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.

FIG. 1.

(A) Inhibition of analogue-sensitive TFIIH-associated kinase by 3-MB-PP1. IP kinase assays from nuclear extract of HCT116 Cdk7^as/as cells immunoprecipitated with anti-TFIIH p62 or anti-green fluorescent protein (anti-GFP) as a control were performed with GST-CTD and [γ-³²P]ATP. An autoradiograph of a sodium dodecyl sulfate-polyacrylamide gel is shown, and the position of phosphorylated GST-CTD (CTD-PO4) is indicated. For the two p62 lanes, a single IP reaction mixture was divided in two for the kinase reactions with 3-MB-PP1 (3MB) (2 μM) (+) or an equal volume of DMSO (−). (B) Cdk7(as) remains associated with TFIIH in the presence of kinase inhibitor. TFIIH was precipitated with anti-p62 from nuclear extracts prepared from control HCT116 Cdk7^as/as cells (−) and cells treated with 3-MB-PP1 (1 μM) for 8 h (+). Immunoprecipitates were analyzed by Western blotting with anti-p62 (top) and anti-Cdk7 (bottom). (C and D) Promoter-proximal pausing is depressed by inhibition of Cdk7 kinase. ChIP signals for total Pol II along c-myc in control (DMSO) and 3-MB-PP1 (3MB)-treated Cdk7^as/as cells. Values are normalized to the maximum signal for the control data set. Note the broadening of the 5′ peaks of Pol II and the shift of the 3′ peaks into the downstream flanking sequences when Cdk7 was inhibited. In panel D, Pol II ChIP signals from panel C are normalized to the maximum signal for each data set. (E) Cdk7 inhibition alters the 5′-3′ profile of Spt5 in parallel with Pol II. ChIP signals are normalized to the maximal signal for each data set. In this and all subsequent figures, ChIP signals are represented by the means ± SEMs (error bars). The arrow pointing down and asterisk indicate RNA 3′ ends in all subsequent figures.

FIG. 2.

(A and B) TFIIH is distributed throughout the c-myc gene before and after inhibiting Cdk7. Relative ChIP signals along the c-myc gene are plotted for TFIIH subunits Cdk7 and p62 in HCT116 Cdk7^as/as cells in the presence of 3-MB-PP1 (3MB) and in the absence of 3-MB-PP1 (Control). Values are normalized to the maximum signal for the 3MB data sets. (C) Cdk7 inhibition does not reduce TFIIB recruitment to the c-myc promoter. Relative TFIIB ChIP signals on c-myc in the presence and absence of 3-MB-PP1 in HCT116 Cdk7^as/as cells are normalized to the maximal signal for the 3MB data set. Means ± SEMs (error bars) are shown. (D) Cdk7 inhibition reduces polymerase pausing on activated p21. Relative Pol II ChIP signals in the presence and absence of 3-MB-PP1 in Cdk7^as/as cells treated with doxorubicin (Dox) normalized to the maximal signals in each data set are shown.

FIG. 3.

Cdk7 is required for recruitment of the negative elongation factor NELF. (A and B) ChIP signals of NELF-A along GAPDH and c-myc, respectively, in control Cdk7^as/as cells and Cdk7^as/as cells treated with 3-MB-PP13 (3MB). For reference, total Pol II profiles are shown as lines. (C) Cdk7 inhibition causes a downstream shift in the peak of CTD S2-phosphorylated Pol II. Phospho-S2 (Ser2-PO₄) ChIP signals along the c-myc gene in control and 3-MB-PP1-treated Cdk7^as/as cells normalized to the highest value in the control data set are shown. (D) Cdk7 inhibition inhibits poly(A) site cleavage and termination of β-globin transcripts. RNase protection analysis of total RNA from Cdk7^as/as cells transfected with CMV-β-globin plasmid in the presence (+) and absence (−) of 3-MB-PP1. Antisense RNA probe A (lanes 1 and 2) distinguishes correctly initiated 5′ ends and transcripts that read around the plasmid. Probe B (lanes 3 and 4) distinguishes cleaved and uncleaved transcripts at the poly(A) site. The ratios of correct 5′ ends/read-around 5′ ends and cleaved/uncleaved transcripts from three RNase protection assay experiments are summarized in the graph.

FIG. 4.

(A and B) Cdk7 inhibition increases H4 acetylation and decreases H3 K36 trimethylation. Ratios of acetylated H4 (Acetyl H4) and trimethylated H3 K36 to total H3 ChIP signals on c-myc in Cdk7^as/as cells in the presence and absence of 3-MB-PP1 (3MB) are shown. (C) Cdk7 and Cdk9 are both CTD Ser5 kinases on c-myc. Ratios of phospho-S5 (Ser5-PO₄) to total Pol II ChIP signals along c-myc for control Cdk7^as/as cells or Cdk7^as/as cells treated with 3-MB-PP1, DRB, or 3-MB-PP1 plus DRB are depicted. Mean values normalized to the maximal signal of the control at amplicon +593 are shown. Note the partial reduction in phospho-S5 when both Cdk7 and Cdk9 are inhibited.

FIG. 5.

(A and B) Cdk7 inhibition reduces CTD S5 and S7 phosphorylation on U2 snRNA and c-fos genes. Ratios of ChIP signals for phospho-S5 and -phospho-S7 relative to total Pol II are shown in Cdk7^as/as cells in the presence and absence of 3-MB-PP1 (3MB). Values are normalized to the maximum in the control data set. Ser-PO₄, phosphorylated serine. (C) Detection of TFIIH, but not Cdk9, on the U2 snRNA gene. ChIP signals for TFIIH subunits p62 and Cdk9 are shown. (D) Cdk7 inhibition causes readthrough transcription of the U2 snRNA gene. Ratios of real-time RT-PCR products for amplicon B at +458 downstream of the gene relative to amplicon A at +87 within the gene are shown for random-primed cDNA from control and 3MB-PP1-treated HCT116 Cdk7^as/as cells.

FIG. 6.

(A) Recombinant Cdk7 and Cdk9 both phosphorylate CTD S5 and S7. Kinase assays were performed with GST-CTD substrate, and the products were immunoblotted with antibodies specific to total CTD, phospho-S2, phospho-S5 (Bethyl), or phospho-S7 (4E12). α-CTD, antibody to total CTD; α-S5-PO₄, antibody to phosphorylated serine 5. The positions of molecular size markers (in kilodaltons) are shown to the left of the gels. (B) Immunoprecipitated TFIIH phosphorylates CTD S7. Immunoprecipitates from HeLa nuclear extract with control antibody, anti-p62 or anti-Cdk7 were incubated with GST-CTD substrate and ATP and immunoblotted for phospho-S7 (4E12) and the ERCC2 subunit of TFIIH as a loading control. S7 phosphorylation was dependent on the addition of ATP (not shown). α-S7-PO4, antibody to phosphorylated serine 7. (C) Summary of Cdk7 effects on CTD phosphorylation, pausing, and chromatin modification. When Cdk7 is inhibited, CTD S5 and S7 phosphorylation (S5-P and S7-P, respectively) is reduced to different extents on different genes. In addition, promoter-proximal pausing is suppressed and the 3′-flanking region pause, where CTD S2 phosphorylation is maximal, shifts further downstream. During elongation within the gene, H4 acetylation (H4 Ac) is enhanced and H3K36 trimethylation (K36me3) is reduced when Cdk7 is inhibited.