Brd4 is a mammalian bromodomain protein that binds to acetylated chromatin. Proteomic analysis revealed that Brd4 interacts with cyclinT1 and Cdk9 that constitutes core positive transcription elongation factor b (P-TEFb). Brd4 interacted with P-TEFb in the living nucleus through its bromodomain. About half of P-TEFb was bound to the inhibitory subunit and functionally inactive. Brd4 interacted with P-TEFb that was free of the inhibitory subunit. An increase in Brd4 expression led to increased P-TEFb-dependent phosphorylation of RNA polymerase II (RNAPII) CTD and stimulation of transcription from promoters in vivo. Conversely, a reduction in Brd4 expression by siRNA reduced CTD phosphorylation and transcription, revealing that Brd4 is a positive regulatory component of P-TEFb. In chromatin immunoprecipitation (ChIP) assays, the recruitment of P-TEFb to a promoter was dependent on Brd4 and was enhanced by an increase in chromatin acetylation. Together, P-TEFb alternately interacts with Brd4 and the inhibitory subunit to maintain functional equilibrium in the cell.

Tat stimulates human immunodeficiency virus type 1 (HIV-1) transcriptional elongation by recruitment of carboxyl-terminal domain (CTD) kinases to the HIV-1 promoter. Using an immobilized DNA template assay, we have analyzed the effect of Tat on kinase activity during the initiation and elongation phases of HIV-1 transcription. Our results demonstrate that cyclin-dependent kinase 7 (CDK7) (TFIIH) and CDK9 (P-TEFb) both associate with the HIV-1 preinitiation complex. Hyperphosphorylation of the RNA polymerase II (RNAP II) CTD in the HIV-1 preinitiation complex, in the absence of Tat, takes place at CTD serine 2 and serine 5. Analysis of preinitiation complexes formed in immunodepleted extracts suggests that CDK9 phosphorylates serine 2, while CDK7 phosphorylates serine 5. Remarkably, in the presence of Tat, the substrate specificity of CDK9 is altered, such that the kinase phosphorylates both serine 2 and serine 5. Tat-induced CTD phosphorylation by CDK9 is strongly inhibited by low concentrations of 5,6-dichloro-1-␤-D-ribofuranosylbenzimidazole, an inhibitor of transcription elongation by RNAP II. Analysis of stalled transcription elongation complexes demonstrates that CDK7 is released from the transcription complex between positions ؉14 and ؉36, prior to the synthesis of transactivation response (TAR) RNA. In contrast, CDK9 stays associated with the complex through ؉79. Analysis of CTD phosphorylation indicates a biphasic modification pattern, one in the preinitiation complex and the other between ؉36 and ؉79. The second phase of CTD phosphorylation is Tatdependent and TAR-dependent. These studies suggest that the ability of Tat to increase transcriptional elongation may be due to its ability to modify the substrate specificity of the CDK9 complex.Human immunodeficiency virus type 1 (HIV-1) encodes a transactivator protein, Tat, which stimulates transcription elongation through interaction with the transactivation response (TAR) RNA element located at the 5Ј end of nascent transcripts (12,28,68,75). In view of the observations that hyperphosphorylation of the carboxyl-terminal domain (CTD) of the large subunit of RNA polymerase II (RNAP II) correlates with the formation of processive elongation complexes (11) and that Tat transactivation requires the CTD (6,44,46,79), it has been proposed that a critical step in Tat transactivation is mediated through a cellular kinase(s) (68, 80). Two cyclin-dependent kinase (CDK)-cyclin pairs, present in two distinct transcription factor complexes, have been implicated as Tat cofactors which could phosphorylate the CTD (54, 68). TFIIH, a general transcription factor which contains nine polypeptides (ERCC3/XPB, ERCC2/XPD, p62, p54, p44, CDK7 [MO15], cyclin H, MAT1, and p34) (13, 24), possesses CTD kinase activity (14, 37). The kinase activity of TFIIH resides in the CDK7 subunit (15,58,61,62). In association with cyclin H and Mat1, CDK7 forms the CDK-activating kinase (CAK) complex that phosphorylates CDKs involved in the regulation of the cell cycle (19,42,43,53,67). The assoc...

