Positive Transcription Elongation Factor B Research Articles

Acetylation of cyclin T1 regulates the equilibrium between active and inactive P-TEFb in cells

The elongation competence of the RNA polymerase II complex is critically dependent on the positive transcription elongation factor b (P-TEFb). P-TEFb exists in two forms in cells, an active form composed of cyclin T1 and CDK9 and an inactive form, in which cyclin T1/CDK9 is sequestered by Hexim1 and 7SK snRNA. Here, we report that partitioning of active and inactive P-TEFb is regulated by acetylation of cyclin T1. Cyclin T1 acetylation triggers dissociation of Hexim1 and 7SK snRNA from cyclin T1/CDK9 and activates the transcriptional activity of P-TEFb. This activation is lost in P-TEFb complexes containing cyclin T1 that can no longer be acetylated. An acetylation-deficient cyclin T1 mutant dominantly suppresses NF-kappaB-mediated activation of the interleukin-8 promoter but continues to synergize normally with the HIV Tat protein to transactivate the HIV long terminal repeat. These findings support the model that acetylation of cyclin T1 serves as a physiological switch that liberates P-TEFb from its endogenous inhibitors Hexim1 and 7SK snRNA, but is not required for the cooperative action with HIV Tat.

TFIIH and P-TEFb Coordinate Transcription with Capping Enzyme Recruitment at Specific Genes in Fission Yeast

Cyclin-dependent kinases (CDKs) are subunits of transcription factor (TF) IIH and positive transcription elongation factor b (P-TEFb). To define their functions, we mutated the TFIIH-associated kinase Mcs6 and P-TEFb homologs Cdk9 and Lsk1 of fission yeast, making them sensitive to inhibition by bulky purine analogs. Selective inhibition of Mcs6 or Cdk9 blocks cell division, alters RNA polymerase (Pol) II carboxyl-terminal domain (CTD) phosphorylation, and represses specific, overlapping subsets of transcripts. At a common target gene, both CDKs must be active for normal Pol II occupancy, and Spt5-a CDK substrate and regulator of elongation-accumulates disproportionately to Pol II when either kinase is inhibited. In contrast, Mcs6 activity is sufficient-and necessary-to recruit the Cdk9/Pcm1 (mRNA cap methyltransferase) complex. In vitro, phosphorylation of the CTD by Mcs6 stimulates subsequent phosphorylation by Cdk9. We propose that TFIIH primes the CTD and promotes recruitment of P-TEFb/Pcm1, serving to couple elongation and capping of select pre-mRNAs.

ARC (NSC 188491) has identical activity to Sangivamycin (NSC 65346) including inhibition of both P-TEFb and PKC

BackgroundThe nucleoside analog, ARC (NSC 188491) is a recently characterized transcriptional inhibitor that selectively kills cancer cells and has the ability to perturb angiogenesis in vitro. In this study, the mechanism of action of ARC was further investigated by comparing in vitro and in vivo activity with other anti-neoplastic purines.MethodsStructure-based homology searches were used to identify those compounds with similarity to ARC. Comparator compounds were then evaluated alongside ARC in the context of viability, cell cycle and apoptosis assays to establish any similarities. Following this, biological overlap was explored in detail using gene-expression analysis and kinase inhibition assays.ResultsResults demonstrated that sangivamycin, an extensively characterized pro-apoptotic nucleoside isolated from Streptomyces, had identical activity to ARC in terms of 1) cytotoxicity assays, 2) ability to induce a G2/M block, 3) inhibitory effects on RNA/DNA/protein synthesis, 4) transcriptomic response to treatment, 5) inhibition of protein kinase C, 6) inhibition of positive transcription elongation factor b (P-TEFb), 7) inhibition of VEGF secretion, and 8) activity within hollow fiber assays. Extending ARC activity to PKC inhibition provides a molecular basis for ARC cancer selectivity and anti-angiogenic effects. Furthermore, functional overlap between ARC and sangivamycin suggests that development of ARC may benefit from a retrospective of previous sangivamycin clinical trials. However, ARC was found to be inactive in several xenograft models, likely a consequence of rapid serum clearance.ConclusionOverall, these data expand on the biological properties of ARC but suggest additional studies are required before it can be considered a clinical trials candidate.

