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PubMed2001

Control of RNA polymerase II activity by dedicated CTD kinases and phosphatases.

The elongation phase of eukaryotic transcription by RNA polymerase II (RNAPII) is an important target for regulation of gene expression. An interplay of positive and negative elongation factors determines the elongation activity of RNAPII in different promoters. The phosphorylation status of the carboxyl-terminal-domain (CTD) of the larger subunit of RNAPII appears to be the regulatory focus of different factors regulating mRNA processivity. The emerging model of the transcription cycle proposes that the phosphorylation state of the CTD is dynamic during elongation with different forms predominating at different stages of transcription. Shortly after initiation RNA polymerase II comes under the control of negative elongation factors and enters abortive elongation. Escape from the action of these negative controls requires the action of at least one positive elongation factor identified in the P-TEFb complex composed of the Cyclin-Dependent Kinase CDK9 and its regulatory subunit cyclin T. Finally, the requirement of CTD phosphatase activity, identified in the FCP1 protein, has been invoked as necessary to recycle the hypophosphorylated form of the RNA polymerase II competent to reinitiate the transcription cycle.

Animals↗
PubMed2003

Structure-function analysis of human Spt4: evidence that hSpt4 and hSpt5 exert their roles in transcriptional elongation as parts of the DSIF complex.

BACKGROUND: The human Spt4/Spt5 complex, termed DRB-sensitivity inducing factor (DSIF) is a dual regulator of transcription that stimulates, or, when cooperating with negative elongation factor (NELF), represses RNA polymerase II (RNAPII) elongation. Spt4 and Spt5 are also thought to be involved in mRNA capping, homologous DNA recombination, and transcription-coupled DNA repair. As a first step to understanding how these proteins regulate diverse cellular processes, we investigated the structure and function of hSpt4 in vitro. RESULTS: Immunodepletion of hSpt5 from HeLa nuclear extracts resulted in the efficient co-depletion of hSpt4. Using DSIF-depleted nuclear extracts and a series of Spt4 mutants, we examined the amino acid sequence of hSpt4 which was important for hSpt5 binding and for transcriptional repression and activation by DSIF. Unexpectedly, the zinc finger of hSpt4, which is critical for the yeast counterpart to function in vivo, was dispensable for hSpt5 binding and for transcriptional regulation in vitro. CONCLUSION: These and other results suggest: (i) that the central region of hSpt4 is necessary and sufficient for its function in vitro and (ii) that there is no free hSpt4 or hSpt5 in cells. We propose that hSpt4 and hSpt5 exert their roles in transcriptional regulation, and possibly in other nuclear processes, as parts of the DSIF complex.

Amino Acid Sequence↗
PubMed2006

Controlling the elongation phase of transcription with P-TEFb.

The positive transcription elongation factor b (P-TEFb) is a cyclin-dependent kinase that controls the elongation phase of transcription by RNA polymerase II (RNAPII). This process is made possible by the reversal of effects of negative elongation factors that include NELF and DSIF. In complex organisms, elongation control is critical for the regulated expression of most genes. In those organisms, the function of P-TEFb is influenced negatively by HEXIM proteins and 7SK snRNA and positively by a variety of recruiting factors. Phylogenetic analyses of the components of the human elongation control machinery indicate that the number of mechanisms utilized to regulate P-TEFb function increased as organisms developed more complex developmental patterns.

Animals↗
PubMed2004

VP16 and ubiquitin; binding of P-TEFb via its activation domain and ubiquitin facilitates elongation of transcription of target genes.

Acidic or type IIB transcriptional activation domains (AADs) increase rates of initiation as well as elongation of transcription. For the former effects, AADs bind general transcription factors and larger coactivator complexes, which position RNA polymerase II (RNAPII) at sites of initiation of transcription. For the latter effects, their ubiquitylation plays an important role. In this study, this posttranslational modification increased the binding between a prototypic AAD and the positive transcription elongation factor b (P-TEFb), which contains a C-type cyclin (CycT1, CycT2, or CycK) and Cdk9. By phosphorylating negative elongation factors and the C-terminal domain of RNAPII, P-TEFb modifies the transcription complex for efficient elongation and cotranscriptional processing of mRNA. Indeed, the activation domain of VP16 and ubiquitin bound the cyclin boxes and the C terminus in CycT1, respectively. Moreover, the artificial fusion of ubiquitin with VP16 not only increased its activity via DNA and RNA, which was reflected in increased ratios of elongated to initiated transcripts, but rescued the deleterious substitution of alanine for phenylalanine at position 442 in its AAD. Thus, the ubiquitylation of AADs increases their interaction with P-TEFb and augments rates of elongation of transcription.

