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:4.1.1.32 (phosphoenolpyruvate carboxykinase)
4,204 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

We have identified DNA elements in the phosphoenolpyruvate carboxykinase (PEPCK) gene promoter which are bound 'in vivo' by proteins under conditions of basal level gene expression and have evaluated several hypothesis to account for the tissue specific expression of the gene. In vitro DNase I footprinting demonstrated that factors which bind to basal expression elements of the PEPCK promoter, the BSE/CRE and NFI/CCAAT sites, are also present in HTC and XC cells which do not express the PEPCK gene. 'In vivo' DNase I footprinting demonstrated that the BSE/CRE, NFI/CCAAT, and three additional sites are bound by protein in H4IIE cells which express the PEPCK gene but not in the HTC or XC cells. No evidence for a repressor protein or for phased nucleosome binding to the PEPCK promoter in HTC or XC cells could be detected. Genomic sequencing was used to determine if differential methylation of the PEPCK promoter could account for the lack of factor binding in HTC and XC nuclei. None of the 14 cytosine residues in CpG dinucleotides was methylated in H4IIE or rat liver DNA, all were methylated in rat sperm DNA, and 6 were methylated in HTC DNA; including the cytosine at position--90 within the BSE/CRE. Only one cytosine residue, at position--90, was methylated in XC DNA. Treatment of XC cells with 5-azacytidine resulted in loss of methylation at the--90 position yet this was insufficient to allow synthesis of a detectable amount of PEPCK mRNA.
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PMID:The interplay of ubiquitous DNA-binding factors, availability of binding sites in the chromatin, and DNA methylation in the differential regulation of phosphoenolpyruvate carboxykinase gene expression. 171 57

Previous studies have identified a region in the promoter of the gene for phosphoenolpyruvate carboxykinase (GTP) (PEPCK) (positions -460 to +73) containing the regulatory elements which respond to cyclic AMP, glucocorticoids, and insulin and confer the tissue- and developmental stage-specific properties to the gene. We report that CCAAT/enhancer-binding protein (C/EBP) binds to the cyclic AMP-responsive element CRE-1 as well as to two regions which have been previously shown to bind proteins enriched in liver nuclei. The DNase I footprint pattern provided by the recombinant C/EBP was identical to that produced by a 43-kDa protein purified from rat liver nuclear extracts, using a CRE oligonucleotide affinity column, which was originally thought to be the CRE-binding protein CREB. Transient contransfection experiments using a C/EBP expression vector demonstrated that C/EBP could trans activate the PEPCK promoter. The trans activation occurred through both the upstream, liver-specific protein-binding domains and the CRE. The CRE-binding protein bound only to CRE-1 and not to the upstream C/EBP-binding sites. The results of this study, along with physiological properties of C/EBP, indicate an important role for this transcription factor in providing the PEPCK gene with several of its regulatory characteristics.
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PMID:The role of the CCAAT/enhancer-binding protein in the transcriptional regulation of the gene for phosphoenolpyruvate carboxykinase (GTP). 214 22

We have previously identified a series of five DNase-I hypersensitive (HS) sites within and around the rat phosphoenolpyruvate carboxykinase (PEPCK) gene. The far upstream region has now been sequenced, and the tissue-specific HS site has been mapped more precisely at 4,800 base pairs upstream of the transcription start site of the PEPCK gene. DNA fragments that include the HS site were cloned upstream of various promoters to test whether these regions modulate transcription of the chloramphenicol acetyltransferase reporter gene. Chloramphenicol acetyltransferase activity was enhanced when the DNA fragment encompassing the upstream HS site was linked to various lengths of the PEPCK promoter or to the heterologous simian virus 40 promoter. This upstream region in conjunction with the proximal promoter, which may contain a tissue-specific element, conferred maximum activation in H4IIE hepatoma cells, which express the endogenous PEPCK gene. When these experiments were performed in XC cells, in which the gene is not expressed, transcriptional activation by the upstream element was still significant. Evidence of a specific protein-DNA interaction, using DNA mobility shift and DNase I footprinting assays, was obtained only when using H4IIE cell nuclear extracts. Competition assay showed that the interacting factor may be similar or identical to the liver-specific factor HNF3. We suggest that this protein factor binds to DNA within the HS site and interacts with the proximal promoter region to control tissue-specific high-level expression of the PEPCK gene.
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PMID:Interaction of a liver-specific factor with an enhancer 4.8 kilobases upstream of the phosphoenolpyruvate carboxykinase gene. 235 22

