Transcriptional Regulation Study Of The Human ORMDL3Gene

Objective: To elucidate the molecular mechanisms involved in human ORMDL3regulation and identify the splicing isoform of ORMDL3gene.Methods: Transcriptional start site (TSS) and new splicing isoform of ORMDL3genewere revealed by5’-RACE and3’-RACE. The proximal minimal promoter wasidentified using a series of5’ deletion promoter plasmids in luciferase reporter assays.Transcription factors regulating ORMDL3gene were demonstrated by mutationalanalysis, RNA interference and overexpression experiments, ChIP and SequentialChIP assays. The CDS of ORMDL3and ORMDL3V1(a new splicing isoform ofORMDL3gene) were subcloned into the pEGFP-C1expression vector to produceORMDL3or ORMDL3V1protein fused to enhanced green fluorescent protein(EGFP) at the N-terminus. The expressions of fusion proteins of EGFP-ORMDL3and EGFP-ORMDL3V1were detected by Western blot analysis. Subcellularlocalization of the protein of ORMDL3and ORMDL3V1were analyzed byfluorescence microscopy.Results: In HEK293cells, ORMDL3gene used multiple TSSs located between34and143bp upstream of the ATG. We constructed series5’ deletions of luciferasereporter plasmids and transfected into HEK293, HeLa and A549cells. Luciferaseassays revealed a60~201-fold increased promoter activity of the pGL-1318/+58ascompared to the empty vector in three cell lines. When the sequences were deletedfrom-1318bp to-84bp, the-84bp truncated fragment still exhibited the hightranscriptional activity. Further5’deletion from-84to-8bp, this construct(pGL-8/+58) had little promoter activity in three cell lines, these results indicated thatthe proximal minimal promoter of ORMDL3gene was located within the region-84/+58relative to the TSS. Using web software TFSEARCH and Genomatix, wefound that it contained Ets-1, SRY, p300, WHNF, SP1(A and B), AhR/Arnt, EIk-1,NRF-1and CREB transcription factor binding sites. Mutational analysisdemonstrated that Ets-1, p300or CREB binding site were essential for maintaining the basal transcriptional activity of the human ORMDL3promoter. RNA interferenceand overexpression experiments, ChIP and Sequential ChIP assays revealed thatORMDL3gene was cooperatively regulated by multiple transcription factors,including Ets-1, p300and CREB. Applying5’-RACE and3’-RACE, we isolated andcharacterized a splicing isoform of ORMDL3, ORMDL3V1, from Hela cells.ORMDL3V1skipped the second exon of the wild-type ORMDL3gene. The predictedprotein sequences of this isoform lack59amino acids in the N-terminus of thewild-type ORMDL3protein. RT-PCR assay showed that the mRNA levels ofORMDL3V1are higher in leukocyte, spleen, thymus and Hela cells, lower in liver,brain, colon, lung, kidney, ovary and testis. No mRNA expression is found inpancreas, heart, placenta, skeletal muscle, prostatel, small intestine, HEK293, HL60,fibroblast and endometrial cells. Western blot analysis detected a~38kDaEGFP-ORMDL3V1fusion protein. Fluorescence microscopy demonstrated that bothORMDL3V1and ORMDL3are almost exclusively expressed and localized in thecytoplasm of HEK293cells.Conclusion: The proximal minimal promoter of ORMDL3gene was located withinthe region-84/+58relative to the TSS. Ets-1, p300and CREB binding to thepromoter region drive the ORMDL3transcription. A splicing isoform of ORMDL3,ORMDL3V1, was identified. Fluorescence microscopy demonstrated that it localizedin the cytoplasm.