HIV-1 Tat assembles a multifunctional transcription elongation complex and stably associates with the 7SK snRNP.
ABSTRACT: HIV-1 transactivator Tat has greatly contributed to our understanding of transcription elongation by RNAPII. We purified HIV-1 Tat-associated factors from HeLa nuclear extract and show that Tat forms two distinct and stable complexes. Tatcom1 consists of the core active P-TEFb, MLL-fusion partners involved in leukemia (AF9, AFF4, AFF1, ENL, and ELL), and PAF1 complex. Importantly, Tatcom1 formation relies on P-TEFb while optimal CDK9 CTD-kinase activity is AF9 dependent. MLL-fusion partners and PAF1 are required for Tat transactivation. Tatcom2 is composed of CDK9, CycT1, and 7SK snRNP lacking HEXIM. Tat remodels 7SK snRNP by interacting directly with 7SK RNA, leading to the formation of a stress-resistant 7SK snRNP particle. Besides the identification of factors required for Tat transactivation and important for P-TEFb function, our data show a coordinated control of RNAPII elongation by different classes of transcription elongation factors associated in a single complex and acting at the same promoter.
Project description: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.
Project description:The HIV-1 Tat protein hijacks P-TEFb kinase to activate paused RNA polymerase II (RNAP II) at the viral promoter. Tat binds additional host factors, but it is unclear how they regulate RNAP II elongation. Here, we identify the cytoplasmic ubiquitin ligase UBE2O as critical for Tat transcriptional activity. Tat hijacks UBE2O to ubiquitinate the P-TEFb kinase inhibitor HEXIM1 of the 7SK snRNP, a fraction of which also resides in the cytoplasm bound to P-TEFb. HEXIM1 ubiquitination sequesters it in the cytoplasm and releases P-TEFb from the inhibitory 7SK complex. Free P-TEFb then becomes enriched in chromatin, a process that is also stimulated by treating cells with a CDK9 inhibitor. Finally, we demonstrate that UBE2O is critical for P-TEFb recruitment to the HIV-1 promoter. Together, the data support a unique model of elongation control where non-degradative ubiquitination of nuclear and cytoplasmic 7SK snRNP pools increases P-TEFb levels for transcriptional activation.
Project description:HIV-1 Tat activates RNA Polymerase II (RNAP II) elongation of the integrated provirus by recruiting a protein kinase known as P-TEFb to TAR RNA at the 5' end of nascent viral transcripts. The catalytic core of P-TEFb contains CDK9 and Cyclin T1 (CCNT1). A human endogenous complexome has recently been described - the set of multi-protein complexes in HeLa cell nuclei. We mined this complexome data set and identified 12 distinct multi-protein complexes that contain both CDK9 and CCNT1. We have termed these complexes CCAPs for CDK9/CCNT1-associated protein complexes. Nine CCAPs are novel, while three were previously identified as Core P-TEFb, the 7SK snRNP, and the Super-Elongation Complex. We have investigated the role of five newly identified CCAPs in Tat function and viral gene expression.We examined five CCAPs that contain: 1) PPP1R10/TOX3/WDR82; 2) TTF2; 3) TPR; 4) WRNIP1; 5) FBXO11/CUL1/SKP1. SiRNA depletions of protein subunits of the five CCAPs enhanced Tat activation of an integrated HIV-1 LTR-Luciferase reporter in TZM-bl cells. Using plasmid transfection assays in HeLa cells, we also found that siRNA depletions of TTF2, FBXO11, PPP1R10, WDR82, and TOX3 enhanced Tat activation of an HIV-1 LTR-luciferase reporter, but the depletions did not enhance expression of an NF-?B reporter plasmid with the exception of PPP1R10. We found no evidence that depletion of CCAPs perturbed the level of CDK9/CCNT1 in the 7SK snRNP. We also found that the combination of siRNA depletions of both TTF2 and FBXO11 sensitized a latent provirus in Jurkat cells to reactivation by sub-optimal amounts of ?CD3/CD28 antibodies.Our results identified five novel CDK9/CCNT1 complexes that are capable of negative regulation of HIV-1 Tat function and viral gene expression. Because siRNA depletions of CCAPs enhance Tat function, it is possible that these complexes reduce the level of CDK9 and CCNT1 available for Tat, similar to the negative regulation of Tat by the 7SK snRNP. Our results highlight the complexity in the biological functions of CDK9 and CCNT1.