Cyclic AMP responsive elements (CRE) have been identified in several genes, including those encoding the alpha-subunit of glycoprotein hormones and the cytosolic form of phosphoenolpyruvate carboxykinase (PEPCK) from the rat. Common to the CRE in these genes is the palindromic sequence T(G/T)ACGTCA. Based upon the strong conservation of this element, we hypothesize that the CRE functions by binding a protein that has been conserved across tissue and species lines. Scatchard analysis of gel mobility shift assays indicate that a nuclear protein in extracts prepared from rat liver and from a human choriocarcinoma cell line binds with high affinity to the cAMP responsive element from either gene (Kd approximately 10(-10) M). In order to identify the critical nucleotides within the CRE from these two genes, a series of oligodeoxynucleotides containing systematic mutations was synthesized and tested for protein binding and transcriptional function. Mutations within the palindromic core of either CRE resulted in a marked loss of binding to the nuclear proteins. Sequences outside the 8-base pair element were less important for nuclear protein binding to the PEPCK CRE and were not important for the alpha-subunit CRE. The relative binding, as determined by gel shift assays, correlated with the ability to confer cAMP responsive transcription to a viral promoter in transfected choriocarcinoma cells. DNase I protection assays suggest that binding of the nuclear factor from rat liver to the PEPCK CRE is more efficient when the core sequence is present in the intact PEPCK promoter regulatory region as compared to the isolated CRE oligodeoxynucleotide. Collectively, these results indicate that the nuclear factors necessary for cAMP induction of transcription of the alpha-subunit and PEPCK genes are conserved between tissues and species. In addition to the conserved features of these cis- and trans-active elements, nonconserved sequences and other elements of the promoter regulatory region influence the affinity of the protein-DNA interaction.
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PMID:Characterization of the cAMP responsive elements from the genes for the alpha-subunit of glycoprotein hormones and phosphoenolpyruvate carboxykinase (GTP). Conserved features of nuclear protein binding between tissues and species. 246 42

Transcription of the gene for the cytosolic form of phosphoenolpyruvate carboxykinase (GTP) (EC 4.1.1.32) (PEPCK) from the rat is acutely regulated by a number of hormones, including glucagon (acting via cAMP), glucocorticoids, and insulin. In this study we demonstrate by DNase I footprinting that a region of the PEPCK promoter, extending from -460 to +73, contained eight protein binding domains. Two nuclear proteins protected adjacent sites from -121 to -99 and -96 to -77, which have been previously shown to be involved in maintaining the level of basal gene transcription and conferring cAMP responsiveness, respectively. Oligonucleotide competition studies suggested that the protein(s) binding to the cAMP-responsive element (CRE) occupies a second site at -147 to -130, which has a high degree of sequence homology to the CRE, and also binds to two other elements that show partial sequence homologies. The protein(s) which bound to these four elements copurified through oligonucleotide affinity chromatography, suggesting that the PEPCK promoter has four binding sites for the CRE-binding protein(s). Potential tissue-specific elements in the PEPCK promoter were identified by footprinting with nuclear extracts prepared from rat liver, kidney, brain, and spleen. The multiple protein-binding sites in this promoter-regulatory region reflect the complex transcriptional regulation that is characteristic of this gene.
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PMID:Identification of multiple protein binding domains in the promoter-regulatory region of the phosphoenolpyruvate carboxykinase (GTP) gene. 254 17

Promoter elements important for basal and cyclic AMP (cAMP)-regulated expression of the phosphoenolpyruvate carboxykinase (PEPCK) gene have been identified by analysis of a series of PEPCK promoter mutations in transfection experiments. Fusion genes containing wild-type and mutated PEPCK promoter sequences from -600 to +69 base pairs (bp) fused to the coding sequence for chloramphenicol acetyltransferase were studied. Internal deletion mutations that replaced specific bases with a 10-bp linker within the region from -129 bp to -18 bp of the PEPCK promoter were examined. In addition, wild-type and mutated DNA templates were used as probes in DNase I protection experiments to determine sites of protein-DNA interaction. The PEPCK promoter contains a binding site for nuclear factor 1-CAAT. Deletion of the 5' end of this binding site reduced the size of the DNase I footprint in this region but had no effect on promoter activity. In contrast, deletion or disruption of the 3' end of this binding site completely eliminated protein binding and reduced promoter activity by 50%. Deletion of core sequences of the cAMP regulatory element (CRE) resulted in loss of cAMP responsiveness and an 85% decrease in basal promoter activity, indicating that the CRE also functions as a basal stimulatory element. Mutation of the core sequence of the CRE resulted in loss of the DNase I footprint over the CRE. Internal deletions flanking the CRE showed no loss of induction by cAMP but did have reduced promoter activity. This delimits the CRE to an 18-bp region between nucleotides -100 and -82. Analysis of mutations that disrupted bases between the CRE and the initiation site identified a basal inhibitory element adjacent to a basal stimulatory element, both located just 3' of the CRE, as well as a basal stimulatory element coincident with the TATA consensus sequence centered at -27. These data demonstrate that several cis-acting elements are located within 130 nucleotides of the initiation site of the PEPCK gene and that the CRE is essential for both basal promoter activity and cAMP-regulated expression of this gene.
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PMID:Identification of basal and cyclic AMP regulatory elements in the promoter of the phosphoenolpyruvate carboxykinase gene. 285 Apr 95

