Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:2.7.7.6 (RNA polymerase)
 34,946 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Premessenger RNA (pre-mRNA) splicing can occur within an individual pre-mRNA (cis-splicing) or between separate pre-mRNAs (trans-splicing). Although a number of examples of mammalian trans-splicing have been reported, the molecular mechanisms involved are poorly understood. Here, we investigate the mechanisms of Sp1 pre-mRNA trans-splicing with human cells expressing modified Sp1 transgenes. We find that the presence of a long intron or the insertion of an RNA polymerase II pause site within an intron promotes trans-splicing. We also add examples of naturally occurring trans-splicing. We propose that Sp1 trans-splicing, and other examples of mammalian trans-splicing, are a consequence of low-frequency disruption of the normal mechanisms that couple transcription and splicing.
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PMID:Delay in synthesis of the 3' splice site promotes trans-splicing of the preceding 5' splice site. 1583 27

The acidic-rich activation domain of VP16 and the proline-rich activation domains of human AP2 and human CTF are able to activate transcription in a whole cell extract from Schizosaccharomyces pombe, whereas the glutamine-rich domains of Sp1 and Oct2 are unable to activate transcription in this system. Immunodepletion experiments of the whole cell extracts using specific antibodies against pombe TAF110, pombe TAF 72, pombe TBP and Srb4 shows that the activation of transcription by VP16, AP2 and CTF is through the mediator, since depletion of Srb4 inhibits activated transcription but does not inhibit basal transcription. Immunodepletion of TBP causes inhibition of both activated and basal transcription. On the other hand, immunodepletion of TAFs does not have an effect on either activated or basal transcription. Purified RNA polymerase holoenzyme is able to rescue the transcriptional activation activity of the anti-Srb4 immunodepleted extract. Moreover, we demonstrate that the mediator is needed for basal transcription of a TATA-less promoter.
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PMID:Mammalian transcription activation domains of VP16, AP2 and CTF activate transcription in a whole cell extract from Schizosaccharomyces pombe through the SRB/mediator. 1594 25

In this study, the roles of Sp1/Sp3 transcription factors in the regulation of the activity of human telomerase reverse transcriptase (hTERT) promoter in response to ceramide were examined in the A549 human lung adenocarcinoma cells. The activity of the N-terminal truncated hTERT promoter, lacking the c-Myc recognition (E-box) region but containing multiple Sp1/Sp3 sites, was also significantly inhibited by C6-ceramide, indicating a role for ceramide in the regulation of Sp1/Sp3 function. Partial inhibition of Sp1 expression using small interfering RNA resulted in a significant inhibition of the hTERT promoter. Treatment with C6-ceramide inhibited the trans-activation function of overexpressed Sp1, whereas it induced the repressor effects of exogenous Sp3 on the hTERT promoter. The interaction between Sp1 and hTERT promoter DNA was significantly reduced in response to ceramide as assessed by chromatin immunoprecipitation analysis. In contrast, the promoter DNA-binding activity of Sp3 was slightly increased in response to C6-ceramide, resulting in the increased ratio of Sp3/Sp1 on the hTERT promoter, which was concomitant with the reduced recruitment of RNA polymerase II to the promoter. Furthermore, mutations of various Sp1/Sp3 recognition sequences significantly attenuated the activity of the promoter in the presence or absence of ceramide, demonstrating the importance of multiple Sp1/Sp3 recognition sites for the promoter activity. Mechanistically, the data demonstrated that C6-ceramide reduced the acetylation of Sp3 protein and partially blocked the activation of the hTERT promoter by the histone deacetylase inhibitor trichostatin A. The roles of endogenous long chain ceramide generated in response to gemcitabine in the inhibition of hTERT promoter activity and the regulation of Sp3 acetylation were also demonstrated.
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PMID:Sp1/Sp3-dependent regulation of human telomerase reverse transcriptase promoter activity by the bioactive sphingolipid ceramide. 1595 64

