Transcriptional regulation of the rat apelin receptor gene: promoter cloning and identification of an Sp1 site necessary for promoter activity.
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.
O'Carroll
Anne-Marie
AM
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
Lolait
Stephen J
SJ
Howell
Gillian M
GM
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
D000075244:Apelin Receptors; C491284:Aplnr protein, rat; D043562:Receptors, G-Protein-Coupled; D016329:Sp1 Transcription Factor; D004958:Estradiol; D003907:Dexamethasone; D004247:DNA