YY1 is a positive regulator of transcription of the Col1a1 gene.

Both cell-specific and ubiquitous transcription factors in fibroblasts have been identified as critical for expression of the Col1a1 gene, which encodes the alpha1 chain of type I collagen. Here, we report that Yin Yang 1 (YY1) binds to the Col1a1 promoter immediately upstream of the TATA box, and we examine the functional implications of YY1 binding for regulation of Col1a1 gene expression in BALBc/3T3 fibroblasts. The Col1a1 promoter region spanning base pairs (bp) -56 to -9 bound purified recombinant YY1 and the corresponding binding activity in nuclear extracts was supershifted using a YY1-specific antibody. Mutation of the TATA box to TgTA enhanced YY1 complex formation. Mutation analysis revealed two YY1 core binding sites at -40/-37 bp (YY1A) and, on the reverse strand, at -32/-29 bp (YY1B) immediately adjacent to the TATA box. In transfections using Col1a1-luciferase constructs, mutation of YY1A decreased activity completely (wild-type p350 (p350wt), -222/+113 bp) or partially (p130wt, -84 bp/+13 bp), whereas mutation of YY1B blocked the expression of both promoter constructs. Cotransfection with pCMV-YY1 increased p350wt and p130wt activities by as much as 10-fold, whereas antisense YY1 decreased constitutive expression and blocked the increased activity due to pCMV-YY1 overexpression. The mTgTA constructs were devoid of activity, arguing for a requirement for cognate binding of the TATA box-binding protein (TBP). Electrophoretic mobility shift assays performed under conditions permitting TBP binding showed that recombinant TBP/TFIID and YY1 could bind to the -56/-9 bp fragment and that YY1B was the preferred site for YY1 binding. Our results indicate that YY1 binds to the Col1a1 proximal promoter and functions as a positive regulator of constitutive activity in fibroblasts. Although YY1 is not sufficient for transcriptional initiation, it is a required component of the transcription machinery in this promoter.

Suppression of type I collagen gene expression by prostaglandins in fibroblasts is mediated at the transcriptional level.

BACKGROUND: Tissues undergoing a chronic inflammatory process, such as the synovium in rheumatoid arthritis, are characterized by the infiltration of lymphocytes of different subsets and activation of monocyte/macrophages. Interleukin-1 (IL-1), a monocyte/ macrophage product that stimulates synovial fibroblasts to produce matrix metalloproteinases (MMPs), prostaglandins, and other cytokines, also has profound effects on the synthesis of extracellular matrix components such as type I collagen. In previous studies, we have shown that synovial fibroblasts and chondrocytes isolated from human joint tissues are particularly sensitive to prostaglandins, which modulate the effects of IL-1 on collagen gene expression in an autocrine manner. MATERIALS AND METHODS: BALBc/3T3 fibroblasts were treated with IL-1 and prostaglandins in the absence and presence of indomethacin to inhibit endogenous prostaglandin biosynthesis. Collagen synthesis was analyzed by SDS-PAGE as [3H]proline-labeled, secreted proteins, and prostaglandin production and cyclic adenosine 3',5'-cyclic monophosphate (camp) content were assayed. The expression of type I collagen gene (Col1a1) promoter-reporter gene constructs was examined in transient transfection experiments, and the binding of nuclear factors to the Col1a1 promoter region spanning -222 bp/+ 116 bp was analyzed by DNase I footprinting and electrophoretic mobility shift (EMSA) assays. RESULTS: IL-1 increased the synthesis of type I and type III collagens in BALBc/3T3 fibroblasts; greater increases were observed when IL-1-stimulated synthesis of PGE2 was blocked by indomethacin. Transient transfection experiments demonstrated dose-dependent inhibition of the-222 bp Col1a1 promoter by exogenously added prostaglandins with the order of potency of PGF2alpha > PGE2 > PGE1 DNase I footprinting showed increased protection, which extended from the region immediately upstream of the TATA box, owing to the binding of nuclear factors from PGE2- or PGE1-treated BALBc/3T3 cells. EMSA analysis showed zinc-dependent differences in the binding of nuclear factors from untreated and prostaglandin-treated cells to the -84 bp/-29 bp region of the Col1a1 promoter. CONCLUSIONS: These results show that the inhibition of Col1a1 expression by IL-1 in fibroblasts is mediated by prostaglandins at the transcriptional level and suggest that PGE-responsive factors may interact directly or indirectly with basal regulatory elements in the proximal promoter region of the Col1a1 gene.