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  • Title: Identification of control elements 3' to the human keratin 1 gene that regulate cell type and differentiation-specific expression.
    Author: Huff CA, Yuspa SH, Rosenthal D.
    Journal: J Biol Chem; 1993 Jan 05; 268(1):377-84. PubMed ID: 7677999.
    Abstract:
    To define DNA regulatory elements that mediate the response of the keratin 1 (K1) gene to Ca(2+)-induced differentiation, regions spanning the 5'- and 3'-flanking sequences, coding regions, and introns from the human K1 gene were cloned into vectors containing the chloramphenicol acetyltransferase (CAT) reporter gene and transfected into cultured mouse keratinocytes. A 4.3-kilobase (kb) region located 3' to the K1 gene stimulated CAT activity in response to increasing Ca2+ concentrations from 0.05 mM (basal cells) to 1.2 mM (differentiated cells). The 4.3-kb fragment was also active in human epidermal cells but inactive in NIH 3T3 cells and primary mouse fibroblasts. Deletion analysis localized the activity to the terminal 1682 base pairs (bp) of the flanking sequence which retained Ca2+ sensitivity in epidermal cells but was not active in mesenchymal cells. Removal of a 207-base pair element created an enhancer which was active in both epidermal and mesenchymal cells but was still Ca(2+)-inducible. Further deletions identified two elements which functioned synergistically to give maximal Ca(2+)-sensitive activity. Stably transfected epidermal cell lines expressed CAT under the direction of these elements when grafted onto nude mice to reconstitute an intact epidermis. Previously reported keratin regulatory motifs were not contained in the 1682-bp fragment, but an AP-1 site was identified in one of the synergistic subunits.
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