Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:2.7.7.6 (RNA polymerase)
34,946 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

For most genetic deficiencies manifested in the liver, maximization of gene expression in hepatocytes will be an important factor in achieving successful gene therapy. A rapid, highly efficient, and nontoxic method for transfecting DNA into hepatocytes was used to compare directly promoter strengths of various cellular and viral promoters. Conditions are described here for transfecting 5-10% of primary hepatocytes using the positively charged liposomes, Lipofectin. Cells are not damaged by this method as they continue to transcribe genes controlled by liver specific promoters and can survive for over 2 weeks in culture. We find that the cytomegalovirus, SR alpha, and beta-actin promoters are more active than the SV40, RSV, RNA polymerase II, albumin, alpha 1-antitrypsin, or phosphoenolpyruvate carboxykinase promoters. A simple TK promoter and a TK promoter with the polyoma enhancer (MCI) were almost completely inactive. This information will be useful in the construction of vectors designed to express genes efficiently in primary hepatocytes for purposes of gene therapy, although the stability of expression from these promoters will need to be demonstrated in hepatocytes in vivo.
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PMID:Evaluation of relative promoter strength in primary hepatocytes using optimized lipofection. 186 38

The methylation of nuclear and chloroplast DNAs has been examined in relation to the known differential expression of C4 photosynthesis genes in the bundle sheath and mesophyll cells of etiolated, greening, and fully green maize leaves. We have focused our research on phosphoenolpyruvate carboxylase, pyruvate, orthophosphate dikinase, and the small subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase (RBUp2Case) which are coded by nuclear genes, and on the large subunit of RBUp2Case which is coded by a plastid gene. Reversed-phase high performance liquid chromatography revealed several kinds of methylated bases in DNAs of both photosynthetic cell types, with the largest amounts in fully green leaves. The occurrence of selective DNA methylation was investigated by employing an isoschizomeric pair of methyl-sensitive and -insensitive endonucleases followed by Southern hybridizations with specific DNA probes. Notably, there was an inverse correlation between the relative abundance of specific transcripts in a given cell type during greening and the methylation status of the corresponding nuclear or chloroplast gene. Furthermore, a heterologous in vitro transcription system using Escherichia coli RNA polymerase revealed that the plastid gene encoding the RBUp2Case large subunit in both cell types was active as a template in the unmethylated state, whereas it was inactive when methylated. Thus, the selective methylation of both chloroplast and nuclear DNA is likely one component of a multilevel control mechanism for the differential regulation of cell-specific C4 photosynthesis gene expression in greening maize leaves.
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PMID:DNA methylation and the differential expression of C4 photosynthesis genes in mesophyll and bundle sheath cells of greening maize leaves. 254 70

The primary structure of the messenger RNA coding for cytosolic phosphoenolpyruvate carboxykinase was determined by sequencing cDNA and genomic DNA and by primer extension of the mRNA. The molecule is 2624 nucleotides in length; this includes 143 nontranslated nucleotides at the 5' end and 615 nontranslated nucleotides at the 3' end. The 3' nontranslated sequence contains a 102-base pair region of alternating purine-pyrimidine nucleotides (the majority of which are UpG dinucleotides), several direct repeats and palindromic sequences, and 8 CpG dinucleotides. The corresponding segment of the phosphoenolpyruvate carboxykinase gene thus has characteristics which favor the formation of Z-DNA. The amino acid sequence of phosphoenolpyruvate carboxykinase was deduced from the mRNA sequence and confirmed by fast atom bombardment mass spectrometric analysis of peptides generated with trypsin and Staphylococcus aureus V8 protease. The protein consists of 621 amino acids and has a molecular weight of 69,289. Charon 4A lambda bacteriophage clones containing genomic DNA coding for phosphoenolpyruvate carboxykinase were isolated from a library of partial HaeIII digests of rat liver DNA. Two clones, lambda PC112 and lambda PC103, contained the entire coding region in 15-kilobase inserts and were used to subclone the gene into pBR322 as EcoRI, BamHI, or SstI-KpnI fragments. Using these subclones, the structure of the phosphoenolpyruvate carboxykinase gene was determined by S1 nuclease mapping, R-loop analysis, and DNA sequencing. The gene is composed of 10 exons and 9 introns with a total length of 6.0 kilobases. The transcription initiation site of the gene was determined by a combination of in vitro transcription in a HeLa cell lysate system, primer extension of mRNAPEPCK, and S1 nuclease mapping. In vitro transcription of purified DNA templates revealed three RNA polymerase II-dependent start sites. Two sites were separated by 600 base pairs on the coding strand and the third site was on the noncoding strand. The products of S1 nuclease mapping and primer extension from a BglII site were compared in order to determine which of the coding strand initiation sites was expressed in vivo. In both cases a 69-base pair fragment was generated and the 5' end of this corresponded to a thymidine residue identified in a sequence ladder of the genomic DNA coding strand. We conclude that mRNAPEPCK synthesis initiates with an adenine residue 69 base pairs 5' of the BglII site; this corresponds to the 3' most transcription initiation site determined in vitro.
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PMID:Rat hepatic cytosolic phosphoenolpyruvate carboxykinase (GTP). Structures of the protein, messenger RNA, and gene. 299 87

