Purpose: To isolate and characterize the promoter of the human βIGH3 gene. Methods: Primer extension and CapSite Hunting methods were used to determine the transcription start sites (TSS) of the human βIGH3 gene. Putative transcription factor-binding sites and potential promoter regions were identified by online tools. Two clones containing 3 Kb and 1 Kb of the 5′-flanking region of the βIGH3 gene were isolated and their respective promoter activities were characterized. Various fusion constructs of βIGH3 promoter-luciferase reporter were made to transfect A549 cells. The responses of these fragments to TGF-β1 were also measured after being treated with TGF-β1 at different concentrations. Several human and nonhuman cell lines were also transfected with the 1 Kb βIGH3 promoter-reporter construct to compare the activity of the βIGH3 promoter in these cells. Results: The transcription start site of human βIGH3 mRNA was determined to be 65 bp upstream of the ATG start codon. Both the 3 Kb (-3011 to -1) and 1 Kb (-1000 to -1) fragments displayed strong and comparable promoter activity in transfected cells. Truncation analyses in A549 cells identified the nucleotide region from -336 to -1 as having high promoter activity (minimal promoter). The results also indicated that the nucleotide fragment from -1000 to -646 contained negative regulatory elements. Twenty ng/ml TGF-β1 upregulated the activity of the 1 Kb construct, but did not upregulate the activity of the -336 to -1 construct, suggesting that TGF-β1 responsive elements existed in the region from -1000 to -336. The 1 Kb construct universally demonstrated promoter activity in all cell lines tested. Conclusions: We identified the βIGH3 gene promoter with a distinct regulatory pattern in the 1 Kb region upstream of the ATG start codon. Further elucidation of the functions of this promoter region may facilitate understanding of βIGH3 and its related corneal dystrophies.
|Number of pages||10|
|State||Published - May 18 2004|