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

Type VII collagen is the major component of anchoring fibrils, structu ral elements that stabilize the attachment of the basement membrane to the underlying dermis. In this study, we have dissected the human typ e VII collagen gene (COL7A1) promoter to characterize the cis elements responsible for the expression of the gene in cultured fibroblasts an d keratinocytes. Using transient cell transfections with various 5' en d deletion COL7A1 promoter/chloramphenicol acetyltransferase reporter gene plasmid constructs, we determined that the region between nucleot ides -524 and -456, relative to the transcription start site, is criti cal for high promoter activity in both cell types studied, Gel mobilit y shift assays using several DNA fragments spanning this region identi fied 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 assay s 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 c ompetition experiments with oligonucleotides containing consensus sequ ences for Sp1 and AP-1 binding identified Sp1 as the transcription fac tor binding to this region of the COL7A1 promoter, Indeed, recombinant human Sp1 was shown to bind the COL7A1 promoter GT-rich element but n ot its mutated form in gel mobility shift assays, In addition, co-tran sfection of pPacSp1, an expression vector for Sp1, together with the C OL7A1 promoter/chloramphenicol acetyltransferase construct into Sp1-de ficient Drosophila Schneider SL2 cells unequivocally demonstrated that Sp1 is essential for high expression of the COL7A1 gene, These data r epresent the first in-depth analysis of the human COL7A1 promoter tran scriptional control.