Positive transcription elongation factor b (P-TEFb), composed of cyclin-dependent kinase 9 (CDK9) and cyclin T, is a global transcription factor for eukaryotic gene expression, as well as a key factor for human immunodeficiency virus (HIV) transcription elongation. P-TEFb phosphorylates the carboxyl-terminal domain (CTD) of the large subunit of RNA polymerase II (RNAP II), facilitating the transition from nonprocessive to processive transcription elongation. Recently, the bromodomain protein Brd4 has been shown to interact with the low-molecular-weight, active P-TEFb complex and recruit P-TEFb to the HIV type 1 long terminal repeat (LTR) promoter. However, the subsequent events through which Brd4 regulates CDK9 kinase activity and RNAP II-dependent transcription are not clearly understood. Here we provide evidence that Brd4 regulates P-TEFb kinase activity by inducing a negative pathway. Moreover, by analyzing stepwise initiation and elongation complexes, we demonstrate that P-TEFb activity is regulated in the transcription complex. Brd4 induces phosphorylation of CDK9 at threonine 29 (T29) in the HIV transcription initiation complex, inhibiting CDK9 kinase activity. P-TEFb inhibition is transient, as Brd4 is released from the transcription complex between positions ؉14 and ؉36. Removal of the phosphate group at T29 by an incoming phosphatase released P-TEFb activity, resulting in increased RNAP II CTD phosphorylation and transcription. Finally, we present chromatin immunoprecipitation studies showing that CDK9 with phosphorylated T29 is associated with the HIV promoter region in the integrated and transcriptionally silent HIV genome.The largest subunit of eukaryotic RNA polymerase II (RNAP II) has a carboxyl-terminal domain (CTD) consisting of tandem repeats of the consensus heptad peptide Tyr-SerPro-Thr-Ser-Pro-Ser, which is conserved among most eukaryotes (3,17,30). The CTD is phosphorylated at Ser 2 (Ser 2P) and Ser 5 (Ser 5P) as RNAP II progresses through the transcription initiation and elongation. At least three cyclindependent kinases are capable of phosphorylating the RNAP II CTD in the regulation of different stages of mRNA synthesis. The first kinase, cyclin-dependent kinase 7 (CDK7), along with MAT1 and cyclin H, forms a complex known as CDKactivating kinase which is part of the general transcription factor TFIIH (9, 23, 35, 45). As analyzed both in vitro and in vivo, TFIIH facilitates promoter clearance by preferentially phosphorylating Ser 5 of the RNAP II CTD within the promoter region (18,36,40). The second kinase, CDK9, is a cdc2-like serine/threonine kinase that was initially isolated by screening a human cDNA library by using oligonucleotide probes to identify CDK-related proteins (12). Consistent with the process of selection, the 43-kDa CDK9 protein shares a high degree of homology with cdc2, cdk2, cdk3, and cdk5. Understanding the function of CDK9 was facilitated by the discovery that CDK9 was the kinase subunit of positive transcription elongation factor b (P-TEFb), which supports transcri...

Positive transcription elongation factor (P-TEFb), which is composed of CDK9 and cyclin T1, plays an important role in cellular and viral gene expression. Our lab has recently demonstrated that P-TEFb is required for Tax transactivation of the viral long terminal repeat (LTR). P-TEFb is found in two major complexes: the inactive form, which is associated with inhibitory subunits 7SK snRNA and HEXIM1, and the active form, which is associated with, at least in part, Brd4. In this study, we analyzed the effect of Brd4 on human T-lymphotropic virus type 1 (HTLV-1) transcription. Overexpression of Brd4 repressed Tax transactivation of the HTLV-1 LTR in a dose-dependent manner. In vitro binding studies suggest that Tax and Brd4 compete for binding to P-TEFb through direct interaction with cyclin T1. Tax interacts with cyclin T1 amino acids 426 to 533, which overlaps the region responsible for Brd4 binding. In vivo, overexpression of Tax decreased the amount of 7SK snRNA associated with P-TEFb and stimulates serine 2 phosphorylation of the RNA polymerase II carboxyl-terminal domain, suggesting that Tax regulates the functionality of P-TEFb. Our results suggest the possibility that Tax may compete and functionally substitute for Brd4 in P-TEFb regulation.Human T-lymphotropic virus type 1 (HTLV-1) is the causative agent of adult T-cell leukemia (ATL) (37, 50, 51), chronic diseases such as HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP) (13, 32, 35), HTLV-1-associated arthropathy (7,22,34,45), uveitis (31), and Sjögren's syndrome (44). The transactivator Tax protein, encoded by the pX region of HTLV-1, is essential for viral replication, transformation, and gene regulation (2,4,10,15,21,33,42). Three highly conserved 21-bp repeat elements located within the long terminal repeat (LTR), Tax-responsive elements (TRE), are critical for Tax-mediated viral transcriptional activation (5,9,40,41,50). Association of Tax with CREB on the HTLV-1 LTR (1, 43, 52) facilitates recruitment of cellular cofactors CBP (14, 17, 23, 52), p300 (16, 52), and PCAF (19), thereby mediating the activation of HTLV-1 gene expression. Tax also regulates the binding of histone deacetylases, a repressor of transactivation, from the HTLV-1-LTR promoter (25, 27).P-TEFb is a protein kinase involved in RNA polymerase (Pol) II elongation of many genes in mammalian cells (39). P-TEFb is composed of catalytic subunit CDK9 and either regulatory subunit cyclin T1, -T2, or -K (11, 36). The transcriptional activity of P-TEFb depends on the CDK9 kinase activity, which hyperphosphorylates the Ser 2 residue of the carboxylterminal domain (CTD) of the largest subunit of RNA Pol II (28, 53). P-TEFb also phosphorylates negative elongation factors DSIF and NELF, which cooperatively inhibit RNA Pol II processivity, to stimulate the elongation of transcription (3,12,38,39). In a previous study, our lab found that CDK9 was an essential factor for transactivation of the HTLV-1 LTR by Tax (54). Our results further suggested that P-TEFb was recruited to the...