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Volume 272, Number 15, Issue of April 11, 1997 pp. 10196-10204
©1997 by The American Society for Biochemistry and Molecular Biology, Inc.

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A GT-rich Sequence Binding the Transcription Factor Sp1 Is Crucial for High Expression of the Human Type VII Collagen Gene (COL7A1) in Fibroblasts and Keratinocytes

(Received for publication, September 19, 1996, and in revised form, December 11, 1996)

Laurence Vindevoghel , Kee-Yang Chung , Angelique Davis , David Kouba , Sirpa Kivirikko , Hansjüerg Alder ^§ , Jouni Uitto ^§¶ and Alain Mauviel

From the Departments of  Dermatology and Cutaneous Biology and  Biochemistry and Molecular Pharmacology, Jefferson Medical College, the ^¶ Jefferson Institute of Molecular Medicine, and the ^§ Kimmel Cancer Center, Thomas Jefferson University, Philadelphia, Pennsylvania 19107

Type VII collagen is the major component of anchoring fibrils, structural elements that stabilize the attachment of the basement membrane to the underlying dermis. In this study, we have dissected the human type VII collagen gene (COL7A1) promoter to characterize the cis-elements responsible for the expression of the gene in cultured fibroblasts and keratinocytes. Using transient cell transfections with various 5 end deletion COL7A1 promoter/chloramphenicol acetyltransferase reporter gene plasmid constructs, we determined that the region between nucleotides 524 and 456, relative to the transcription start site, is critical for high promoter activity in both cell types studied. Gel mobility shift assays using several DNA fragments spanning this region identified a GT-rich sequence between residues 512 and 505, necessary for the binding of nuclear proteins to this region of the promoter. Point mutations abolished the binding of nuclear proteins in gel shift assays and drastically diminished the activity of the promoter in transient cell transfections. Supershift assays with antibodies against various transcription factors including Sp1, Sp3, c-Jun/AP-1, and AP-2, and competition experiments with oligonucleotides containing consensus sequences for Sp1 and AP-1 binding identified Sp1 as the transcription factor binding to this region of the COL7A1 promoter. Indeed, recombinant human Sp1 was shown to bind the COL7A1 promoter GT-rich element but not its mutated form in gel mobility shift assays. In addition, co-transfection of pPacSp1, an expression vector for Sp1, together with the COL7A1 promoter/chloramphenicol acetyltransferase construct into Sp1-deficient Drosophila Schneider SL2 cells unequivocally demonstrated that Sp1 is essential for high expression of the COL7A1 gene. These data represent the first in-depth analysis of the human COL7A1 promoter transcriptional control.

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