The Experts below are selected from a list of 282093 Experts worldwide ranked by ideXlab platform
Andrew P. Rice - One of the best experts on this subject based on the ideXlab platform.
-
ral ssBioMed CentRetrovirology Open AcceResearch Effects of prostratin on Cyclin T1/P-TEFb function and the gene
2016Co-Authors: Tzu-ling Sung, Andrew P. RiceAbstract:Background: The latent reservoir of human immunodeficiency virus type 1 (HIV-1) in resting CD4+ T cells is a major obstacle to the clearance of infection by highly active antiretroviral therapy (HAART). Recent studies have focused on searches for adjuvant therapies to activate this reservoir under conditions of HAART. Prostratin, a non tumor-promoting phorbol ester, is a candidate for such a strategy. Prostratin has been shown to reactivate latent HIV-1 and Tat-mediated transactivation may play an important role in this process. We examined resting CD4+ T cells from healthy donors to determine if prostratin induces Cyclin T1/P-TEFb, a cellular Kinase composed of Cyclin T1 and Cyclin-Dependent Kinase-9 (CDK9) that mediates Tat function. We also examined effects of prostratin on Cyclin T2a, an alternative regulatory subunit for CDK9, and 7SK snRNA and the HEXIM1 protein, two factors that associate with P-TEFb and repress its Kinase activity. Results: Prostratin up-regulated Cyclin T1 protein expression, modestly induced CDK9 protein expression, and did not affect Cyclin T2a protein expression. Although the Kinase activity of CDK9 in vitro was up-regulated by prostratin, we observed a large increase in the association of 7SK snRNA and the HEXIM1 protein with CDK9. Using HIV-1 reporter viruses with and without
-
Limited redundancy in genes regulated by Cyclin T2 and Cyclin T1.
BMC research notes, 2011Co-Authors: Rajesh Ramakrishnan, Andrew P. RiceAbstract:The elongation phase, like other steps of transcription by RNA Polymerase II, is subject to regulation. The positive transcription elongation factor b (P-TEFb) complex allows for the transition of mRNA synthesis to the productive elongation phase. P-TEFb contains Cdk9 (Cyclin-Dependent Kinase 9) as its catalytic subunit and is regulated by its Cyclin partners, Cyclin T1 and Cyclin T2. The HIV-1 Tat transactivator protein enhances viral gene expression by exclusively recruiting the Cdk9-Cyclin T1 P-TEFb complex to a RNA element in nascent viral transcripts called TAR. The expression patterns of Cyclin T1 and Cyclin T2 in primary monocytes and CD4+ T cells suggests that Cyclin T2 may be generally involved in expression of constitutively expressed genes in quiescent cells, while Cyclin T1 may be involved in expression of genes up-regulated during macrophage differentiation, T cell activation, and conditions of increased metabolic activity To investigate this issue, we wished to identify the sets of genes whose levels are regulated by either Cyclin T2 or Cyclin T1. We used shRNA lentiviral vectors to stably deplete either Cyclin T2 or Cyclin T1 in HeLa cells. Total RNA extracted from these cells was subjected to cDNA microarray analysis. We found that 292 genes were down- regulated by depletion of Cyclin T2 and 631 genes were down-regulated by depletion of Cyclin T1 compared to cells transduced with a control lentivirus. Expression of 100 genes was commonly reduced in either knockdown. Additionally, 111 and 287 genes were up-regulated when either Cyclin T2 or Cyclin T1 was depleted, respectively, with 45 genes in common. These results suggest that there is limited redundancy in genes regulated by Cyclin T1 or Cyclin T2.