HIV-1 gene expression is regulated by a viral transactivator protein (Tat) which induces transcriptional elongation of HIV-1 long tandem repeat (LTR). This induction requires hyperphosphorylation of the C-terminal domain (CTD) repeats of RNA polymerase II (Pol II). To achieve CTD hyperphosphorylation, Tat stimulates CTD kinases associated with general transcription factors of the promoter complex, specifically TFIIH-associated CDK7 and positive transcription factor b-associated CDK9 (cyclin-dependent kinase 9). Other studies indicate that Tat may bind an additional CTD kinase that regulates the target-specific phosphorylation of RNA Pol II CTD. We previously reported that Tat-associated T-cell-derived kinase (TTK), purified from human primary T-cells, stimulates Tat-dependent transcription of HIV-1 LTR in vivo [Nekhai, Shukla, Fernandez, Kumar and Lamb (2000) Virology 266, 246-256]. In the work presented here, we characterized the components of TTK by biochemical fractionation and the function of TTK in transcription assays in vitro. TTK uniquely co-purified with CDK2 and not with either CDK9 or CDK7. Tat induced the TTK-associated CDK2 kinase to phosphorylate CTD, specifically at Ser-2 residues. The TTK fraction restored Tat-mediated transcription activation of HIV-1 LTR in a HeLa nuclear extract immunodepleted of CDK9, but not in the HeLa nuclear extract double-depleted of CDK9 and CDK7. Direct microinjection of the TTK fraction augmented Tat transactivation of HIV-1 LTR in human primary HS68 fibroblasts. The results argue that TTK-associated CDK2 may function to maintain target-specific phosphorylation of RNA Pol II that is essential for Tat transactivation of HIV-1 promoter. They are also consistent with the observed cell-cycle-specific induction of viral gene transactivation.

Transcription consists of a series of highly regulated steps: assembly of a preinitiation complex (PIC) at the promoter nucleated by TFIID, followed by initiation, elongation, and termination. The present study has focused on the role of the TFIID component, TAF7, in regulating transcription initiation. In TFIID, TAF7 binds to TAF1 and inhibits its intrinsic acetyl transferase activity. We now report that although TAF7 remains bound to TAF1 and associated with TFIID during the formation of the PIC, TAF7 dissociates from the PIC upon transcription initiation. Entry of polymerase II into the assembling PIC is associated with TAF1 and TAF7 phosphorylation, coincident with TAF7 release. We propose that the TFIID composition is dynamic and that TAF7 functions as a check-point regulator suppressing premature transcription initiation until PIC assembly is complete.MHC class I ͉ regulation ͉ preinitiation complex ͉ TFIID I n eukaryotic cells, expression of most protein-encoding genes depends on the RNA polymerase II (Pol II)-dependent transcription machinery. The RNA Pol II machinery assembled on the promoter is composed of distinct complexes that are responsible for effecting the sequential steps of transcription initiation and elongation (1-5). The first step in transcription is the recognition of the core promoter by the TFIID complex and the assembly of a preinitiation complex (PIC) through an ordered recruitment of the general transcription factors (GTFs) TFIIB and TFIIA, followed by the RNA Pol II holoenzyme, the mediator and the remaining GTFs, TFIIF, TFIIE, and TFIIH. Once the PIC has been assembled, transcription initiation ensues: local melting of the promoter DNA, formation of the first phosphodiester bond, followed by the synthesis of a short nascent RNA at which point the polymerase pauses. The initiation of transcription is accompanied by the phosphorylation of serine 5 in the C-terminal domain (CTD) heptad repeat of RNA Pol II (CTD) by the kinase subunit of TFIIH, CDK7 (6-9).Despite the extensive understanding of the general requirements of transcription, many details remain unresolved and there is considerable variation among different promoters and cell types. Promoter recognition is mediated by members of either the TFIID complex family (TFIID, TFIID-like) or the SAGA complex family (e.g., TFTC, PCAF, SAGA) (10-12). In yeast, 90% of gene expression is TFIID-dependent transcription; the remaining 10% is largely dependent on the SAGA complex (13). The TFIID complexes are composed of either the TATAbinding protein (TBP) or a TBP related protein (TRF1, TRF2) and several TBP-associated factors (TAFs) (14-17). The SAGA family complexes do not contain TBP or TBP-related proteins; rather, they contain a GCN5-related histone acetyl transferase (AT) subunit, several adapter and Spt proteins and a subset of TAFs. The composition of the TAFs present in these different complexes varies depending on the structure of the promoter and the cell cycle and tissue-specific gene expression requirement. For example, in yeas...

