Regulation of the human secretin gene is controlled by the combined effects of CpG methylation, Sp1/Sp3 ratio, and the E-box element.
To unravel the mechanisms that regulate the human secretin gene expression, in this study, we have used secretin-expressing (HuTu-80 cells, human duodenal adenocarcinoma) and non-secretin-expressing [PANC-1 (human pancreatic ductile carcinoma) and HepG2 (human hepatocellular carcinoma) cells] cell models for in vitro and in vivo analyses. By transient transfection assays, within the promoter region (-11 to -341 from ATG, relative to the ATG initiation codon), we have initially identified several functional motifs including an E-box and 2 GC-boxes. Results from gel mobility shift and chromatin immunoprecipitation assays confirmed further that NeuroD, E2A, Sp1, and Sp3 bind to these E- and GC-boxes in HuTu-80 cells in vitro and in vivo, whereas only high levels of Sp3 is observed to bind the promoter in HepG2 cells. In addition, overexpression of Sp3 resulted in a dose-dependent repression of the Sp1-mediated transactivation. Collectively, these data suggest that the Sp1/Sp3 ratio is instrumental to controlling secretin gene expression in secretin-producing and non-secretin-producing cells. The functions of GC-box and Sp proteins prompted us to investigate the possible involvement of DNA methylation in regulating this gene. Consistent with this idea, we found a putative CpG island (-336 to 262 from ATG) that overlaps with the human secretin gene promoter. By methylation-specific PCR, all the CpG dinucleo-tides (26 of them) within the CpG island in HuTu-80 cells are unmethylated, whereas all these sites are methylated in PANC-1 and HepG2 cells. The expressions of secretin in PANC-1 and HepG2 cells were subsequently found to be significantly activated by a demethylation agent, 5'-Aza-2' deoxycytidine. Taken together, our data indicate that the human secretin gene is controlled by the in vivo Sp1/Sp3 ratio and the methylation status of the promoter.
Lee
Leo Tsz-On
LT
Department of Zoology, University of Hong Kong, Pokfulam Road, Hong Kong, People's Republic of China.
Tan-Un
Kian-Cheng
KC
Pang
Ronald Ting-Kai
RT
Lam
David Tai-Wai
DT
Chow
Billy Kwok-Chong
BK
D024506:5' Flanking Region; D000818:Animals; D001483:Base Sequence; D051792:Basic Helix-Loop-Helix Proteins; D018899:CpG Islands; D019175:DNA Methylation; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D003841:Deoxycytidine / Q000494:pharmacology; D004330:Drosophila / Q000166:cytology / Q000235:genetics; D024721:E-Box Elements; D005786:Gene Expression Regulation / Q000187:drug effects; D006801:Humans; D008969:Molecular Sequence Data; D009419:Nerve Tissue Proteins / Q000235:genetics / Q000378:metabolism; D008926:Plicamycin / Q000494:pharmacology; D011401:Promoter Regions, Genetic; D012633:Secretin / Q000187:drug effects / Q000235:genetics* / Q000378:metabolism; D016329:Sp1 Transcription Factor / Q000235:genetics / Q000378:metabolism*; D051705:Sp3 Transcription Factor; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism*; D014407:Tumor Cells, Cultured
D051792:Basic Helix-Loop-Helix Proteins; D004268:DNA-Binding Proteins; D003841:Deoxycytidine; D009419:Nerve Tissue Proteins; D008926:Plicamycin; D012633:Secretin; D016329:Sp1 Transcription Factor; D051705:Sp3 Transcription Factor; D014157:Transcription Factors; C497121:SP3 protein, human; C407909:TCF3 protein, human; C093564:Neurogenic differentiation factor 1