-
limited redundancy in genes regulated by cyclin t2 and cyclin t1
BMC Research Notes, 2011Co-Authors: Rajesh Ramakrishnan, Andrew P. RiceAbstract:The elongation phase, like other steps of transcription by RNA Polymerase II, is subject to regulation. The positive transcription elongation factor b (P-TEFb) complex allows for the transition of mRNA synthesis to the productive elongation phase. P-TEFb contains Cdk9 (Cyclin-Dependent Kinase 9) as its catalytic subunit and is regulated by its Cyclin partners, Cyclin T1 and Cyclin T2. The HIV-1 Tat transactivator protein enhances viral gene expression by exclusively recruiting the Cdk9-Cyclin T1 P-TEFb complex to a RNA element in nascent viral transcripts called TAR. The expression patterns of Cyclin T1 and Cyclin T2 in primary monocytes and CD4+ T cells suggests that Cyclin T2 may be generally involved in expression of constitutively expressed genes in quiescent cells, while Cyclin T1 may be involved in expression of genes up-regulated during macrophage differentiation, T cell activation, and conditions of increased metabolic activity To investigate this issue, we wished to identify the sets of genes whose levels are regulated by either Cyclin T2 or Cyclin T1.
-
effects of prostratin on cyclin t1 p tefb function and the gene expression profile in primary resting cd4 t cells
Retrovirology, 2006Co-Authors: Tzu-ling Sung, Andrew P. RiceAbstract:Background The latent reservoir of human immunodeficiency virus type 1 (HIV-1) in resting CD4+ T cells is a major obstacle to the clearance of infection by highly active antiretroviral therapy (HAART). Recent studies have focused on searches for adjuvant therapies to activate this reservoir under conditions of HAART. Prostratin, a non tumor-promoting phorbol ester, is a candidate for such a strategy. Prostratin has been shown to reactivate latent HIV-1 and Tat-mediated transactivation may play an important role in this process. We examined resting CD4+ T cells from healthy donors to determine if prostratin induces Cyclin T1/P-TEFb, a cellular Kinase composed of Cyclin T1 and Cyclin-Dependent Kinase-9 (CDK9) that mediates Tat function. We also examined effects of prostratin on Cyclin T2a, an alternative regulatory subunit for CDK9, and 7SK snRNA and the HEXIM1 protein, two factors that associate with P-TEFb and repress its Kinase activity.
-
Induction of the HIV-1 Tat co-factor cyclin T1 during monocyte differentiation is required for the regulated expression of a large portion of cellular mRNAs
Retrovirology, 2006Co-Authors: Yan Wang, Chad A. Shaw, Xiao Feng Qin, Andrew P. RiceAbstract:P-TEFb, a general RNA polymerase II elongation factor, is composed of CDK9 (Cyclin-Dependent Kinase 9) as a catalytic unit and either cyclin T1, T2 or K as a regulatory subunit. The cyclin T1/P-TEFb complex is targeted by HIV to mediate Tat transactivation. Cyclin T1 protein expression is induced during early macrophage differentiation, suggesting a role in regulation of mRNA expression during the differentiation process. To study the functional significance of cyclin T1 induction during differentiation, we utilized the human Mono Mac 6 (MM6) monocytic cell line. We found that cyclin T1 protein expression is induced by a post-transcriptional mechanism following PMA treatment of MM6 cells, similar to its induction in primary monocytes and macrophages. Also in agreement with findings in primary cells, cyclin T2a is present at relatively high levels in MM6 cells and is not induced by PMA. Although the knock-down of cyclin T1 in MM6 cells by shRNA inhibited HIV-1 Tat transactivation, MM6 cell growth was not affected by the depletion of cyclin T1. Using DNA microarray technology, we found that more than 20% of genes induced by PMA require cyclin T1 for their normal level of induction, and approximately 15% of genes repressed by PMA require cyclin T1 for their normal level of repression. Gene ontology analysis indicates that many of these cyclin T1-dependent genes are related to immune response and signal transduction. These results suggest that cyclin T1 serves a critical role in the program of macrophage differentiation, and this raises questions about the feasibility of cyclin T1 serving as an antiviral therapeutic target.
Allan R. Brasier - One of the best experts on this subject based on the ideXlab platform.
