Identification of the promoter region of the human βIGH3 gene

Ching Yuan, Mei Chuan Yang, Emily J. Zins, Christopher S. Boehlke, Andrew J W Huang

Research output: Contribution to journalArticlepeer-review

11 Scopus citations

Abstract

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.

Original languageEnglish (US)
Pages (from-to)351-360
Number of pages10
JournalMolecular Vision
Volume10
StatePublished - May 18 2004

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