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.
Riquet
F B
FB
Beth Israel Deaconess Medical Center, Division of Rheumatology, and New England Baptist Bone & Joint Institute, Harvard Institutes of Medicine, Boston, Massachusetts 02115, USA.
Tan
L
L
Choy
B K
BK
Osaki
M
M
Karsenty
G
G
Osborne
T F
TF
Auron
P E
PE
Goldring
M B
MB
D016475:3T3 Cells; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D002467:Cell Nucleus / Q000378:metabolism; D003094:Collagen / Q000235:genetics* / Q000378:metabolism*; D024042:Collagen Type I; D000090602:Collagen Type I, alpha 1 Chain; D004252:DNA Mutational Analysis; D004268:DNA-Binding Proteins / Q000378:metabolism*; D050981:Erythroid-Specific DNA-Binding Factors; D005786:Gene Expression Regulation; D008156:Luciferases / Q000378:metabolism; D051379:Mice; D008969:Molecular Sequence Data; D009154:Mutation; D016376:Oligonucleotides, Antisense / Q000494:pharmacology; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D035362:Transcription Factor TFIID; D014157:Transcription Factors / Q000378:metabolism*; D019473:Transcription Factors, TFII / Q000378:metabolism; D014158:Transcription, Genetic; D014162:Transfection; D050986:YY1 Transcription Factor
D024042:Collagen Type I; D000090602:Collagen Type I, alpha 1 Chain; D004268:DNA-Binding Proteins; D050981:Erythroid-Specific DNA-Binding Factors; D016376:Oligonucleotides, Antisense; D035362:Transcription Factor TFIID; D014157:Transcription Factors; D019473:Transcription Factors, TFII; D050986:YY1 Transcription Factor; C494699:Yy1 protein, mouse; D003094:Collagen; D008156:Luciferases