Identification of a decidua-specific enhancer on the human prolactin gene with two critical activator protein 1 (AP-1) binding sites

Deletion analysis of the human PRL promoter in endometrial stromal cells de cidualized in vitro revealed a 536-bp enhancer located between nucleotide ( nt) -2,040 to -1,505 in the 5'-flanking region. The 536-bp enhancer fragmen t ligated into a thymidine kinase (TK) promoter-luciferase reporter plasmid conferred enhancer activity in decidual-type cells but not nondecidual cel ls. DNase I footprint analysis of decidualized endometrial stromal cells re vealed three protected regions, FP1-FP3. Transfection of overlapping 100-bp fragments of the 536-bp enhancer indicated that FP1 and FP3 each conferred enhancer activity. Gel shift assays indicated that both FP1 and FP3 bind a ctivator protein 1 (AP-1), and JunD and Fra-2 are components of the AP-1 co mplex in decidual fibroblasts. Mutation of the AP-1 binding site in either FP1 or FP3 decreased enhancer activity by approximately 50%, while mutation of both sites almost completely abolished activity. Coexpression of the 53 6-bp enhancer and A-fos, a dominant negative to AP-1, decreased enhancer ac tivity by approximately 70%. Conversely, coexpression of Fra-2 in combinati on with JunD or c-Jun and p300 increased enhancer activity 6- to 10-fold. I ntroduction of JunD and Fra-2 into nondecidual cells is sufficient to confe r enhancer activity. JunD and Fra-2 protein expression was markedly increas ed in secretory phase endometrium and decidua of early pregnancy (high PRL content) compared with proliferative phase endometrium (no PRL). These inve stigations indicate that the 5'-flanking region of the human PRL gene conta ins a decidua-specific enhancer between nt -2,040/-1,505 and AP-1 binding s ites within this enhancer region are critical for activity.