactly how S2, S5, and S7 phosphorylation affect initiation, elongation, and termination remains poorly understood. It is generally thought that the positive transcription elongation factor PTEFb (Cdk9/cyclin T [CycT]) is the principal S2 kinase and that Cdk7 associated with TFIIH is the principal S5 kinase (31, 32, 38). How Cdk7 affects CTD phosphorylation on metazoan genes in vivo is still an unresolved question, and the S7 kinase is yet to be identified. In the only previous investigation of Cdk7 effects on CTD phosphorylation at the site of transcription in a multicellular organism, phospho-S5 and total Pol II levels were reduced equivalently at the Drosophila melanogaster Hsp70 gene in a Cdk7 temperature-sensitive (TS) mutant, and it was suggested that multiple S5 kinases may operate in vivo (36). Indeed, there is evidence that Cdk9 can phosphorylate CTD S5 from RNA interference knockdown of this kinase in Drosophila (11) and from the fact that the Cdk9 inhibitor 5,6-5,6-dichloro-1-β-d-ribofuranosylbenzimidazole (DRB) reduced S5 phosphorylation on the human p21 gene (14). Promoter-proximal pausing is an obligate step in the RNA Pol II transcription cycle for thousands of human genes and constitutes a rate-limiting step in mRNA synthesis with particular importance for inducible genes and in stem cells (8, 16, 21). Paused Pol II complexes have recently been shown to regulate enhancer function by acting as insulators (7). The mechanisms responsible for establishment of the pause are not fully understood; however, two key regulators have been identified: negative elongation factor NELF and DRB sensitivity-inducing factor DSIF (Spt4/5) (30, 33, 41). Knockdown of NELF inhibits transcription of some genes, suggesting that the promoter-proximal pause can facilitate gene activation (12). Pausing is antagonized by the positive transcription elongation factor PTEFb (Cdk9/CycT), which can phosphorylate DSIF, NELF, and the Pol II CTD and is sensitive to the inhibitor, DRB (27, 30, 33, 41-43). Exactly how the TFIIH-associated Cdk7 activity affects Pol II transcription remains unclear. The kinase is dispensable for initiation in vitro in many cases (26, 37, 40) but has been implicated in early elongation of dihydrofolate reductase transcripts (3). Interestingly, in Drosophila, inactivation of TS Cdk7 reduced the amount of Pol II accumulated at the 5′ end prior to gene activation, suggesting that TFIIH helps establish promoter-proximal pausing (36). However, it is difficult to distinguish in this case whether reduced amounts of paused Pol II are specifically caused by loss of Cdk7 activity or by a secondary effect on initiation due to destabilization of TFIIH, which requires Cdk7 for its integrity (40). Pol II encounters a second prominent pause site on many human genes in the 3′-flanking region several hundred base pairs past the poly(A) site (13, 15), and following this pause, termination occurs. S2 phosphorylation is maximal at the 3′ pause, and DRB inhibition of the S2 kinase Cdk9 inhibits 3′ pausing (13). Whether TFIIH-associated Cdk7 might also function in 3′ pausing and termination is unknown. In this report we investigated the roles of Cdk7 in CTD phosphorylation and polymerase pausing on human genes using mutant HCT116 cells in which both copies of the Cdk7 gene driven by its endogenous promoter were replaced by homologous recombination with an analogue-sensitive (as) mutant of the ATP-binding site (23). This strategy permits specific inhibition of kinases with cell-permeable ATP analogues and has been used to target budding yeast Cdk7 (18), human Cdk7 (23), and many other kinases in vivo (19). This strategy is preferable to TS or deletion mutations or RNA interference-mediated knockdown because it permits one to distinguish between the catalytic and structural functions of kinases (19). We combined targeted inhibition of Cdk7(as) with high-resolution chromatin immunoprecipitation (ChIP) analysis of Pol II and associated elongation factors to show that this kinase can phosphorylate S7 and S5 in vivo and affects both the 5′ and 3′ pausing events that are characteristic features of transcription on many human genes. While this work was in progress, it was independently reported that budding yeast TFIIH phosphorylates CTD Ser7 residues (2).