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Query: UNIPROT:P23193 (transcription elongation factor)
 739 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

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.
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PMID:HEXIM2, a HEXIM1-related protein, regulates positive transcription elongation factor b through association with 7SK. 1571 62

Tat activates transcription by interacting with Sp1, NF-kappaB, positive transcription elongation factor b, and trans-activator-responsive element (TAR). Tat and Sp1 play major roles in transcription by protein-protein interactions at human immunodeficiency virus, type 1 (HIV-1) long terminal repeat. Sp1 activates transcription by interacting with cyclin T1 in the absence of Tat. To disrupt the transcription activation by Tat and Sp1, we fused Sp1-inhibiting polypeptides, zinc finger polypeptide, and the TAR-binding mutant Tat (TatdMt) together. A designed or natural zinc finger and Tat mutant fusion was used to target the fusion to the key regulatory sites (GC box and TAR) on the long terminal repeat and nascent short transcripts to disrupt the molecular interaction that normally result in robust transcription. The designed zinc finger and TatdMt fusions were targeted to the TAR, and they potently repressed both transcription and replication of HIV-1. The Sp1-inhibiting POZ domain, TatdMt, and zinc fingers are key functional domains important in repression of transcription and replication. The designed artificial zinc fingers were targeted to the high affinity Sp1-binding site, and by being fused with TatdMt and POZ domain, they strongly block both Sp1-cyclin T1-dependent transcription and Tat-dependent transcription, even in the presence of excess expressed Tat.
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PMID:Artificial zinc finger fusions targeting Sp1-binding sites and the trans-activator-responsive element potently repress transcription and replication of HIV-1. 1574 74

Positive transcription elongation factor b (P-TEFb) controls the fraction of initiated RNA polymerase II molecules that make full length transcripts. This important factor is a heterodimer of cyclin-dependent kinase 9 (Cdk9) and one of four cyclin partners, cyclin T1, T2a, T2b or K. There are two isoforms of Cdk9 in mammalian cells, Cdk9(42) and Cdk9(55). Cdk9(55) has a 117 residue amino terminal extension not present in Cdk9(42). An expression vector with a tetracycline-responsive promoter driving FLAG-tagged Cdk9(55) and a HeLa 37 Tet-Off cell line were constructed. FLAG-tagged Cdk9(55) was inducibly expressed and was found to be localized to the nucleus by immunofluorescence. Western analysis of murine tissues showed that the relative abundance of the two forms of Cdk9 varied across different tissues with liver having more Cdk9(55) than Cdk9(42). During adaptation of primary rat hepatocytes to culture the ratio of the two forms of Cdk9 changed. Initially, Cdk9(55) was the predominate form, but as the cells began to enter the cell cycle Cdk9(42) became the major form. During this change, expression of Cdk9(42) was induced, while Cdk9(55) remained relatively constant.
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PMID:Characterization of Cdk9(55) and differential regulation of two Cdk9 isoforms. 1578 Sep 80

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.
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PMID:Identification of a cyclin T-binding domain in Hexim1 and biochemical analysis of its binding competition with HIV-1 Tat. 1585 66

Estrogen receptor alpha (ERalpha) regulates transcription of specific genes and is believed to play a major role in breast tumorigenesis. We previously identified estrogen down regulated gene 1 (EDG1 (also known as HEXIM1)) using the C-terminus of ERalpha (E/F domain) as bait in yeast two-hybrid screenings. Here we report on the role of EDG1 as a coregulator of ERalpha transcriptional activity. We observe an interaction between EDG1 and ERalpha. EDG1 inhibits the transcriptional activity of ERalpha and this is dependent upon the C-terminus of EDG1. The C-terminus of EDG1/HEXIM1 was recently shown to inhibit the positive transcription elongation factor b (P-TEFb) by interacting with the cyclin T1 subunit. Here we show that ERalpha interacts with cyclin T1, cyclin T1 and ER co-occupancy on the promoter region of an ER target gene, and that this interaction plays an important role in ERalpha-induced gene expression. The interaction of ERalpha with cyclin T1 also allows ERalpha to compete with EDG1 for cyclin T1, and may release cyclin T1 from EDG1 repression. Conversely, increased EDG1 expression results in inhibition of cyclin T1 recruitment and ERalpha DNA binding. Our results support a novel functional interaction between ERalpha and cyclin T1 that is modulated by EDG1.
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PMID:The breast cell growth inhibitor, estrogen down regulated gene 1, modulates a novel functional interaction between estrogen receptor alpha and transcriptional elongation factor cyclin T1. 1594 Feb 64