-
1 Systematic Determination of Human Cyclin Dependent Kinase (CDK)-9 Interactome Identifies Novel Functions in RNA Splicing Mediated by the
2016Co-Authors: Chukwudi B. Edeh, John E Wiktorowicz, Andrzej Kudlicki, Allan R. BrasierAbstract:The abbreviations used are: BRD4, bromodomain containing 4; CAN508, 4-(3,5-Diamino-1H-pyrazol-4-ylazo)-phenol; CDK9, cyclin dependent Kinase 9; DDX5/17, DEAD-box RNA helicase 5/17; FDR, false discovery rate; GO, gene ontology; HEXIM, hexamethylene bis-acetamide inducible; HOA1, hydroxyacid oxidase 1; IP, immunoprecipitation;LC-MS/MS, Liquid chromatography- tandem mass spectrometry; NF-B, nuclear factor kappa-light-chain-enhancer of activated B cells; Poly (I:C), polyinosinic:polycytidylic acid; P-TEFb, the positive transcription elongation factor complex; PPI, protein-protein interactions; Q-gPCR, quantitative genomic real time PCR; Q-RT-PCR, quantitative reverse transcription real time PCR; RFESD, Rieske (Fe-S) domain containing; ROS, reactive oxygen species; RP, ribosomal protein; SAEC, small airway epithelial cells; shRNA, small or short hairpin RNA; SOD3, extracellular superoxide dismutase 3; SRM, selected reaction monitoring; STAT1, signal transducer and activator of transcription 1; XChIP, two-step cross-linking chromatin immunoprecipitation.
-
systematic determination of human cyclin dependent Kinase cdk 9 interactome identifies novel functions in rna splicing mediated by the dead box ddx 5 17 rna helicases
Molecular & Cellular Proteomics, 2015Co-Authors: Jun Yang, Bing Tian, Mohammad Jamaluddin, Chukwudi B. Edeh, John E Wiktorowicz, Andrzej Kudlicki, Yingxin Zhao, Mridul Kalita, Allan R. BrasierAbstract:Inducible transcriptional elongation is a rapid, stereotypic mechanism for activating immediate early immune defense genes by the epithelium in response to viral pathogens. Here, the recruitment of a multifunctional complex containing the cyclin dependent Kinase 9 (CDK9) triggers the process of transcriptional elongation activating resting RNA polymerase engaged with innate immune response (IIR) genes. To identify additional functional activity of the CDK9 complex, we conducted immunoprecipitation (IP) enrichment-stable isotope labeling LC-MS/MS of the CDK9 complex in unstimulated cells and from cells activated by a synthetic dsRNA, polyinosinic/polycytidylic acid [poly (I:C)]. 245 CDK9 interacting proteins were identified with high confidence in the basal state and 20 proteins in four functional classes were validated by IP-SRM-MS. These data identified that CDK9 interacts with DDX 5/17, a family of ATP-dependent RNA helicases, important in alternative RNA splicing of NFAT5, and mH2A1 mRNA two proteins controlling redox signaling. A direct comparison of the basal versus activated state was performed using stable isotope labeling and validated by IP-SRM-MS. Recruited into the CDK9 interactome in response to poly(I:C) stimulation are HSPB1, DNA dependent Kinases, and cytoskeletal myosin proteins that exchange with 60S ribosomal structural proteins. An integrated human CDK9 interactome map was developed containing all known human CDK9- interacting proteins. These data were used to develop a probabilistic global map of CDK9-dependent target genes that predicted two functional states controlling distinct cellular functions, one important in immune and stress responses. The CDK9-DDX5/17 complex was shown to be functionally important by shRNA-mediated knockdown, where differential accumulation of alternatively spliced NFAT5 and mH2A1 transcripts and alterations in downstream redox signaling were seen. The requirement of CDK9 for DDX5 recruitment to NFAT5 and mH2A1 chromatin target was further demonstrated using chromatin immunoprecipitation (ChIP). These data indicate that CDK9 is a dynamic multifunctional enzyme complex mediating not only transcriptional elongation, but also alternative RNA splicing and potentially translational control.
