A functional retinoic acid response element (RARE) is present within the distal promoter of the rat gonadotropin-releasing hormone (GnRH) gene

Abstract

We previously demonstrated that all- trans-retinoic acid (all- trans-RA) regulates gonadotropin-releasing hormone (GnRH) release and gene expression in rat hypothalamic fragments and GT1-1 neuronal cells. Promoter analysis of rat GnRH gene revealed that the enhancing effect of all- trans-RA on GnRH transcription is mediated by cis-elements localized within −1640/−1438 of the rat GnRH promoter. In the present study, we attempted to localize functional retinoic acid response elements (RAREs) within the all- trans-RA-responsive region of the rat GnRH gene. Sequence analysis showed that there exist three putative repeats of AGGTCA-related sequences (−1637/−1617, −1579/−1562, and −1494/−1470) within this promoter sequence. Among them, only the −1494/−1470 sequence could compete the specific binding of GT1-1 nuclear extracts to the consensus RARE (direct repeat of AGGTCA with a 5-bp spacer, DR-5) and vice versa in electrophoretic mobility shift assays. In addition, like consensus RARE, the −1494/−1470 sequence could confer all- trans-RA responsiveness when inserted into the upstream region of SV40 promoter. Treatment of GT1-1 cells with all- trans- or 9- cis-RA increased the specific bindings of GT1-1 nuclear extracts to the consensus RARE and to the −1494/−1470 sequence while not affecting the specific binding to the cAMP response element (CRE). Both retinoids induced RARβ gene expression in GT1-1 cells. The −1494/−1470 sequence (5′-TCTT A G GA C T CTGTG T G AC C T AAGA) is similar to the direct repeat of T G AC C T (complementary sequence of A G GT C A) with a spacer of 5 bp (i.e. DR-5 in the reverse orientation). A mutation of the second core recognition motif of the −1494/−1470 sequence to a more divergent one from consensus RARE (from T GAC CT to T TAC AT) abolished the responsiveness to all- trans-RA, whereas a mutation of first core recognition motif to a more TGACCT-like sequence (from AG GACT to TG AACT) increased the responsiveness to all- trans-RA. These results indicate that the −1494/−1470 sequence is indeed a weak but functional RARE of the modified DR-5 type. Taken together, these data indicate that all- trans-RA enhances GnRH transcription via functional RARE present in the distal region of the GnRH promoter.