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David H Price - One of the best experts on this subject based on the ideXlab platform.
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release of positive transcription elongation factor b p tefb from 7sk small nuclear ribonucleoprotein snrnp activates hexamethylene bisacetamide inducible protein hexim1 transcription
Journal of Biological Chemistry, 2014Co-Authors: Pingyang Liu, David H Price, Koh Fujinaga, Koen Bartholomeeusen, Yanhui Xiang, Kyle A Nilson, Matija B PeterlinAbstract:By phosphorylating negative elongation factors and the C-terminal domain of RNA polymerase II (RNAPII), positive transcription elongation factor b (P-TEFb), which is composed of CycT1 or CycT2 and CDK9, activates eukaryotic transcription elongation. In growing cells, it is found in active and inactive forms. In the former, free P-TEFb is a potent transcriptional coactivator. In the latter, it is inhibited by HEXIM1 or HEXIM2 in the 7SK small nuclear ribonucleoprotein (snRNP), which contains, additionally, 7SK snRNA, methyl phosphate-capping enzyme (MePCE), and La-related protein 7 (LARP7). This P-TEFb equilibrium determines the state of growth and proliferation of the cell. In this study, the release of P-TEFb from the 7SK snRNP led to increased synthesis of HEXIM1 but not HEXIM2 in HeLa cells, and this occurred only from an unannotated, proximal promoter. ChIP with sequencing revealed P-TEFb-sensitive poised RNA polymerase II at this proximal but not the previously annotated distal HEXIM1 promoter. Its immediate upstream sequences were fused to luciferase reporters and were found to be responsive to many P-TEFb-releasing compounds. The superelongation complex subunits AF4/FMR2 family member 4 (AFF4) and elongation factor RNA polymerase II 2 (ELL2) were recruited to this proximal promoter after P-TEFb release and were required for its transcriptional effects. Thus, P-TEFb regulates its own equilibrium in cells, most likely to maintain optimal cellular homeostasis.
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PKC phosphorylates HEXIM1 and regulates P-TEFb activity - eScholarship
2012Co-Authors: David H Price, Koh Fujinaga, Zeping Luo, Matjaz Barboric, B. Matija PeterlinAbstract:The positive transcription elongation factor b (P-TEFb) regulates RNA polymerase II elongation. In cells, P-TEFb partitions between small active and larger inactive states. In the latter, HEXIM1 binds to 7SK snRNA and recruits as well as inactivates P-TEFb
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PKC phosphorylates HEXIM1 and regulates P-TEFb activity
Nucleic Acids Research, 2012Co-Authors: Koh Fujinaga, David H Price, Zeping Luo, Matjaz Barboric, B. Matija PeterlinAbstract:The positive transcription elongation factor b (P-TEFb) regulates RNA polymerase II elongation. In cells, P-TEFb partitions between small active and larger inactive states. In the latter, HEXIM1 binds to 7SK snRNA and recruits as well as inactivates P-TEFb in the 7SK snRNP. Several stimuli can affect this P-TEFb equilibrium. In this study, we demonstrate that protein kinase C (PKC) phosphorylates the serine at position158 (S158) in HEXIM1. This phosphorylated HEXIM1 protein neither binds to 7SK snRNA nor inhibits P-TEFb. Phorbol esters or the engagement of the T cell antigen receptor, which activate PKC and the expression of the constitutively active (CA) PKCθ protein, which is found in T cells, inhibit the formation of the 7SK snRNP. All these stimuli increase P-TEFb-dependent transcription. In contrast, the kinase-negative PKCθ and the mutant HEXIM1 (S158A) proteins block effects of these PKC-activating stimuli. These results indicate that the phosphorylation of HEXIM1 by PKC represents a major regulatory step of P-TEFb activity in cells.