-
RelA Ser276 Phosphorylation Is Required for Activation of a Subset of NF-κB-Dependent Genes by Recruiting Cyclin-Dependent Kinase 9/Cyclin T1 Complexes
Molecular and cellular biology, 2008Co-Authors: David E. Nowak, Bing Tian, Mohammad Jamaluddin, Istvan Boldogh, Leoncio A. Vergara, Sanjeev Choudhary, Allan R. BrasierAbstract:NF-κB 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-κB/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-κB-dependent genes, serine 276 (Ser276)-phosphorylated RelA binding is seen primarily on a subset of genes that are rapidly induced by tumor necrosis factor (TNF), including Gro-β, interleukin-8 (IL-8), and IκBα. Previous work has shown that TNF-inducible RelA Ser276 phosphorylation is controlled by a reactive oxygen species (ROS)-protein Kinase A signaling pathway. To further understand the role of phospho-Ser276 RelA in target gene expression, we inhibited its formation by ROS scavengers and antioxidants, treatments that disrupt phospho-Ser276 formation but not the translocation and DNA binding of nonphosphorylated RelA. Here we find that phospho-Ser276 RelA is required only for activation of IL-8 and Gro-β, with IκBα being unaffected. These data were confirmed in experiments using RelA−/− murine embryonic fibroblasts reconstituted with a RelA Ser276Ala mutation. In addition, we observe that phospho-Ser276 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-Ser276 RelA-P-TEFb complex is required for IL-8 and Gro-β gene activation but not for IκBα gene activation. These studies indicate that TNF induces target gene expression by heterogeneous mechanisms. One is mediated by phospho-Ser276 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-β genes. The second involves a phospho-Ser276 RelA-independent activation of genes preloaded with Pol II, exemplified by the IκBα 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.
Robert P Fisher - One of the best experts on this subject based on the ideXlab platform.
-
a cdk9 pp1 switch regulates the elongation termination transition of rna polymerase ii
Nature, 2018Co-Authors: Pabitra K Parua, Gregory T Booth, Miriam Sanso, Bradley Benjamin, Jason C Tanny, John T Lis, Robert P FisherAbstract:The end of the RNA polymerase II (Pol II) transcription cycle is strictly regulated to prevent interference between neighbouring genes and to safeguard transcriptome integrity 1 . The accumulation of Pol II downstream of the cleavage and polyadenylation signal can facilitate the recruitment of factors involved in mRNA 3′-end formation and termination 2 , but how this sequence is initiated remains unclear. In a chemical–genetic screen, human protein phosphatase 1 (PP1) isoforms were identified as substrates of positive transcription elongation factor b (P-TEFb), also known as the Cyclin-Dependent Kinase 9 (Cdk9)–cyclin T1 (CycT1) complex 3 . Here we show that Cdk9 and PP1 govern phosphorylation of the conserved elongation factor Spt5 in the fission yeast Schizosaccharomyces pombe. Cdk9 phosphorylates both Spt5 and a negative regulatory site on the PP1 isoform Dis2 4 . Sites targeted by Cdk9 in the Spt5 carboxy-terminal domain can be dephosphorylated by Dis2 in vitro, and dis2 mutations retard Spt5 dephosphorylation after inhibition of Cdk9 in vivo. Chromatin immunoprecipitation and sequencing analysis indicates that Spt5 is dephosphorylated as transcription complexes traverse the cleavage and polyadenylation signal, concomitant with the accumulation of Pol II phosphorylated at residue Ser2 of the carboxy-terminal domain consensus heptad repeat 5 . A conditionally lethal Dis2-inactivating mutation attenuates the drop in Spt5 phosphorylation on chromatin, promotes transcription beyond the normal termination zone (as detected by precision run-on transcription and sequencing 6 ) and is genetically suppressed by the ablation of Cdk9 target sites in Spt5. These results suggest that the transition of Pol II from elongation to termination coincides with a Dis2-dependent reversal of Cdk9 signalling—a switch that is analogous to a Cdk1–PP1 circuit that controls mitotic progression 4 . The Kinase Cdk9 and the phosphatase Dis2 regulate the termination of transcription in fission yeast in part by controlling the phosphorylation state of the elongation factor Spt5.