P-TEFb- the final frontier

Regulation of gene expression is essential to all aspects of physiological processes in single-cell as well as multicellular organisms. It gives ultimately cells the ability to efficiently respond to extra- and intracellular stimuli participating in cell cycle, growth, differentiation and survival. Regulation of gene expression is executed primarily at the level of transcription of specific mRNAs by RNA polymerase II (RNAPII), typically in several distinct phases. Among them, transcription elongation is positively regulated by the positive transcription elongation factor b (P-TEFb), consisting of CDK9 and cyclin T1, T2 or K. P-TEFb enables transition from abortive to productive transcription elongation by phosphorylating carboxyl-terminal domain (CTD) in RNAPII and negative transcription elongation factors. Over the years, we have learned a great deal about molecular composition of P-TEFb complexes, their assembly and their role in transcription of specific genes, but function of P-TEFb in other physiological processes was not apparent until just recently. In light of emerging discoveries connecting P-TEFb to regulation of cell cycle, development and several diseases, I would like to discuss these observations as well as future perspectives.

The 3′UTR of HIC mRNA improves the production of recombinant proteins in Chinese hamster ovary cells

Positive transcription elongation factor b (P-TEFb) is an important transcriptional regulator which controls 70–80% of RNA polymerase II transcription. It has been reported that the human I-mfa (inhibitor of MyoD family a) domain-containing protein (HIC) interacts with P-TEFb and that expression of HIC cDNA stimulates P-TEFb-dependent transcription. Interestingly, our recent study shows that transcriptional stimulation by HIC is predominately due to the 3′ untranslated region (3′UTR) of HIC mRNA rather than its coding region. In this report, we investigate the effects of HIC 3′UTR on recombinant protein expression in mammalian cells. In transient transfections, overexpression of HIC 3′UTR stimulates transgene expression in several mammalian cell lines and significantly increases the production of human erythropoietin and interferon-γ in Chinese hamster ovary (CHO) cells. This is the first report that demonstrates the improvement of expression of biopharmaceutical proteins by overexpressing a non-coding 3′UTR in CHO cells.

Phosphatase PPM1A Regulates Phosphorylation of Thr-186 in the Cdk9 T-loop

Cdk9 is the catalytic subunit of a general RNA polymerase II elongation factor known as positive transcription elongation factor b (P-TEFb). The kinase function of P-TEFb requires phosphorylation of Thr-186 in the T-loop of Cdk9 to allow substrates to access the catalytic core of the enzyme. To identify human phosphatases that dephosphorylate the T-loop of Cdk9, we used a Thr-186-phosphospecific antiserum to screen a phosphatase expression library. Overexpression of PPM1A and the related PPM1B greatly reduced Cdk9 T-loop phosphorylation in vivo. PPM1A and Cdk9 appear to associate in vivo as the proteins could be co-immunoprecipitated. The short hairpin RNA depletion of PPM1A resulted in an increase in Cdk9 T-loop phosphorylation. In phosphatase reactions in vitro, purified PPM1A could dephosphorylate Thr-186 both with and without the association of 7SK RNA, a small nuclear RNA that is bound to approximately 50% of total cellular P-TEFb. PPM1B only efficiently dephosphorylated Cdk9 Thr-186 in vitro when 7SK RNA was depleted from P-TEFb. Taken together, our data indicate that PPM1A and to some extent PPM1B are important negative regulators of P-TEFb function.

