TY - JOUR
T1 - Transcriptional regulation of the rat apelin receptor gene
T2 - Promoter cloning and identification of an Sp1 site necessary for promoter activity
AU - O'Caroll, Anne Marie
AU - Lolait, Stephen J.
AU - Howell, Gillian M.
PY - 2006/2
Y1 - 2006/2
N2 - 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.
AB - 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.
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U2 - 10.1677/jme.1.01927
DO - 10.1677/jme.1.01927
M3 - Article
C2 - 16461940
AN - SCOPUS:33244469279
SN - 0952-5041
VL - 36
SP - 221
EP - 235
JO - Journal of Molecular Endocrinology
JF - Journal of Molecular Endocrinology
IS - 1
ER -