Project description:The HIV transactivator protein, Tat, enhances HIV transcription by recruiting P-TEFb from the inactive 7SK snRNP complex and directing it to proviral elongation complexes. To test the hypothesis that T-cell receptor (TCR) signaling induces critical post-translational modifications leading to enhanced interactions between P-TEFb and Tat, we employed affinity purification-tandem mass spectrometry to analyze P-TEFb. TCR or phorbal ester (PMA) signaling strongly induced phosphorylation of the CDK9 kinase at Ser175. Molecular modeling studies based on the Tat/P-TEFb X-ray structure suggested that pSer175 strengthens the intermolecular interactions between CDK9 and Tat. Mutations in Ser175 confirm that this residue could mediate critical interactions with Tat and with the bromodomain protein BRD4. The S175A mutation reduced CDK9 interactions with Tat by an average of 1.7-fold, but also completely blocked CDK9 association with BRD4. The phosphomimetic S175D mutation modestly enhanced Tat association with CDK9 while causing a 2-fold disruption in BRD4 association with CDK9. Since BRD4 is unable to compete for binding to CDK9 carrying S175A, expression of CDK9 carrying the S175A mutation in latently infected cells resulted in a robust Tat-dependent reactivation of the provirus. Similarly, the stable knockdown of BRD4 led to a strong enhancement of proviral expression. Immunoprecipitation experiments show that CDK9 phosphorylated at Ser175 is excluded from the 7SK RNP complex. Immunofluorescence and flow cytometry studies carried out using a phospho-Ser175-specific antibody demonstrated that Ser175 phosphorylation occurs during TCR activation of primary resting memory CD4+ T cells together with upregulation of the Cyclin T1 regulatory subunit of P-TEFb, and Thr186 phosphorylation of CDK9. We conclude that the phosphorylation of CDK9 at Ser175 plays a critical role in altering the competitive binding of Tat and BRD4 to P-TEFb and provides an informative molecular marker for the identification of the transcriptionally active form of P-TEFb.
Project description:Eukaryotic gene expression is commonly controlled at the level of RNA polymerase II (RNAPII) pausing subsequent to transcription initiation. Transcription elongation is stimulated by the positive transcription elongation factor b (P-TEFb) kinase, which is suppressed within the 7SK small nuclear ribonucleoprotein (7SK snRNP). However, the biogenesis and functional significance of 7SK snRNP remain poorly understood. Here, we report that LARP7, BCDIN3, and the noncoding 7SK small nuclear RNA (7SK) are vital for the formation and stability of a cell stress-resistant core 7SK snRNP. Our functional studies demonstrate that 7SK snRNP is not only critical for controlling transcription elongation, but also for regulating alternative splicing of pre-mRNAs. Using a transient expression splicing assay, we find that 7SK snRNP disintegration promotes inclusion of an alternative exon via the increased occupancy of P-TEFb, Ser2-phosphorylated (Ser2-P) RNAPII, and the splicing factor SF2/ASF at the minigene. Importantly, knockdown of larp7 or bcdin3 orthologues in zebrafish embryos destabilizes 7SK and causes severe developmental defects and aberrant splicing of analyzed transcripts. These findings reveal a key role for P-TEFb in coupling transcription elongation with alternative splicing, and suggest that maintaining core 7SK snRNP is essential for vertebrate development.