-
a cdk9 pp1 Kinase phosphatase switch regulates the elongation termination transition of rna polymerase ii
bioRxiv, 2017Co-Authors: Pabitra K Parua, Gregory T Booth, Miriam Sanso, Bradley Benjamin, Jason C Tanny, John T Lis, Robert P FisherAbstract:The end of the RNA polymerase II (Pol II) transcription cycle is strictly regulated to ensure proper mRNA maturation and prevent interference between neighboring genes 1 . Pol II slowing downstream of the cleavage and polyadenylation signal (CPS) leads to recruitment of cleavage and polyadenylation factors and termination 2 , but how this chain of events is initiated remains unclear. In a chemical-genetic screen, we identified protein phosphatase 1 (PP1) isoforms as substrates of human positive transcription elongation factor b (P-TEFb), the Cyclin-Dependent Kinase 9 (Cdk9)-cyclin T1 complex 3 . Here we show that Cdk9 and PP1 govern phosphorylation of the conserved transcription factor Spt5 in the fission yeast Schizosaccharomyces pombe. Cdk9 phosphorylates both Spt5 and a negative regulatory site on the PP1 isoform Dis2 4 . Sites phosphorylated by Cdk9 in the Spt5 carboxy-terminal domain (CTD) are dephosphorylated by Dis2 in vitro , and Cdk9 inhibition in vivo leads to rapid Spt5 dephosphorylation that is retarded by concurrent Dis2 inactivation. Chromatin immunoprecipitation and sequencing (ChIP-seq) analysis indicates that Spt5 is dephosphorylated as transcription complexes traverse the CPS, prior to or concomitant with slowing of Pol II 5 . A Dis2-inactivating mutation stabilizes Spt5 phosphorylation (pSpt5) on chromatin, promotes transcription beyond the normal termination zone detected by precision run-on transcription and sequencing (PRO-seq) 6 , and is suppressed by ablation of Cdk9 target sites in Spt5. These results support a model whereby the transition of Pol II from elongation to termination is regulated by opposing activities of Cdk9 and Dis2 towards their common substrate Spt5−a bistable switch analogous to a Cdk1-PP1 module that controls mitotic progression 4 .
-
a cdk9 pp1 switch regulates the elongation termination transition of rna polymerase ii
bioRxiv, 2017Co-Authors: Pabitra K Parua, Gregory T Booth, Miriam Sanso, Bradley Benjamin, Jason C Tanny, John T Lis, Robert P FisherAbstract:The end of the RNA polymerase II (Pol II) transcription cycle is strictly regulated to ensure proper mRNA maturation and prevent interference between neighboring genes. Pol II slowing downstream of the cleavage and polyadenylation signal (CPS) leads to recruitment of cleavage and polyadenylation factors and termination, but how this chain of events is initiated remains unclear. In a chemical-genetic screen, we identified protein phosphatase 1 (PP1) isoforms as substrates of human positive transcription elongation factor b (P-TEFb), the Cyclin-Dependent Kinase 9 (Cdk9)-cyclin T1 complex. Here we show that Cdk9 and PP1 govern phosphorylation of the conserved transcription factor Spt5 in the fission yeast Schizosaccharomyces pombe. Cdk9 phosphorylates both Spt5 and a negative regulatory site on the PP1 isoform Dis2. Sites phosphorylated by Cdk9 in the Spt5 carboxy-terminal domain (CTD) are dephosphorylated by Dis2 in vitro, and Cdk9 inhibition in vivo leads to rapid Spt5 dephosphorylation that is retarded by concurrent Dis2 inactivation. Chromatin immunoprecipitation and sequencing (ChIP-seq) analysis indicates that Spt5 is dephosphorylated as transcription complexes traverse the CPS, prior to or concomitant with slowing of Pol II. A Dis2-inactivating mutation stabilizes Spt5 phosphorylation (pSpt5) on chromatin, promotes transcription beyond the normal termination zone detected by precision run-on transcription and sequencing (PRO-seq), and is suppressed by ablation of Cdk9 target sites in Spt5. These results support a model whereby the transition of Pol II from elongation to termination is regulated by opposing activities of Cdk9 and Dis2 towards their common substrate Spt5−a bistable switch analogous to a Cdk1-PP1 module that controls exit from mitosis.
