Transcriptional Regulation Study Of The New Promoter In The Fisrt Intron Of ORMDL3Gene

Objective To construct a luciferase reporter plasmid containing the new promoter in the first intron of ORMDL3gene, evaluate the promoter activity in eukaryotic cells, and to further investigate its transcriptional regulation.Methods The776bp fragment in the first intron of the new transcription start site upstream640bp to downstream136bp was amplified by PCR with human genomic DNA as a template and was directionally cloned into pGL3-Basic plasmid without promoter activity to construct the luciferase reporter plasmid pGL3B-p776. Transfection of HEK-293cells and Hela cells with the promoter-driven luciferase construct was performed to induce luciferase gene expression and calculate the relative luciferase activity unit (RLU). Promoter sequence around the transcription initiation site in the fist intron of ORMDL3gene was analyzed by using Promoter2.0Prediction software. With point mutations, chromatin immuno-precipitation (ChIP), transcriptional factor interference and overexpression plasmids transfected into Hela cells, we identified the location and features of the core promoter and its upstream regulatory region of alternative splicing isoforms of ORMDL3gene.Results It is successful to construct the plasmid containing the promoter in the first intron of ORMDL3gene. Promoter activity analysis showed that compared with normal pGL3-Basic plasmid, the new promoter activity was increased. The core promoter is located in front of new transcription start sites between-363bp to-166bp. The-363bp to-166bp fragment exhibited the maximal transcriptional activity. Spl/3sites were essential for maintaining the basal transcriptional activity of the promoter in the fist intron of ORMDL3gene. ChIP assays demonstrate the occupancy of the promoter by Sp1、Sp3in vivo. Overexpression of Spl or Sp3transactivated the promoter of the alternative splicing isoforms of ORMDL3gene, whereas Sp1or Sp3interference plasmid inhibited markedly the promoter’s activity.Conclusion The fragment upstream the transcription start site in the first intron of ORMDL3has promoter activity. The necessary regulatory elements area exists in the-363bp to-166bp region. Sp1and Sp3transcription factors were involved in their transcriptional regulation.