Coordination of transcription factor phosphorylation and histone methylation by the P-TEFb kinase during human immunodeficiency virus type 1 transcription.
(65/294)
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. (+info)
Granulin and granulin repeats interact with the Tat.P-TEFb complex and inhibit Tat transactivation.
(66/294)
The cellular positive transcription elongation factor b (P-TEFb), containing cyclin T1 and cyclin-dependent kinase 9 (CDK9), interacts with the human immunodeficiency virus, type 1 (HIV-1) regulatory protein Tat to enable viral transcription and replication. Cyclin T1 is an unusually long cyclin and is engaged by cellular regulatory proteins. Previous studies showed that the granulin/epithelin precursor (GEP) binds the histidine-rich region of cyclin T1 and inhibits P-TEFb function. GEP is composed of repeats that vary in sequence and properties. GEP also binds directly to Tat. Here we show that GEP and some of its constituent granulin repeats can inhibit HIV-1 transcription via Tat without directly binding to cyclin T1. The interactions of granulins with Tat and cyclin T1 differ with respect to their binding sites and divalent cation requirements, and we identified granulin repeats that bind differentially to Tat and cyclin T1. Granulins DE and E bind Tat but do not interact directly with cyclin T1. These granulins are present in complexes with Tat and P-TEFb in which Tat forms a bridge between the cellular proteins. Granulins DE and E repress transcription from the HIV-1 LTR and gene expression from the viral genome, raising the possibility of developing granulin-based inhibitors of viral infection. (+info)
Compensatory contributions of HEXIM1 and HEXIM2 in maintaining the balance of active and inactive positive transcription elongation factor b complexes for control of transcription.
(67/294)
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 7SK.HEXIM1.P-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 P-TEFb. 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. (+info)
HEXIM2, a HEXIM1-related protein, regulates positive transcription elongation factor b through association with 7SK.
(68/294)
The kinase activity of positive transcription elongation factor b (P-TEFb), composed of cyclin-dependent kinase 9 and cyclin T1 or T2, is required for the transition of RNA polymerase II into productive elongation. P-TEFb activity has been shown to be negatively regulated by association with the small nuclear RNA 7SK and the HEXIM1 protein. Here, we characterize HEXIM2, a previously predicted protein with sequence similarity to HEXIM1. HEXIM2 is expressed in HeLa and Jurkat cells, and glycerol gradient analysis and immunoprecipitations indicate that HEXIM2, like HEXIM1, has a regulated association with P-TEFb. As HEXIM1 is knocked down, HEXIM2 functionally compensates for its association with P-TEFb. Electrophoretic mobility shift assays and in vitro kinase assays demonstrate that HEXIM2 forms complexes containing 7SK and P-TEFb and, in conjunction with 7SK, inhibits P-TEFb kinase activity. Our results provide strong evidence that HEXIM2 is a regulator of P-TEFb function. Furthermore, our results support the idea that the utilization of HEXIM1 or HEXIM2 to bind and inhibit P-TEFb can be differentially regulated in vivo. (+info)
Identification of a cyclin T-binding domain in Hexim1 and biochemical analysis of its binding competition with HIV-1 Tat.
(69/294)
The active form of the positive transcription elongation factor b (P-TEFb) consists of cyclin T and the kinase Cdk9. P-TEFb stimulates transcription by phosphorylating the C-terminal domain of RNA polymerase II. It becomes inactivated when associated in a tetrameric complex with the abundant 7SK small nuclear RNA and the recently identified protein Hexim1. In this study, we identified a stable and soluble C-terminal domain (residues 255-359) in Hexim1 of 12.5-kDa size that binds the cyclin boxes of Cyclin T1. Functional assays in HeLa cells showed that this cyclin T-binding domain (TBD) is required for the binding of Hexim1 to P-TEFb and inhibition of transcriptional activity in vivo. Analytical gel filtration and GST pull-down experiments revealed that both full-length Hexim1 and the TBD are homodimers. Isothermal titration calorimetry yielded a weak multimer for the TBD with a multimerization constant of 1.3 x 10(3) m. The binding affinity between the TBD and cyclin T1 was analyzed with fluorescence spectroscopy methods, using a dansyl-based fluorescence label at position G257C. Equilibrium fluorescence titration and stopped flow fast kinetics yield a dissociation constant of 1.2 mum. Finally, we tested the effect of the HIV-1 Tat protein on the cyclin T1-TBD complex formation. GST pull-down experiments and size exclusion chromatography exhibit a mutually exclusive binding of the two effectors to cyclin T1. Our data suggest a model where HIV-1 Tat competes with Hexim1 for cyclin T1 binding, thus releasing P-TEFb from the inactive complex to stimulate the transcription of HIV-1 gene expression. (+info)
The glucocorticoid receptor blocks P-TEFb recruitment by NFkappaB to effect promoter-specific transcriptional repression.
