TFIIH-associated Cdk7 kinase functions in phosphorylation of C-terminal domain Ser7 residues, promoter-proximal pausing, and termination by RNA polymerase II.
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.
Glover-Cutter
Kira
K
Department of Biochemistry and Molecular Genetics, University of Colorado School of Medicine, UCHSC, MS 8101, P.O. Box 6511, Aurora, CO 80045, USA.
Larochelle
Stéphane
S
Erickson
Benjamin
B
Zhang
Chao
C
Shokat
Kevan
K
Fisher
Robert P
RP
Bentley
David L
DL
D045744:Cell Line, Tumor; D002843:Chromatin / Q000378:metabolism; D042863:Cyclin-Dependent Kinase 9 / Q000378:metabolism; D018844:Cyclin-Dependent Kinases / Q000378:metabolism*; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases / Q000378:metabolism; D006657:Histones / Q000378:metabolism*; D006801:Humans; D009687:Nuclear Proteins / Q000378:metabolism*; D010749:Phosphoprotein Phosphatases / Q000378:metabolism*; D010766:Phosphorylation / Q000502:physiology; D016271:Proto-Oncogene Proteins c-myc / Q000378:metabolism; D012319:RNA Polymerase II / Q000378:metabolism*; D012694:Serine / Q000378:metabolism; D051758:Transcription Factor TFIIH / Q000378:metabolism*; D020741:rho GTP-Binding Proteins / Q000378:metabolism; D000095043:Cyclin-Dependent Kinase-Activating Kinase
D002843:Chromatin; D006657:Histones; C489427:MYC protein, human; D009687:Nuclear Proteins; D016271:Proto-Oncogene Proteins c-myc; D051758:Transcription Factor TFIIH; D012694:Serine; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases; C467969:CDK9 protein, human; D042863:Cyclin-Dependent Kinase 9; D018844:Cyclin-Dependent Kinases; D012319:RNA Polymerase II; C413483:CTDSP1 protein, human; D010749:Phosphoprotein Phosphatases; D020741:rho GTP-Binding Proteins; D000095043:Cyclin-Dependent Kinase-Activating Kinase; C000722416:CDK7 protein, human