IDENTIFICATION AND CHARACTERIZATION OF A FUNCTIONAL RETINOIC ACID THYROID HORMONE-RESPONSE ELEMENT UPSTREAM OF THE HUMAN INSULIN GENE ENHANCER

A deletion analysis of the human insulin gene extending to 2 kb upstre am of the transcription start site provided evidence of regulatory seq uences located upstream of the insulin-linked polymorphic region (ILPR ). Within this ILPR-distal region is a sequence (Ink, for insulin kilo base upstream) which contains three potential nuclear hormone-receptor half-sites, closely matching the consensus sequence AGGTCA. These seq uences are arranged as a palindromic element with zero spacing overlap ping a direct repeat with 2 bp spacing, The Ink sequence was used in e lectrophoretic mobility-shift assays within nuclear extracts from COS- 7 cells overexpressing the vitamin D, thyroid hormone or retinoic acid receptors, or from an insulin-expressing hamster cell line, HIT-T15. These studies suggest that the insulin-expressing cell line contains t hyroid hormone and retinoic acid receptors at least, and that these re ceptors are able to recognize the Ink sequence. Three copies of the In k sequence were placed upstream of the thymidine kinase promoter and f irefly luciferase reporter gene. In COS-7 cells expressing the appropr iate nuclear hormone receptor, this construct was responsive to both t hyroid hormone (18-fold) and all-trans-retinoic acid (31-fold). In HIT -T15 cells the same construct responded to all-trans-retinoic acid, bu t not to thyroid hormone. Within the context of a 2 kb insulin gene fr agment, the Ink sequence was shown to be activated by retinoic acid an d by the retinoic acid receptor, but acted as a negative element in th e presence of both retinoic acid and the retinoic acid receptor. Mutag enesis studies demonstrated that the palindromic sequence was importan t for the retinoic acid response, and for binding of complexes contain ing retinoic acid receptor. In human islets of Langerhans, retinoic ac id was shown to stimulate insulin mRNA levels. These results demonstra te that a functional nuclear hormone-receptor-response element is loca ted upstream of the human ILPR. As retinoic acid and thyroid hormone a re frequently involved in developmental regulatory processes, it is po ssible that this element may be important in the process of islet cell differentiation.