Chloramphenicol O-Acetyltransferase↗
PubMed2006

Cofactor of BRCA1: a novel transcription factor regulator in upper gastrointestinal adenocarcinomas.

Cofactor of BRCA1 (COBRA1) is a newly characterized member of the negative elongation factor (NELF) complex. In this work, we show that COBRA1 is overexpressed in the majority of primary upper gastrointestinal adenocarcinomas (UGC), and its overexpression correlates with down-regulation of TFF1. We have detected overexpression of COBRA1 mRNA using quantitative real-time reverse transcription-PCR in 28 (79%) primary UGCs. Immunohistochemical analysis of UGC tissue arrays that contained 70 tumor samples showed moderate-strong staining for COBRA1 in 60 (84%) tumors. Interestingly, the tumor samples showed absent-weak staining for TFF1 in 45 (65%) of the tumors. Simultaneous loss of TFF1 expression and overexpression of COBRA1 was observed in 42 of 70 (60%) tumors. Using small interfering RNA technology with gastric cancer cells, we have shown that COBRA1 inhibition leads to increased TFF1 promoter activity and gene expression. Promoter analysis of TFF1 indicated that regulation of TFF1 by COBRA1 is estrogen independent in contrast to breast cancer. Moreover, COBRA1 regulation of TFF1 in gastric cancer cells was independent of NELF-E. Using several truncated mutants and site mutants of the TFF1 promoter, we have shown that COBRA1 can negatively regulate the activator protein-1 (AP-1) complex at the TFF1 promoter and thus down-regulate TFF1 expression in gastric cancer cell lines. Electrophoretic mobility shift assay showed that COBRA1 attenuates AP-1 binding to DNA. Our results suggest COBRA1 as a novel oncogene in UGCs that regulate AP-1 binding and the expression of TFF1 in upper gastric epithelia.

Adenocarcinoma↗
PubMed2006

The Yin and Yang of P-TEFb regulation: implications for human immunodeficiency virus gene expression and global control of cell growth and differentiation.

The positive transcription elongation factor b (P-TEFb) stimulates transcriptional elongation by phosphorylating the carboxy-terminal domain of RNA polymerase II and antagonizing the effects of negative elongation factors. Not only is P-TEFb essential for transcription of the vast majority of cellular genes, but it is also a critical host cellular cofactor for the expression of the human immunodeficiency virus (HIV) type 1 genome. Given its important role in globally affecting transcription, P-TEFb's activity is dynamically controlled by both positive and negative regulators in order to achieve a functional equilibrium in sync with the overall transcriptional demand as well as the proliferative state of cells. Notably, this equilibrium can be shifted toward either the active or inactive state in response to diverse physiological stimuli that can ultimately affect the cellular decision between growth and differentiation. In this review, we examine the mechanisms by which the recently identified positive (the bromodomain protein Brd4) and negative (the noncoding 7SK small nuclear RNA and the HEXIM1 protein) regulators of P-TEFb affect the P-TEFb-dependent transcriptional elongation. We also discuss the consequences of perturbations of the dynamic associations of these regulators with P-TEFb in relation to the pathogenesis and progression of several major human diseases, such as cardiac hypertrophy, breast cancer, and HIV infection.

Animals↗
PubMed2006

Modulation of a P-TEFb functional equilibrium for the global control of cell growth and differentiation.

P-TEFb phosphorylates RNA polymerase II and negative elongation factors to stimulate general transcriptional elongation. It is kept in a functional equilibrium through alternately interacting with its positive (the Brd4 protein) and negative (the HEXIM1 protein and 7SK snRNA) regulators. To investigate the physiological significance of this phenomenon, we analyzed the responses of HeLa cells and murine erythroleukemia cells (MELC) to hexamethylene bisacetamide (HMBA), which inhibits growth and induces differentiation of many cell types. For both cell types, an efficient, albeit temporary disruption of the 7SK-HEXIM1-P-TEFb snRNP and enhanced formation of the Brd4-P-TEFb complex occurred soon after the treatment started. When the P-TEFb-dependent HEXIM1 expression markedly increased as the treatment continued, the abundant HEXIM1 pushed the P-TEFb equilibrium back toward the 7SK/HEXIM1-bound state. For HeLa cells, as HMBA produced only a minor, temporary effect on their growth, the equilibrium gradually returned to its pretreatment level. In contrast, long-term treatment of MELC induced terminal division and differentiation. Concurrently, the P-TEFb equilibrium was shifted overwhelmingly toward the 7SK snRNP side. Together, these data link the P-TEFb equilibrium to the intracellular transcriptional demand and proliferative/differentiated states of cells.