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7sk snrna a noncoding rna that plays a major role in regulating eukaryotic transcription
Wiley Interdisciplinary Reviews - Rna, 2012Co-Authors: Matija B Peterlin, John E Brogie, David H PriceAbstract:The human 7SK small nuclear RNA (snRNA) is an abundant noncoding RNA whose function has been conserved in evolution from invertebrates to humans. It is transcribed by RNA polymerase III (RNAPIII) and is located in the nucleus. Together with associated cellular proteins, 7SK snRNA regulates the activity of the positive transcription elongation factor, P-TEFb. In humans, this regulation is accomplished by the recruitment of P-TEFb by the 7SK snRNA-binding proteins, HEXIM1 or HEXIM2, which inhibit the kinase activity of P-TEFb. P-TEFb regulates the transition of promoter proximally paused RNA polymerase II (RNAPII) into productive elongation, thereby, allowing efficient mRNA production. The protein composition of the 7SK small nuclear ribonucleoprotein (snRNP) is regulated dynamically. Whereas the La related protein LARP7 is a constitutive component, the methyl phosphate capping enzyme MePCE associates secondarily to phosphorylate the 5' end of 7SK snRNA. The release of active P-TEFb is closely followed by release of HEXIM proteins and both are replaced by heterogeneous nuclear ribonucleoproteins (hnRNPs). The released P-TEFb activates the expression of most cellular and viral genes. Regulated release of P-TEFb determines the expression pattern of many of the genes that respond to environmental stimuli and regulate growth, proliferation and differentiation of cells.
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The P-TEFb inhibitors DRB, seliciclib and flavopiridol release P-TEFb from the large form
2011Co-Authors: Sebastian Biglione, David H Price, Sarah A. Byers, Olivier Bensaude, Jason P. Price, Van Trung Nguyen, Wendy MauryAbstract:Copyright information:Taken from "Inhibition of HIV-1 replication by P-TEFb inhibitors DRB, seliciclib and flavopiridol correlates with release of free P-TEFb from the large, inactive form of the complex"http://www.retrovirology.com/content/4/1/47Retrovirology 2007;4():47-47.Published online 11 Jul 2007PMCID:PMC1948018. Low-salt cytosolic extract (CE) containing the large form of P-TEFb and high-salt nuclear extracts (NE) containing the free form of P-TEFb were generated from (A) DRB-treated HeLa cells, (B) DRB treated Jurkat cells, (C) seliciclib-treated HeLa37 cells or (D) flavopiridol-treated Jurkat cells. Quantitative western blotting was performed on low salt cytosolic extracts (CE) and high-salt nuclear extracts (NE) to detect the percentage of Cdk9 and cyclin T1 present in the free and large form of the P-TEFb complex. The percent of P-TEFb in the large form of the complex (low-salt or CE) was calculated as a fraction of the total amount of P-TEFb (low-salt + high-salt P-TEFb) and plotted as a function of the concentration of P-TEFb inhibitor
Qiang Zhou - One of the best experts on this subject based on the ideXlab platform.
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HEXIM1 controls P-TEFb processing and regulates drug sensitivity in triple-negative breast cancer.
Molecular Biology of the Cell, 2020Co-Authors: Hengyi Shao, Qiang Zhou, Qingwei Zhu, Amanda Chang, Carol Gao, Kunxin LuoAbstract:The positive transcription elongation factor b (P-TEFb), composed of CDK9 and cyclin T, stimulates transcriptional elongation by RNA polymerase (Pol) II and regulates cell growth and differentiation. Recently, we demonstrated that P-TEFb also controls the expression of EMT regulators to promote breast cancer progression. In the nucleus, more than half of P-TEFb are sequestered in the inactive-state 7SK snRNP complex. Here, we show that the assembly of the 7SK snRNP is preceded by an intermediate complex between HEXIM1 and P-TEFb that allows transfer of the kinase active P-TEFb from Hsp90 to 7SK snRNP for its suppression. Down-regulation of HEXIM1 locks P-TEFb in the Hsp90 complex, keeping it in the active state to enhance breast cancer progression, but also rendering the cells highly sensitive to Hsp90 inhibition. Because HEXIM1 is often down-regulated in human triple-negative breast cancer (TNBC), these cells are particularly sensitive to Hsp90 inhibition. Our study provides a mechanistic explanation for the increased sensitivity of TNBC to Hsp90 inhibition.