The human immunodeficiency virus type 1 (HIV-1) Tat protein recruits positive transcription elongation factor b (P-TEFb) to the transactivation response (TAR) RNA structure to facilitate formation of processive transcription elongation complexes (TECs). Here we examine the role of the Tat/TAR-specified cyclin-dependent kinase 9 (CDK9) kinase activity in regulation of HIV-1 transcription elongation and histone methylation. In HIV-1 TECs, P-TEFb phosphorylates the RNA polymerase II (RNAP II) carboxyl-terminal domain (CTD) and the transcription elongation factors SPT5 and Tat-SF1 in a Tat/TAR-dependent manner. Using in vivo chromatin immunoprecipitation analysis, we demonstrate the following distinct properties of the HIV-1 transcription complexes. First, the RNAP II CTD is phosphorylated at Ser 2 and Ser 5 near the promoter and at downstream coding regions. Second, the stable association of SPT5 with the TECs is dependent upon P-TEFb kinase activity. Third, P-TEFb kinase activity is critical for the induction of methylation of histone H3 at lysine 4 and lysine 36 on HIV-1 genes. Flavopiridol, a potent P-TEFb kinase inhibitor, inhibits CTD phosphorylation, stable SPT5 binding, and histone methylation, suggesting that its potent antiviral activity is due to its ability to inhibit several critical and unique steps in HIV-1 transcription elongation.The Tat protein encoded by human immunodeficiency virus type 1 (HIV-1) stimulates transcription elongation through its interaction with the transactivation response (TAR) RNA structure located at the 5Ј end of nascent viral transcripts (2,16,30,67). In view of the observations that hyperphosphorylation of the carboxyl-terminal domain (CTD) of the large subunit of RNA polymerase II (RNAP II) correlates with the formation of processive elongation complexes (14) and that Tat transactivation requires RNAP II CTD (12,48,71), it has been proposed that a critical step in Tat transactivation is mediated through cellular kinases which are capable of phosphorylating RNAP II CTD (54,62,72). The RNAP II CTD is composed of multiple heptad repeats of Tyr-Ser-Pro-ThrSer-Pro-Ser, whose consensus is conserved among most eukaryotes. Ser 2 and Ser 5 are targets for phosphorylation and dephosphorylation during transcription (34).Positive transcription elongation factor b (P-TEFb) (42-44) or Tat-associated kinase (23, 25, 26), composed of cyclin-dependent kinase 9 (CDK9) and cyclin T1 (23,41,52,68,70,76,78), is essential for Tat transactivation. Formation of the tripartite complex between Tat, cyclin T1, and TAR depends on the 5Ј bulge and central loop in the TAR RNA structure (3,18,20,29,38,68). CDK9 autophosphorylation promotes highaffinity binding of the Tat/P-TEFb complex to the TAR RNA structure (17, 19) as well as CDK9 activation (75). TAR-bound P-TEFb induces a Tat/TAR-dependent phosphorylation of RNAP II CTD to stimulate transcription elongation (33, 74). P-TEFb preferentially phosphorylates Ser 2 of the RNAP II CTD, but its substrate specificity can be modulated to target both Ser 2 an...