Acetamides↗
PubMed2004

Cellular control of gene expression by T-type cyclin/CDK9 complexes.

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. This review is centered on CDK9, which is activated by T-type cyclins and cyclin K generating distinct Positive-Transcription Elongation Factors termed P-TEFb. P-TEFb positively regulates transcriptional elongation by phosphorylating the C-terminal domain (CTD) of RNA polymerase II (RNA pol II), as well as negative elongation factors, which block elongation by RNA pol II shortly after the initiation of transcription. Work over the past few years has led to a dramatic increase in our understanding of how productive transcriptional elongation occurs. This review will briefly describe the mechanisms regulating the activity of T-type cyclin/CDK9 complexes and discuss how these complexes regulate gene expression. For further information, the reader is directed to excellent existing reviews on transcriptional elongation and HIV transcription.

Animals↗
PubMed1998

Evidence that P-TEFb alleviates the negative effect of DSIF on RNA polymerase II-dependent transcription in vitro.

Recently, a positive and a negative elongation factor, implicated in 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole (DRB) inhibition of transcription elongation, has been identified. P-TEFb is a positive transcription elongation factor and the DRB-sensitive kinase that phosphorylates the C-terminal domain (CTD) of the largest subunit of RNA polymerase II (Pol II). PITALRE, a member of the Cdc2 family of protein kinases, is the catalytic subunit of P-TEFb. DSIF is a human homolog of the yeast Spt4-Spt5 complex and renders elongation of transcription sensitive to DRB. DRB sensitivity-inducing factor (DSIF) binds to RNA Pol II and may directly regulate elongation. Here we show a functional interaction between P-TEFb and DSIF. The reduction of P-TEFb activity induced by either DRB, antibody against PITALRE, or immunodepletion resulted in a negative effect of DSIF on transcription. DSIF acts at an early phase of elongation, and the prior action of P-TEFb makes transcription resistant to DSIF. The state of phosphorylation of CTD determines the DSIF-RNA Pol II interaction, and may provide a direct link between P-TEFb and DSIF. Taken together, this study reveals a molecular basis for DRB action and suggests that P-TEFb stimulates elongation by alleviating the negative action of DSIF.

Cyclin-Dependent Kinase 9↗
PubMed2025

Evolution of promoter-proximal pausing enabled a new layer of transcription control.

Promoter-proximal pausing of RNA polymerase (Pol) II is a key regulatory step during transcription. Despite the central role of pausing in gene regulation, we do not understand the evolutionary processes that led to the emergence of Pol II pausing or its transition to a rate-limiting step actively controlled by transcription factors. Here, we analyzed transcription in species across the tree of life. Unicellular eukaryotes display an accumulation of Pol II near transcription start sites, which we propose transitioned to the longer-lived, focused pause observed in metazoans. This transition coincided with the evolution of new subunits in the negative elongation factor (NELF) and 7SK complexes. Depletion of NELF in mammals shifted the promoter-proximal buildup of Pol II from the pause site into the early gene body and compromised transcriptional activation for a set of heat-shock genes. Our work details the evolutionary history of Pol II pausing and sheds light on how new transcriptional regulatory mechanisms evolve.

RNA Polymerase II↗
PubMed2006

Regulation of clustered gene expression by cofactor of BRCA1 (COBRA1) in breast cancer cells.