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LARP7 suppresses P-TEFb activity to inhibit breast cancer progression and metastasis
eLife, 2014Co-Authors: Qiang Zhou, Kunxin LuoAbstract:Transcriptional elongation by RNA polymerase (Pol) II is essential for gene expression during cell growth and differentiation. The positive transcription elongation factor b (P-TEFb) stimulates transcriptional elongation by phosphorylating Pol II and antagonizing negative elongation factors. A reservoir of P-TEFb is sequestered in the inactive 7SK snRNP where 7SK snRNA and the La-related protein LARP7 are required for the integrity of this complex. Here, we show that P-TEFb activity is important for the epithelial-mesenchymal transition (EMT) and breast cancer progression. Decreased levels of LARP7 and 7SK snRNA redistribute P-TEFb to the transcriptionally active super elongation complex, resulting in P-TEFb activation and increased transcription of EMT transcription factors, including Slug, FOXC2, ZEB2, and Twist1, to promote breast cancer EMT, invasion, and metastasis. Our data provide the first demonstration that the transcription elongation machinery plays a key role in promoting breast cancer progression by directly controlling the expression of upstream EMT regulators.
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AFF1 is a ubiquitous P-TEFb partner to enable Tat extraction of P-TEFb from 7SK snRNP and formation of SECs for HIV transactivation
Proceedings of the National Academy of Sciences of the United States of America, 2013Co-Authors: Yuhua Xue, Ursula Schulze-gahmen, Jeffrey R. Johnson, Nevan J. Krogan, Tom Alber, Qiang ZhouAbstract:The positive transcription elongation factor b (P-TEFb) stimulates RNA polymerase elongation by inducing the transition of promoter proximally paused polymerase II into a productively elongating state. P-TEFb itself is regulated by reversible association with various transcription factors/cofactors to form several multisubunit complexes [e.g., the 7SK small nuclear ribonucleoprotein particle (7SK snRNP), the super elongation complexes (SECs), and the bromodomain protein 4 (Brd4)–P-TEFb complex] that constitute a P-TEFb network controlling cellular and HIV transcription. These complexes have been thought to share no components other than the core P-TEFb subunits cyclin-dependent kinase 9 (CDK9) and cyclin T (CycT, T1, T2a, and T2b). Here we show that the AF4/FMR2 family member 1 (AFF1) is bound to CDK9–CycT and is present in all major P-TEFb complexes and that the tripartite CDK9–CycT–AFF1 complex is transferred as a single unit within the P-TEFb network. By increasing the affinity of the HIV-encoded transactivating (Tat) protein for CycT1, AFF1 facilitates Tat’s extraction of P-TEFb from 7SK snRNP and the formation of Tat–SECs for HIV transcription. Our data identify AFF1 as a ubiquitous P-TEFb partner and demonstrate that full Tat transactivation requires the complete SEC.
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The AFF4 scaffold binds human P-TEFb adjacent to HIV Tat
eLife, 2013Co-Authors: Ursula Schulze-gahmen, Qiang Zhou, Nevan J. Krogan, Heather Upton, Andrew Birnberg, Katherine Bao, Seemay Chou, Tom AlberAbstract:Human positive transcription elongation factor b (P-TEFb) phosphorylates RNA polymerase II and regulatory proteins to trigger elongation of many gene transcripts. The HIV-1 Tat protein selectively recruits P-TEFb as part of a super elongation complex (SEC) organized on a flexible AFF1 or AFF4 scaffold. To understand this specificity and determine if scaffold binding alters P-TEFb conformation, we determined the structure of a tripartite complex containing the recognition regions of P-TEFb and AFF4. AFF4 meanders over the surface of the P-TEFb cyclin T1 (CycT1) subunit but makes no stable contacts with the CDK9 kinase subunit. Interface mutations reduced CycT1 binding and AFF4-dependent transcription. AFF4 is positioned to make unexpected direct contacts with HIV Tat, and Tat enhances P-TEFb affinity for AFF4. These studies define the mechanism of scaffold recognition by P-TEFb and reveal an unanticipated intersubunit pocket on the AFF4 SEC that potentially represents a target for therapeutic intervention against HIV/AIDS. DOI:http://dx.doi.org/10.7554/eLife.00327.001.
