The Transcriptional Inhibitors, Actinomycin D and α-Amanitin, Activate the HIV-1 Promoter and Favor Phosphorylation of the RNA Polymerase II C-terminal Domain

Self Cite

The carboxyl-terminal domain (CTD) of the largest RNA polymerase (RNAP) II subunit undergoes reversible phosphorylation throughout the transcription cycle. The unphosphorylated form of RNAP II is referred to as IIA, whereas the hyperphosphorylated form is known as IIO. Phosphorylation occurs predominantly at serine 2 and serine 5 within the CTD heptapeptide repeat and has functional implications for RNAP II with respect to initiation, elongation, and transcription-coupled RNA processing. In an effort to determine the role of the major CTD phosphatase (FCP1) in regulating events in transcription that appear to be influenced by serine 2 and serine 5 phosphorylation, the specificity of FCP1 was examined. FCP1 is capable of dephosphorylating heterogeneous RNAP IIO populations of HeLa nuclear extracts. The extent of dephosphorylation at specific positions was assessed by immunoreactivity with monoclonal antibodies specific for phosphoserine 2 or phosphoserine 5. As an alternative method to assess FCP1 specificity, RNAP IIO isozymes were prepared in vitro by the phosphorylation of purified calf thymus RNAP IIA with specific CTD kinases and used as substrates for FCP1. FCP1 dephosphorylates serine 2 and serine 5 with comparable efficiency. Accordingly, the specificity of FCP1 is sufficiently broad to dephosphorylate RNAP IIO at any point in the transcription cycle irrespective of the site of serine phosphorylation within the consensus repeat. Reversible phosphorylation of the carboxyl-terminal domain (CTD)1 of the largest RNA polymerase (RNAP) II subunit plays an important role in the regulation of gene expression. The CTD of mammalian RNAP II is comprised of 52 repeats of the consensus sequence 1 YSPTSPS 7 (for a review, see Ref. 1). RNAP IIA, which contains an unmodified CTD, is actively recruited to the promoter as part of the preinitiation complex (2-5), whereas RNAP IIO, which contains a hyperphosphorylated CTD, is responsible for transcript elongation (6, 7). Therefore, protein kinases and phosphatases that alter the state of CTD phosphorylation can serve as transcriptional activators or repressors depending on the point in the transcription cycle at which they function.CTD phosphorylation occurs predominantly at serines 2 and 5 within the heptapeptide repeat. Genetic evidence indicates that the roles of serines in positions 2 and 5 are different. First, the partial substitution of serines in either position 2 or 5 have different effects on viability (8). Second, SRB (suppressors of RNA Polymerase IIB) mutations suppress the lethal effect of position 2 substitutions but not position 5 substitutions (9). Biochemical evidence has also confirmed differences between the two predominant serine positions. Serine 5 but not serine 2 phosphorylation recruits and activates the 5Ј-capping machinery (10, 11). Furthermore, nutritional stress and heat shock can independently alter the pattern of CTD phosphorylation, indicating that phosphoserine 2 and phosphoserine 5 are functionally different (12)(13)(14).A recent study using...

Section: Fcp1 Dephosphorylates Phosphoserine 2 and Phosphoserine 5 Wimentioning

confidence: 99%

TFIIF-associating Carboxyl-terminal Domain Phosphatase Dephosphorylates Phosphoserines 2 and 5 of RNA Polymerase II

Lin

Dubois

Dahmus

2002

Self Cite

“…␣-Amanitin (Sigma) was dissolved in water, and increasing concentrations were added to the culture medium for 24 h, as previously described (26). Inhibition of uPA transcription was estimated by quantitative reverse transcription-PCR of uPA mRNA at different ␣-amanitin concentrations.…”

Section: Methodsmentioning

confidence: 99%

“…Thus we treated PC3 cells with ␣-amanitin, an inhibitor of RNAP II (26,37), and asked what was the fate of the DAF amplicons in drug-treated PC3 cells, assuming that complexes not involved in transcription would persist despite the treatment. ChIP-ready chromatin was prepared from ␣-amanitin-treated cells and then used for the MNase digestion time-course experiment in which bulk chromatin yielded a digestion pattern similar to that of untreated cells (Fig.…”

Section: Presence Of Daf-a -B and -Bx Depends On Ongoing Transcriptmentioning

confidence: 99%

A Transcription-dependent Micrococcal Nuclease-resistant Fragment of the Urokinase-type Plasminogen Activator Promoter Interacts with the Enhancer

