Identification of the promoter region of the human βIGH3gene

[摘要] Purpose: To isolate and characterize the promoter of the humanβIGH3 gene.Methods: Primer extension and CapSite Hunting methods were used todetermine the transcription start sites (TSS) of the human βIGH3 gene. Putative transcription factor-binding sites and potentialpromoter regions were identified by online tools. Two clones containing3 Kb and 1 Kb of the 5'-flanking region of the βIGH3 gene wereisolated and their respective promoter activities were characterized.Various fusion constructs of βIGH3 promoter-luciferase reporterwere made to transfect A549 cells. The responses of these fragments toTGF-β1 were also measured after being treated with TGF-β1 atdifferent concentrations. Several human and nonhuman cell lines werealso transfected with the 1 Kb βIGH3 promoter-reporterconstruct to compare the activity of the βIGH3 promoter inthese cells.Results: The transcription start site of human βIGH3 mRNAwas determined to be 65 bp upstream of the ATG start codon. Both the 3Kb (-3011 to -1) and 1 Kb (-1000 to -1) fragments displayed strong andcomparable promoter activity in transfected cells. Truncation analysesin A549 cells identified the nucleotide region from -336 to -1 as havinghigh promoter activity (minimal promoter). The results also indicatedthat the nucleotide fragment from -1000 to -646 contained negativeregulatory elements. Twenty ng/ml TGF-β1 upregulated the activityof the 1 Kb construct, but did not upregulate the activity of the -336to -1 construct, suggesting that TGF-β1 responsive elements existedin the region from -1000 to -336. The 1 Kb construct universallydemonstrated promoter activity in all cell lines tested.Conclusions: We identified the βIGH3 gene promoter with adistinct regulatory pattern in the 1 Kb region upstream of the ATG startcodon. Further elucidation of the functions of this promoter region mayfacilitate understanding of βIGH3 and its related cornealdystrophies.

[学科分类] 生物化学/生物物理