Regulation of the Human Secretin Gene Is Controlled by the Combined Effects of CpG Methylation, Sp1/Sp3 Ratio, and the E-Box Element

• Published in: Molecular endocrinology (Baltimore, Md.) Md.), 2004-07, Vol.18 (7), p.1740-1755
• Main Authors: Lee, Leo Tsz-On, Tan-Un, Kian-Cheng, Pang, Ronald Ting-Kai, Lam, David Tai-Wai, Chow, Billy Kwok-Chong
• Format: Article
• Language: English
• Subjects: 5' Flanking Region; Animals; Base Sequence; Basic Helix-Loop-Helix Transcription Factors; CpG Islands; Deoxycytidine - pharmacology; DNA Methylation; DNA-Binding Proteins - genetics; DNA-Binding Proteins - metabolism; Drosophila - cytology; Drosophila - genetics; E-Box Elements; Gene Expression Regulation - drug effects; Humans; Molecular Sequence Data; Nerve Tissue Proteins - genetics; Nerve Tissue Proteins - metabolism; Plicamycin - pharmacology; Promoter Regions, Genetic; Secretin - drug effects; Secretin - genetics; Secretin - metabolism; Sp1 Transcription Factor - genetics; Sp1 Transcription Factor - metabolism; Sp3 Transcription Factor; Transcription Factors - genetics; Transcription Factors - metabolism; Tumor Cells, Cultured
• ISSN: 0888-8809; 1944-9917
• DOI: 10.1210/me.2003-0461

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.