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 (Kahäri et al., 1990). In the present study, using transient cell transfections of human dermal fibroblast cultures 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 identified a segment of the proximal promoter region, located between nucleotides -161 and -125 relative to the transcription start site, as critical for down-regulation of COL1A2 promoter activity by IFN-gamma. This IFN-gamma response element (IgRE) is clearly distinct from the previously described tumor necrosis factor-alpha response element (TaRE) located between nucleotides -265 and -241 of the COL1A2 promoter, a difference which is likely to explain the additive inhibitory effect of these two cytokines. The inhibitory effect of IFN-gamma was dose-dependent and rapidly induced, requiring less than 5 min exposure of fibroblast cultures. Gel mobility shift assays indicated that a highly specific nuclear protein complex bound to this 37-base pair region of promoter. Competition 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 that this element plays a crucial role not only in the IFN-gamma response, but also in the basal activity of the proximal promoter. Substitution mutations within the IgRE of the -161/CAT construct attenuated the promoter response to IFN-gamma, as measured in transient cell transfections, and eliminated specific DNA/protein complex formation, as measured by gel mobility shift assay. UV-crosslinking experiments indicated that two DNA/protein complexes were formed with the IgRE, with molecular weights around 55 kDa and 30 kDa, corresponding to proteins of approximately 30 kDa and approximately 6 kDa, respectively. Our results further clarify the molecular mechanisms involved in the regulation of type I collagen gene expression by IFN-gamma.
Higashi
K
K
Department of Dermatology and Cutaneous Biology, Jefferson Medical College, Philadelphia, Pennsylvania 19107, USA.
Kouba
D J
DJ
Song
Y J
YJ
Uitto
J
J
Mauviel
A
A
D001667:Binding, Competitive / Q000235:genetics / Q000528:radiation effects; D002478:Cells, Cultured; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism / Q000528:radiation effects; D005347:Fibroblasts; D006801:Humans; D007371:Interferon-gamma / Q000378:metabolism / Q000494:pharmacology* / Q000528:radiation effects; D008297:Male; D016297:Mutagenesis, Site-Directed; D011401:Promoter Regions, Genetic / Q000187:drug effects / Q000528:radiation effects; D011485:Protein Binding / Q000235:genetics / Q000528:radiation effects; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism; D014158:Transcription, Genetic / Q000187:drug effects* / Q000528:radiation effects; D014409:Tumor Necrosis Factor-alpha / Q000235:genetics*; D014466:Ultraviolet Rays
D004268:DNA-Binding Proteins; D014157:Transcription Factors; D014409:Tumor Necrosis Factor-alpha; D007371:Interferon-gamma