The cyclinT1/Cdk9 heterodimer that constitutes core P-TEFb is generally presumed to be the transcriptionally active form for stimulating RNA polymerase II elongation. About half of cellular P-TEFb also exists in an inactive complex with the 7SK snRNA and the HEXIM1 protein. Here, we show that the remaining half associates with the bromodomain protein Brd4. In stress-induced cells, the 7SK/HEXIM1-bound P-TEFb is quantitatively converted into the Brd4-associated form. The association with Brd4 is necessary to form the transcriptionally active P-TEFb, recruits P-TEFb to a promoter, and enables P-TEFb to contact the Mediator complex, a potential target for the Brd4-mediated recruitment. Although generally required for transcription, the P-TEFb-recruitment function of Brd4 can be substituted by that of HIV-1 Tat, which recruits P-TEFb directly for activated HIV-1 transcription. Brd4, HEXIM1, and 7SK are all implicated in regulating cell growth, which may result from their dynamic control of the general transcription factor P-TEFb.

The positive transcriptional elongation factor b (P-TEFb), consisting of CDK9 and cyclin T, stimulates transcription by phosphorylating RNA polymerase II. It becomes inactivated when associated with the abundant 7SK snRNA. Here, we show that the 7SK binding alone was not sufficient to inhibit P-TEFb. P-TEFb was inhibited by the HEXIM1 protein in a process that specifically required 7SK for mediating the HEXIM1:P-TEFb interaction. This allowed HEXIM1 to inhibit transcription both in vivo and in vitro. P-TEFb dissociated from HEXIM1 and 7SK in cells undergoing stress response, increasing the level of active P-TEFb for stress-induced transcription. P-TEFb was the predominant HEXIM1-associated protein factor, and thus likely to be the principal target of inhibition coordinated by HEXIM1 and 7SK. Since HEXIM1 expression is induced in cells treated with hexamethylene bisacetamide, a potent inducer of cell differentiation, targeting the general transcription factor P-TEFb by HEXIM1/7SK may contribute to the global control of cell growth and differentiation.

The positive transcription elongation factor b (P-TEFb), consisting of Cdk9 and cyclin T, stimulates RNA polymerase II elongation and cotranscriptional pre-mRNA processing. To accommodate different growth conditions and transcriptional demands, a reservoir of P-TEFb is kept in an inactive state in the multisubunit 7SK snRNP. Under certain stress or disease conditions, P-TEFb is released to activate transcription, although the signaling pathway(s) that controls this is largely unknown. Here, through analyzing the UV-or hexamethylene bisacetamide (HMBA)-induced release of P-TEFb from 7SK snRNP, an essential role for the calcium ion (Ca 2+ )-calmodulin-protein phosphatase 2B (PP2B) signaling pathway is revealed. However, Ca 2+ signaling alone is insufficient, and PP2B must act sequentially and cooperatively with protein phosphatase 1␣ (PP1␣) to disrupt 7SK snRNP. Activated by UV/HMBA and facilitated by a PP2B-induced conformational change in 7SK snRNP, PP1␣ releases P-TEFb through dephosphorylating phospho-Thr186 in the Cdk9 T-loop. This event is also necessary for the subsequent recruitment of P-TEFb by the bromodomain protein Brd4 to the preinitiation complex, where Cdk9 remains unphosphorylated and inactive until after the synthesis of a short RNA. Thus, through cooperatively dephosphorylating Cdk9 in response to Ca 2+ signaling, PP2B and PP1␣ alter the P-TEFb functional equilibrium through releasing P-TEFb from 7SK snRNP for transcription.[Keywords: P-TEFb; CDK activation; transcriptional elongation; Ca 2+ signal transduction; protein phosphatases] Supplemental material is available at http://www.genesdev.org. Received November 21, 2007; revised version accepted March 31, 2008. In eukaryotes, the transcription of protein-coding genes is performed by RNA polymerase (Pol) II in a cyclic process consisting of several tightly regulated stages (Sims et al. 2004). During the elongation stage, the C-terminal domain of the largest subunit of Pol II is phosphorylated by the positive transcription elongation factor b (P-TEFb) (Peterlin and Price 2006;Zhou and Yik 2006). This modification is crucial for Pol II to change from abortive to productive elongation and produce full-length RNA transcripts. Consisting of Cdk9 and cyclin T1 (or the minor forms T2 and K), P-TEFb is considered a general transcription factor required for the expression of a vast array of protein-coding genes (Chao and Price 2001;Shim et al. 2002). Not only is P-TEFb critical for cellular gene transcription, it is also a specific host cofactor for the HIV-1 Tat protein. Tat recruits P-TEFb to the TAR RNA element located at the 5Ј end of nascent viral transcripts, allowing P-TEFb to phosphorylate stalled Pol II and enhance HIV-1 elongation (Peterlin and Price 2006).However, not every P-TEFb in the nucleus is in a transcriptionally active state. A major reservoir of P-TEFb (∼50% of total P-TEFb in HeLa cells) actually exists in an inactive complex termed 7SK snRNP that also contains the 7SK snRNA (Nguyen et al. 2001;Yang et al. 2001) and three nuclear...