The HIV type 1 (HIV-1) Tat protein stimulates transcription elongation by recruiting P-TEFb (CDK9͞cyclin T1) to the transactivation response (TAR) RNA structure. Tat T he capping of mRNA occurs cotranscriptionally by a series of three enzymatic reactions in which the 5Ј triphosphate terminus of the nascent transcript is cleaved to a diphosphate by RNA 5Ј triphosphatase, capped with GMP by RNA guanylyltransferase (GT) and methylated at the N7 position of guanine by RNA (guanine-7) methyltransferase. The three activities are present in all eukaryotes examined. Yeast species encode three proteins corresponding to each enzyme activity, whereas in metazoans the first two activities are part of one protein consisting of N-terminal triphosphatase and C-terminal GT domains (1, 2).Targeting of cap formation to RNA polymerase II (RNAP II) transcripts is achieved through physical interaction of components of the capping apparatus with the phosphorylated Cterminal domain (CTD) of the largest subunit of RNAP II (3-6). The mammalian RNAP II CTD is composed of 52 tandemly repeated heptads with a consensus, Tyr-1-Ser-2-Pro-3-Thr-4-Ser-5-Pro-6-Ser-7, conserved in most eukaryotes. Ser-2 and Ser-5 are targets of phosphorylation and dephosphorylation during transcription (7). Principal kinases responsible for RNAP II CTD phosphorylation during transcription include transcription factor IIH (TFIIH) (CDK7͞cyclin H) and positive transcription elongation factor P-TEFb (CDK9͞cyclin T1). Phosphorylation of the CTD Ser-2 and Ser-5 residues has differential effects on recruitment and activation of capping enzyme (CE) (8, 9). Although Ser-2 phosphorylation of CTD heptads is sufficient for mammalian GT binding, its activation requires Ser-5-phosphorylated CTD heptads (10). RNAP II CTD is phosphorylated at Ser-5 by TFIIH during transcription initiation through the promoter clearance stage and changes to Ser-2 phosphorylation when the polymerase is associated with the coding region (11). However, RNAP II CTD Ser-5 phosphorylation is sustained by Tat͞transactivation response (TAR)-induced P-TEFb following release of TFIIH from HIV-1 transcription elongation complexes (TECs) at the promoter clearance stage (12, 13). Chromatin immunoprecipitation (ChIP) assays show an accumulation of RNAP II phosphorylated at Ser-5 in the promoter proximal regions of a number of genes (14, 15), suggesting that the role of RNAP II CTD phosphorylation in RNA processing is more than mere anchoring of transcription factors.HIV-1 Tat transactivation provides a particularly useful model to study regulation of processive transcription elongation and mRNA capping. Tat stimulates HIV-1 transcription elongation by recruitment of P-TEFb to the TAR RNA structure, and Tat͞TAR-associated CDK9 then phosphorylates RNAP II CTD and other RNAP II-associated proteins, leading to a transition from nonprocessive to processive transcription (16). P-TEFb phosphorylates both Ser-2 and Ser-5 of RNAP II CTD in the presence of Tat, whereas P-TEFb alone phosphorylates only . In this study, w...

Human T-lymphotropic virus type 1 (HTLV-1) encodes a transcriptional activator, Tax, whose function is essential for viral transcription and replication. Tax transactivates the viral long-terminal repeat through a series of protein-protein interactions which facilitate CREB and CBP/p300 binding. In addition, Tax dissociates transcription repressor histone deacetylase 1 interaction with the CREB response element. The subsequent events through which Tax interacts and communicates with RNA polymerase II and cyclin-dependent kinases (CDKs) are not clearly understood. Here we present evidence that Tax recruits positive transcription elongation factor b (P-TEFb) (CDK9/cyclin T1) to the viral promoter. This recruitment likely involves proteinprotein interactions since Tax associates with P-TEFb in vitro as demonstrated by glutathione S-transferase fusion protein pull-down assays and in vivo as shown by coimmunoprecipitation assays. Functionally, small interfering RNA directed toward CDK9 inhibited Tax transactivation in transient assays. Consistent with these findings, the depletion of CDK9 from nuclear extracts inhibited Tax transactivation in vitro. Reconstitution of the reaction with wild-type P-TEFb, but not a kinase-dead mutant, recovered HTLV-1 transcription. Moreover, the addition of the CDK9 inhibitor flavopiridol blocked Tax transactivation in vitro and in vivo. Interestingly, we found that Tax regulates CDK9 kinase activity through a novel autophosphorylation pathway.Human T-lymphotropic virus type 1 (HTLV-1) is the causative agent of adult T-cell leukemia and the neurological disorder HTLV-1-associated myelopathy/tropical spastic paraparesis (22,34,51,56,76). Studies have shown that the transactivator protein Tax, which is encoded by the pX region of HTLV-1, is a potent activator of the HTLV-1 long terminal repeat (LTR) (4, 16-18, 31, 33, 55, 58). Three highly conserved 21-bp repeat elements located in the LTR, termed Tax response elements (TRE), are critical to Tax-mediated transcription activation (5,14,61,62,75). Tax facilitates binding and associates with the LTR through interaction with CREB (1,2,23,25,69,80). The formation of the Tax-CREB promoter complex serves as a binding site for the recruitment of multifunctional cellular coactivators CBP, p300, and PCAF (24,28,32,38,40). Tax has also been shown to interfere with the binding of transcription inhibitors, such as histone deacetylase 1, to the viral LTR (45).At least three cyclin-dependent kinases are involved in the regulation of different stages of mRNA synthesis. Cyclin-dependent kinase 7 (CDK7), along with cyclin H and MAT1, forms a complex known as CAK, which is part of the general transcription factor TFIIH (13,46,63,73). The second kinase, CDK9, is a cdc2-like serine/threonine kinase initially isolated by screening a human cDNA library using oligonucleotide probes to identify CDK-related proteins (26). Consistently, the 43-kDa CDK9 protein shares a high degree of homology with cdc2, cdk2, cdk3, and cdk5. Understanding of the CDK9 function was ...

Transcription consists of a series of highly regulated steps: assembly of the preinitiation complex (PIC) at the promoter, initiation, elongation, and termination. PIC assembly is nucleated by TFIID, a complex composed of the TATA-binding protein (TBP) and a series of TBP-associated factors (TAFs). One component, TAF7, is incorporated in the PIC through its interaction with TFIID but is released from TFIID upon transcription initiation. We now report that TAF7 interacts with the transcription factors, TFIIH and P-TEFb, resulting in the inhibition of their Pol II CTD kinase activities. Importantly, in in vitro transcription reactions, TAF7 inhibits steps after PIC assembly and formation of the first phosphodiester bonds. Further, in vivo TAF7 coelongates with P-TEFb and Pol II downstream of the promoter. We propose a model in which TAF7 contributes to the regulation of the transition from PIC assembly to initiation and elongation.MHC class I genes ͉ regulation ͉ transcription initiation I n eukaryotic cells, expression of protein-encoding genes depends on the ordered recruitment of the general transcription factors (GTF) TFIID, TFIIB, and TFIIA to the promoter, followed by association of Pol II, the mediator, and the remaining GTFs, TFIIF, TFIIE, and TFIIH, to form a preinitiation complex (PIC) (1). Once PIC assembly is complete, transcription initiation ensues; Pol II with the elongation complex dissociate from the PIC (2, 3). A required step during transcription initiation is the phosphorylation of serine 5 in the carboxy terminal domain (CTD) heptad repeat of Pol II by the kinase subunit of TFIIH, CDK7 (4, 5), after which Pol II pauses to ensure proper pre-mRNA capping (6-9). The transition from pausing to elongation is facilitated by the P-TEFb elongation complex, which also mediates efficient elongation (10). P-TEFb consists of two subunits, cyclin T1 and the kinase CDK9, which phosphorylates serine 2 of the CTD, required for productive elongation and the recruitment of complexes involved in mRNA processing (splicing and polyadenylation) (10-15).Although the general mechanics of transcription have been characterized, relatively little is known about how the transitions from PIC assembly to initiation/pausing to elongation are regulated. Promoter recognition is largely mediated by TFIID, which is composed of the TATA binding protein (TBP) and over a dozen TBP associated factors (TAFs) (16,17,18). The largest TFIID component, TAF1, has both acetyltransferase (AT) and kinase activities (19,20). We demonstrated that TAF1 and its intrinsic acetyltransferase activity are essential for transcription of an MHC class I gene (21). Importantly, MHC class I transcription is inhibited both in vitro and in vivo by the viral transactivator, HIV Tat, which binds to the TAF1 AT domain, inhibiting its enzymatic activity (22, 23). TAF7, a cellular 55-kDa TFIID component (24,25), also binds to TAF1 inhibiting its AT activity and repressing MHC class I transcription (26) Significantly, we have demonstrated that TAF7 remains boun...