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

Abstract

The function of human TFIIH-associated Cdk7 in RNA polymerase II (Pol II) transcription and C-terminal domain (CTD) phosphorylation was investigated in analogue-sensitive Cdk7 as/as mutant cells where the kinase can be inhibited without disrupting TFIIH. We show that both Cdk7 and Cdk9/PTEFb contribute to phosphorylation of Pol II CTD Ser5 residues on transcribed genes. Cdk7 is also a major kinase of CTD Ser7 on Pol II at the c-fos and U snRNA genes. Furthermore, TFIIH and recombinant Cdk7-CycH-Mat1 as well as recombinant Cdk9-CycT1 phosphorylated CTD Ser7 and Ser5 residues in vitro. Inhibition of Cdk7 in vivo suppressed the amount of Pol II accumulated at 5 ends on several genes including c-myc, p21, and glyceraldehyde-3-phosphate dehydrogenase genes, indicating reduced promoter-proximal pausing or polymerase "leaking" into the gene. Consistent with a 5 pausing defect, Cdk7 inhibition reduced recruitment of the negative elongation factor NELF at start sites. A role of Cdk7 in regulating elongation is further suggested by enhanced histone H4 acetylation and diminished histone H4 trimethylation on lysine 36-two marks of elongation-within genes when the kinase was inhibited. Consistent with a new role for TFIIH at 3 ends, it was detected within genes and 3-flanking regions, and Cdk7 inhibition delayed pausing and transcription termination.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 Y 1 S 2 P 3 T 4 S 5 P 6 S 7 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 3Ј-end formation by cleavage/polyadenylation (1, 4, 29). Ser7 phosphorylation has been specifically implicated in formation of U snRNA 3Ј 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 t...

Structural and functional characterization of an atypical activation domain in erythroid Krüppel-like factor (EKLF)

“…Recent studies have demonstrated that TFIIH is a rate-limiting factor during the elongation phase of β-globin gene expression (28,29), and this is consistent with the fact that patients with mutations in TFIIH suffer from β-thalassemia (30). Together, these studies indicate that TFIIH plays a crucial role in β-globin gene expression.…”

TFIIB dephosphorylation links transcription inhibition with the p53-dependent DNA damage response

“…Our results add to the growing body of evidence that suggests alternate RNAPII CTD kinases may be required at some genes, that is particularly evident with the p21 gene (15,24). Indeed, the role of CDK7 in the phosphorylation of TFIIB correlates with the differential requirement for CDK7 and TFIIB phosphorylation at the AREG gene versus the p21 gene.…”

PHF13: A new player involved in RNA polymerase II transcriptional regulation and co-transcriptional splicing

“…7 The PIC is phosphorylated by Cdk7, a component of the general transcription factor TFIIH, at position S5 and S7 of RNAPII CTD, causing its release from the mediator complex and leading to its translocation to the TSS or just downstream of the TSS. 4,8,9 RNAPII S5P/S7P is transcriptionally competent but is paused before elongation, a delay that is stabilized by the recruitment of two negative regulators of transcription, namely negative elongation factor (NELF) and DRB sensitivity inducing factor (DSIF). 10 In order for productive transcriptional elongation to commence, Cdk9 phosphorylates both NELF and DSIF converting them into positive regulators of transcription and concomitantly phosphorylates S2.…”

“…4,8,[14][15][16] Thus, RNAPII S5P localizes to different chromatin regions, including repressed bivalent genes, 14 active promoters/promoter proximal regions 3 and at splice site junctions. 3,15 Furthermore, most of these RNAPII S5P contexts are affiliated with a pausing of RNAPII, 14,17,18 indicating that S5P is additionally important for co-transcriptional processing of pre-mRNA.…”

“…RNAPII S5P/S7P is associated with promoter/promoter proximal pausing that allows for 5 0 capping of the nascent RNA transcript, 8 which can be visualized in ChIP sequencing as a bimodal accumulation of reads near the TSS and at the C1 nucleosome. 22 Interestingly, the mean exon length in mouse and humans is 147 nt and corresponds to the length of DNA wrapped around a nucleosome.…”