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hiv 1 tat and host aff4 recruit two transcription elongation factors into a bifunctional complex for coordinated activation of hiv 1 transcription
Molecular Cell, 2010Co-Authors: Min Liu, Yuhua Xue, Nevan J. Krogan, Tom Alber, Seemay Chou, Joanne Hsu, Alma L Burlingame, Qiang ZhouAbstract:Recruitment of the P-TEFb kinase by HIV-1 Tat to the viral promoter triggers the phosphorylation and escape of RNA polymerase II from promoter-proximal pausing. It is unclear, however, if Tat recruits additional host factors that further stimulate HIV-1 transcription. Using a sequential affinity-purification scheme, we have identified human transcription factors/coactivators AFF4, ENL, AF9, and elongation factor ELL2 as components of the Tat-P-TEFb complex. Through the bridging functions of Tat and AFF4, P-TEFb and ELL2 combine to form a bifunctional elongation complex that greatly activates HIV-1 transcription. Without Tat, AFF4 can mediate the ELL2-P-TEFb interaction, albeit inefficiently. Tat overcomes this limitation by bringing more ELL2 to P-TEFb and stabilizing ELL2 in a process that requires active P-TEFb. The ability of Tat to enable two different classes of elongation factors to cooperate and coordinate their actions on the same polymerase enzyme explains why Tat is such a powerful activator of HIV-1 transcription.
Olivier Bensaude - One of the best experts on this subject based on the ideXlab platform.
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A Cyclin T1 point mutation that abolishes positive transcription elongation factor (P-TEFb) binding to Hexim1 and HIV tat
Retrovirology, 2014Co-Authors: Nina Verstraete, Olivier Bensaude, Van Trung Nguyen, Alona Kuzmina, Gaelle Diribarne, Lydia Kobbi, Monika Ludanyi, Ran TaubeAbstract:Background The positive transcription elongation factor b (P-TEFb) plays an essential role in activating HIV genome transcription. It is recruited to the HIV LTR promoter through an interaction between the Tat viral protein and its Cyclin T1 subunit. P-TEFb activity is inhibited by direct binding of its subunit Cyclin T (1 or 2) with Hexim (1 or 2), a cellular protein, bound to the 7SK small nuclear RNA. Hexim1 competes with Tat for P-TEFb binding.
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The P-TEFb inhibitors DRB, seliciclib and flavopiridol release P-TEFb from the large form
2011Co-Authors: Sebastian Biglione, David H Price, Sarah A. Byers, Olivier Bensaude, Jason P. Price, Van Trung Nguyen, Wendy MauryAbstract:Copyright information:Taken from "Inhibition of HIV-1 replication by P-TEFb inhibitors DRB, seliciclib and flavopiridol correlates with release of free P-TEFb from the large, inactive form of the complex"http://www.retrovirology.com/content/4/1/47Retrovirology 2007;4():47-47.Published online 11 Jul 2007PMCID:PMC1948018. Low-salt cytosolic extract (CE) containing the large form of P-TEFb and high-salt nuclear extracts (NE) containing the free form of P-TEFb were generated from (A) DRB-treated HeLa cells, (B) DRB treated Jurkat cells, (C) seliciclib-treated HeLa37 cells or (D) flavopiridol-treated Jurkat cells. Quantitative western blotting was performed on low salt cytosolic extracts (CE) and high-salt nuclear extracts (NE) to detect the percentage of Cdk9 and cyclin T1 present in the free and large form of the P-TEFb complex. The percent of P-TEFb in the large form of the complex (low-salt or CE) was calculated as a fraction of the total amount of P-TEFb (low-salt + high-salt P-TEFb) and plotted as a function of the concentration of P-TEFb inhibitor
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Caffeine prevents transcription inhibition and P-TEFb/7SK dissociation following UV-induced DNA damage.
PLOS ONE, 2010Co-Authors: Giuliana Napolitano, Olivier Bensaude, Barbara Gargano, Stefano Amente, Barbara Majello, Virginia Castiglia, Vera M. Ruda, Xavier Darzacq, Luigi LaniaAbstract:Author(s): Napolitano, Giuliana; Amente, Stefano; Castiglia, Virginia; Gargano, Barbara; Ruda, Vera; Darzacq, Xavier; Bensaude, Olivier; Majello, Barbara; Lania, Luigi | Abstract: BACKGROUND: The mechanisms by which DNA damage triggers suppression of transcription of a large number of genes are poorly understood. DNA damage rapidly induces a release of the positive transcription elongation factor b (P-TEFb) from the large inactive multisubunit 7SK snRNP complex. P-TEFb is required for transcription of most class II genes through stimulation of RNA polymerase II elongation and cotranscriptional pre-mRNA processing. METHODOLOGY/PRINCIPAL FINDINGS: We show here that caffeine prevents UV-induced dissociation of P-TEFb as well as transcription inhibition. The caffeine-effect does not involve PI3-kinase-related protein kinases, because inhibition of phosphatidylinositol 3-kinase family members (ATM, ATR and DNA-PK) neither prevents P-TEFb dissociation nor transcription inhibition. Finally, caffeine prevention of transcription inhibition is independent from DNA damage. CONCLUSION/SIGNIFICANCE: Pharmacological prevention of P-TEFb/7SK snRNP dissociation and transcription inhibition following UV-induced DNA damage is correlated.