The positive transcription elongation factor b (PTEFb), comprising CDK9 and cyclin T, stimulates transcription of cellular and viral genes by phosphorylating RNA polymerase II. A major portion of nuclear P-TEFb is sequestered and inactivated by the coordinated actions of the 7SK snRNA and the HEXIM1 protein, whose induced dissociation from P-TEFb is crucial for stressinduced transcription and pathogenesis of cardiac hypertrophy. The 7SK⅐P-TEFb interaction, which can occur independently of HEXIM1 and does not by itself inhibit P-TEFb, recruits HEXIM1 for P-TEFb inactivation. To study the control of this interaction, we established an in vitro system that reconstituted the specific interaction of P-TEFb with 7SK but not other snRNAs. Using this system, together with an in vivo binding assay, we show that the phosphorylation of CDK9, on possibly the conserved Thr-186 in the T-loop, was crucial for the 7SK⅐P-TEFb interaction. This phosphorylation was not caused by CDK9 autophosphorylation or the general CDK-activating kinase CAK, but rather by a novel HeLa nuclear kinase. Furthermore, the stress-induced disruption of the 7SK⅐P-TEFb interaction was not caused by any prohibitive changes in 7SK but by the dephosphorylation of P-TEFb, leading to the loss of the key phosphorylation important for 7SK binding. Thus, the phosphorylated P-TEFb is tagged for inhibition through association with 7SK. We discuss the implications of this mechanism in controlling P-TEFb activity during normal and stress-induced transcription.7SK is an abundant (2 ϫ 10 5 copies/cell) and evolutionarily conserved small nuclear RNA (snRNA) 1 of ϳ330 nucleotides (1-4). It is transcribed by RNA polymerase (Pol) III from one or more genes belonging to a family of interspersed repeats in the mammalian genome (2, 5). The high conservation and abundance of 7SK suggest an important physiological function of this RNA. In searching for nuclear factors that can bind to and regulate the activity of human positive transcription elongation factor b (P-TEFb), we and others (6, 7) have identified 7SK as a specific P-TEFb-associated factor. Consisting of CDK9 and cyclin T1 (CycT1) (8, 9), P-TEFb strongly enhances the processivity of RNA Pol II by phosphorylating the C-terminal domain (CTD) of Pol II and antagonizing the actions of negative elongation factors (10 -13). Studies employing RNA interference in Caenorhabditis elegans and a pharmacological inhibitor of CDK9 in mammalian cells implicate P-TEFb as essential for expression of most protein-coding genes (10, 14).P-TEFb also functions as a specific host cellular cofactor for the HIV-1 Tat protein. Stimulation of Pol II elongation is essential for HIV transcription, during which P-TEFb is recruited to the nascent mRNA by Tat (15, 16). Tat binds to CycT1 and recruits P-TEFb through formation of a stable ternary complex containing P-TEFb, Tat, and the HIV TAR RNA structure located at the 5Ј-end of the nascent viral transcript. Once recruited, P-TEFb phosphorylates the CTD and stimulates the production of the full-l...

Human immunodeficiency virus type 1 (HIV-1) transcriptional transactivator (Tat) recruits the positive transcription elongation factor b (P-TEFb) to the viral promoter. Consisting of cyclin dependent kinase 9 (Cdk9) and cyclin T1, P-TEFb phosphorylates RNA polymerase II and the negative transcription elongation factor to stimulate the elongation of HIV-1 genes. A major fraction of nuclear P-TEFb is sequestered into a transcriptionally inactive 7SK small nuclear ribonucleoprotein (snRNP) by the coordinated actions of the 7SK small nuclear RNA (snRNA) and hexamethylene bisacetamide (HMBA) induced protein 1 (HEXIM1). In this study, we demonstrate that Tat prevents the formation of and also releases P-TEFb from the 7SK snRNP in vitro and in vivo. This ability of Tat depends on the integrity of its N-terminal activation domain and stems from the high affinity interaction between Tat and cyclin T1, which allows Tat to directly displace HEXIM1 from cyclin T1. Furthermore, we find that in contrast to the Tat-independent activation of the HIV-1 promoter, Tat-dependent HIV-1 transcription is largely insensitive to the inhibition by HEXIM1. Finally, primary blood lymphocytes display a reduced amount of the endogenous 7SK snRNP upon HIV-1 infection. All these data are consistent with the model that Tat not only recruits but also increases the active pool of P-TEFb for efficient HIV-1 transcription.