Dominant negative mutant cyclin T1 proteins that inhibit HIV transcription by forming a kinase inactive complex with Tat

Transcription of the human immunodeficiency virus type 1 (HIV) requires the interaction of the cyclin T1 (CycT1) subunit of a host cellular factor, the positive transcription elongation factor b (P-TEFb), with the viral Tat protein, at the transactivation response element (TAR) of nascent transcripts. Because of this virus-specific interaction, CycT1 may potentially serve as a target for the development of anti-HIV therapies. Here we report the development of a mutant CycT1 protein, containing three threonine-to-alanine substitutions in the linker region between two of the cyclin boxes, which displays a potent dominant negative effect on HIV transcription. Investigation into the inhibitory mechanism revealed that this mutant CycT1 interacted with Tat and the cyclin-dependent kinase 9 (Cdk9) subunit of P-TEFb, but failed to stimulate the Cdk9 kinase activity critical for elongation. This mutant CycT1 protein may represent a novel class of specific inhibitors of HIV transcription which could lead to development of new antiviral therapies.

The kinase activity of Cdk9 is required for phosphorylation of p300 and acetylation of GATA4 as well as cardiomyocyte hypertrophy

A zinc finger protein GATA-4 is one of the factors involved in transcriptional regulation during myocardial cell hypertrophy. In response to hypertrophic stimuli, GATA-4 forms a complex with an intrinsic histone acetyltransferase (HAT), p300, and increases its transcriptional activity by acetylation. By tandem affinity purification and mass spectrometric analyses, we identified cyclin-dependent kinase-9 (Cdk9), a component of positive transcription elongation factor b (P-TEFb), as a novel GATA4-binding partner. Cdk9 also forms a complex with p300 as well as GATA-4. However, precise functional relationship between p300/GATA-4 and P-TEFb is unknown. Intact p300 induced not only the acetylation of GATA-4 but also the interaction of GATA-4 with P-TEFb. Furthermore, p300 induced the hyperphosphorylation of RNA Pol II, suggesting that p300 is involved in regulating the kinase activity of Cdk9. All of these effects were inhibited by a dominant-negative form of (DN-) p300. Meanwhile, DN-Cdk9, which loses its kinase activity by a single amino acid substitution, inhibited p300-induced hyperphophorylation of RNA Pol II. Notably, DN-Cdk9 inhibited phosphorylation of p300 as well as p300-induced acetylation and DNA binding of GATA-4, indicating a requirement of Cdk9 in the HAT activity of p300. Finally, both DN-p300 and DN-Cdk9 inhibited phenylephrine-induced hypertrophic responses in cardiac myocytes. These findings demonstrate that Cdk9 forms a functional complex with the p300/GATA4 and is required for p300/GATA4-transcriptional pathway during cardiomyocyte hypertrophy.

HEXIM1 Regulates 17β-Estradiol/Estrogen Receptor-α–Mediated Expression of Cyclin D1 in Mammary Cells via Modulation of P-TEFb

Estrogen receptor alpha (ERalpha) plays a key role in mammary gland development and is implicated in breast cancer through the transcriptional regulation of genes linked to proliferation and apoptosis. We previously reported that hexamethylene bisacetamide inducible protein 1 (HEXIM1) inhibits the activity of ligand-bound ERalpha and bridges a functional interaction between ERalpha and positive transcription elongation factor b (P-TEFb). To examine the consequences of a functional HEXIM1-ERalpha-P-TEFb interaction in vivo, we generated MMTV/HEXIM1 mice that exhibit mammary epithelial-specific and doxycycline-inducible expression of HEXIM1. Increased HEXIM1 expression in the mammary gland decreased estrogen-driven ductal morphogenesis and inhibited the expression of cyclin D1 and serine 2 phosphorylated RNA polymerase II (S2P RNAP II). In addition, increased HEXIM1 expression in MCF-7 cells led to a decrease in estrogen-induced cyclin D1 expression, whereas down-regulation of HEXIM1 expression led to an enhancement of estrogen-induced cyclin D1 expression. Studies on the mechanism of HEXIM1 regulation on estrogen action indicated a decrease in estrogen-stimulated recruitment of ERalpha, P-TEFb, and S2P RNAP II to promoter and coding regions of ERalpha-responsive genes pS2 and CCND1 with increased HEXIM1 expression in MCF-7 cells. Notably, increased HEXIM1 expression decreased only estrogen-induced P-TEFb activity. Whereas there have been previous reports on HEXIM1 inhibition of P-TEFb activity, our studies add a new dimension by showing that E(2)/ER is an important regulator of the HEXIM1/P-TEFb functional unit in breast cells. Together, these studies provide novel insight into the role of HEXIM1 and ERalpha in mammary epithelial cell function.

