Identification of the promoter region of the human betaIGH3 gene

Mol Vis. 2004 May 18:10:351-60.


Purpose: 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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Base Sequence
  • Cell Line
  • Cloning, Molecular
  • Extracellular Matrix Proteins / genetics*
  • Extracellular Matrix Proteins / metabolism
  • Humans
  • Molecular Sequence Data
  • Polymerase Chain Reaction
  • Promoter Regions, Genetic / genetics*
  • RNA, Messenger / genetics*
  • Transcription Factors / genetics
  • Transcription Initiation Site*
  • Transcription, Genetic
  • Transfection
  • Transforming Growth Factor beta / genetics*
  • Transforming Growth Factor beta / metabolism
  • Transforming Growth Factor beta / pharmacology
  • Transforming Growth Factor beta1


  • Extracellular Matrix Proteins
  • RNA, Messenger
  • TGFB1 protein, human
  • Transcription Factors
  • Transforming Growth Factor beta
  • Transforming Growth Factor beta1
  • betaIG-H3 protein