A PROXIMAL ELEMENT WITHIN THE HUMAN ALPHA-2(I) COLLAGEN (COL1A2) PROMOTER, DISTINCT FROM THE TUMOR-NECROSIS-FACTOR-ALPHA RESPONSE ELEMENT, MEDIATES TRANSCRIPTIONAL REPRESSION BY INTERFERON-GAMMA

Previous studies have shown that interferon-gamma (IFN-gamma) inhibits type I collagen gene expression through both transcriptional and post -transcriptional mechanisms (Kahari et al., 1990). In the present stud y, using transient cell transfections of human dermal fibroblast cultu res with a series of 5' deletion promoter/CAT reporter gene constructs , we have identified the IFN-gamma-response element of the human alpha 2(I) collagen gene (COL1A2) promoter. Specifically, we have identifie d a segment of the proximal promoter region, located between nucleotid es -161 and -125 relative to the transcription start site, as critical for down-regulation of COL1A2 promoter activity by IFN-gamma. This IF N-gamma response element (IgRE) is clearly distinct from the previousl y described tumor necrosis factor-a response element (TaRE) located be tween nucleotides -265 and -241 of the COL1A2 promoter, a difference w hich is likely to explain the additive inhibitory effect of these two cytokines. The inhibitory effect of IFN-gamma was dose-dependent and r apidly induced, requiring less than 5 min exposure of fibroblast cultu res. Gel mobility shift assays indicated that a highly specific nuclea r protein complex bound to this 37-base pair region of promoter. Compe tition experiments with oligonucleotides spanning discrete segments of this promoter region mapped the binding element within a distinctive pyrimidine-rich sequence. Point mutations within the latter revealed t hat this element plays a crucial role not only in the IFN-gamma respon se, but also in the basal activity of the proximal promoter. Substitut ion mutations within the IgRE of the -161/CAT construct attenuated the promoter response to IFN-gamma, as measured in transient cell transfe ctions, and eliminated specific DNA/protein complex formation, as meas ured by gel mobility shift assay. UV-crosslinking experiments indicate d that two DNA/protein complexes were formed with the IgRE, with molec ular weights around 55 kDa and 30 kDa, corresponding to proteins of si milar to 30 kDa and similar to 6 kDa, respectively. Our results furthe r clarify the molecular mechanisms involved in the regulation of type I collagen gene expression by IFN-gamma.