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 Colla1 gene, which encod es the alpha1 chain of type I collagen. Here, we report that Yin Yang 1 (YY 1) binds to the Colla1 promoter immediately upstream of the TATA box, and w e examine the functional implications of YY1 binding for regulation of Coll a1 gene expression in BALBc/3T3 fibroblasts. The Colla1 promoter region spa nning base pairs (bp) -56 to -9 bound purified recombinant YY1 and the corr esponding binding activity in nuclear extracts was supershifted using a YY1 -specific antibody. Mutation of the TATA box to TgTA enhanced YY1 complex f ormation. Mutation analysis revealed two YY1 core binding sites at -40/-37 by (YY1A) and, on the reverse strand, at -32/-29 by (YY1B) immediately adja cent to the TATA box. In transfections using Colla1-luciferase constructs, mutation of YY1A decreased activity completely (wild-type p350 (p350wt), -2 22/+113 bp) or partially (p130wt, -84 bp/+13 bp), whereas mutation of YY1B blocked the expression of both promoter constructs. Cotransfection with pCM V-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 o f activity, arguing for a requirement for cognate binding of the TATA box-b inding protein (TBP). Electrophoretic mobility shift assays performed under conditions permitting TBP binding showed that recombinant TBP/TFIID and YY 1 could bind to the -56/-9 by fragment and that YY1B was the preferred site for YY1 binding. Our results indicate that YY1 binds to the Colla1 proxima l promoter and functions as a positive regulator of constitutive activity i n fibroblasts. Although YY1 is not sufficient for transcriptional initiatio n, it is a required component of the transcription machinery in this promot er.