Cyclin-dependent kinases (Cdks) 1 are key regulators of a variety of cellular processes, such as cell cycle progression, transcription, and neuronal differentiation. The kinase activity of Cdks in turn is tightly regulated. Association with a cyclin partner and phosphorylation of the T-loop is needed for activation of Cdks. They are also subject to negative regulation via phosphorylation or through interaction with a family of Cdk inhibitory proteins (1-4).P-TEFb plays a key role in RNA polymerase II elongation control (5-7). It is comprised of one of two isoforms of Cdk9 (8, 9) and one of three cyclins, T1, T2 (10), or K (11) in human. One of the major targets of the kinase activity of P-TEFb is the carboxyl-terminal domain of the largest subunit of RNA polymerase II (12), and this phosphorylation of the carboxyl-terminal domain by P-TEFb occurs during transcription elongation (13). P-TEFb controls gene expression by regulating the fraction of RNA polymerase II molecules that generate full-length mRNAs (6). In addition to its normal cellular role, P-TEFb has been shown to be recruited by the viral transactivator Tat to the promoter to enhance viral transcription, which is required for efficient HIV-1 replication (8, 14 -18). P-TEFb is uniquely regulated by the reversible association of a small nuclear RNA, 7SK (19,20),. Glycerol gradient analyses of cell lysates indicate that two forms of P-TEFb exist in the cell: a large inactive form containing 7SK and HEXIM proteins, and a smaller active form comprised of just P-TEFb subunits (9,19,21,23,24). When cells are treated with P-TEFb inhibitors, such as 5,6-dichloro-1-␤-Dribofuranosylbenzimidazole, or other agents that block transcription elongation, the large form is converted into the small active form (21). This form of P-TEFb regulation is physiologically significant, because it has been shown that all signals that trigger cardiac hypertrophy converge at the critical step of activating P-TEFb through dissociation of 7SK and HEXIM. This activation causes increased cellular transcription, and an increase in the size of cardiomyocytes (25)(26)(27). Several studies have uncovered some of the important interactions in the 7SK⅐HEXIM1⅐P-TEFb complex. Two regions of HEXIM1 have been characterized. The region centered upon KHRR (amino acids 152-155) is involved in binding of 7SK, and contains nuclear localization signals (21,28,29). A second region centered upon PYNT (amino acids 202-205) is involved in interaction with P-TEFb (23, 29). In addition, regions involved in interactions have been narrowed down to amino acids 1-254 of 726 of cyclin T1, all of Cdk9, and nucleotide 1-175 of 7SK (19,21,22). Furthermore, phosphorylation of the T-loop of Cdk9 has been implicated in activation of P-TEFb, and is required for the formation of the 7SK⅐HEXIM1⅐P-TEFb complex (30).In this report, we analyzed the stoichiometry of the 7SK⅐HEXIM1⅐P-TEFb complex. We identified residues critical for inhibition of the kinase activity of P-TEFb, and formation of the 7SK⅐HEXIM1⅐P-TEFb complex....

Regulation of the elongation phase of RNA polymerase II transcription by P-TEFb is a critical control point for gene expression. The activity of P-TEFb is regulated, in part, by reversible association with one of two HEXIMs and the 7SK snRNP. A recent proteomics survey revealed that P-TEFb and the HEXIMs are tightly connected to two previously-uncharacterized proteins, the methyphosphate capping enzyme, MEPCE, and a La-related protein, LARP7. Glycerol gradient sedimentation analysis of lysates from cells treated with P-TEFb inhibitors, suggested that the 7SK snRNP reorganized such that LARP7 and 7SK remained associated after P-TEFb and HEXIM1 were released. Immunodepletion of LARP7 also depleted most of the 7SK regardless of the presence of P-TEFb, HEXIM or hnRNP A1 in the complex. Small interfering RNA knockdown of LARP7 in human cells decreased the steady-state level of 7SK, led to an initial increase in free P-TEFb and increased Tat transactivation of the HIV-1 LTR. Knockdown of LARP7 or 7SK ultimately caused a decrease in total P-TEFb protein levels. Our studies have identified LARP7 as a 7SK-binding protein and suggest that free P-TEFb levels are determined by a balance between release from the large form and reduction of total P-TEFb.

