title: Transcriptional regulation of the rat apelin receptor gene: promoter cloning and identification of an Sp1 site necessary for promoter activity.
abstract: The genomic structure and transcriptional regulation of the rat apelin receptor (APJR) were analysed by rapid amplification of 5' cDNA ends (5'-RACE), transient expression assays and DNA-protein interaction. Analysis of the 5'-flanking region of a rat genomic clone shows no TATA box, but a putative CAAT box and several putative binding sites for transcription factors are present. Two transcriptional start sites were identified by 5'-RACE, RNase protection and primer extension analyses. Promoter activity was exhibited in the APJR- expressing SH-SY5Y cell line as well as in COS-7 and Chinese hamster ovary (CHO-K1) cells. Consecutive 5'-deletion analysis revealed the highest promoter activity in a region between bp -966 and -165. DNaseI footprint analysis revealed seven protected regions and electrophoretic mobility shift, super-shift and competition assays identified individual DNA-protein complexes capable of binding Sp1, estrogen receptor (ER)alpha, glucocorticoid receptor and CCAAT enhancer binding protein (C/EBP)gamma transcription factors. Site-directed mutagenesis identified an individual Sp1 motif that plays a major role in activation of the APJR promoter and also demonstrated constitutive transcriptional regulation of the promoter by estrogen and glucocorticoid receptors. Promoter regulation by the cAMP-dependent signal cascade was also shown.
authors.affiliation: Henry Wellcome Laboratories for Integrative Neuroscience and Endocrinology (LINE), Dorothy Hodgkin Building, Whitson Street, University of Bristol, Bristol BS1 3NY, UK. A.M.OCarroll@bristol.ac.uk
authors.forename: Anne-Marie
authors.initials: AM
authors.lastname: O'Carroll
authors.forename: Stephen J
authors.initials: SJ
authors.lastname: Lolait
authors.forename: Gillian M
authors.initials: GM
authors.lastname: Howell
mesh_terms: D000818:Animals; D000075244:Apelin Receptors; D001483:Base Sequence; D016466:CHO Cells; D019556:COS Cells; D002522:Chlorocebus aethiops; D006224:Cricetinae; D004247:DNA; D018983:DNA Footprinting; D003907:Dexamethasone / Q000494:pharmacology; D024202:Electrophoretic Mobility Shift Assay; D004958:Estradiol / Q000494:pharmacology; D005786:Gene Expression Regulation / Q000187:drug effects; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011401:Promoter Regions, Genetic; D051381:Rats; D043562:Receptors, G-Protein-Coupled / Q000235:genetics*; D016329:Sp1 Transcription Factor / Q000502:physiology*; D014158:Transcription, Genetic / Q000187:drug effects
chemical_list: D000075244:Apelin Receptors; C491284:Aplnr protein, rat; D043562:Receptors, G-Protein-Coupled; D016329:Sp1 Transcription Factor; D004958:Estradiol; D003907:Dexamethasone; D004247:DNA