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

17 Citations (Scopus)

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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Glucocorticoid Receptors
Estrogen Receptors
Organism Cloning
Transcription Factors
CCAAT-Enhancer-Binding Proteins
TATA Box
CHO Cells
5' Flanking Region
DNA
Site-Directed Mutagenesis
Cricetulus
Genes
Ovary
Proteins
Complementary DNA
Clone Cells
Binding Sites
Cell Line
rat Aplnr protein
angiogenin

ASJC Scopus subject areas

  • Molecular Biology
  • Endocrinology

Cite this

Transcriptional regulation of the rat apelin receptor gene : Promoter cloning and identification of an Sp1 site necessary for promoter activity. / O'Caroll, Anne Marie; Lolait, Stephen J.; Howell, Gillian M.

In: Journal of Molecular Endocrinology, Vol. 36, No. 1, 01.02.2006, p. 221-235.

Research output: Contribution to journalArticle

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