Regulation of a human epidermal keratin gene: sequences and nuclear factors involved in keratinocyte-specific transcription.

Abstract

The keratinocyte is a major cell type of the body, and in epidermis, keratinocytes have potential for future gene targeting and drug therapy. Despite the importance of keratinocytes in cell biology and medicine, little is known about the molecular mechanisms underlying keratinocyte-specific gene expression. Here, we report the first detailed characterization of the sequences and factors controlling expression of a human gene expressed specifically in keratinocytes. Using 5' upstream sequence of the human K14 keratin gene coupled to one of two reporter genes, we examined sequences necessary and sufficient for expression of K14 in both cultured human keratinocytes and in mitotically active basal keratinocytes of transgenic mouse epidermis. We demonstrated the existence of distal and proximal elements located 5' from the transcription initiation site of the hK14 gene, which when combined with a TATA box element, appear to act in concert to drive keratinocyte-specific expression. We examined the proximal region in detail. After using CAT assays to narrow a transcriptional activation element to within 110 bp, we demonstrated the existence of a keratinocyte nuclear factor which binds to a 10-bp palindrome, 5'-GCCTGCAGGC-3', within this domain. Using methylation interference analysis, we identified the G residues important for factor binding, and showed that point mutations in these G residues not only blocked factor binding but also resulted in decreased transcriptional activity of an hK14-CAT gene. The factor was most abundant in keratinocytes, was expressed at lower levels in some simple epithelial cell lines, and was not detected in fibroblasts or lymphoma cells. Moreover, the 10-bp sequence was similar to sequences found in the 5' upstream sequences of several other genes expressed in keratinocytes, and at least one of these genes, the human K1 gene, contained a sequence that competed with the hK14 proximal element for binding factor. Collectively, our data suggest that both the sequence and the nuclear factor that we have identified may be involved in controlling keratinocyte-specific expression in vitro and in vivo.