The HEXIM1 protein has been shown to form a protein-RNA complex composed of 7SK small nuclear RNA and positive transcription elongation factor b (P-TEFb), which is composed of cyclin-dependent kinase 9 (CDK9) and cyclin T1, and to inhibit the kinase activity of CDK9, thereby suppressing RNA polymerase II-dependent transcriptional elongation. Here, we biochemically demonstrate that HEXIM1 forms a distinct complex with glucocorticoid receptor (GR) without RNA, CDK9, or cyclin T1. HEXIM1, through its arginine-rich nuclear localization signal, directly associates with the ligand-binding domain of GR. Introduction of HEXIM1 short interfering RNA and adenovirus-mediated exogenous expression of HEXIM1 positively and negatively modulated glucocorticoid-responsive gene activation, respectively. In the nucleus, HEXIM1 was shown to localize in a distinct compartment from that of the p160 coactivator transcriptional intermediary factor 2. Overexpression of HEXIM1 decreased ligand-dependent association between GR and transcriptional intermediary factor 2. Antisense-mediated disruption of 7SK blunted the negative effect of HEXIM1 on arylhydrocarbon receptor-dependent transcription but not on GR-mediated one, indicating that a class of transcription factors are direct targets of HEXIM1. These results indicate that HEXIM1 has dual roles in transcriptional regulation: inhibition of transcriptional elongation dependent on 7SK RNA and positive transcription elongation factor b and interference with the sequence-specific transcription factor GR via a direct protein-protein interaction. Moreover, the fact that the central nuclear localization signal of HEXIM1 is essential for both of these actions may argue the crosstalk of these functions.
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PMID:HEXIM1 forms a transcriptionally abortive complex with glucocorticoid receptor without involving 7SK RNA and positive transcription elongation factor b. 1594 32

Human immunodeficiency virus type 1 (HIV-1) Tat transactivation is an essential step in the viral life cycle. Over the past several years, it has become widely accepted that Tat exerts its transcriptional effect by binding the transactivation-responsive region (TAR) and enhancing transcriptional elongation. Consistent with this hypothesis, it has been shown that Tat promotes the binding of P-TEFb, a transcription elongation factor composed of cyclin T1 and cdk9, and the interaction of Tat with P-TEFb and TAR leads to hyperphosphorylation of the C-terminal domain (CTD) of RNA Pol II and increased processivity of RNA Pol II. A recent report, however, has generated renewed interest that Tat may also play a critical role in transcription complex (TC) assembly at the preinitiation step. Using in vivo chromatin immunoprecipitation assays, the authors reported that the HIV TC contains TBP but not TBP-associated factors. The stimulatory effect involved the direct interaction of Tat and P-TEFb and was evident at the earliest step of TC assembly, the TBP-TATA box interaction. In this article, we will review this data in context of earlier data which also support Tat's involvement in transcriptional complex assembly. Specifically, we will discuss experiments which demonstrated that Tat interacted with TBP and increased transcription initiation complex stability in cell free assays. We will also discuss studies which demonstrated that over expression of TBP alone was sufficient to obtain Tat activated transcription in vitro and in vivo. Finally, studies using self-cleaving ribozymes which suggested that Tat transactivation was not compatible with pausing of the RNA Pol II at the TAR site will be discussed.
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PMID:Tat gets the "green" light on transcription initiation. 1628 76