The sensitivity to DNase I digestion of the gene encoding rat phosphoenolpyruvate carboxykinase (GTP) (EC 4.1.1.32) was assessed during development and prior to the onset of expression. This gene is resistant to DNase I digestion in nuclei isolated from livers of 19-day rat fetuses. Gradual acquisition of sensitivity of the phosphoenolpyruvate carboxykinase gene, which starts later than the 19th day of gestation and is completed by the 21st day, occurs before initiation of gene expression. As transcription of the phosphoenolpyruvate carboxykinase gene is not detected until birth, the events observed may represent a shift from a dormant to an active gene. Injection of N6,O2-dibutyryladenosine 3',5'-cyclic monophosphate into fetuses on the 19th day of gestation induces gene expression and sensitivity to DNase I digestion within 3 hr of treatment. While this short treatment does not affect the methylation pattern of the gene, longer treatment of fetuses (2 days) with dibutyryl-cAMP results in premature hypomethylation of the gene. A hierarchy of modifications of the phosphoenolpyruvate carboxykinase gene during development is discussed.
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PMID:Developmental acquisition of DNase I sensitivity of the phosphoenolpyruvate carboxykinase (GTP) gene in rat liver. 302 67

Previous studies have shown that the adipose tissue of young genetically obese Zucker rats was characterized by a coordinate overtranscription of lipogenic genes, suggesting that the fa mutation triggers transcription factor(s) acting in common on the promoters of these genes. To test this hypothesis, we developed a system of transient transfection of rat adipocytes with constructs containing glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and fatty acid synthetase (FAS) promoters fused to gene reporter CAT. Those transfected cells expressed high levels of promoter-driven chloramphenicol acetyltransferase (CAT) activity through correctly initiated transcription as shown by primer extension analysis. Using this system we found a direct effect of insulin on GAPDH and FAS gene expression in rat adipocytes. In transfected adipocytes from obese compared to lean rats, activity of GAPDH and FAS promoters fused to CAT, was 2.6- and 8-fold increased, respectively. In contrast when reporter gene activity was driven by either phosphoenolpyruvate carboxykinase or beta-actin promoter, no difference could be observed between lean and obese, pointing out the promoter specificity of genotype effect. 5' deletion analysis of GAPDH promoter allowed us to narrow down the fa responsive region to nucleotide -488-329. As assessed by gel retardation and DNase I footprinting analysis, adipocyte nuclear protein interactions to this 159-bp fragment were found to be identical and to footprint the same 20-bp sequence. This study pointed out that overexpression of GAPDH and FAS genes in adipose tissue of genetically obese rats relies on promoter activation, through a 159-bp cis-acting region within the GAPDH promoter. The effects of the fa mutation on trans-acting factors binding to this region remain to be identified.
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PMID:Evidence of increased glyceraldehyde-3-phosphate dehydrogenase and fatty acid synthetase promoter activities in transiently transfected adipocytes from genetically obese rats. 783 67

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

Previous, in vivo experiments have shown that an appropriate hormonal environment (high plasma insulin, low plasma glucagon) was unable to induce the accumulation of glucokinase mRNA in term fetal rat liver, whereas it was very efficient in the newly born rat. We have confirmed in the present study that insulin induced the accumulation of glucokinase mRNA in cultured hepatocytes from 1-day-old newborn rats, but not in cultured hepatocytes from 21-day-old fetuses. To identify regulatory regions of the glucokinase gene involved in the insulin response, we have scanned the glucokinase locus for DNase I hypersensitive sites in its in vivo conformation. We confirmed the presence of four liver-specific DNase I hypersensitive sites located in the 5' flanking region of the gene. Moreover, two additional hypersensitive sites, located at 2.5 kb and 3.5 kb upstream of the cap site were found but none of these new sites displayed inducibility by insulin. Finally, an increase of the sensitivity of hypersensitive site-1 and hypersensitive site-2 to DNase I correlates with the ability of insulin to induce glucokinase gene expression in cultured hepatocytes from 1-day-old rats, as observed in previous in vivo studies. This suggests that neither a prior exposure to insulin nor a simple aging of the fetal cells in the presence of the hormone in culture are instrumental for the full DNase-I hypersensitivity of the two proximal sites necessary for the neonatal response of the glucokinase gene to insulin. The proximal hypersensitive site-1, which is close to the transcription start site in the liver, does coincide with a sequence (designated IRSL) that is 80% identical to the phosphoenolpyruvate carboxykinase IRS and with a DNase-I footprint that has been identified overlapping this sequence. Nevertheless, functional analysis of this sequence suggested that it is unlikely that the insulin-response sequence like alone is sufficient to mediate the transcriptional effect of insulin on the hepatic glucokinase gene.
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PMID:Induction of the glucokinase gene by insulin in cultured neonatal rat hepatocytes. Relationship with DNase-I hypersensitive sites and functional analysis of a putative insulin-response element. 861 67


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