-
cyclin dependent Kinase 9 cdk9 of fission yeast is activated by the cdk activating Kinase csk1 overlaps functionally with the tfiih associated Kinase mcs6 and associates with the mrna cap methyltransferase pcm1 in vivo
Molecular and Cellular Biology, 2006Co-Authors: Hongyan Du, Stewart Shuman, Juliet Singer, Courtney V St Amour, Selena Granitto, Robert P FisherAbstract:Cyclin-Dependent Kinases (CDKs) first emerged as controllers of cell division but have also been implicated in processes not strictly coupled to the cell cycle, most notably transcription by RNA polymerase II (Pol II) (4). In metazoans, Cdk9 and cyclin T constitute positive transcription elongation factor b (P-TEFb), which phosphorylates both the carboxyl-terminal domain (CTD) of Rpb1, the largest subunit of Pol II, and Spt5, a subunit of the elongation factor DRB sensitivity-inducing factor, to overcome kinetic blocks to elongation (21). The requirement for Cdk9 in facilitating elongation is probably a general one (66) and is posited to be a quality control mechanism to ensure that nascent transcripts are not elongated unless and until mRNA-capping enzymes and other processing machinery can be recruited (9, 10, 49, 52). In the budding yeast Saccharomyces cerevisiae, the Bur1/Bur2 CDK-cyclin pair and a heterotrimeric CDK, CTDK-I, show roughly equal homology between their catalytic subunits (Bur1 and Ctk1, respectively) and metazoan Cdk9 (46). The BUR1 gene is essential (56), whereas neither BUR2 nor any of the genes encoding CTDK-I subunits is required for viability (68, 77). Cdk9 of the fission yeast Schizosaccharomyces pombe was identified in a two-hybrid interaction screen with Pct1, the RNA triphosphatase component of the mRNA-capping apparatus (52). Cdk9 can form an active Kinase complex with the essential fission yeast cyclin Pch1, and the two proteins expressed together in S. cerevisiae complemented a bur1 deletion (19, 52), but the physiologic cyclin partner of Cdk9 in S. pombe remained to be identified. CDKs depend to various degrees on phosphorylation within the activation segment, or T loop, of the catalytic subunit by a CDK-activating Kinase (CAK). The dedicated cell cycle CDKs, exemplified by Cdk1, absolutely require T-loop phosphorylation (22, 37), whereas CDKs involved in transcription need this modification for full catalytic activity and/or for stability but not for their essential functions (29, 31, 78). The CAKs fall into two classes. In metazoans, the major CAK is a heterotrimeric complex of Cdk7, cyclin H, and the RING finger protein Mat1, which is also part of general transcription factor IIH (TFIIH), which phosphorylates the Pol II CTD (23). In contrast, the sole CAK in budding yeast is Cak1, a monomeric enzyme related only distantly to the CDK family (27). Cak1 activates both Cdk1 and the Cdk7 ortholog Kin28 (17, 29), which unlike its metazoan counterpart is a dedicated TFIIH-associated CTD Kinase that does not activate CDKs (12, 73). S. pombe has one CAK from each class: the Mcs6/Mcs2/Pmh1 complex, which is homologous to Cdk7/cyclin H/Mat1, and Csk1, a single-subunit enzyme most closely related to Cak1 (2, 5, 14, 24, 33, 34, 67). Both enzymes can activate Cdk1 (34, 63), a redundancy that probably explains why csk1+ is dispensable for viability (43) and why mutations in genes encoding Mcs6 complex subunits do not impair CDK activation or impede entry into mitosis unless combined with other mutations, such as csk1Δ (5, 14, 24, 33, 34, 43, 63). The Mcs6 complex is required for viability, however, suggesting it has another essential target, which is likely to be Pol II. The growth defects caused by csk1 deletion (3, 25, 63) might reflect specialized requirements for Csk1-mediated activation of Cdk1 and/or activity of Csk1 towards proteins that the Mcs6 complex does not phosphorylate. Activation of metazoan P-TEFb by a CAK has not been demonstrated, but mutating Thr-186 in the human Cdk9 T loop abolished activity and, paradoxically, binding to a ribonucleoprotein inhibitor (7). In budding yeast, a temperature-sensitive bur1 mutation was suppressed by overexpression of CAK1, and phosphorylation by Cak1 stimulated the Kinase activity of Bur1 in vitro, dependent on the Thr-240 residue of the Bur1 T loop. That stimulation is apparently important in vivo; a bur1T240A allele only partially complemented bur1Δ (78). More recently, Cak1 was shown to activate Ctk1, and a mutation in ctk1 preventing T-loop phosphorylation caused a defect in the entry into stationary phase (50). Changing Thr-212 within the T loop of fission yeast Cdk9 to alanine abolished heterologous complementation of bur1Δ, whereas a mutation of the same residue to glutamic acid rendered it cold and temperature sensitive (52, 53). The