Original Article

Journal of Investigative Dermatology (1991) 97, 281–285; doi:10.1111/1523-1747.ep12480380

Human Nidogen Gene: Structural and Functional Characterization of the 5'-Flanking Region

Michael J Fazio, Joan O'Leary1,2, Veli-Matti Kähäri1,2, Yue Qiu Chen1,2, Biagio Saitta1,2 and Jouni Uitto1,2

  1. 1Departments of Dermatology, and Biochemistry and Molecular Biology, Jefferson Medical College, and Section of Molecular, Philadelphia, Pennsylvania, U.S.A.
  2. 2Dermatology, Jefferson Institute of Molecular Medicine, Thomas Jefferson University, Philadelphia, Pennsylvania, U.S.A.

Received 9 July 1990; Accepted 14 February 1991.

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Abstract

Nidogen is a sulfated multifunctional glycoprotein present in basement membranes. In this study, we have cloned the 5'-flanking region of the human nidogen gene. Initially, an approx 35-kb DNA clone (NCos4) was isolated from a human cosmid genomic library. Southern hybridization of EcoRI-digested NCos4 allowed isolation of a 3.7-kb fragment, which was shown to contain a portion of intron 1, the entire exon 1, and approx 0.9 kb of 5'-flanking sequences of the nidogen gene. Nucleotide sequencing of the 5'-flanking DNA revealed the presence of two canonic CCAAT consensus sequences in the antisense strand and a potential variant of the TATA motif, TATTT, in the sense strand. One putative AP-2 and six putative SP1 binding sites were also present. To test the functional promoter activity of the 5'-flanking genomic DNA, two nidogen promoter/CAT reporter gene constructs, with the promoter segment spanning from –864 to –1 and from –534 to –1, respectively, were developed and analyzed in transient transfections of human and mouse cell cultures. Both constructs showed clearly detectable promoter activity, and the activity of the larger construct could be up-regulated by 12-O-tetradecanoyl phorbol 13-acetate up to 2.5 times. The results indicate that the nidogen promoter/CAT gene constructs developed in this study provide a means to examine the transcriptional regulation of nidogen gene expression in human diseases of the basement membrane zone.

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