Transcriptional regulation of the rat apelin receptor gene: Promoter cloning and identification of an Sp1 site necessary for promoter activity

Anne Marie O'Caroll, Stephen J. Lolait, Gillian M. Howell

Research output: Contribution to journalArticle

18 Scopus citations

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 by -966 and -165. DNasel 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)α, glucocorticoid receptor and CCAAT enhancer binding protein (C/EBP)γ 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.

Original languageEnglish (US)
Pages (from-to)221-235
Number of pages15
JournalJournal of Molecular Endocrinology
Volume36
Issue number1
DOIs
StatePublished - Feb 1 2006

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ASJC Scopus subject areas

  • Molecular Biology
  • Endocrinology

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