Positive transcription elongation factor b (P-TEFb) is an RNA polymerase II elongation factor which exists as multiple complexes in human cells. These complexes contain cyclin-dependent kinase 9 as the catalytic subunit and different cyclin subunits-cyclin T1, T2a, T2b, or K. Cyclin T1 is targeted by the human immunodeficiency virus (HIV) Tat protein to activate transcription of the HIV provirus. Expression of this P-TEFb subunit is highly regulated in monocyte-derived macrophages (MDMs). Cyclin T1 is induced early during differentiation and is shut off later by proteasome-mediated proteolysis. Cyclin T1 can be reinduced by pathogen-associated molecular patterns (PAMPs) or HIV infection. In this study, we analyzed regulation of P-TEFb in MDMs by examining 7SK small nuclear RNA and the HEXIM1 protein; these factors associate with P-TEFb and are thought to regulate its function. 7SK and HEXIM1 were induced early during differentiation, and this correlates with increased overall transcription. 7SK expression remained high, but HEXIM1 was shut off later during differentiation by proteasome-mediated proteolysis. Significantly, the cyclin T2a subunit of P-TEFb was not shut off during differentiation, and it was not induced by activation. Induction of cyclin T1 by PAMPs was found to be a slow process and did not involve an increase in cyclin T1 mRNA levels. Treatment of MDMs with PAMPs or a proteasome inhibitor induced cyclin T1 to a level equivalent to treatment with both agents together, suggesting that PAMPs and proteasome inhibitors act at a similar rate-limiting step. It is therefore likely that cyclin T1 induction by PAMPs is the result of a reduction in proteasome-mediated proteolysis.
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PMID:Cyclin T1 but not cyclin T2a is induced by a post-transcriptional mechanism in PAMP-activated monocyte-derived macrophages. 1633 May 31

The positive transcription elongation factor b (P-TEFb), a complex of Cdk9 and cyclin T1/T2, stimulates transcription by phosphorylating RNA polymerase II. The 7SK small nuclear RNA, in cooperation with HEXIM1 protein, functions as a general polymerase II transcription regulator by sequestering P-TEFb into a large kinase-inactive 7SK/HEXIM1/P-TEFb complex. Here, determination and characterization of the functionally essential elements of human 7SK snRNA directing HEXIM1 and P-TEFb binding led to a new model for the assembly of the 7SK/HEXIM1/P-TEFb regulatory complex. We demonstrate that two structurally and functionally distinct protein binding elements located in the 5'- and 3'-terminal hairpins of 7SK support the in vivo recruitment of HEXIM1 and P-TEFb. Consistently, a minimal regulatory RNA composed of the 5' and 3' hairpins of 7SK can modulate polymerase II transcription in HeLa cells. HEXIM1 binds independently and specifically to the G24-C48/G60-C87 distal segment of the 5' hairpin of 7SK. Binding of HEXIM1 is a prerequisite for association of P-TEFb with the G302-C324 apical region of the 3' hairpin of 7SK that is highly reminiscent of the human immunodeficiency virus transactivation-responsive RNA.
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PMID:Regulation of polymerase II transcription by 7SK snRNA: two distinct RNA elements direct P-TEFb and HEXIM1 binding. 1638 53

Positive transcription elongation factor b (P-TEFb) phosphorylates the C-terminal domain of RNA polymerase II, facilitating transcriptional elongation. In addition to its participation in general transcription, P-TEFb is recruited to specific promoters by some transcription factors such as c-Myc or MyoD. The P-TEFb complex is composed of a cyclin-dependent kinase (cdk9) subunit and a regulatory partner (cyclin T1, cyclin T2, or cyclin K). Because cdk9 has been shown to participate in differentiation processes, such as muscle cell differentiation, we studied a possible role of cdk9 in adipogenesis. In this study we show that the expression of the cdk9 p55 isoform is highly regulated during 3T3-L1 adipocyte differentiation at RNA and protein levels. Furthermore, cdk9, as well as cyclin T1 and cyclin T2, shows differences in nuclear localization at distinct stages of adipogenesis. Overexpression of cdk9 increases the adipogenic potential of 3T3-L1 cells, whereas inhibition of cdk9 by specific cdk inhibitors, and dominant-negative cdk9 mutant impairs adipogenesis. We show that the positive effects of cdk9 on the differentiation of 3T3-L1 cells are mediated by a direct interaction with and phosphorylation of peroxisome proliferator-activated receptor gamma (PPARgamma), which is the master regulator of this process, on the promoter of PPARgamma target genes. PPARgamma-cdk9 interaction results in increased transcriptional activity of PPARgamma and therefore increased adipogenesis.
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PMID:Peroxisome proliferator-activated receptor gamma recruits the positive transcription elongation factor b complex to activate transcription and promote adipogenesis. 1648 39


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