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LARP7 is a stable component of the 7SK snRNP while P-TEFb, HEXIM1 and hnRNP A1 are reversibly associated
Nucleic Acids Research, 2008Co-Authors: Brian J. Krueger, Célia Jeronimo, Bibhuti Bhusan Roy, Annie Bouchard, Charlotte Barrandon, Sarah A. Byers, Courtney E. Searcey, Jeffrey J. Cooper, Olivier Bensaude, Éric A. CohenAbstract: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.
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RNA-driven cyclin-dependent kinase regulation: when CDK9/cyclin T subunits of P-TEFb meet their ribonucleoprotein partners.
Biotechnology journal, 2008Co-Authors: Annemieke A Michels, Olivier BensaudeAbstract:The positive transcription elongation factor (P-TEFb) consists of CDK9, a cyclin-dependent kinase and its cyclin T partner. It is required for transcription of most class II genes. Its activity is regulated by non-coding RNAs. The 7SK cellular RNA turns the HEXIM cellular protein into a P-TEFb inhibitor that binds its cyclin T subunit. Thus, P-TEFb activity responds to variations in global cellular transcriptional activity and to physiological conditions linked to cell differentiation, proliferation or cardiac hypertrophy. In contrast, the Tat activation region RNA plays an activating role. This feature at the 5' end of the human immunodeficiency (HIV) viral transcript associates with the viral protein Tat that in turn binds cyclin T1 and recruits active P-TEFb to the HIV promoter. This results in enhanced P-TEFb activity, which is critical for an efficient production of viral transcripts. Although discovered recently, the regulation of P-TEFb becomes a paradigm for non-coding RNAs that regulate transcription factors. It is also a unique example of RNA-driven regulation of a cyclindependent kinase.
Keiko Ozato - One of the best experts on this subject based on the ideXlab platform.
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DNA Damage Induces Dynamic Associations of BRD4/P-TEFb With Chromatin and Modulates Gene Transcription in a BRD4-Dependent and -Independent Manner
Frontiers in molecular biosciences, 2020Co-Authors: Yawei Song, Keiko Ozato, Jinping Jia, Mingze Yao, Xiaoshan Wang, Andrew P. Hutchins, Jiekai Chen, Hongjie YaoAbstract:The bromodomain-containing protein BRD4 has been thought to transmit epigenetic information across cell divisions by binding to both mitotic chromosomes and interphase chromatin. UV-released BRD4 mediates the recruitment of active P-TEFb to the promoter, which enhances transcriptional elongation. However, the dynamic associations between BRD4 and P-TEFb and BRD4-mediated gene regulation after UV stress are largely unknown. In this study, we found that BRD4 dissociates from chromatin within 30 min after UV treatment and thereafter recruits chromatin. However, P-TEFb binds tightly to chromatin right after UV treatment, suggesting that no interactions occur between BRD4 and P-TEFb within 30 min after UV stress. BRD4 knockdown changes the distribution of P-TEFb among nuclear soluble and chromatin and downregulates the elongation activity of RNA polymerase II. Inhibition of JNK kinase but not other MAP kinases impedes the interactions between BRD4 and P-TEFb. RNA-seq and ChIP assays indicate that BRD4 both positively and negatively regulates gene transcription in cells treated with UV stress. These results reveal previously unrecognized dynamics of BRD4 and P-TEFb after UV stress and regulation of gene transcription by BRD4 acting as either activator or repressor in a context-dependent manner.