Transcription initiation of the gene encoding phosphoenolpyruvate carboxykinase (PEPCK) is stimulated by glucocorticoids and glucagon, via cAMP, and dominantly inhibited by insulin in rat liver and H4IIE cells. Lysolecithin-permeabilized H4IIE cells recover completely and continue to multiply, yet are transiently penetrable by macromolecules. These cells, after various hormonal treatments, were utilized for in situ DNase I protection studies of the PEPCK promoter. Nearly all of the sites of protein interaction observed in vitro are protected in vivo as well as several additional sites. The DNase I protection pattern is the same in cells without or with any of the hormone treatments, suggesting that hormonal modulation of transcription does not involve addition or removal of factors from the hormone response elements of the promoter. We focused on the organization and stability of the transcription initiation complex as well as the dynamic nature of distal promoter factors in their interaction with DNA. The transcription initiation complex was detected, and it appears to be co-existent with a short region of naked single-stranded DNA over the TATA box on the template strand, as determined by potassium permanganate reactivity. This complex is quite stable, even under conditions of much reduced RNA synthesis, which suggests that the complex is not broken down and reformed with each round of initiation by RNA polymerase II. Other factors bind to the PEPCK promoter with half-lives ranging from a few minutes to more than 40 min. The cAMP response element apparently involves transcriptional modulation achieved through modification of a bound factor (presumably cAMP response element-binding protein), whereas the glucocorticoid/insulin-responsive region of the promoter functions through factors which are involved in a rapid exchange, suggesting quite different modes of transcriptional regulation.
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PMID:Dynamic aspects of DNA/protein interactions in the transcriptional initiation complex and the hormone-responsive domains of the phosphoenolpyruvate carboxykinase promoter in vivo. 822 59

Transcription of the phosphoenolpyruvate carboxykinase (PEPCK) gene is induced upon activation of protein kinase A by cAMP and phosphorylation of Ser-133 in the transcription factor, cAMP-response element binding protein (CREB), and this induction is inhibited by insulin. We show here that insulin does not act by dephosphorylating CREB or by affecting heterologous kinases that phosphorylate Ser-129 or Ser-142 in CREB. In addition, insulin inhibition of minimal PEPCK promoter activity induced by CREB-GAL4 + protein kinase A was equivalent to inhibition of basal transcription, and thus cAMP-independent. On the other hand, nearly complete insulin inhibition is observed with the full PEPCK promoter (-600/+69), indicating that other factors are involved. The additional promoter elements required for induction by protein kinase A lie within -271 nucleotides of the start site and correspond to putative binding sites for activator protein-1 and CAAT/enhancer-binding protein (C/EBP), first identified by Roesler et al. (Roesler, W. J., McFie, P. J., and Puttick, D. M., (1993) J. Biol. Chem. 268, 3791-3796). This tripartite array of binding sites for CREB, C/EBP, and activator protein-1 (AP-1) factors forms a cAMP response unit that, together with the minimal promoter, can mediate both induction by cAMP and inhibition by insulin. Thus, for the PEPCK gene with a single CREB site, the CREB.CBP.RNA polymerase II complex cannot mediate either induction by cAMP or inhibition by insulin.
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PMID:A tripartite array of transcription factor binding sites mediates cAMP induction of phosphoenolpyruvate carboxykinase gene transcription and its inhibition by insulin. 966 47