Basal transcription of the HIV LTR is highly repressed and requires Tat to recruit the positive transcription elongation factor, P-TEFb, which functions to promote the transition of RNA polymerase II from abortive to productive elongation. P-TEFb is found in two forms in cells, a free, active form and a large, inactive complex that also contains 7SK RNA and HEXIM1 or HEXIM2. Here we show that HIV infection of cells led to the release of P-TEFb from the large form. Consistent with Tat being the cause of this effect, transfection of a FLAG-tagged Tat in 293T cells caused a dramatic shift of P-TEFb out of the large form to a smaller form containing Tat. In vitro, Tat competed with HEXIM1 for binding to 7SK, blocked the formation of the P-TEFb–HEXIM1–7SK complex, and caused the release P-TEFb from a pre-formed P-TEFb–HEXIM1–7SK complex. These findings indicate that Tat can acquire P-TEFb from the large form. In addition, we found that HEXIM1 binds tightly to the HIV 5′ UTR containing TAR and recruits and inhibits P-TEFb activity. This suggests that in the absence of Tat, HEXIM1 may bind to TAR and repress transcription elongation of the HIV LTR.

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.Transcription by RNA polymerase II is a highly regulated process, influenced by many factors and a cycle of RNA polymerase II phosphorylation (1, 2). The default state of RNA polymerase II transcription is abortive, with RNA polymerase II falling under the influence of negative factors such as DRB 1 sensitivity-inducing factor (DSIF), and negative elongation factor (NELF), resulting in the production of short transcripts (3-5). The transition to the productive elongation of full-length transcripts requires positive transcription elongation factor b (P-TEFb) (6, 7), which phosphorylates serine 2 residues in the heptad repeats of the carboxyl-terminal domain of the largest subunit of RNA polymerase II (7,8). P-TEFb is required for the proper expression of RNA polymerase II genes. The knockdown of cyclin-dependent kinase 9 or both of the cyclin Ts in Caenorhabditis elegans reduces the expression of early embryonic genes and the level of serine 2 phosphorylation of the carboxylterminal domain and leads to the death of the embryo at an early stage (9). Inhibition of P-TEFb kinase activity through treatment with flavopiridol blocks the transcription of RNA polymerase II genes in HeLa cells (10, 11), with complete inhibition causing apoptosis. P-TEFb activity can be targeted to specific genes through association with activators, such as human immunodeficiency virus-1 Tat (12, 13), CIITA (14), nuclear factor-B (15), and Myc (16, 17).There are two distinct P-TEFb complexes, which differ in size, composition, and activity (18, 19). The originally identified, smaller P-TEFb complex has kinase activity (7) and is composed of Cdk9 42 or Cdk9 55 (20) and a cyclin partner T1, T2, or K (21, 22). A large P-TEFb complex with reduced kinase activity was found to contain the small nuclear RNA 7SK (18, 19) and HEXIM1 (23,24), in addition to P-TEFb subunits. Independently, HEXIM1 and 7SK have limited inhibitory effect...

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Background: The positive transcription elongation factor, P-TEFb, comprised of cyclin dependent kinase 9 (Cdk9) and cyclin T1, T2 or K regulates the productive elongation phase of RNA polymerase II (Pol II) dependent transcription of cellular and integrated viral genes. P-TEFb containing cyclin T1 is recruited to the HIV long terminal repeat (LTR) by binding to HIV Tat which in turn binds to the nascent HIV transcript. Within the cell, P-TEFb exists as a kinase-active, free form and a larger, kinase-inactive form that is believed to serve as a reservoir for the smaller form.

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MLN944 is a novel compound currently being codeveloped by Millennium Pharmaceuticals and Xenova Ltd. as a cancer therapeutic and is in a phase I clinical trial for solid tumors. Although MLN944 was originally proposed to function as a topoisomerase I and II inhibitor, more recent data has shown that it is a DNA-intercalating agent that does not inhibit the catalytic activity of topoisomerase I or II. We show here that MLN944 inhibits incorporation of radiolabeled precursors into RNA preferentially over incorporation into DNA and protein in HCT116 and H460 cells. To determine if MLN944 inhibits transcription, a human RNA polymerase II in vitro transcription system was used. MLN944 inhibited initiation when added before or after the formation of preinitiation complexes and inhibited elongation at higher concentrations. The preferential inhibition of initiation differentiates MLN944 from actinomycin D, which more strongly inhibits elongation. Transcription of all RNA polymerases was inhibited in nuclei isolated from HeLa cells treated with low concentrations of MLN944. Our data are consistent with transcription as the target of the potent cytotoxic effects of MLN944. [Mol Cancer Ther 2005;4(8):1260 -7]