regulation of Cdk9 by upstream Kinases (CAKs) in fission yeast has not been investigated. Moreover, its role(s) in regulating gene expression and possibly coordinating mRNA-processing events with transcription remains to be elucidated. Here we show specificity within the CAK-CDK network of S. pombe; Csk1, but not the Mcs6 complex, activates Cdk9/Pch1 complexes in vitro and in vivo. The nonphosphorylatable T-loop mutant cdk9T212A grows poorly on minimal media and is cold sensitive, essentially phenocopying csk1Δ. We observe a synthetic interaction between cdk9T212A and the analogous mcs6S165A T-loop mutation, suggesting that the essential Cdk9 and Mcs6 complexes, which have partially overlapping substrate specificities in vitro, have redundant as well as unique functions in controlling gene expression in vivo. Finally, we provide support for the idea that Cdk9 couples transcription to mRNA capping (9, 10, 49, 52) by demonstrating (i) that the carboxyl-terminal, Pct1-interacting region of Cdk9 (52) is required for viability and (ii) that Cdk9 stably associates in vivo with the guanine-N7 methyltransferase component of the fission yeast mRNA-capping apparatus in ∼500-kDa complexes that are released from larger complexes by RNase digestion.
Debashis Mitra - One of the best experts on this subject based on the ideXlab platform.
-
Cyclin K Inhibits HIV-1 Gene Expression and Replication by Interfering with Cyclin-Dependent Kinase 9 (CDK9)-Cyclin T1 Interaction in Nef-dependent Manner
The Journal of biological chemistry, 2011Co-Authors: Sohrab Zafar Khan, Debashis MitraAbstract:Human immunodeficiency virus-1 (HIV-1) exploits a number of host cellular factors for successful survival and propagation. The viral protein Nef plays an important role in HIV-1 pathogenesis by interacting with various cellular proteins. In the present work, we identified Cyclin K (CycK) as a novel Nef-interacting protein, and for the first time, we showed that CycK inhibits HIV-1 gene expression and replication in a Nef-dependent manner. The positive elongation factor b complex comprising Cyclin-Dependent Kinase 9 (CDK9) and Cyclin T1 is a critical cellular complex required for viral gene expression and replication. Enhanced expression of CycK in the presence of Nef induced CycK-CDK9 binding, which prevented CDK9-Cyclin T1 complex formation and nuclear translocation of CDK9, resulting in inhibition of HIV-1 long terminal repeat-driven gene expression. Furthermore, this effect of CycK was not observed with Nef-deleted virus, indicating the importance of Nef in this phenomenon. Finally, silencing of CycK in HIV-1-infected cells resulted in increased translocation of CDK9 into the nucleus, leading to increased viral gene expression and replication. These data also suggest that endogenous CycK might act as an inhibitory factor for HIV-1 gene expression and replication in T-cells. Thus, our results clearly demonstrate that CycK utilizes HIV-1 Nef protein to displace CycT1 from the positive elongation factor b complex, resulting in inhibition of HIV-1 gene expression and replication.
-
heat shock protein 40 is necessary for human immunodeficiency virus 1 nef mediated enhancement of viral gene expression and replication
Journal of Biological Chemistry, 2005Co-Authors: Manish Kumar, Debashis MitraAbstract:The human immunodeficiency virus-1 (HIV-1) Nef protein, originally identified as a negative factor, has now emerged as one of the most important viral proteins necessary for viral pathogenesis and disease progression. Nef has been also implicated in viral infectivity and replication, however, the molecular mechanism of Nef-induced viral gene expression and replication is not clearly understood. Although involvement of heat shock proteins in viral pathogenesis has been reported earlier, a clear understanding of their role remains to be elucidated. Here we report for the first time that Nef not only interacts with heat shock protein 40 (Hsp40) but it also induces the expression of Hsp40 in HIV-1-infected cells. The interaction between Nef and Hsp40 is important for increased Hsp40 translocation into the nucleus of infected cells, which seems to facilitate viral gene expression by becoming part of the Cyclin-Dependent Kinase 9-associated transcription complex regulating long terminal repeat-mediated gene expression. The finding is consistent with the failure of the nef-deleted virus to induce Hsp40, resulting in reduced virus production. Our data further shows that, whereas, Hsp40 overexpression induces viral gene expression, silencing of Hsp40 reduces the gene expression in a Nef-dependent manner. Thus our results clearly indicate that Hsp40 is crucial for Nef-mediated enhancement of viral gene expression and replication.
