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Volume 272, Number 3, Issue of January 17, 1997 pp. 1753-1760
©1997 by The American Society for Biochemistry and Molecular Biology, Inc.

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Binding of Upstream Stimulatory Factor to an E-box in the 3-Flanking Region Stimulates 1(I) Collagen Gene Transcription

(Received for publication, March 26, 1996, and in revised form, October 30, 1996)

Richard A. Rippe , Akihiro Umezawa , Jon P. Kimball , Michael Breindl ^¶ and David A. Brenner

From the Department of  Medicine and  Biochemistry and Biophysics, The University of North Carolina, Chapel Hill, North Carolina 27599-7038 and the ^¶ Department of Biology and Molecular Biology Institute, San Diego State University, San Diego, California 92182

Since several lines of evidence implicate the 3-flanking region in regulating 1(I) collagen gene transcription, we analyzed 12.4-kilobase pairs of 3-flanking sequence of the murine 1(I) collagen gene for transcriptional elements. A region of the 3-flanking region stimulated expression of the heterologous -globin gene promoter in an enhancer trap plasmid and of the 1(I) collagen gene promoter in a collagen-luciferase reporter gene construct when located 3 to the luciferase reporter gene. DNase I footprinting analysis demonstrated the presence of three regions where DNA binding proteins specifically interact within this 3-stimulatory region. Inspection of the DNA sequence revealed a consensus E-box, a binding site for basic helix-loop-helix proteins, in one of the protein binding sites. Mobility shift assays demonstrated that upstream stimulatory factors (USF) USF-1 and USF-2 bind to this E-box. Mutating the E-box in the context of the 3-flanking region confirmed that it contributes to the enhancement of transcriptional activity of the 1(I) collagen gene promoter. Mutations in all three protein binding sites abolished transcriptional activation by the 3-flanking region, suggesting a complex interaction among the trans-acting factors in enhancing transcriptional activity. Thus, a region of the 3-flanking region of the 1(I) collagen gene stimulates transcription of the 1(I) collagen gene promoter, and USF-1 and USF-2 contribute to this transcriptional stimulation.

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