Interaction of nuclear factors with the promoter of the human pro-1̉(I) collagen gene

Abstract

At least thirteen types of collagen have been identified, each having a characteristic tissue distribution. Type I, the most - abundant collagen, consists of two a 1 and one a2 polypeptides. There is considerable evidence that expression of the genes coding for the type I subunits is regulated on the level of transcription. In the pro-a 1 (I) collagen gene, regulatory regions have been identified both upstream of the transcription initiation site and in the first intron, with the minimal promoter contained in 250 bp immediately upstream of the start site for transcription. This 250 bp region has been shown to be sufficient to direct transcription of heterologous genes in different systems (Rossouw et al, 1987; Rippe et al, 1989; Liska et al, 1990 and Karsentry and de Crombrugghe, 1990). In this study, the minimal promoter of the human pro-al(I) collagen gene is characterized. Transfection of human growth hormone under control of different promoters, revealed that even in the absence of an enhancer the pro-a 1 (I) collagen promoter 1s a strong promoter. The results also suggest that the promoter sequences contain information for cell-specific modulation of expression of the gene. Several DNA-protein binding assays performed with nuclear extracts from both expressing (fibroblasts) and non-expressing cells (lymphocytes) revealed differences in DNA-protein interactions with different cell extracts. These differences were observed at a pyrimidine stretch upstream of the CCAAT-boxes, around the duplicated and inverted CCAAT-boxes, and at a putative AP-2 site. Whereas DNAase I protection assays indicate that the pyrimidine stretch is bound by a factor present in fibroblasts, several DNAase I hypersensitive sites are induced here in the case of lymphocyte extracts. This DNAase I hypersensitivity suggests an alteration in DNA conformation in this region. The different but overlapping binding of nuclear factors to the CCAAT-boxes is suggestive of mutually exclusive binding of CCAAT-binding factors

as a possible mechanism for down-regulating the gene in nonexpressing cells. In addition, we observed binding at a putative AP- 2 site in the case of fibroblast extracts but not lymphocyte extracts. Similarities between fibroblast and lymphocyte extracts include binding to a putative Spl site and a factor of about 40 kd, named col-B, binding to a previously unidentified element upstream of the pyrimidine stretch.