Eucaryotic genes that are coordinately expressed tend to be clustered. Furthermore, gene clusters across chromosomal regions are often upregulated in various tumors. However, relatively little is known about how gene clusters are coordinately expressed in physiological or pathological conditions. Cofactor of BRCA1 (COBRA1), a subunit of the human negative elongation factor, has been shown to repress estrogen-stimulated transcription of trefoil factor 1 (TFF1 or pS2) by stalling RNA polymerase II. Here, we carried out a genome-wide study to identify additional physiological target genes of COBRA1 in breast cancer cells. The study identified a total of 134 genes that were either activated or repressed upon small hairpin RNA-mediated reduction of COBRA1. Interestingly, many COBRA1-regulated genes reside as clusters on the chromosomes and have been previously implicated in cancer development. Detailed examination of two such clusters on chromosome 21 (21q22) and chromosome X (Xp11) reveals that COBRA1 is physically associated with a subset of its regulated genes in each cluster. In addition, COBRA1 was shown to regulate both estrogen-dependent and -independent transcription of the gene cluster at 21q22, which encompasses the previously identified COBRA1-regulated TFF1 (pS2) locus. Thus, COBRA1 plays a critical role in the regulation of clustered gene expression at preferred chromosomal domains in breast cancer cells.

Biomarkers, Tumor↗
PubMed2006

Mechanisms controlling CDK9 activity.

This review primarily focuses on the mechanisms that modulate CDK9 activity and its recruitment to cellular genes, where it phosphorylates the C-terminal domain of RNA polymerase II (RNAPII) as well as negative elongation factors. CDK9 associates with each of four cyclins (T1, T2a, T2b and K), forming distinct positive transcription elongation factors (P-TEFb). Research done during the past decade has demonstrated a role for P-TEFb in stimulating elongation of otherwise paused RNAPII transcripts. Recent work suggests that P-TEFb also positively modulates other steps during transcription. In addition, "abnormal" CDK9 function is associated with certain diseases. Specifically, the activity of the cyclin T1/CDK9 complex is essential for HIV-1 replication and CDK9 upregulation is associated with cardiac hypertrophy. Thus, the role of CDK9 in these processes, and the possibility of therapeutically targeting CDK9, will also be briefly discussed.

Cell Cycle Proteins↗
PubMed2000

C-terminal domain phosphatase sensitivity of RNA polymerase II in early elongation complexes on the HIV-1 and adenovirus 2 major late templates.

The fate of RNA polymerase II in early elongation complexes is under the control of factors that regulate and respond to the phosphorylation state of the C-terminal domain (CTD). Phosphorylation of the CTD protects early elongation complexes from negative transcription elongation factors such as NELF, DSIF, and factor 2. To understand the relationship between transcript elongation and the sensitivity of RNA polymerase IIO to dephosphorylation, elongation complexes at defined positions on the Ad2-ML and human immunodeficiency virus type 1 (HIV-1) templates were purified, and their sensitivity to CTD phosphatase was determined. Purified elongation complexes treated with 1% Sarkosyl and paused at U(14)/G(16) on an HIV-1 template and at G(11) on the Ad2-ML template are equally sensitive to dephosphorylation by CTD phosphatase. Multiple elongation complexes paused at more promoter distal sites are more resistant to dephosphorylation than are U(14)/G(16) and G(11) complexes. The HIV-1 long terminal repeat and adenovirus 2 major late promoter do not appear to differentially influence the CTD phosphatase sensitivity of stringently washed complexes. Subsequent elongation by 1% Sarkosyl-washed U(14)/G(16) complexes is unaffected by prior CTD phosphatase treatment. This result is consistent with the hypothesis that CTD phosphatase requires the presence of specific elongation factors to propagate a negative effect on transcript elongation. The action of CTD phosphatase on elongation complexes is inhibited by HIV-1 Tat protein. This observation is consistent with the idea that Tat suppression of CTD phosphatase plays a role in transactivation.

Adenoviruses, Human↗
PubMed2003

The fragile X mental retardation protein FMRP binds elongation factor 1A mRNA and negatively regulates its translation in vivo.

Loss of the RNA-binding protein FMRP (fragile X mental retardation protein) leads to fragile X syndrome, the most common form of inherited mental retardation. Although some of the messenger RNA targets of this protein, including FMR1, have been ascertained, many have yet to be identified. We have found that Xenopus elongation factor 1A (EF-1A) mRNA binds tightly to recombinant human FMRP in vitro. Binding depended on protein determinants located primarily in the C-terminal end of hFMRP, but the hnRNP K homology domain influenced binding as well. When hFMRP was expressed in cultured cells, it dramatically reduced endogenous EF-1A protein expression but had no effect on EF-1A mRNA levels. In contrast, the translation of several other mRNAs, including those coding for dynamin and constitutive heat shock 70 protein, was not affected by the hFMRP expression. Most importantly, EF-1A mRNA and hFMR1 mRNA were coimmunoprecipitated with hFMRP. Finally, in fragile X lymphoblastoid cells in which hFMRP is absent, human EF-1A protein but not its corresponding mRNA is elevated compared with normal lymphoblastoid cells. These data suggest that hFMRP binds to EF-1A mRNA and also strongly argue that FMRP negatively regulates EF-1A expression in vivo.

