| Objective:To predict one of cancer-testis antigens OY-TES-1 promoter region and confirm its core promoter area,understand the regulation of transcription factor Spl to the OY-TES-1 gene promoter in hepatoma cells and influence of methylation to the OY-TES-1 promoter activity.Method:(1)By using a variety of bioinformatics softwares online,we accessed the 5' up stream sequences and full-length sequences of OY-TES-1,then predicted OY-TES-1 promoter region and transcription factor binding sites.(2)By detecting the luciferase reporter gene vector activity,'we confirmed the OY-TES-1 core promoter area and analyzed the regulation of the transcription factor Spl to the OY-TES-1 promoter;Using Chromatin immunoprecipitation method to understand the regulatory mechanisms of Spl to OY-TES-1.(3)Using CpG Methyltransferase(M.SssI)treat the OY-TES-1 core promoter vector and compare the activity change between the treated OY-TES-1 promoter vector and untreated promoter.Result:(1)According to various bioinformatics softwares analysis,we designed four OY-TES-1 truncated promoter and found-184bp?+ 67bp region had the most activity,so we considered it was the core promoter region.(2)According to the luciferase reporter gene activity analysis,it showed that the negative regulation of transcription factor Sp1 to the OY-TES-1 promoter.Chromatin immunoprecipitation results revealed that Sp1 was directly binded on OY-TES-1 promoter region,it suggested that the OY-TES-1 promoter was directly regulated by transcription factor Sp1.(3)After OY-TES-1 promoter was treated by methylation transferase,its promoter activity was significantly decreased,it confirmed that methylation had a major impact on OY-TES-1 promoter activity.Conclusion:OY-TES-1 core promoter region is located on-184bp?+ 67bp;The transcription factor Spl has a certain inhibition to OY-TES-1 promoter and promoter methylation can significantly inhibit OY-TES-1 promoter activity. |
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