Functional analysis of the interaction of a tissue-specific factor with an upstream enhancer element of the rat prolactin gene.

Abstract

A nuclear factor which binds to an upstream element of the rat PRL gene has been identified and the functional properties of the factor-DNA interaction have been assessed by mutagenesis of the factor binding sites. Gel mobility shift assays have been used to identify a factor which binds to a fragment from the -1712 to -1494 region of the rat PRL gene. The DNA binding factor is present in nuclear extracts from PRL-producing GH3 cells, but not in nuclear extracts from several other cell lines. Although previous studies have shown that the estrogen receptor binds to this region of DNA, chromatography on heparin-agarose demonstrated that the factor detected by mobility shift assay is probably not the estrogen receptor. Nuclease protection experiments demonstrate that the factor binds to a discrete region at positions -1666 to -1652. The protected region includes half of a palindrome, TCATTAT ... ATAATGA. Mutagenesis by T to G transversions of either both halves of this symmetrical sequence, or only the upstream portion shown to interact with the factor substantially reduced factor binding as assessed by gel mobility shift assay. Transfer of fusion genes containing this region of DNA into GH3 cells demonstrated that the -1712 to -1494 region has a basal enhancer activity which is reduced severalfold by T to G mutagenesis of the complete dyad symmetry at positions -1665 to -1644. The results suggest that the -1712 to -1494 region of the rat PRL gene contains two relatively independent elements. One element, located at positions -1582 to -1569, interacts with the estrogen receptor and mediates estrogenic stimulation of transcription.(ABSTRACT TRUNCATED AT 250 WORDS)