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brd4 coordinates recruitment of pause release factor p tefb and the pausing complex nelf dsif to regulate transcription elongation of interferon stimulated genes
Molecular and Cellular Biology, 2013Co-Authors: Mira C. Patel, Maxime Debrosse, Matthew Smith, Anup Dey, Walter Huynh, Naoyuki Sarai, Tom D. Heightman, Tomohiko Tamura, Keiko OzatoAbstract:RNA polymerase II (Pol II) and the pausing complex, NELF and DSIF, are detected near the transcription start site (TSS) of many active and silent genes. Active transcription starts when the pause release factor P-TEFb is recruited to initiate productive elongation. However, the mechanism of P-TEFb recruitment and regulation of NELF/DSIF during transcription is not fully understood. We investigated this question in interferon (IFN)-stimulated transcription, focusing on BRD4, a BET family protein that interacts with P-TEFb. Besides P-TEFb, BRD4 binds to acetylated histones through the bromodomain. We found that BRD4 and P-TEFb, although not present prior to IFN treatment, were robustly recruited to IFN-stimulated genes (ISGs) after stimulation. Likewise, NELF and DSIF prior to stimulation were hardly detectable on ISGs, which were strongly recruited after IFN treatment. A shRNA-based knockdown assay of NELF revealed that it negatively regulates the passage of Pol II and DSIF across the ISGs during elongation, reducing total ISG transcript output. Analyses with a BRD4 small-molecule inhibitor showed that IFN-induced recruitment of P-TEFb and NELF/DSIF was under the control of BRD4. We suggest a model where BRD4 coordinates both positive and negative regulation of ISG elongation.
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BRD4 Coordinates Recruitment of Pause Release Factor P-TEFb and the Pausing Complex NELF/DSIF To Regulate Transcription Elongation of Interferon-Stimulated Genes
Molecular and cellular biology, 2013Co-Authors: Mira C. Patel, Maxime Debrosse, Matthew Smith, Anup Dey, Walter Huynh, Naoyuki Sarai, Tom D. Heightman, Tomohiko Tamura, Keiko OzatoAbstract:RNA polymerase II (Pol II) and the pausing complex, NELF and DSIF, are detected near the transcription start site (TSS) of many active and silent genes. Active transcription starts when the pause release factor P-TEFb is recruited to initiate productive elongation. However, the mechanism of P-TEFb recruitment and regulation of NELF/DSIF during transcription is not fully understood. We investigated this question in interferon (IFN)-stimulated transcription, focusing on BRD4, a BET family protein that interacts with P-TEFb. Besides P-TEFb, BRD4 binds to acetylated histones through the bromodomain. We found that BRD4 and P-TEFb, although not present prior to IFN treatment, were robustly recruited to IFN-stimulated genes (ISGs) after stimulation. Likewise, NELF and DSIF prior to stimulation were hardly detectable on ISGs, which were strongly recruited after IFN treatment. A shRNA-based knockdown assay of NELF revealed that it negatively regulates the passage of Pol II and DSIF across the ISGs during elongation, reducing total ISG transcript output. Analyses with a BRD4 small-molecule inhibitor showed that IFN-induced recruitment of P-TEFb and NELF/DSIF was under the control of BRD4. We suggest a model where BRD4 coordinates both positive and negative regulation of ISG elongation.
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Modulation of the Brd4/P-TEFb Interaction by the Human T-Lymphotropic Virus Type 1 Tax Protein
Journal of Virology, 2007Co-Authors: Won-kyung Cho, M Jang, Keiko Ozato, Meisheng Zhou, Keven Huang, Soo-jin Jeong, John N. BradyAbstract:Positive transcription elongation factor (P-TEFb), which is composed of CDK9 and cyclin T1, plays an important role in cellular and viral gene expression. Our lab has recently demonstrated that P-TEFb is required for Tax transactivation of the viral long terminal repeat (LTR). P-TEFb is found in two major complexes: the inactive form, which is associated with inhibitory subunits 7SK snRNA and HEXIM1, and the active form, which is associated with, at least in part, Brd4. In this study, we analyzed the effect of Brd4 on human T-lymphotropic virus type 1 (HTLV-1) transcription. Overexpression of Brd4 repressed Tax transactivation of the HTLV-1 LTR in a dose-dependent manner. In vitro binding studies suggest that Tax and Brd4 compete for binding to P-TEFb through direct interaction with cyclin T1. Tax interacts with cyclin T1 amino acids 426 to 533, which overlaps the region responsible for Brd4 binding. In vivo, overexpression of Tax decreased the amount of 7SK snRNA associated with P-TEFb and stimulates serine 2 phosphorylation of the RNA polymerase II carboxyl-terminal domain, suggesting that Tax regulates the functionality of P-TEFb. Our results suggest the possibility that Tax may compete and functionally substitute for Brd4 in P-TEFb regulation.
