CpG methylation and transcription factors Sp1 and Sp3 regulate the expression of the human secretin receptor gene.
The human secretin receptor (hSR) is an important glycoprotein receptor for regulating the secretion of pancreatic bicarbonate, water, and electrolytes. In this study we investigated the transcriptional regulation of the hSR gene. A minimal 106-bp promoter was identified, and it contains two GC boxes (GC box-A, -240 to -226; and GC box-B, -203 to -194, from the translation start site). EMSA and supershift analyses showed that both GC boxes interact with Sp1 and Sp3 transcription factors. Transient transfection in pancreas-derived human pancreatic ductule carcinoma (PANC)-1 and bovine pancreatic duct-1 cells showed that mutation of either GC box-A or -B reduced the promoter strength by 56-67%, whereas mutation of both GC boxes caused more than 90% reduction of promoter activity. Cotransfections of the hSR promoter with Sp1 and Sp3 expression vectors in Sp-deficient Drosophila SL-2 Schneider cells further demonstrated that the ratio of Sp1 to Sp3 is the key mechanism to modulate hSR gene expression. The methylation statuses of 27 CpG sites within the promoter region (-400 to -151 bp) were assessed in various human pancreas and liver cell lines. The hSR promoter is unmethylated (CAPAN-1, human pancreatic adenocarcinoma) or partially methylated (PANC-1 and HPAC, human pancreatic adenocarcinoma) in hSR-expressing cell lines but is completely methylated in hSR nonexpressing HepG2 cells. Methyltransferase inhibitor 5-aza-2'deoxycytidine increased hSR gene expression level in PANC-1 cells and induced hSR gene expression in HepG2 cells. Together, our study shows that, in addition to Sp1 and Sp3, promoter methylation also plays a role in the regulation of hSR gene expression.
Pang
Ronald Ting-Kai
RT
Department of Zoology, University of Hong Kong, Special Administrate Region, People's Republic of China.
Lee
Leo Tsz-On
LT
Ng
Samuel Sai-Ming
SS
Yung
Wing-Ho
WH
Chow
Billy Kwok-Chong
BK
D000230:Adenocarcinoma / Q000188:drug therapy / Q000235:genetics; D000818:Animals; D001374:Azacitidine / Q000494:pharmacology; D001482:Base Composition; D001483:Base Sequence; D002417:Cattle; D002478:Cells, Cultured; D018899:CpG Islands; D003596:Cytosine / Q000378:metabolism; D019175:DNA Methylation; D015254:DNA Modification Methylases / Q000037:antagonists & inhibitors; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D004330:Drosophila / Q000166:cytology / Q000235:genetics; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005786:Gene Expression Regulation; D006801:Humans; D008969:Molecular Sequence Data; D010190:Pancreatic Neoplasms / Q000188:drug therapy / Q000235:genetics; D011401:Promoter Regions, Genetic / Q000235:genetics; D043562:Receptors, G-Protein-Coupled; D011964:Receptors, Gastrointestinal Hormone / Q000187:drug effects / Q000235:genetics* / Q000378:metabolism; D012045:Regulatory Sequences, Nucleic Acid; D016329:Sp1 Transcription Factor / Q000235:genetics / Q000378:metabolism*; D051705:Sp3 Transcription Factor; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism*
D004268:DNA-Binding Proteins; D004791:Enzyme Inhibitors; D043562:Receptors, G-Protein-Coupled; D011964:Receptors, Gastrointestinal Hormone; C497121:SP3 protein, human; D016329:Sp1 Transcription Factor; D014157:Transcription Factors; C029937:secretin receptor; D051705:Sp3 Transcription Factor; D003596:Cytosine; D015254:DNA Modification Methylases; D001374:Azacitidine