Ferrai

Munari

Luraghi

et al. 2007

We show the interaction between the enhancer and the minimal promoter of urokinase-type plasminogen activator gene during active transcription by coupling micrococcal nuclease digestion of cross-linked, sonicated chromatin, and chromatin immunoprecipitation. This approach allowed the precise identification of the interacting genomic fragments, one of which is resistant to micrococcal nuclease cleavage. The interacting fragments form a single transcriptional control unit, as indicated by their common protein content. Furthermore, we show that the enhancer-MP interaction persists during the early stages of transcription and is lost upon ␣-amanitin treatment, indicating the requirement for active transcription. Our results support a looping model of interaction between the enhancer and the MP of the urokinase-type plasminogen activator gene.Transcription regulation in eukaryotic cells is a multistep process that involves the assembly of multiprotein complexes on gene regulatory regions (1). These regulatory regions contain two types of sequences: enhancers/silencers, which recruit a complex array of transcription factors and chromatin-modifying activities, and core promoter elements to which the general transcriptional machinery, including RNAP II, 2 is recruited. Understanding the molecular mechanism(s) involved in transcriptional regulation over long distances is of fundamental importance, because in most cases the activities of remote control elements are essential in turning on or off specific subsets of genes in a temporally and spatially regulated manner (2). The main models to explain distal enhancer function invoke enhancer-promoter communication, either through proteinprotein interactions resulting in the formation of DNA/chromatin loops (looping model), the free sliding of proteins recruited on the enhancer along the DNA (scanning model), or the establishment of modified chromatin domains between the enhancer and the promoter by facilitator proteins, which generate a progressive chain of higher order complexes along the chromatin fiber (3-9). The looping model, however, is supported by recent results in a number of different experimental systems, showing that the close physical proximity of distant regulatory sequences is required for proper gene expression (10 -15). The interaction between enhancer elements and proximal promoter culminates in the transcription activation event. However it is not yet clarified if the activation of transcription stems from the transient interaction of regulatory elements or through a more stable structure working as a single control unit.The uPA gene codes for a serine protease involved in the degradation of the extracellular matrix and in cell motility (16). The expression of this gene is normally dependent on the presence of inducers, but in cancer cells it is often constitutive. The regulatory region of the uPA gene contains an MP and an enhancer located 2 kb upstream (17). In highly invasive human prostate adenocarcinoma (PC3) cells this gene is constitutively expresse...

“…In fact, treatment of cells with several stress-inducing agents rapidly releases 7SK and HEXIM1 from P-TEFb (6,7,17,18). These agents have been shown to stimulate CTD phosphorylation and HIV transcription (21,22). Thus, the induced release of 7SK/HEXIM1 and activation of P-TEFb may contribute directly to the stress-induced HIV and cellular gene expression.…”

mentioning

confidence: 99%

Phosphorylated Positive Transcription Elongation Factor b (P-TEFb) Is Tagged for Inhibition through Association with 7SK snRNA

Chen

Yang

Zhou

2004

162

216

The positive transcription elongation factor b (PTEFb), 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 stressinduced transcription and pathogenesis of cardiac hypertrophy. The 7SK⅐P-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 7SK⅐P-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 7SK⅐P-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.7SK is an abundant (2 ϫ 10 5 copies/cell) and evolutionarily conserved small nuclear RNA (snRNA) 1 of ϳ330 nucleotides (1-4). It is transcribed by RNA polymerase (Pol) III from one or more genes belonging to a family of interspersed repeats in the mammalian genome (2, 5). The high conservation and abundance of 7SK suggest an important physiological function of this RNA. In searching for nuclear factors that can bind to and regulate the activity of human positive transcription elongation factor b (P-TEFb), we and others (6, 7) have identified 7SK as a specific P-TEFb-associated factor. Consisting of CDK9 and cyclin T1 (CycT1) (8, 9), P-TEFb strongly enhances the processivity of RNA Pol II by phosphorylating the C-terminal domain (CTD) of Pol II and antagonizing the actions of negative elongation factors (10 -13). Studies employing RNA interference in Caenorhabditis elegans and a pharmacological inhibitor of CDK9 in mammalian cells implicate P-TEFb as essential for expression of most protein-coding genes (10, 14).P-TEFb also functions as a specific host cellular cofactor for the HIV-1 Tat protein. Stimulation of Pol II elongation is essential for HIV transcription, during which P-TEFb is recruited to the nascent mRNA by Tat (15, 16). Tat binds to CycT1 and recruits P-TEFb through formation of a stable ternary complex containing P-TEFb, Tat, and the HIV TAR RNA structure located at the 5Ј-end of the nascent viral transcript. Once recruited, P-TEFb phosphorylates the CTD and stimulates the production of the full-l...