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recruitment of p tefb for stimulation of transcriptional elongation by the bromodomain protein brd4
Molecular Cell, 2005Co-Authors: Zhiyuan Yang, Jasper H N Yik, Ruichuan Chen, M Jang, Keiko Ozato, Qiang ZhouAbstract: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.
Ruichuan Chen - One of the best experts on this subject based on the ideXlab platform.
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P-TEFb: Finding its ways to release promoter-proximally paused RNA polymerase II
Transcription, 2018Co-Authors: Min Liu, Lin Feng Chen, Ruichuan ChenAbstract:The release of a paused Pol II depends on the recruitment of P-TEFb. Recent studies showed that both active P-TEFb and inactive P-TEFb (7SK snRNP) can be recruited to the promoter regions of global genes by different mechanisms. Here, we summarize the recent advances on these distinct recruitment mechanisms.
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Isolation and functional characterization of P-TEFb-associated factors that control general and HIV-1 transcriptional elongation
Methods, 2010Co-Authors: Ruichuan Chen, Min Liu, Qiang Zhou, Kai ZhangAbstract:Originally identified as a factor crucial for RNA polymerase (Pol) II transcriptional elongation of cellular genes, the P-TEFb kinase was subsequently shown to also serve as a specific host co-factor required for HIV-1 transcription. Recruited by either the bromodomain protein Brd4 to cellular promoters for general transcription or the HIV-1 Tat protein to the viral LTR for activated HIV-1 transcription, P-TEFb stimulates the processivity of Pol II through phosphorylating the C-terminal domain of Pol II and a pair of negative elongation factors, leading to the synthesis of full-length transcripts. However, abundant evidence indicates that P-TEFb does not act alone in the cell and that all of its known biological functions are likely mediated through the interactions with various regulators. Although a number of P-TEFb-associated factors have already been identified, there are likely more yet to be discovered. Given that P-TEFb plays an essential role in HIV-1 transcription, a major challenge facing the field is to identify all the P-TEFb-associated factors and determine how they may modulate Tat-transactivation and HIV-1 replication. Described here is a set of experimental procedures that have not only enabled us to isolate and identify several P-TEFb-associated factors, but also provided the means to characterize their biochemical functions in HIV-1 transcriptional control. In light of the recent demonstrations that transcriptional elongation plays a much more important role in controlling metazoan gene expression than previously thought, the techniques presented here will also be useful for analyzing Pol II elongation of cellular genes.
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PP2B and PP1α cooperatively disrupt 7SK snRNP to release P-TEFb for transcription in response to Ca2+ signaling
Genes & Development, 2008Co-Authors: Ruichuan Chen, Min Liu, Yuhua Xue, Wanichaya N. Ramey, Haohong Luo, Ying Zhu, Nan ZhouAbstract: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 5′ 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 proteins, HEXIM1 (Michels et al. 2003; Yik et al. 2003), BCDIN3 (Jeronimo et al. 2007), and PIP7S/LARP7 (He et al. 2008; Krueger et al. 2008). Within this complex, HEXIM1 inhibits the Cdk9 kinase in a 7SK-dependent manner (Yik et al. 2003). Underscoring the importance of 7SK as a molecular scaffold to maintain the integrity of 7SK snRNP, this RNA is protected from both 5′–3′ and 3′–5′ exonucleases by the respective actions of BCDIN3, a methylphosphate capping enzyme specific for 7SK (Jeronimo et al. 2007), and PIP7S/LARP7, a La-related protein bound to the 3′ UUU-OH sequence of 7SK (He et al. 2008). Besides P-TEFb sequestered in 7SK snRNP, a separate population of P-TEFb exists in a complex together with the bromodomain protein Brd4, which recruits P-TEFb to chromatin templates through interacting with acetylated histones and the mediator complex (Jang et al. 2005; Yang et al. 2005). Recent evidence shows that this recruitment occurs mostly at late mitosis and is essential to promote G1 gene expression and cell cycle