Expression of human snRNA genes from beginning to end

In addition to protein-coding genes, mammalian pol II (RNA polymerase II) transcribes independent genes for some non-coding RNAs, including the spliceosomal U1 and U2 snRNAs (small nuclear RNAs). snRNA genes differ from protein-coding genes in several key respects and some of the mechanisms involved in expression are gene-type-specific. For example, snRNA gene promoters contain an essential PSE (proximal sequence element) unique to these genes, the RNA-encoding regions contain no introns, elongation of transcription is P-TEFb (positive transcription elongation factor b)-independent and RNA 3'-end formation is directed by a 3'-box rather than a cleavage and polyadenylation signal. However, the CTD (C-terminal domain) of pol II closely couples transcription with RNA 5' and 3' processing in expression of both gene types. Recently, it was shown that snRNA promoter-specific recognition of the 3'-box RNA processing signal requires a novel phosphorylation mark on the pol II CTD. This new mark plays a critical role in the recruitment of the snRNA gene-specific RNA-processing complex, Integrator. These new findings provide the first example of a phosphorylation mark on the CTD heptapeptide that can be read in a gene-type-specific manner, reinforcing the notion of a CTD code. Here, we review the control of expression of snRNA genes from initiation to termination of transcription.

The Positive Transcription Elongation Factor b Is an Essential Cofactor for the Activation of Transcription by Myocyte Enhancer Factor 2

The positive transcription elongation factor b (P-TEFb), composed of cyclin-dependent kinase 9 and cyclin T1, stimulates the elongation of transcription by hyperphosphorylating the C-terminal region of RNA polymerase II. Aberrant activation of P-TEFb results in manifestations of cardiac hypertrophy in mice, suggesting that P-TEFb is an essential factor for cardiac myocyte function and development. Here, we present evidence that P-TEFb selectively activates transcription mediated by the myocyte enhancer factor 2 (MEF2) family of transcription factors, key regulatory factors for myocyte development. Knockdown of endogenous cyclin T1 in murine C2C12 cells abolishes MEF2-dependent reporter gene expression as well as transcription of endogenous MEF2 target genes, whereas overexpression of P-TEFb enhances MEF2-dependent transcription. P-TEFb interacts with MEF2 both in vitro and in vivo. Activation of MEF2-dependent transcription induced by serum starvation is mediated by a rapid dissociation of P-TEFb from its inhibitory subunit, HEXIM1, and a subsequent recruitment of P-TEFb to MEF2 binding sites in the promoter region of MEF2 target genes. These results indicate that recruitment of P-TEFb is a critical step for stimulation of MEF2-dependent transcription, therefore providing a fundamentally important regulatory mechanism underlying the transcriptional program in muscle cells.

Dominant negative mutant Cyclin T1 proteins inhibit HIV transcription by specifically degrading Tat

BackgroundThe positive transcription elongation factor b (P-TEFb) is an essential cellular co-factor for the transcription of the human immunodeficiency virus type 1 (HIV-1). The cyclin T1 (CycT1) subunit of P-TEFb associates with a viral protein, Tat, at the transactivation response element (TAR). This represents a critical and necessary step for the stimulation of transcriptional elongation. Therefore, CycT1 may serve as a potential target for the development of anti-HIV therapies.ResultsTo create effective inhibitors of HIV transcription, mutant CycT1 proteins were constructed based upon sequence similarities between CycT1 and other cyclin molecules, as well as the defined crystal structure of CycT1. One of these mutants, termed CycT1-U7, showed a potent dominant negative effect on Tat-dependent HIV transcription despite a remarkably low steady-state expression level. Surprisingly, the expression levels of Tat proteins co-expressed with CycT1-U7 were significantly lower than Tat co-expressed with wild type CycT1. However, the expression levels of CycT1-U7 and Tat were restored by treatment with proteasome inhibitors. Concomitantly, the dominant negative effect of CycT1-U7 was abolished by these inhibitors.ConclusionThese results suggest that CycT1-U7 inhibits HIV transcription by promoting a rapid degradation of Tat. These mutant CycT1 proteins represent a novel class of specific inhibitors for HIV transcription that could potentially be used in the design of anti-viral therapy.

