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 speci®c 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 e ect. 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.
FCP1, a phosphatase specific of the carboxyl-terminal-domain of the large subunit of the RNA polymerase II (RNAPII), stimulates transcription elongation and it is required for general transcription and cell viability. To identify novel interacting proteins of FCP1, we used a human cell line expressing an epitope flagged FCP1 and proteins, which formed complexes with FCP1, were identified by mass spectrometry. We identified four proteins: RPB2 subunit of the RNAPII, the nuclear kinase, NDR1, the methyltransferase PRMT5 and the enhancer of rudimentary homologue (ERH) proteins. Intriguingly, both the PRMT5 and ERH proteins are interacting partners of the SPT5 elongation factor. Interactions of RPB2, ERH, NDR1 and PRMT5 with FCP1 were confirmed by co-immunoprecipitation or in vitro pull-down assays. Interaction between PRMT5 and FCP1 was further confirmed by co-immunoprecipitation of endogenous proteins. We found that FCP1 is a genuine substrate of PRMT5-methylation both in vivo and in vitro, and FCP1-associated PRMT5 can methylate histones H4 in vitro.
The positive transcription elongation factor (P-TEFb) comprises a kinase, CDK9, and a Cyclin T1 or T2. Its activity is inhibited by association with the HEXIM1 or HEXIM2 protein bound to 7SK small nuclear RNA. HEXIM1 and HEXIM2 were found to form stable homoand hetero-oligomers. Using yeast two-hybrid and transfection assays, we have now shown that the C-terminal domains of HEXIM proteins directly interact with each other. Hydrodynamic parameters measured by glycerol gradient ultracentrifugation and gel-permeation chromatography demonstrate that both purified recombinant and cellular HEXIM1 proteins form highly anisotropic particles. Chemical cross-links suggest that HEXIM1 proteins form dimers. The multimeric nature of HEXIM1 is maintained in P-TEFb⅐HEXIM1⅐7SK RNA complexes. Multiple P-TEFb modules are found in the inactive P-TEFb⅐HEXIM1⅐7SK complexes. It is proposed that 7SK RNA binding to a HEXIM1 multimer promotes the simultaneous recruitment and hence inactivation of multiple P-TEFb units.The positive transcription elongation factor (P-TEFb) 1 comprises a protein kinase, CDK9, and a Cyclin T1, T2, or K (1, 2). It is required for transcription elongation of most class II genes. P-TEFb phosphorylates numerous substrates, including the C-terminal domain (CTD) of RNA polymerase II and the Spt5 subunit of the DRB sensitivity-inducing factor (DSIF). The DSIF prevents transcription from proceeding efficiently after initiation. The kinase activity of P-TEFb antagonizes this inhibitory effect. Several class II genes such as heat-shock or U2 small nuclear RNA genes do not have a strong requirement for P-TEFb activity to elongate, but rather to terminate, transcription properly (3, 4).Recent studies have indicated that two major forms of PTEFb are present in equivalent amounts in HeLa cell lysates (5). The active form consists of "core" P-TEFb, CDK9 and Cyclin T1 or T2. The inactive form consists of CDK9, Cyclin T1 or T2, MAQ1/HEXIM1, and 7SK RNA (5-8). The 7SK RNA is an abundant class III noncoding RNA (9) detected in vertebrates, cephalochordates, and mollusks.2 Binding of 7SK RNA to HEXIM1 turns this protein into a P-TEFb inhibitor (10). In response to treatments that arrest transcription (5-8) or following cardiac hypertrophic stimuli (11, 12), HEXIM1 and 7SK RNA dissociate from P-TEFb. As a consequence, the P-TEFb activity is up-regulated.HEXIM1 expression is up-regulated in smooth muscle cells treated with hexamethylene-bisacetamide (13) or down-regulated by estrogen in breast cancer cells and therefore named EDG-1 (14). HEXIM1 has also been reported as a protein accumulating in heart tissues during early embryogenesis and therefore named CLP-1 (cardiac lineage protein) (15). Disruption of the HEXIM1/CLP1 gene results in heart defects leading to death at birth in homozygote CLP-1(Ϫ/Ϫ) mice (16). Because P-TEFb plays a general role in class II gene expression, such a late developmental defect suggests the existence of a compensatory mechanism at the cellular level. Indeed, a BLAST search within genome and Express...
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