progression (Yang et al. 2008). Importantly, the two populations of P-TEFb are kept in a functional equilibrium that can be perturbed by conditions that impact cell growth. For example, treating cells with global transcription inhibitors DRB and actinomycin D or the DNA-damaging agent UV disrupts 7SK snRNP and converts P-TEFb into the Brd4-bound form for stress-induced gene expression (Nguyen et al. 2001; Yang et al. 2001, 2005). Similarly, in cardiac myocytes, hypertrophic signals release P-TEFb from 7SK snRNP, leading to an overall increase in cellular protein and RNA contents, enlarged cells, and hypertrophic growth (Sano et al. 2002). Finally, RNAi-mediated depletion of PIP7S/LARP7 compromises the integrity of 7SK snRNP, resulting in P-TEFb-dependent transformation of mammary epithelial cells (He et al. 2008). Recent data indicate that the P-TEFb functional equilibrium can also be affected by HMBA (hexamethylene bisacetamide), which is known to inhibit growth and induce differentiation of many cell types (Marks et al. 1994). Interestingly, the response to HMBA displays a biphasic nature (He et al. 2006; Contreras et al. 2007). Shortly after the treatment begins, a disruption of 7SK snRNP and enhanced formation of the Brd4–P-TEFb complex occur. However, when the P-TEFb-dependent HEXIM1 expression markedly increases as the treatment continues, the elevated HEXIM1 levels eventually push the P-TEFb equilibrium back toward the 7SK snRNP side to accommodate an overall reduced transcriptional demand in terminally differentiated cells (He et al. 2006). Accumulating evidence indicates that 7SK snRNP represents a major reservoir of activity where P-TEFb can be recruited to activate transcription (Zhou and Yik 2006). However, the signaling pathway(s) that controls this process is mostly unknown. Of note, in the course of our studies, it was reported that the activity of PI3K/Akt is required for HMBA to release P-TEFb from 7SK snRNP (Contreras et al. 2007). However, since neither the treatment with various pharmacological activators of the PI3K/Akt pathway nor the expression of a CA form of Akt induces 7SK snRNP disruption (Supplemental Fig. S1), it is unlikely that PI3K/Akt alone is sufficient to accomplish this task. Here, through analyzing the disruption of 7SK snRNP by UV or short-term HMBA treatment, we show that both agents cause calcium ion (Ca2+) influx and activation of a Ca2+–calmodulin–PP2B (protein phosphatase 2B, also known as calcineurin) signaling pathway that is necessary although insufficient to cause the disruption. To dissociate HEXIM1 from P-TEFb, PP2B must act sequentially and cooperatively with PP1α (protein phosphatase 1α). Facilitated by a PP2B-induced conformational change in 7SK snRNP, PP1α releases P-TEFb from 7SK snRNP through dephosphorylating phospho-Thr186 located in the Cdk9 T-loop. This event is also necessary for the subsequent recruitment of P-TEFb by Brd4 to the preinitiation complex (PIC), where Cdk9 has been reported to remain unphosphorylated and inactive until after the synthesis of a short RNA transcript (Zhou et al. 2001). Together, our data are consistent with a model that PP2B and PP1α act cooperatively and in response to Ca2+ signaling to dephosphorylate Cdk9 T-loop, disrupt 7SK snRNP, and generate a pool of P-TEFb that can be recruited to the PIC.
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recruitment of p tefb for stimulation of transcriptional elongation by the bromodomain protein brd4
Molecular Cell, 2005Co-Authors: Zhiyuan Yang, Jasper H N Yik, Ruichuan Chen, M Jang, Keiko Ozato, Qiang ZhouAbstract: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.
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Phosphorylated Positive Transcription Elongation Factor b (P-TEFb) Is Tagged for Inhibition through Association with 7SK snRNA
Journal of Biological Chemistry, 2003Co-Authors: Ruichuan Chen, Zhiyuan Yang, Qiang ZhouAbstract:The positive transcription elongation factor b (P-TEFb), 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 stress-induced transcription and pathogenesis of cardiac hypertrophy. The 7SK.P-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 7SK.P-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 7SK.P-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.