The structure of P-TEFb (CDK9/cyclin T1), its complex with flavopiridol and regulation by phosphorylation

The positive transcription elongation factor b (P-TEFb) (CDK9/cyclin T (CycT)) promotes mRNA transcriptional elongation through phosphorylation of elongation repressors and RNA polymerase II. To understand the regulation of a transcriptional CDK by its cognate cyclin, we have determined the structures of the CDK9/CycT1 and free cyclin T2. There are distinct differences between CDK9/CycT1 and the cell cycle CDK CDK2/CycA manifested by a relative rotation of 26 degrees of CycT1 with respect to the CDK, showing for the first time plasticity in CDK cyclin interactions. The CDK9/CycT1 interface is relatively sparse but retains some core CDK-cyclin interactions. The CycT1 C-terminal helix shows flexibility that may be important for the interaction of this region with HIV TAT and HEXIM. Flavopiridol, an anticancer drug in phase II clinical trials, binds to the ATP site of CDK9 inducing unanticipated structural changes that bury the inhibitor. CDK9 activity and recognition of regulatory proteins are governed by autophosphorylation. We show that CDK9/CycT1 autophosphorylates on Thr186 in the activation segment and three C-terminal phosphorylation sites. Autophosphorylation on all sites occurs in cis.

RelA Ser276 Phosphorylation Is Required for Activation of a Subset of NF-κB-Dependent Genes by Recruiting Cyclin-Dependent Kinase 9/Cyclin T1 Complexes

NF-kappaB plays a central role in cytokine-inducible inflammatory gene expression. Previously we empirically determined the identity of 92 members of the genetic network under direct NF-kappaB/RelA control that show marked heterogeneity in magnitude of transcriptional induction and kinetics of peak activation. To investigate this network further, we have applied a recently developed two-step chromatin immunoprecipitation assay that accurately reflects association and disassociation of RelA binding to its chromatin targets. Although inducible RelA binding occurs with similar kinetics on all NF-kappaB-dependent genes, serine 276 (Ser(276))-phosphorylated RelA binding is seen primarily on a subset of genes that are rapidly induced by tumor necrosis factor (TNF), including Gro-beta, interleukin-8 (IL-8), and IkappaBalpha. Previous work has shown that TNF-inducible RelA Ser(276) phosphorylation is controlled by a reactive oxygen species (ROS)-protein kinase A signaling pathway. To further understand the role of phospho-Ser(276) RelA in target gene expression, we inhibited its formation by ROS scavengers and antioxidants, treatments that disrupt phospho-Ser(276) formation but not the translocation and DNA binding of nonphosphorylated RelA. Here we find that phospho-Ser(276) RelA is required only for activation of IL-8 and Gro-beta, with IkappaBalpha being unaffected. These data were confirmed in experiments using RelA(-/-) murine embryonic fibroblasts reconstituted with a RelA Ser(276)Ala mutation. In addition, we observe that phospho-Ser(276) RelA binds the positive transcription elongation factor b (P-TEFb), a complex containing the cyclin-dependent kinase 9 (CDK-9) and cyclin T1 subunits. Inhibition of P-TEFb activity by short interfering RNA (siRNA)-mediated knockdown shows that the phospho-Ser(276) RelA-P-TEFb complex is required for IL-8 and Gro-beta gene activation but not for IkappaBalpha gene activation. These studies indicate that TNF induces target gene expression by heterogeneous mechanisms. One is mediated by phospho-Ser(276) RelA formation and chromatin targeting of P-TEFb controlling polymerase II (Pol II) recruitment and carboxy-terminal domain phosphorylation on the IL-8 and Gro-beta genes. The second involves a phospho-Ser(276) RelA-independent activation of genes preloaded with Pol II, exemplified by the IkappaBalpha gene. Together, these data suggest that the binding kinetics, selection of genomic targets, and mechanisms of promoter induction by RelA are controlled by a phosphorylation code influencing its interactions with coactivators and transcriptional elongation factors.

