Characterization of the human involucrin promoter using a transient β-galactosidase assay

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Abstract
Involucrin, a component of the cornified cell envelope, is expressed specifically in differentiating keratinocytes of stratified squamous epithelia. To explore the regulation of involucrin expression, 3.7 kb of upstream sequences of the human involucrin gene was cloned into a plasmid containing a -galactosidase reporter gene and transfected into early passage keratinocytes and a variety of human cell types. The full-length construct gave maximal and tissue-specific expression. Deletion analysis showed that sequences between 900 and 2500 bp upstream of the transcriptional start site and the intron located between the transcriptional and translational start sites were required for maximal expression. Further analysis of the intron indicated that its effects on expression were independent of it being present in nascent RNA and suggested that sequences within the intron have regulatory activity. These results suggest that the involucrin intron operates in vivo to regulate expression in the epidermis. Abbreviations used: ADH, alcohol dehydrogenase; β-gal, β-galactosidase; DME, Dulbecco’s modified Eagle’s medium; DDAB, dimethyldioctyldecylammonium bromide; FCS, fetal calf serum; kb, kilobase; ONPG, O-nitrophenyl β-D-galactopyranoside; PBS, phosphate buffered saline without calcium/magnesium salts; PCR, polymerase chain reaction; PtdEtn, dioleoyl-L-α-phosphatidylethanolamine; RSV, Rous sarcoma virus; SV40, simian virus 40.