title: 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.
abstract: 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.
authors: Department of Pharmacology, Case Western Reserve University School of Medicine, Cleveland, Ohio 44106.
authors: J A
authors: JA
authors: Bokar
authors: W J
authors: WJ
authors: Roesler
authors: G R
authors: GR
authors: Vandenbark
authors: D M
authors: DM
authors: Kaetzel
authors: R W
authors: RW
authors: Hanson
authors: J H
authors: JH
authors: Nilson
mesh_terms: D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism; D002822:Choriocarcinoma; D000242:Cyclic AMP / Q000502:physiology*; D004268:DNA-Binding Proteins / Q000378:metabolism*; D005260:Female; D005796:Genes; D015292:Glycoprotein Hormones, alpha Subunit / Q000235:genetics*; D006801:Humans; D008099:Liver / Q000378:metabolism; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000378:metabolism*; D010729:Phosphoenolpyruvate Carboxykinase (GTP) / Q000235:genetics*; D011247:Pregnancy; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D051381:Rats; D014594:Uterine Neoplasms
chemical_list: D004268:DNA-Binding Proteins; D015292:Glycoprotein Hormones, alpha Subunit; D009687:Nuclear Proteins; D000242:Cyclic AMP; D010729:Phosphoenolpyruvate Carboxykinase (GTP)