Mechanisms underlying the induction of the putative human tumor suppressor GPRC5A by retinoic acid.
Retinoic acid regulates the expression of genes involved in cell proliferation, differentiation and survival by direct control of gene transcription via activation of nuclear retinoid receptors bound to response elements in the promoters of target genes or by indirect mechanisms. Herein, we investigated the mechanism by which retinoic acid induces the expression of the human tumor suppressor GPRC5A. The proximal 5' upstream region of the GPRC5A gene was found to contain two potential RAR/RXR binding sites (RAREs) and one VDR/RXR binding site with direct repeat 5 (DR5) motifs designated DR5I (-489 to -473), DR5II (-136 to -120) and DR5III (-81 to -65). DR5II and DR5III but not DR5I were conserved among vertebrates. However, only DR5III (5'-TGT CCC TCT GCT CAC CC-3') was found to be the functional RARE for mediating induction of GPRC5A as indicated by electrophoretic mobility shift assay using wild type and mutated synthetic oligonucleotides representing different fragments of the promoter for competition with retinoic acid receptor beta RARE. Chromatin immunoprecipitation assay confirmed the binding of retinoic acid receptors alpha and gamma and retinoid X receptors alpha and beta to DR5III in intact cells. These results demonstrate the importance of functional analysis for validating the activity of predicted response elements.
Ye
Xiaofeng
X
Department of Thoracic/Head and Neck Medical Oncology, University of Texas M.D. Anderson Cancer Center, Houston, TX 77030, USA.
Tao
Qingguo
Q
Wang
Yafan
Y
Cheng
Yijun
Y
Lotan
Reuben
R
D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D045744:Cell Line, Tumor; D017124:Conserved Sequence; D004247:DNA / Q000235:genetics / Q000378:metabolism; D005091:Exons; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects; D017930:Genes, Reporter; D006801:Humans; D007438:Introns; D008156:Luciferases / Q000378:metabolism; D008969:Molecular Sequence Data; D009281:Naphthalenes / Q000494:pharmacology; D009363:Neoplasm Proteins / Q000235:genetics / Q000378:metabolism*; D010957:Plasmids / Q000235:genetics; D011401:Promoter Regions, Genetic / Q000235:genetics; D011485:Protein Binding / Q000235:genetics; D012333:RNA, Messenger / Q000235:genetics; D043562:Receptors, G-Protein-Coupled / Q000235:genetics / Q000378:metabolism*; D018168:Receptors, Retinoic Acid / Q000037:antagonists & inhibitors / Q000378:metabolism; D000072482:Retinoic Acid Receptor alpha; D047488:Retinoid X Receptors / Q000235:genetics / Q000378:metabolism; D012689:Sequence Homology, Nucleic Acid; D016385:TATA Box / Q000235:genetics; D014157:Transcription Factors / Q000378:metabolism; D024363:Transcription Initiation Site; D014158:Transcription, Genetic / Q000187:drug effects; D014162:Transfection; D014212:Tretinoin / Q000378:metabolism / Q000494:pharmacology*; D000097768:Retinoic Acid Receptor gamma
D004247:DNA; D008156:Luciferases; D009281:Naphthalenes; D009363:Neoplasm Proteins; D012333:RNA, Messenger; D043562:Receptors, G-Protein-Coupled; D018168:Receptors, Retinoic Acid; D000072482:Retinoic Acid Receptor alpha; D047488:Retinoid X Receptors; D014157:Transcription Factors; D014212:Tretinoin; D000097768:Retinoic Acid Receptor gamma; C116760:GPRC5A protein, human; C000607071:RARA protein, human; C096920:AGN 193109