Hormones regulate glucose homeostasis, in part, by controlling the expression of gluconeogenic enzymes, such as phosphoenolpyruvate carboxykinase (PEPCK). Insulin and glucocorticoids reciprocally regulate PEPCK expression primarily at the level of gene transcription. We demonstrate here that glucocorticoids promote, whereas insulin disrupts, the association of CREB-binding protein (CBP) and RNA polymerase II with the hepatic PEPCK gene promoter in vivo. We also show that accessory factors, such as CCAAT/enhancer-binding protein beta (C/EBP beta), can recruit CBP to drive transcription. Insulin increases protein levels of liver-enriched transcriptional inhibitory protein (LIP), an inhibitory form of C/EBP beta, in a phosphatidylinositol 3-kinase-dependent manner. LIP concomitantly replaces liver-enriched transcriptional activator protein on the PEPCK gene promoter, which can abrogate the recruitment of CBP and polymerase II, culminating in the repression of PEPCK expression and the attenuation of hepatocellular glucose production.
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PMID:Insulin inhibits hepatocellular glucose production by utilizing liver-enriched transcriptional inhibitory protein to disrupt the association of CREB-binding protein and RNA polymerase II with the phosphoenolpyruvate carboxykinase gene promoter. 1207 Jan 72

The expression of the insulin-like growth factor II (IGF-II) gene (Igf2) in rodents is completely abrogated in almost all adult tissues. A prominent exception are neoplasms in which IGF-II frequently serves as an autocrine growth factor. We have investigated the potential role of Igf2 expression during liver carcinogenesis. After application of diethylnitrosamine (DEN) preneoplastic foci and adenomas emerged in liver tissue of wild-type and phosphoenolpyruvate carboxykinase (PEPCK)-IGF-II transgenic mice. Surprisingly, number and size of preneoplastic foci were not significantly increased in PEPCK-IGF-II mice as compared with wild-type animals. In situ preparation showed that early adenomas expressed Igf2 transcripts. Reverse transcriptase polymerase chain reaction (RT-PCR) and restriction enzyme analysis confirmed that DEN treatment had indeed reactivated the hepatic expression of murine Igf2 in control mice in a dose-dependent manner. This re-expression of Igf2 persisted for at least 18 months. Species-specific RT-PCR analyses also revealed the presence of murine Igf2 mRNAs in some PEPCK-IGF-II mice. A similar reactivation of Igf2 was detected in bovine growth hormone transgenic mice which develop hepatocellular neoplasms with high frequency. Our results suggest that reactivation of Igf2 is an early event during hepatocarcinogenesis in mice. Its appearance in two independent animal models suggests that Igf2 may be important at pivotal checkpoints of hepatocarcinogenesis.
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PMID:Diethylnitrosamine induces long-lasting re-expression of insulin-like growth factor II during early stages of liver carcinogenesis in mice. 1212 4

