Riquet F B, Tan L, Choy B K, Osaki M, Karsenty G, Osborne T F, Auron P E, Goldring M B
Beth Israel Deaconess Medical Center, Division of Rheumatology, and New England Baptist Bone & Joint Institute, Harvard Institutes of Medicine, Boston, Massachusetts 02115, USA.
J Biol Chem. 2001 Oct 19;276(42):38665-72. doi: 10.1074/jbc.M009881200. Epub 2001 Aug 20.
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.
成纤维细胞中细胞特异性和普遍存在的转录因子均已被确定为对Col1a1基因表达至关重要，该基因编码I型胶原的α1链。在此，我们报告阴阳1（YY1）结合到TATA盒上游紧邻的Col1a1启动子，并且我们研究了YY1结合对BALBc/3T3成纤维细胞中Col1a1基因表达调控的功能影响。跨越碱基对（bp）-56至-9的Col1a1启动子区域结合了纯化的重组YY1，并且使用YY1特异性抗体使核提取物中的相应结合活性发生超迁移。TATA盒突变为TgTA增强了YY1复合物的形成。突变分析揭示了紧邻TATA盒的位于-40/-37 bp（YY1A）以及在反向链上位于-32/-29 bp（YY1B）的两个YY1核心结合位点。在使用Col1a1-荧光素酶构建体的转染中，YY1A的突变完全降低活性（野生型p350（p350wt），-222/+113 bp）或部分降低活性（p130wt，-84 bp/+13 bp），而YY1B的突变阻断了两种启动子构建体的表达。与pCMV-YY1共转染使p350wt和p130wt活性增加多达10倍，而反义YY1降低组成型表达并阻断由于pCMV-YY1过表达导致的活性增加。mTgTA构建体无活性，这表明需要TATA盒结合蛋白（TBP）的同源结合。在允许TBP结合的条件下进行的电泳迁移率变动分析表明，重组TBP/TFIID和YY1可以结合到-56/-9 bp片段，并且YY1B是YY1结合的优选位点。我们的结果表明，YY1结合到Col1a1近端启动子并作为成纤维细胞中组成型活性的正调节因子发挥作用。虽然YY1不足以启动转录，但它是该启动子转录机制的必需组分。
