title: Evidence that an additional conserved element with the consensus C/GAGA/C is essential for maximal responsiveness of the cyclic AMP enhancer.
abstract: We analyzed the ability of cyclic AMP-response element binding proteins (CREBs) to interact with the CRE sequences derived from different genes and examined the role of sequences flanking the core CRE element in rendering cAMP-responsiveness to the enhancer. We were able to detect reproducibly, sing the Southwestern blotting technique, five major CREB factors of molecular weights 56, 47, 40, and 36-34 kDa which were present in various rat tissues and cultured cells. The 34-40 kDa proteins (CREB-327/341) were able to bind to the CRE of cAMP-inducible genes (somatostatin, c-fos, E2A), but not to genes whose expression is not controlled by cAMP (glucagon, parathyroid hormone). The novel 47 kDa CREB had a high specificity for the core octameric CRE sequence and it bound equally well to the consensus CRE of cAMP-inducible and noninducible genes. On the other hand, the 47 kDa CREB did not bind at all to the phorbol ester response element (TRE), whereas the 56 kDa protein, reminiscent of the CRE-BP1 protein, could bind to both elements. A computer aided sequence analysis of cAMP-inducible gene promoters revealed the presence of an additional conserved element starting 4-6 nucleotides 3' to the octomer with the consensus C/GAGA/C. We have shown this element to be essential for maximal cAMP-responsiveness of the enhancer in transient expression assays of CRE-CAT plasmid constructs indicating that the functional interaction of CREB proteins with the cAMP-inducible enhancer involves an additional 8-10 base pairs immediately downstream from the CRE core element.
authors.affiliation: Institute for Biological Sciences, National Research Council, Ottawa, Canada.
authors.forename: J
authors.initials: J
authors.lastname: Kwast-Welfeld
authors.forename: P R
authors.initials: PR
authors.lastname: Walker
authors.forename: J F
authors.initials: JF
authors.lastname: Whitfield
authors.forename: M
authors.initials: M
authors.lastname: Sikorska
mesh_terms: D000818:Animals; D001483:Base Sequence; D001667:Binding, Competitive; D015500:Chloramphenicol O-Acetyltransferase / Q000378:metabolism; D016384:Consensus Sequence; D000242:Cyclic AMP / Q000378:metabolism; D017362:Cyclic AMP Response Element-Binding Protein / Q000378:metabolism*; D004247:DNA; D004742:Enhancer Elements, Genetic; D004790:Enzyme Induction; D006801:Humans; D008969:Molecular Sequence Data; D016760:Proto-Oncogene Proteins c-fos / Q000235:genetics / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D012045:Regulatory Sequences, Nucleic Acid; D013004:Somatostatin / Q000378:metabolism; D014162:Transfection
chemical_list: D017362:Cyclic AMP Response Element-Binding Protein; D016760:Proto-Oncogene Proteins c-fos; D013004:Somatostatin; D004247:DNA; D000242:Cyclic AMP; D015500:Chloramphenicol O-Acetyltransferase