Identification of control elements 3' to the human keratin 1 gene that regulate cell type and differentiation-specific expression

C. A. Huff, S. H. Yuspa, D. Rosenthal

Research output: Contribution to journalArticlepeer-review

Abstract

To define DNA regulatory elements that mediate the response of the keratin 1 (K1) gene to Ca2+-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 mesenchumal cells but was still Ca2+-inducible. Further deletions identified two elements which functioned synergistically to give maximal Ca2+-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.

Original languageEnglish (US)
Pages (from-to)377-384
Number of pages8
JournalJournal of Biological Chemistry
Volume268
Issue number1
StatePublished - Jan 1 1993
Externally publishedYes

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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