The 7SK snRNP represents a major reservoir of activity where P-TEFb, a general transcription factor key for RNA polymerase II elongation, can be withdrawn to promote gene expression, cell growth and development. Within this complex, 7SK snRNA is a central scaffold that coordinates key protein–protein interactions and maintains P-TEFb in an inactive state. Although the stability of 7SK directly affects the amount of active P-TEFb in vivo, relatively little is known about how it is maintained and how the 7SK methylphosphate capping enzyme MePCE and LARP7, a La-related protein associated with the 3′-poly(U) of 7SK, contribute to this process. Here, we show that 7SK is capped by the LARP7-free MePCE and in probably a co-transcriptional manner prior to its sequestration into 7SK snRNP. However, upon interacting with LARP7 within 7SK snRNP, MePCE loses its capping activity, probably due to the occlusion of its catalytic center by LARP7. Despite its lack of capping activity in 7SK snRNP, MePCE displays a capping-independent function to promote the LARP7–7SK interaction, which in turn stabilizes 7SK and facilitates the assembly of a stable MePCE–LARP7–7SK subcomplex. Our data indicate that MePCE and LARP7 act cooperatively to stabilize 7SK and maintain the integrity of 7SK snRNP.

The HEXIM1 protein inhibits the kinase activity of P-TEFb (CDK9/cyclin T) to suppress RNA polymerase II transcriptional elongation in a process that specifically requires the 7SK snRNA, which mediates the interaction of HEXIM1 with P-TEFb. In an attempt to define the sequence requirements for HEXIM1 to interact with 7SK and inactivate P-TEFb, we have identified the first 18 amino acids within the previously described nuclear localization signal (NLS) of HEXIM1 as both necessary and sufficient for binding to 7SK in vivo and in vitro. This 7SK-binding motif was essential for HEXIM1's inhibitory action, as the HEXIM1 mutants with this motif replaced with a foreign NLS failed to interact with 7SK and P-TEFb and hence were unable to inactivate P-TEFb. The 7SK-binding motif alone, however, was not sufficient to inhibit P-TEFb. A region C-terminal to this motif was also required for HEXIM1 to associate with P-TEFb and suppress P-TEFb's kinase and transcriptional activities. The 7SK-binding motif in HEXIM1 contains clusters of positively charged residues reminiscent of the arginine-rich RNA-binding motif found in a wide variety of proteins. Part of it is highly homologous to the TAR RNA-binding motif in the human immunodeficiency virus type 1 (HIV-1) Tat protein, which was able to restore the 7SK-binding ability of a HEXIM1 NLS substitution mutant. We propose that a similar RNA-protein recognition mechanism may exist to regulate the formation of both the Tat-TAR-P-TEFb and the HEXIM1-7SK-P-TEFb ternary complexes, which may help convert the inactive HEXIM1/7SK-bound P-TEFb into an active one for Tat-activated and TAR-dependent HIV-1 transcription.During transcription by RNA polymerase II (Pol II), phosphorylation of the carboxy-terminal domain (CTD) of the largest subunit of Pol II by the positive transcriptional elongation factor b (P-TEFb) is crucial for the transition from the abortive to the productive phase of transcriptional elongation, leading to the generation of full-length RNA transcripts (for reviews, see references 7 and 14). Active P-TEFb is composed of CDK9 and its regulatory subunit, cyclin T1 (CycT1) (14). Unlike other CDKs and their cyclin partners whose functions are closely related to the cell cycle regulation, P-TEFb is constitutively expressed and, hence, its protein level varies little throughout the cell cycle (5). Not only is P-TEFb essential for the expression of most protein-encoding genes (2, 16), but also it is indispensable for the replication of human immunodeficiency virus type 1 (HIV-1), since it is a specific host cellular cofactor for the viral Tat protein (7,14). P-TEFb is recruited by Tat to the HIV-1 long terminal repeat (LTR) through the formation of a stable ternary complex consisting of P-TEFb, Tat, and the TAR RNA stem-loop structure located at the 5Ј end of the nascent viral transcript (14). Once recruited, P-TEFb phosphorylates the Pol II CTD and stimulates the production of full-length HIV-1 transcripts.Not every P-TEFb complex in the cell can function as a transcription factor...

Human positive transcriptional elongation factor b (P-TEFb), consisting of a cyclin-dependent kinase 9-cyclin T heterodimer, stimulates general and disease-specific transcriptional elongation by phosphorylating RNA polymerase II. The HEXIM1 protein, aided by the 7SK snRNA, sequesters P-TEFb into an inactive 7SK⅐HEXIM1⅐P-TEFb small nuclear ribonucleic acid particle for inhibition of transcription and, consequently, cell proliferation. Here we show that, like HEXIM1, a highly homologous protein named HEXIM2 also possesses the ability to inactivate P-TEFb to suppress transcription through a 7SK-mediated interaction with PTEFb. Furthermore, HEXIM1 and HEXIM2 can form stable homo-and hetero-oligomers (most likely dimers), which may nucleate the formation of the 7SK small nuclear ribonucleic acid particle. Despite their similar functions, HEXIM1 and HEXIM2 exhibit distinct expression patterns in various human tissues and established cell lines. In HEXIM1-knocked down cells, HEXIM2 can functionally and quantitatively compensate for the loss of HEXIM1 to maintain a constant level of the 7SK/ HEXIM-bound P-TEFb. Our results demonstrate that there is a tightly regulated cellular process to maintain the balance between active and inactive P-TEFb complexes, which controls global transcription as well as cell growth and differentiation.