The La‐related protein LARP7 is a component of the 7SK ribonucleoprotein and affects transcription of cellular and viral polymerase II genes

The positive transcription elongation factor b (P-TEFb) is a heterodimeric complex composed of cyclin-dependent kinase 9 and its regulator cyclin T1/2. It stimulates transcription elongation by phosphorylation of serine 2 residues in the carboxy-terminal domain of polymerase II. 7SK RNA and HEXIM proteins can antagonize transcriptional stimulation by sequestering P-TEFb in a catalytically inactive ribonucleoprotein (RNP). Here, we show that the previously uncharacterized La-related protein 7 (LARP7) has a role in 7SK-mediated regulation of transcription. LARP7 binds to the highly conserved 3'-terminal U-rich stretch of 7SK RNA and is an integral part of the 7SK RNP. On stimulation, LARP7 remains associated with 7SK RNA, whereas P-TEFb is released. Interestingly, reduction of LARP7 by RNA interference enhances transcription from cellular polymerase II promoters, as well as a TAT-dependent HIV-1 promoter. Thus, LARP7 is a negative transcriptional regulator of polymerase II genes, acting by means of the 7SK RNP system.

PP2B and PP1α cooperatively disrupt 7SK snRNP to release P-TEFb for transcription in response to Ca2+ signaling

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 (Ca2+)-calmodulin-protein phosphatase 2B (PP2B) signaling pathway is revealed. However, Ca2+ signaling alone is insufficient, and PP2B must act sequentially and cooperatively with protein phosphatase 1alpha (PP1alpha) to disrupt 7SK snRNP. Activated by UV/HMBA and facilitated by a PP2B-induced conformational change in 7SK snRNP, PP1alpha 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 Ca2+ signaling, PP2B and PP1alpha alter the P-TEFb functional equilibrium through releasing P-TEFb from 7SK snRNP for transcription.

CDK9 a Potential Target for Drug Development

The family of Cyclin-Dependent Kinases (CDKs) can be subdivided into two major functional groups based on their roles in cell cycle and/or transcriptional control. CDK9 is the catalytic subunit of positive transcription elongation factor b (P-TEFb). CDK9 is the kinase of the TAK complex (Tat-associated kinase complex), and binds to Tat protein of HIV, suggesting a possible role for CDK9 in AIDS progression. CDK9 complexed with its regulatory partner cyclin T1, serves as a cellular mediator of the transactivation function of the HIV Tat protein. P-TEFb is responsible for the phosphorylation of the carboxyl-terminal domain of RNA Pol II, resulting in stimulation of transcription. Furthermore, the complexes containing CDK9 induce the differentiation in distinct tissue. The CDK9/cyclin T1 complex is expressed at higher level in more differentiated primary neuroectodermal and neuroblastoma tumors, showing a correlation between the kinase expression and tumor differentiation grade. This may have clinical and therapeutical implications for these tumor types. Among the CDK inhibitors two have shown to be effective against CDK9: Roscovitine and Flavopiridol. These two inhibitors prevented the replication of human immunodeficiency virus (HIV) type 1 by blocking Tat transactivation of the HIV type 1 promoter. These compounds inhibit CDKs by binding to the catalytic domain in place of ATP, preventing transfer of a phosphate group to the substrate. More sensitive therapeutic agents of CDK9 can be designed, and structural studies can add information in the understanding of this kinase. The major features related to CDK9 inhibition will be reviewed in this article.

