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

We previously demonstrated that all-trans-retinoic acid (all-trans-RA) regu lates gonadotropin-releasing hormone (GnRH) release and gene expression in rat hypothalamic fragments and GTI-1 neuronal cells. Promoter analysis of r at GnRH gene revealed that the enhancing effect of all-trans-RA on GnRH tra nscription is mediated by cis-elements localized within -1640/-1438 of the rat GnRH promoter. In the present study, we attempted to localize functiona l retinoic acid response elements (RAREs) within the all-trans-RA-responsiv e region of the rat GnRH gene. Sequence analysis showed that there exist th ree putative repeats of AGGTCA-related sequences (-1637/-1617,-1579/-1562, and -1494/-1470) within this promoter sequence. Among them, only the -1493/ -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) a nd vice versa in electrophoretic mobility shift assays. In addition, like c onsensus RARE, the -1494/-1470 sequence could confer all-trans-RA responsiv eness 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 sequen ce while not affecting the specific binding to the cAMP response element (C RE). Both retinoids induced RAR beta gene expression in GT1-1 cells. The -1 494/-1470 sequence (5'-TCTTAGGACTCTGTGTGACCTAAGA) is similar to the direct repeat of TGACCT (complementary sequence of AGGTCA) with a spacer of 5 bp ( i.e. DR-5 in the reverse orientation). A mutation of the second core recogn ition motif of the -1494/-1470 sequence to a more divergent one from consen sus RARE (from TGACCT to TTACAT) abolished the responsiveness to all-trans- RA, whereas a mutation of first core recognition motif to a more TGACCT-lik e sequence (from AGGACT to TGAACT) increased the responsiveness to all-tran s-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. (C) 2001 Elsevier Scienc e B.V. All rights reserved.