Characterization of the 5' transcriptional regulatory region of chicken HB-EGF gene

• Main Authors: Lei Yen-Ping, 雷燕萍
• Other Authors: Lin Nien-Tsung
• Format: Others
• Language: zh-TW
• Online Access: http://ndltd.ncl.edu.tw/handle/54057541379904632318

碩士 === 慈濟大學 === 醫學研究所 === 89 === The HB-EGF (heparin-binding epidermal growth factor-like growth factor) is a heparin-binding member of the EGF family. HB-EGF has been implicated in a variety of normal physiological processes (such as blastocyst implantation and wound healing) and pathological processes (such as tumorigenesis, and atherosclerosis). We previously demonstrated that HB-EGF was an oncogenic target activated by avian oncogene v-jun. To understand transcriptional regulation of HB-EGF gene, we tried to isolate and characterize the 5’ upstream regulatory region of avian HB-EGF gene. A 12-kb genomic fragment, hybridizing with probes derived from 5’ coding sequence of HB-EGF, was isolated from a genomic DNA library of chicken embryos. Sequence analysis demonstrated this genomic clone is composed of the 4.3-kb 5’ upstream region and the complete coding sequence of HB-EGF gene. RT-PCR results showed that chicken HB-EGF gene is organized into six exons and five introns. The overall structure of the putative chicken HB-EGF promoter region shares low homology with other known HB-EGF promoters, only the absence of TATA and CAT boxes, and the presence of Sp1 sites are common among them. A series of promoter/reporter constructs were created in which either the 4.3-kb putative promoter region or various deletion fragments was cloned into the upstream of the reporter gene. Reporter assays in both JEG-3 cells and DF-1 cells indicate the 4.3-kb fragment can function as a promoter. Although classical AP-1 sites are not found within the 4.3-kb fragment, Jun proteins were shown to enhance the promoter activity. The transcriptional start site for HB-EGF gene was determined by primer extension analysis and S1 protection assay. The results from both experiments conclude that the transcription start site locate at position 236 nucleotide upstream of ATG. To further analyze the potential role of the putative Sp1-binding sites in the regulation of the chicken HB-EGF promoter activity, a series of Sp-1 sites-related mutants were created and tested in reporter assays. The results indicate that the Sp1-binding sites are essential for promoter activity.