Keratinocyte transglutaminase promoter analysis. Identification of a functional response element.
Keratinocyte transglutaminase catalyzes isopeptide bond formation to yield the highly insoluble cross-linked envelope during terminal differentiation of epidermal cells. Transcriptional response elements were identified in the 5'-flanking DNA of the gene for this enzyme by a combination of transient transfection and electrophoretic mobility shift analyses. Since human keratinocytes transcribed ineffectively transfected transglutaminase flanking DNA, a key feature of these experiments was the use of rat bladder epithelial cells as recipients. Serial deletion experiments identified by transient transfection an important response region containing three putative AP2-like response elements approximately 0.5 kilobases from the transcription initiation site. Oligonucleotides, each containing a single one of the elements, formed specific complexes with keratinocyte nuclear proteins. Two of the response elements were found to be functional by transfection in site-specific deletion experiments. Of these one formed specific DNA-protein complexes with nuclear proteins only from cells exhibiting keratinocyte differentiation. UV cross-linking experiments estimated the protein component of the complex to be approximately 85 kDa. This response element alone increased substantially the transcription of a minimal transglutaminase promoter in transient transfections. Further characterization of the putative transcription factor binding to this response element may provide insight into the regulation of keratinocyte transglutaminase.
Mariniello
L
L
Department of Environmental Toxicology, University of California, Davis 95616-8588, USA.
Qin
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Jessen
B A
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Rice
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D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D004591:Electrophoresis, Polyacrylamide Gel; D005347:Fibroblasts / Q000378:metabolism; D006801:Humans; D015603:Keratinocytes / Q000201:enzymology*; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides; D011401:Promoter Regions, Genetic; D051381:Rats; D014162:Transfection; D011503:Transglutaminases / Q000235:genetics*; D014407:Tumor Cells, Cultured
D009838:Oligodeoxyribonucleotides; D011503:Transglutaminases