, Volume 287, Issue 8, pp 740-746

Repression of involucrin gene expression by transcriptional enhancer factor 1 (TEF-1)

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Involucrin is one of the precursor proteins of keratinocyte cornified envelope that is formed beneath the inner surface of the cell membrane during terminal differentiation. Although involucrin is specifically expressed in the upper squamous cells of the epidermis, the precise regulatory mechanism of involucrin gene expression remains unknown. Transcriptional enhancer factor 1 (TEF-1), which binds to SV40 enhancer, is a nuclear protein expressed in various types of cells including keratinocytes. Immunohistochemical study has revealed that TEF-1 protein is highly expressed on the basal cell layer of the epidermis. To examine the possible regulatory mechanism of involucrin gene expression by TEF-1 protein, we analysed involucrin promoter activity of the INV-CAT vector, which was constructed by connecting the 5′ upstream region of the involucrin gene (−801 bp upstream from the transcription start site and downstream including the untranslated first exon) to the chloramphenicol acetyltransferase (CAT) reporter gene. The INV-CAT vector was transfected to SV40-transformed human keratinocytes (SVHK). Cotransfection of the TEF-1 expression vector significantly repressed INV-CAT promoter activity in a dose-dependent manner. The repression was also observed by transfection of the GAL4-TEF-1 vector, which was constructed by replacement of the TEF-1 DNA binding domain by the GAL4 activator domain. This suggests that TEF-1-induced repression is due to interference/squelching of a limiting transcriptional intermediary factor that is essential for involucrin expression. Analysis of the deleted INV-CAT vector suggested that the region from −599 to −495 of the involucrin gene, which contains two possible TEF-1 binding sites, was critical for the repression of the involucrin gene by TEF-1. By gel retardation analysis, the specific DNA binding of SVHK cell nuclear extracts and the recombinant TEF-1 protein was confirmed. TEF-1-dependent repression of involucrin gene expression might explain the suprabasal involucrin expression in the epidermis.