Identification of the promoter region of the human betaIGH3 gene.
OBJECTIVE
To isolate and characterize the promoter of the human betaIGH3 gene.


METHODS
Primer extension and CapSite Hunting methods were used to determine the transcription start sites (TSS) of the human betaIGH3 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 betaIGH3 gene were isolated and their respective promoter activities were characterized. Various fusion constructs of betaIGH3 promoter-luciferase reporter were made to transfect A549 cells. The responses of these fragments to TGF-beta1 were also measured after being treated with TGF-beta1 at different concentrations. Several human and nonhuman cell lines were also transfected with the 1 Kb betaIGH3 promoter-reporter construct to compare the activity of the betaIGH3 promoter in these cells.


RESULTS
The transcription start site of human betaIGH3 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-beta1 upregulated the activity of the 1 Kb construct, but did not upregulate the activity of the -336 to -1 construct, suggesting that TGF-beta1 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 betaIGH3 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 betaIGH3 and its related corneal dystrophies.
Yuan
Ching
C
Department of Ophthalmology, University of Minnesota, Minneapolis, MN 55455, USA.
Yang
Mei-Chuan
MC
Zins
Emily J
EJ
Boehlke
Christopher S
CS
Huang
Andrew J W
AJ
D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D003001:Cloning, Molecular; D016326:Extracellular Matrix Proteins / Q000235:genetics* / Q000378:metabolism; D006801:Humans; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D011401:Promoter Regions, Genetic / Q000235:genetics*; D012333:RNA, Messenger / Q000235:genetics*; D014157:Transcription Factors / Q000235:genetics; D024363:Transcription Initiation Site; D014158:Transcription, Genetic; D014162:Transfection; D016212:Transforming Growth Factor beta / Q000235:genetics* / Q000378:metabolism / Q000494:pharmacology; D053773:Transforming Growth Factor beta1; D000099222:betaIG-H3 Protein
D016326:Extracellular Matrix Proteins; D012333:RNA, Messenger; D014157:Transcription Factors; D016212:Transforming Growth Factor beta; D053773:Transforming Growth Factor beta1; D000099222:betaIG-H3 Protein; C508960:TGFB1 protein, human