Project description:Efficient HIV transcription requires P-TEFb, an essential co-factor for Tat. In actively replicating cells, P-TEFb is incorporated into the 7SK snRNP complex together with the repressor protein HEXIM1. Using an affinity purification-tandem mass spectrometry approach to identify modification sites on HEXIM1 that regulate the sequestration of P-TEFb by 7SK snRNP, we found that HEXIM1 can be phosphorylated on adjacent residues in a region immediately upstream of the coiled-coil dimerization domain (Ser268, Thr270, Tyr271, and Tyr274). Phosphomimetic mutations of Tyr271 and Tyr274 disrupted the assembly of P-TEFb and HEXIM1 into the 7SK snRNP complex. Although Y271E/Y274E did not adversely affect the nuclear localization pattern of HEXIM1, it induced the redistribution of the CDK9 subunit of P-TEFb into the cytoplasm. By contrast, the Y271F/Y274F HEXIM1 mutant assembled normally with P-TEFb within the 7SK snRNP complex but severely reduced proviral gene expression in T cells in response to activation signals and caused a severe growth defect of Jurkat T cells. Thus, Y271F/Y274F, which cannot be phosphorylated on these residues, appears to block the exchange of active P-TEFb from the 7SK complex, thereby limiting the level of P-TEFb below the threshold required to support transcription elongation of the HIV provirus and cellular genes.
Project description: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.
Project description:The transition from transcription initiation into elongation is controlled by transcription factors, which recruit positive transcription elongation factor b (P-TEFb) to promoters to phosphorylate RNA polymerase II. A fraction of P-TEFb is recruited as part of the inhibitory 7SK small nuclear ribonucleoprotein particle (snRNP), which inactivates the kinase and prevents elongation. However, it is unclear how P-TEFb is captured from the promoter-bound 7SK snRNP to activate elongation. Here, we describe a mechanism by which transcription factors mediate the enzymatic release of P-TEFb from the 7SK snRNP at promoters to trigger activation in a gene-specific manner. We demonstrate that Tat recruits PPM1G/PP2C? to locally disassemble P-TEFb from the 7SK snRNP at the HIV promoter via dephosphorylation of the kinase T loop. Similar to Tat, nuclear factor (NF)-?B recruits PPM1G in a stimulus-dependent manner to activate elongation at inflammatory-responsive genes. Recruitment of PPM1G to promoter-assembled 7SK snRNP provides a paradigm for rapid gene activation through transcriptional pause release.
Project description:The 7SK small nuclear RNP (snRNP), composed of the 7SK small nuclear RNA (snRNA), MePCE, and Larp7, regulates the mRNA elongation capacity of RNA polymerase II (RNAPII) through controlling the nuclear activity of positive transcription elongation factor b (P-TEFb). Here, we demonstrate that the human 7SK snRNP also functions as a canonical transcription factor that, in collaboration with the little elongation complex (LEC) comprising ELL, Ice1, Ice2, and ZC3H8, promotes transcription of RNAPII-specific spliceosomal snRNA and small nucleolar RNA (snoRNA) genes. The 7SK snRNA specifically associates with a fraction of RNAPII hyperphosphorylated at Ser5 and Ser7, which is a hallmark of RNAPII engaged in snRNA synthesis. Chromatin immunoprecipitation (ChIP) and chromatin isolation by RNA purification (ChIRP) experiments revealed enrichments for all components of the 7SK snRNP on RNAPII-specific sn/snoRNA genes. Depletion of 7SK snRNA or Larp7 disrupts LEC integrity, inhibits RNAPII recruitment to RNAPII-specific sn/snoRNA genes, and reduces nascent snRNA and snoRNA synthesis. Thus, through controlling both mRNA elongation and sn/snoRNA synthesis, the 7SK snRNP is a key regulator of nuclear RNA production by RNAPII.
Project description:The 7SK snRNA sequesters P-TEFb, a general transcription elongation factor and human co-factor for HIV-1 Tat protein, into the catalytically inactive 7SK snRNP Little is known about how 7SK RNA is regulated to perform this function. Here, we show that most of 7SK is pseudouridylated at position U250 by the predominant cellular pseudouridine synthase machinery, the DKC1-box H/ACA RNP Pseudouridylation is critical to stabilize 7SK snRNP, as its abolishment by either mutation at or around U250 or depletion of DKC1, the catalytic component of the box H/ACA RNP, disrupts 7SK snRNP and releases P-TEFb to form the super elongation complex (SEC) and the Brd4-P-TEFb complex. The SEC is then recruited by Tat to the HIV-1 promoter to stimulate viral transcription and escape from latency. Thus, although 7SK RNA levels remain mostly unchanged, its function is modulated by pseudouridylation, which in turn controls transcription of both HIV-1 and cellular genes.