Bile acid metabolism plays an essential role in cholesterol homeostasis and is critical for the initiation of atherosclerotic disease. However, despite the recent advances, the molecular mechanisms whereby bile acids regulate gene transcription and cholesterol homeostasis in mammals still need further investigations. Here, we show that bile acids suppress transcription of the gene (CYP7A1) encoding cholesterol 7alpha-hydroxylase, the rate-limiting enzyme in bile acid biosynthesis, also through an unusual mechanism not involving the bile acid nuclear receptor, farnesoid X receptor. By performing cell-based reporter assays, protein/protein interaction, and chromatin immunoprecipitation assays, we demonstrate that bile acids impair the recruitment of peroxisome proliferator-activated receptor-gamma coactivator-1alpha and cAMP response element-binding protein-binding protein by hepatocyte nuclear factor-4alpha, a master regulator of CYP7A1. We also show for the first time that bile acids inhibit transcription of the gene (PEPCK) encoding phosphoenolpyruvate carboxykinase, the rate-limiting enzyme in gluconeogenesis, through the same farnesoid X receptor-independent mechanism. Chromatin immunoprecipitation assay revealed that bile acid-induced dissociation of coactivators from hepatocyte nuclear factor-4alpha decreased the recruitment of RNA polymerase II to the core promoter and downstream in the 3'-untranslated regions of these two genes, reflecting the reduction of gene transcription. Finally, we found that Cyp7a1 expression was stimulated in fasted mice in parallel to Pepck, whereas the same genes were repressed by bile acids. Collectively, these results reveal a novel regulatory mechanism that controls gene transcription in response to extracellular stimuli and argue that the transcription regulation by bile acids of genes central to cholesterol and glucose metabolism should be viewed dynamically in the context of the fasted-to-fed cycle.
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PMID:Coordinated control of cholesterol catabolism to bile acids and of gluconeogenesis via a novel mechanism of transcription regulation linked to the fasted-to-fed cycle. 1286 25

Activation of phosphoenolpyruvate carboxykinase (PEPCK) gene transcription in response to all-trans-retinoic acid (RA) or a glucocorticoid such as dexamethasone (Dex) requires a distinct arrangement of DNA-response elements and their cognate transcription activators on the gene promoter. Two of the accessory factor-binding elements involved in the Dex response (gAF1 and gAF3) coincide with the DNA-response elements involved in the RA response. We demonstrate here that the combination of Dex/RA has a synergistic effect on endogenous PEPCK gene expression in rat hepatocytes and H4IIE hepatoma cells. Reporter gene studies show that the gAF3 element and one of the two glucocorticoid receptor-binding elements (GR1) are most important for this effect. Chromatin immunoprecipitation assays revealed that when H4IIE cells were treated with Dex/RA, ligand-activated retinoic acid receptors (retinoic acid receptor/retinoid X receptor) and glucocorticoid receptors are recruited to this gene promoter, as are the transcription coregulators p300, CREB-binding protein, p/CIP, and SRC-1. Notably, the recruitment of p300 and RNA polymerase II to the PEPCK promoter is increased by the combined Dex/RA treatment compared with Dex or RA treatment alone. The functional importance of p300 in the Dex/RA response is illustrated by the observation that selective reduction of this coactivator, but not that of CREB-binding protein, abolishes the synergistic effect in H4IIE cells.
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PMID:The synergistic effect of dexamethasone and all-trans-retinoic acid on hepatic phosphoenolpyruvate carboxykinase gene expression involves the coactivator p300. 1516 31

Thyroid hormone receptors (TRs) are ligand-regulated transcription factors that bind to thyroid hormone response elements of target genes. Upon ligand binding, they recruit coactivator complexes that increase histone acetylation and recruit RNA polymerase II (Pol II) to activate transcription. Recent studies suggest that nuclear receptors and coactivators may have temporal recruitment patterns on hormone response elements, yet little is known about the nature of the patterns at multiple endogenous target genes. We thus performed chromatin immunoprecipitation assays to investigate coactivator recruitment and histone acetylation patterns on the thyroid hormone response elements of four endogenous target genes (GH, sarcoplasmic endoplasmic reticulum calcium-adenosine triphosphatase, phosphoenolpyruvate carboxykinase, and cholesterol 7alpha-hydroxylase) in a rat pituitary cell line that expresses TRs. We found that TRbeta, several associated coactivators (steroid receptor coactivator-1, glucocorticoid receptor interacting protein-1, and TR-associated protein 220), and RNA Pol II were rapidly recruited to thyroid hormone response elements as early as 15 min after T3 addition. When the four target genes were compared, we observed differences in the types and temporal patterns of recruited coactivators and histone acetylation. Interestingly, the temporal pattern of RNA Pol II was similar for three genes studied. Our findings suggest that thyroid hormone-regulated target genes may have distinct patterns of coactivator recruitment and histone acetylation that may enable highly specific regulation.
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PMID:Thyroid hormone-regulated target genes have distinct patterns of coactivator recruitment and histone acetylation. 1625 15


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