Saitta B, Chu M L
Department of Biochemistry and Molecular Biology, Jefferson Institute of Molecular Medicine, Jefferson Medical College, Thomas Jefferson University, Philadelphia, PA 19107.
Eur J Biochem. 1994 Jul 15;223(2):675-82. doi: 10.1111/j.1432-1033.1994.tb19040.x.
Our previous studies have demonstrated that the human alpha 2(VI) collagen gene produces four mRNA species with different 5'-untranslated regions [Saitta, B., Timpl, R. & Chu, M.-L. (1992) J. Biol. Chem. 267, 6188-6196]. The major mRNA species initiates from exon 1, located at the most 5' end, whereas three minor mRNAs start from an alternative exon, 1A, located 657 bp downstream of exon 1. In this study, we have investigated whether or not these different mRNAs are transcribed from two separate promoters. DNA fragments preceding exons 1 and 1A were fused with a reporter gene for chloramphenicol acetyl transferase (CAT) and transfected into human dermal fibroblasts and fibrosarcoma HT1080 cells. Strong CAT activity in both cell types was observed using a construct containing DNA from nucleotide -502 to + 115 preceding exon 1. The CAT activity of a construct containing nucleotide +514 to +894 preceding exon 1A was almost as high as that of the former construct, indicating the presence of two promoters, P1 and P2, preceding exons 1 and 1A, respectively. Transient transfection assays also identified positive and negative regulatory regions for the P1 promoter, located from nucleotide -2152 to -1384 and from nucleotide -1383 to -503, respectively. A negative regulatory region located at nucleotide +116 to +513 was found for the P2 promoter. This region strongly inhibits the P2 promoter in dermal fibroblasts, and thus may be responsible for the low expression of the endogenous exon-1A-containing mRNAs in these cells. Footprinting analysis of the two promoters with purified Sp1 protein and AP2 protein extract showed several sites of DNA-protein interaction. The specificity of these sites was confirmed by competition experiments using consensus Sp1 and AP2 oligonucleotides. The results thus demonstrate that the human alpha 2(VI) collagen gene contains two promoters, which are regulated by positive and negative cis-acting DNA elements and trans-acting factors.
