Transcriptional regulation by targeted recruitment of cyclin-dependent CDK9 kinase in vivo.
The CDK9 kinase in association with Cyclin T is a component of the transcription positive-acting complex pTEFb which facilitates the transition from abortive to productive transcription elongation by phosphorylating the carboxyl-terminal domain of RNA polymerase II. The Cyclin T1/CDK9 complex is implicated in Tat transactivation, and it has been suggested that Tat functions by recruiting this complex to RNAPII through cooperative binding to RNA. Here, we demonstrate that targeted recruitment of Cyclin T1/CDK9 kinase complex to specific promoters, through fusion to a DNA-binding domain of either Cyclin T1 or CDK9 kinase, stimulates transcription in vivo. Transcriptional enhancement was dependent on active CDK9, as a catalytically inactive form had no transcriptional effect. We determined that, unlike conventional activators, DNA-bound CDK9 does not activate enhancerless TATA-promoters unless TBP is overexpressed, suggesting that CDK9 acts in vivo at a step subsequent to TFIID recruitment DNA-bound. Finally, we determined that CDK9-mediated transcriptional activation is mediated by preferentially stimulating productive transcription elongation.
Majello
B
B
Department of Genetics, Molecular and General Biology, University of Naples 'Federico II' and International Institute of Genetics and Biophysics, CNR, Naples, Italy.
Napolitano
G
G
Giordano
A
A
Lania
L
L
D002461:Cell Line, Transformed; D056906:Cyclin T; D042863:Cyclin-Dependent Kinase 9; D018844:Cyclin-Dependent Kinases / Q000235:genetics / Q000378:metabolism*; D016213:Cyclins / Q000235:genetics / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000378:metabolism; D005656:Fungal Proteins / Q000235:genetics / Q000378:metabolism; D005786:Gene Expression Regulation; D006678:HIV / Q000235:genetics; D006367:HeLa Cells; D006801:Humans; D011401:Promoter Regions, Genetic; D016271:Proto-Oncogene Proteins c-myc / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins; D013539:Simian virus 40 / Q000235:genetics; D035181:TATA-Box Binding Protein; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism; D014158:Transcription, Genetic
C503684:CCNT1 protein, human; D056906:Cyclin T; D016213:Cyclins; D004268:DNA-Binding Proteins; D005656:Fungal Proteins; C052130:GAL4 protein, S cerevisiae; D016271:Proto-Oncogene Proteins c-myc; D011993:Recombinant Fusion Proteins; D029701:Saccharomyces cerevisiae Proteins; D035181:TATA-Box Binding Protein; D014157:Transcription Factors; C467969:CDK9 protein, human; D042863:Cyclin-Dependent Kinase 9; D018844:Cyclin-Dependent Kinases