Identification of a retinoic acid response element upstream of the rat alpha-fetoprotein gene

Retinoic acid (RA) is known to have potent effects on development and differentiation. alpha-Fetoprotein (AFP), an oncodevelopmental protein, is transcriptionally activated by RA in several cell lines, but little is known about the mechanism of RA regulation of AFP gene expression. In the present study, we have identified a RA response element (RARE) in the 5'-flanking region of the AFP gene. Using deletion mapping, the RARE was located between -6337 to -6266 of the rat AFP 5'-flanking region, which confers RA responsiveness in a heterologous promoter. Further sequence analysis of this cis-acting element demonstrated a RARE direct repeat sequence of AGGTCA and RARE-like motifs at -6327 and -6319, respectively. This far upstream RARE (AFP-RARE1) can specifically bind to both RAR and RXR proteins in gel mobility shift assays. In co-transfections with RAR alpha, beta, gamma and RXR alpha expression vectors, a reporter gene construct consisting of the AFP-RARE1 sequence ligated upstream of the chloramphenicol acetyltransferase (CAT) gene showed strong RA responsiveness to RAR alpha and RXR alpha with 15- and 25-fold increases in CAT activity, respectively. Furthermore, responsiveness of AFP-RARE1 to RA was independent of orientation. These studies present a novel target for RA action by identifying a RARE in the AFP gene.