The imprinted SNRPN locus is a complex transcriptional unit that encodes the SNURF and SmN polypeptides as well as multiple non-coding RNAs. SNRPN is located within the Prader-Willi and Angelman syndrome (PWS/AS) region that contains multiple imprinted genes, which are coordinately regulated by a bipartite imprinting center (IC). The SNRPN 5' region co-localizes with the PWS-IC and contains two DNase I hypersensitive sites, DHS1 at the SNRPN promoter, and DHS2 within intron 1, exclusively on the paternally inherited chromosome. We have examined DHS1 and DHS2 to identify cis- and trans-acting regulatory elements within the endogenous SNRPN 5' region. Analysis of DHS1 by in vivo footprinting and chromatin immunoprecipitation identified allele-specific interaction with multiple regulatory proteins, including NRF-1, which regulates genes involved in mitochondrial and metabolic functions. DHS2 acted as an enhancer of the SNRPN promoter and contained a highly conserved region that showed allele-specific interaction with unphosphorylated RNA polymerase II, YY1, Sp1 and NRF-1, further suggesting a key role for NRF-1 in regulation of the SNRPN locus. We propose that one or more of the regulatory elements identified in this study may also contribute to PWS-IC function.
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PMID:Characterization of cis- and trans-acting elements in the imprinted human SNURF-SNRPN locus. 1611 39

Lack of maturation of phagosomes containing pathogenic Mycobacterium tuberculosis within macrophages has been widely recognized as a crucial factor for the persistence of mycobacterial pathogen. Host molecule tryptophan-aspartate containing coat protein (TACO) has been shown to play a crucial role in the arrest of such a maturation process. The present study was addressed to understand whether or not polyphenols derived from green tea could down-regulate TACO gene transcription. And if yes, what impact TACO gene down-regulation has on the uptake/survival of M. tuberculosis within macrophages. The reverse-transcriptase polymerase chain reaction and reporter assay technology, employed in this study, revealed that the major component of green tea polyphenols, epigallocatechin-3-gallate had the inherent capacity to down-regulate TACO gene transcription within human macrophages through its ability to inhibit Sp1 transcription factor. We also found out that TACO gene promoter does contain Sp1 binding sequence using bioinformatics tools. The down-regulation of TACO gene expression by epigallocatechin-3-gallate was accompanied by inhibition of mycobacterium survival within macrophages as assessed through flow cytometry and colony counts. Based on these results, we propose that epigallocatechin-3-gallate may be of importance in the prevention of tuberculosis infection.
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PMID:Green tea polyphenol inhibits Mycobacterium tuberculosis survival within human macrophages. 1635 57

In this study, we show that exposure of human hepatocellular HepG2 cells to SP600125 rapidly and dramatically reduced global histone H3-Ser10 phosphorylation, without significantly affecting the global acetylation of neighboring lysines. The loss of phosphorylation is not due to changes in cell cycle distribution and/or apoptosis and is mediated independent of either p46/54(JNK) or MSK-1/2 inhibition. Moreover, SP600125 repressed the basal expression of the endogenous LDL receptor in a gene-specific manner, whereas the expression of squalene synthase, sterol response element-binding protein-1, and beta-actin was not altered by SP600125. Finally, chromatin immunoprecipitation and in vivo footprinting assays provided direct evidence that localized histone H3-Ser10 dephosphorylation at the low-density lipoprotein receptor promoter was associated with a significant decrease in the occupancy of the Sp1 binding site, with a slight reduction in the occupancy of RNA polymerase II. Together, our findings show that SP600125 is an efficient inhibitor of histone H3-Ser10 phosphorylation in vivo, and our results led us to hypothesize that this modification plays a novel role in regulating transcriptional control by modulating promoter accessibility to maintain basal expression in a gene-specific manner.
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PMID:Selective repression of low-density lipoprotein receptor expression by SP600125: coupling of histone H3-Ser10 phosphorylation and Sp1 occupancy. 1644 44

The mammalian transcription factors Sp1 and Sp3 compete for the same DNA binding sites but play different roles in the regulation of expression of numerous genes. It is known that, in the interphase nucleus, Sp1 and Sp3 are organized into distinct foci. In this study, we show that throughout the mitotic process, while being displaced from the condensed chromosomes and dispersed throughout the cell, Sp1 and Sp3 maintain their separate punctate distributions. In metaphase, both Sp1 and Sp3 foci show a high degree of colocalization with microfilaments, suggesting that F-actin is involved in the organization of Sp1 and Sp3 foci during mitosis. Constant Sp1 and Sp3 levels were observed during mitosis, signifying a recovery of the pre-existing Sp1 and Sp3 population in newly formed nuclei. In late telophase, Sp1 and Sp3 are equally segregated between daughter cells, and their subnuclear organization as distinct foci is restored in a sequential fashion with Sp3 regrouping into the newly formed nuclei prior to Sp1. Both Sp1 and Sp3 return to the nuclei ahead of RNA polymerase II. Our results support a model in which entry of Sp1, Sp3 and RNA polymerase II into the newly formed nuclei is an ordered process.
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PMID:Sp1 and Sp3 foci distribution throughout mitosis. 1649 4