(70/294)
To investigate the determinants of promoter-specific gene regulation by the glucocorticoid receptor (GR), we compared the composition and function of regulatory complexes at two NFkappaB-responsive genes that are differentially regulated by GR. Transcription of the IL-8 and IkappaBalpha genes is stimulated by TNFalpha in A549 cells, but GR selectively represses IL-8 mRNA synthesis by inhibiting Ser2 phosphorylation of the RNA polymerase II (pol II) C-terminal domain (CTD). The proximal kappaB elements at these genes differ in sequence by a single base pair, and both recruited RelA and p50. Surprisingly, GR was recruited to both of these elements, despite the fact that GR failed to repress the IkappaBalpha promoter. Rather, the regulatory complexes formed at IL-8 and IkappaBalpha were distinguished by differential recruitment of the Ser2 CTD kinase, P-TEFb. Disruption of P-TEFb function by the Cdk-inhibitor, DRB, or by small interfering RNA selectively blocked TNFalpha stimulation of IL-8 mRNA production. GR competed with P-TEFb recruitment to the IL-8 promoter. Strikingly, IL-8 mRNA synthesis was repressed by GR at a post-initiation step, demonstrating that promoter proximal regulatory sequences assemble complexes that impact early and late stages of mRNA synthesis. Thus, GR accomplishes selective repression by targeting promoter-specific components of NFkappaB regulatory complexes. (+info)
A human splicing factor, SKIP, associates with P-TEFb and enhances transcription elongation by HIV-1 Tat.
(71/294)
HIV-1 Tat binds human CyclinT1 and recruits the CDK9/P-TEFb complex to the viral TAR RNA in a step that links RNA polymerase II (RNAPII) C-terminal domain (CTD) Ser 2 phosphorylation with transcription elongation. Previous studies have suggested a connection between Tat and pre-mRNA splicing factors. Here we show that the splicing-associated c-Ski-interacting protein, SKIP, is required for Tat transactivation in vivo and stimulates HIV-1 transcription elongation, but not initiation, in vitro. SKIP associates with CycT1:CDK9/P-TEFb and Tat:P-TEFb complexes in nuclear extracts and interacts with recombinant Tat:P-TEFb:TAR RNA complexes in vitro, indicating that it may act through nascent RNA to overcome pausing by RNAPII. SKIP also associates with U5snRNP proteins and tri-snRNP110K in nuclear extracts, and facilitates recognition of an alternative Tat-specific splice site in vivo. The effects of SKIP on transcription elongation, binding to P-TEFb, and splicing are mediated through the SNW domain. HIV-1 Tat transactivation is accompanied by the recruitment of P-TEFb, SKIP, and tri-snRNP110K to the integrated HIV-1 promoter in vivo, whereas the U5snRNPs associate only with the transcribed coding region. These findings suggest that SKIP plays independent roles in transcription elongation and pre-mRNA splicing. (+info)
Analysis of the large inactive P-TEFb complex indicates that it contains one 7SK molecule, a dimer of HEXIM1 or HEXIM2, and two P-TEFb molecules containing Cdk9 phosphorylated at threonine 186.
(72/294)
Positive transcription elongation factor b (P-TEFb) regulates eukaryotic gene expression at the level of elongation, and is itself controlled by the reversible association of 7SK RNA and an RNA-binding protein, HEXIM1 or HEXIM2. To further understand how P-TEFb is regulated, we analyzed the stoichiometry of all the known components of the large, inactive P-TEFb complex. Mutational analyses of a putative coiled coil region in the carboxyl-terminal portion of HEXIM1 revealed that the protein is a dimer in solution and remains a dimer after binding to 7SK. Although a HEXIM1 dimer contains two potential RNA binding motifs and ultimately recruits two P-TEFb molecules, it associates with only one molecule of RNA. The first 172 nucleotides of the 330-nucleotide 7SK are sufficient to bind HEXIM1 or HEXIM2, and then recruit and inhibit P-TEFb. Deletion of the first 121 amino acids of HEXIM1 allowed it to inhibit P-TEFb partially in the absence of 7SK RNA. Mutation of a conserved tyrosine (Tyr(271) in HEXIM1) to alanine or glutamate or mutation of a conserved phenylalanine (Phe(208)) to alanine, aspartate, or lysine, resulted in loss of inhibition of P-TEFb, but did not affect formation of the 7SK.HEXIM.P-TEFb complex. Analysis of T-loop phosphorylation in Cdk9 indicated that phosphorylation of Thr(186), but not Ser(175), was essential for kinase activity and for recruitment of P-TEFb to the 7SK.HEXIM complex. A model illustrates what is currently known about how HEXIM proteins, 7SK, and P-TEFb assemble to maintain an activated kinase in a readily available, but inactive form. (+info)