Qiang Zhou - One of the best experts on this subject based on the ideXlab platform.
-
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.
-
Tat competes with HEXIM1 to increase the active pool of P-TEFb for HIV-1 transcription
Nucleic Acids Research, 2007Co-Authors: Matjaz Barboric, Zhiyuan Yang, Ruichuan Chen, B. Matija Peterlin, Matthias Geyer, Jasper H. N. Yik, Nadine Czudnochowski, Xavier Contreras, Qiang ZhouAbstract:Human immunodeficiency virus type 1 (HIV-1) transcriptional transactivator (Tat) recruits the positive transcription elongation factor b (P-TEFb) to the viral promoter. Consisting of cyclin dependent Kinase 9 (Cdk9) and cyclin T1, P-TEFb phosphorylates RNA polymerase II and the negative transcription elongation factor to stimulate the elongation of HIV-1 genes. A major fraction of nuclear P-TEFb is sequestered into a transcriptionally inactive 7SK small nuclear ribonucleoprotein (snRNP) by the coordinated actions of the 7SK small nuclear RNA (snRNA) and hexamethylene bisacetamide (HMBA) induced protein 1 (HEXIM1). In this study, we demonstrate that Tat prevents the formation of and also releases P-TEFb from the 7SK snRNP in vitro and in vivo. This ability of Tat depends on the integrity of its N-terminal activation domain and stems from the high affinity interaction between Tat and cyclin T1, which allows Tat to directly displace HEXIM1 from cyclin T1. Furthermore, we find that in contrast to the Tat-independent activation of the HIV-1 promoter, Tat-dependent HIV-1 transcription is largely insensitive to the inhibition by HEXIM1. Finally, primary blood lymphocytes display a reduced amount of the endogenous 7SK snRNP upon HIV-1 infection. All these data are consistent with the model that Tat not only recruits but also increases the active pool of P-TEFb for efficient HIV-1 transcription.
-
compensatory contributions of hexim1 and hexim2 in maintaining the balance of active and inactive positive transcription elongation factor b complexes for control of transcription
Journal of Biological Chemistry, 2005Co-Authors: Jasper H. N. Yik, Ruichuan Chen, Andrea C Pezda, Qiang ZhouAbstract:Human positive transcriptional elongation factor b (P-TEFb), consisting of a Cyclin-Dependent Kinase 9-cyclin T heterodimer, stimulates general and disease-specific transcriptional elongation by phosphorylating RNA polymerase II. The HEXIM1 protein, aided by the 7SK snRNA, sequesters P-TEFb into an inactive 7SK.HEXIM1.P-TEFb small nuclear ribonucleic acid particle for inhibition of transcription and, consequently, cell proliferation. Here we show that, like HEXIM1, a highly homologous protein named HEXIM2 also possesses the ability to inactivate P-TEFb to suppress transcription through a 7SK-mediated interaction with P-TEFb. Furthermore, HEXIM1 and HEXIM2 can form stable homo- and hetero-oligomers (most likely dimers), which may nucleate the formation of the 7SK small nuclear ribonucleic acid particle. Despite their similar functions, HEXIM1 and HEXIM2 exhibit distinct expression patterns in various human tissues and established cell lines. In HEXIM1-knocked down cells, HEXIM2 can functionally and quantitatively compensate for the loss of HEXIM1 to maintain a constant level of the 7SK/HEXIM-bound P-TEFb. Our results demonstrate that there is a tightly regulated cellular process to maintain the balance between active and inactive P-TEFb complexes, which controls global transcription as well as cell growth and differentiation.