Estrogens, acting through their nuclear receptors have a broad impact on target cells, eliciting a transcriptional response program that involves gene repression as well as gene stimulation. While much is known about the mechanisms by which the estrogen-occupied estrogen receptor (ER) stimulates gene expression, the molecular events that lead to gene repression by the hormone-ER complex are largely unknown. Because estradiol represses expression of the cyclin G2 gene, which encodes a negative regulator of the cell cycle, our aim was to understand the mechanism by which cyclin G2 is repressed by estrogen. We show that cyclin G2 is a primary ER target gene in MCF-7 breast cancer cells that is rapidly and robustly down-regulated by estrogen. Promoter analysis reveals a responsive region containing a half-estrogen response element and GC-rich region that interact with ER and Sp1 proteins. Mutation of the half-ERE abrogates hormone-mediated repression. Mutational mapping of receptor reveals a requirement for its N-terminal region and DNA binding domain to support cyclin G2 repression. Following estradiol treatment of cells, chromatin immunoprecipitation analyses reveal recruitment of ER to the cyclin G2 regulatory region, dismissal of RNA polymerase II, and recruitment of a complex containing N-CoR and histone deacetylases, leading to a hypoacetylated chromatin state. Our study provides evidence for a mechanism by which the estrogen-occupied ER is able to actively repress gene expression in vivo and indicates a role for nuclear receptor corepressors and associated histone deacetylase activity in mediating negative gene regulation by this hormone-occupied nuclear receptor.
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PMID:Estrogen-occupied estrogen receptor represses cyclin G2 gene expression and recruits a repressor complex at the cyclin G2 promoter. 1660 56

A new method called promoter trapping was developed to purify promoter-protein complex using the c-jun promoter (-200+81) as a model, which was shown to have significant promoter activity. Polymerase chain reaction (PCR), lambda exonuclease digestion combined with (AC)(5)-Sepharose DNA affinity chromatography were used to produce c-jun promoter with a (GT)(5) tail at each 3' end. The intact promoter and different length pieces with one or two (GT)(5) tails had almost the same capacity to bind with (AC)(5)-Sepharose. In solution, tailed c-jun promoter (60 nM) and competitor poly dI:dC (30 ng/microl) was incubated with crude HEK293 nuclear extract to form a large protein-promoter complex, and the complex was then trapped by (AC)(5)-Sepharose by centrifugation or on a column. Compared with a popular alternative method, called here the immobilized promoter method, the products of promoter trapping were purer. The preinitiation complex purified by promoter trapping had the expected components including RNA polymerase II, TATA-box binding protein (TBP), TFIIF subunit RAP74, and transcription factor SP1, and transcribed RNA in vitro. Thus, the promoter trapping approach provides a useful tool for the purification and investigation of transcription complexes.
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PMID:Promoter trapping of c-jun promoter-binding transcription factors. 1693 21

The transcription factor (TF) Sp1 is a well-known RNA polymerase II transcription activator that binds to GC-rich recognition sites in a number of essential cellular and viral promoters. In addition, direct interference of Sp1 binding to DNA cognate sites using DNA-interacting compounds may provide promising therapies for suppression of cancer progression and viral replication. In this study, we present a rapid, sensitive and cost-effective evaluation of a GC intercalative drug, doxorubicin (DOX), in dissociating the Sp1-DNA complex using fluorescence correlation spectroscopy (FCS) in a microfluidic system. FCS allows assay miniaturization without compromising sensitivity, making it an ideal analytical method for integration of binding assays into high-throughput, microfluidic platforms. A polydimethylsiloxane (PDMS)-based microfluidic chip with a mixing network is used to achieve specific drug concentrations for drug titration experiments. Using FCS measurements, the IC50 of DOX on the dissociation of Sp1-DNA complex is estimated to be 0.55 microM, which is comparable to that measured by the electrophoretic mobility shift assay (EMSA). However, completion of one drug titration experiment on the proposed microfluidic-FCS platform is accomplished using only picograms of protein and DNA samples and less than 1 h total assay time, demonstrating vast improvements over traditional ensemble techniques.
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PMID:A microfluidic-FCS platform for investigation on the dissociation of Sp1-DNA complex by doxorubicin. 1710 58


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