Animals↗
PubMed2004

Dynamics of human immunodeficiency virus transcription: P-TEFb phosphorylates RD and dissociates negative effectors from the transactivation response element.

The elongation of transcription is a highly regulated process that requires negative and positive effectors. By binding the double-stranded stem in the transactivation response (TAR) element, RD protein from the negative transcription elongation factor (NELF) inhibits basal transcription from the long terminal repeat of the human immunodeficiency virus type 1 (HIVLTR). Tat and its cellular cofactor, the positive transcription elongation factor b (P-TEFb), overcome this negative effect. Cdk9 in P-TEFb also phosphorylates RD at sites next to its RNA recognition motif. A mutant RD protein that mimics its phosphorylated form no longer binds TAR nor represses HIV transcription. In sharp contrast, a mutant RD protein that cannot be phosphorylated by P-TEFb functions as a dominant-negative effector and inhibits Tat transactivation. These results better define the transition from abortive to productive transcription and thus replication of HIV.

Cell Line↗
PubMed1981

Duplication of the tuf gene, which encodes peptide chain elongation factor Tu, is widespread in Gram-negative bacteria.

The tuf gene which encodes peptide chain elongation factor Tu was found to be duplicated in nine enteric and four nonenteric gram-negative bacteria, but present only in one copy in two gram-positive genera. In two of the nonenteric gram-negative genera, Pseudomonas and Caulobacter, the duplicate tuf genes were found to be very close together on the chromosome, which contrasts with the situation in Escherichia coli, where they are more than 660 kilobases apart.

Chromosome Mapping↗
PubMed1997

Drosophila factor 2, an RNA polymerase II transcript release factor, has DNA-dependent ATPase activity.

Drosophila factor 2 has been identified as a component of negative transcription elongation factor (N-TEF) that causes the release of RNA polymerase II transcripts in an ATP-dependent manner (Xie, Z. and Price D. H. (1996) J. Biol. Chem. 271, 11043-11046). We show here that the transcript release activity of factor 2 requires ATP or dATP and that adenosine 5'-O-(thiotriphosphate) (ATPgammaS), adenosine 5'-(beta,gamma-imino)triphosphate (AMP-PNP), or other NTPs do not support the activity. Factor 2 demonstrated a strong DNA-dependent ATPase activity that correlated with its transcript release activity. At 20 microg/ml DNA, the ATPase activity of factor 2 had an apparent Km(ATP) of 28 microM and an estimated Kcat of 140 min-1. Factor 2 caused the release of nascent transcripts associated with elongation complexes generated by RNA polymerase II on a dC-tailed template. Therefore, no other protein cofactors are required for the transcript release activity of factor 2. Using the dC-tailed template assay, it was found that renaturation of the template was required for factor 2 function.

Adenosine Triphosphatases↗
PubMed1998

Characterization of the interactions among vaccinia virus transcription factors G2R, A18R, and H5R.

Prior genetic analysis suggests that there may exist an interaction between the products of the vaccinia virus genes A18R, a putative negative transcription elongation factor, and G2R, a putative positive transcription elongation factor. In addition, affinity purification of polyhistidine-tagged G2R protein overexpressed in vaccinia virus-infected cells, reported here, results in copurification of the vaccinia H5R protein, previously characterized as a late viral transcription factor. We have therefore used several methods to screen further for interactions among the G2R, A18R, and H5R proteins. Methods include copurification or co-immunoprecipitation of proteins overexpressed during vaccinia virus infection, activation of the gal 4 promoter by gal 4 fusions in the yeast two-hybrid system, and co-immunoprecipitation of proteins synthesized in vitro in a rabbit reticulocyte lysate. The results reveal interactions which include all possible pairwise combinations of the three proteins G2R, A18R, and H5R; however, not all possible permutations of the interactions are observed and the interactions are not observed in all environments tested. The results suggest that the vaccinia virus proteins G2R, A18R, and H5R interact as part of a higher order transcription complex.

Adenosine Triphosphatases↗