Identification of an involucrin promoter transcriptional response element with activity restricted to keratinocytes

The involucrin proximal promoter was examined for response elements that co nfer cell-type specificity. Using a segment spanning positions -157 to +41, three possible response elements were identified by their protein-binding activity using DNase I footprinting. From distal to proximal, they were: an activator protein-1 (AP-I) site (previously identified) overlapping an Ets -like site:a second Ets-like site located 13 bp more proximally; and an ext ended region designated footprinted site A (FPA). Mutation of the distal Et s-like site had essentially no effect on the transcriptional activity in tr ansfections, while mutation of the proximal site reduced the activity by ha lf. FPA was shown by electrophoretic mobility-shift assay (EMSA) to be comp rised of two separable binding sites, FPA1 (distal) and FPA2 (proximal). Wh ile mutation of FPA2 had only a modest effect on transcriptional activity i n transient transfections, mutation of FPA1 reduced transcriptional activit y to approx. 20% of that obtained with the intact promoter. Additional muta tions of FPA1 indicated that the active region comprises positions -85 to - 73 (GTGGTGAAACCTGT). The molecular masses of the major proteins binding to this site were shown by UV cross-linking to be approx. 40 and 50 kDa, while minor bands were observed at 80 and 110 kDa. Since the involucrin promoter exhibits much higher transcriptional activity in keratinocytes than in oth er cell types in transfection assays (indicating that cell type specificity of expression is retained), the comparative influence of FPA1 was examined . While mutation of the AP-1 site affected transcriptional activity similar ly in all cell lines tested, mutation of FPA1 decreased activity substantia lly in keratinocytes, but not in NIH-3T3 and HeLa cells, evidence for a con tribution to cell-type specificity of expression. Furthermore, a correlatio n between the sensitivity to FPA1 mutation and amount of involucrin express ion in different keratinocyte cell lines was evident. EMSA showed that NIH- 3T3 and HeLa cells lacked the same FPA1 DNA-protein complex as keratinocyte s. However, the amount of complex formed with nuclear extracts from several keratinocyte lines did not correlate well with the level of involucrin exp ression. Other factors, such as differences in posttranslational modificati on or co-activators, must account for varied transcriptional response media ted by this site among keratinocyte lines.