Role of the hinge region of glucocorticoid receptor for HEXIM1-mediated transcriptional repression

We previously reported that HEXIM1 (hexamethylene bisacetamide-inducible protein 1), which suppresses transcription elongation via sequestration of positive transcription elongation factor b (P-TEFb) using 7SK RNA as a scaffold, directly associates with glucocorticoid receptor (GR) to suppress glucocorticoid-inducible gene activation. Here, we revealed that the hinge region of GR is essential for its interaction with HEXIM1, and that oxosteroid receptors including GR show sequence homology in their hinge region and interact with HEXIM1, whereas the other members of nuclear receptors do not. We also showed that HEXIM1 suppresses GR-mediated transcription in two ways: sequestration of P-TEFb by HEXIM1 and direct interaction between GR and HEXIM1. In contrast, peroxisome proliferator-activated receptor γ-dependent gene expression is negatively modulated by HEXIM1 solely via sequestration of P-TEFb. We, therefore, conclude that HEXIM1 may act as a gene-selective transcriptional regulator via direct interaction with certain transcriptional regulators including GR and contribute to fine-tuning of, for example, glucocorticoid-mediated biological responses.

Acetylation of Conserved Lysines in the Catalytic Core of Cyclin-Dependent Kinase 9 Inhibits Kinase Activity and Regulates Transcription

Promoter clearance and transcriptional processivity in eukaryotic cells are fundamentally regulated by the phosphorylation of the carboxy-terminal domain of RNA polymerase II (RNAPII). One of the kinases that essentially performs this function is P-TEFb (positive transcription elongation factor b), which is composed of cyclin-dependent kinase 9 (CDK9) associated with members of the cyclin T family. Here we show that cellular GCN5 and P/CAF, members of the GCN5-related N-acetyltransferase family of histone acetyltransferases, regulate CDK9 function by specifically acetylating the catalytic core of the enzyme and, in particular, a lysine that is essential for ATP coordination and the phosphotransfer reaction. Acetylation markedly reduces both the kinase function and transcriptional activity of P-TEFb. In contrast to unmodified CDK9, the acetylated fraction of the enzyme is specifically found in the insoluble nuclear matrix compartment. Acetylated CDK9 associates with the transcriptionally silent human immunodeficiency virus type 1 provirus; upon transcriptional activation, it is replaced by the unmodified form, which is involved in the elongating phase of transcription marked by Ser2-phosphorylated RNAPII. Given the conservation of the CDK9 acetylated residues in the catalytic task of virtually all CDK proteins, we anticipate that this mechanism of regulation might play a broader role in controlling the function of other members of this kinase family.

Nucleophosmin Interacts with HEXIM1 and Regulates RNA Polymerase II Transcription

Hexamethylene bis-acetamide-inducible protein 1 (HEXIM1) was identified earlier as an inhibitor of positive transcription elongation factor b (P-TEFb), which is a key transcriptional regulator of RNA polymerase II (Pol II). Studies show that more than half of P-TEFb in cells is associated with HEXIM1, which results in the inactivation of P-TEFb. Here, we identify a nucleolar protein, nucleophosmin (NPM), as a HEXIM1-binding protein. NPM binds to HEXIM1 in vitro and in vivo, and functions as a negative regulator of HEXIM1. Over-expression of NPM leads to proteasome-mediated degradation of HEXIM1, resulting in activation of P-TEFb-dependent transcription. In contrast, an increase in HEXIM1 protein levels and a decrease in transcription are detected when NPM is knocked down. We show that a cytoplasmic mutant of NPM, NPMc+, associates with and sequesters HEXIM1 in the cytoplasm resulting in higher RNA Pol II transcription. Correspondingly, cytoplasmic localization of endogenous HEXIM1 is detected in an acute myeloid leukemia (AML) cell line containing the NPMc+ mutation, suggesting the physiological importance of HEXIM1-NPMc+ interaction. Over-expression of NPM has been detected in tumors of various histological origins and our results may provide a possible molecular mechanism for the proto-oncogenic function of NPM. Furthermore, considering that 35% of AML patients are diagnosed with NPMc+ mutation, our findings suggest that in some cases of AML, RNA Pol II transcription may be disregulated by the malfunction of NPM and the mislocation of HEXIM1.