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) 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 RARbeta gene expression in GT1-1 cells. The --1494/--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 recognition motif of the --1494/--1470 sequence to a more divergent one from consensus RARE (from TGACCT to TTACAT) abolished the responsiveness to all-trans-RA, whereas a mutation of first core recognition motif to a more TGACCT-like sequence (from AGGACT to TGAACT) 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.
Cho
S
S
School of Biological Sciences and Research Center for Cell Differentiation, Seoul National University, Seoul, 151-742, South Korea.
Chung
J J
JJ
Choe
Y
Y
Choi
H S
HS
Han Kim
D
D
Rhee
K
K
Kim
K
K
D000818:Animals; D002478:Cells, Cultured; D005786:Gene Expression Regulation / Q000502:physiology; D005822:Genetic Vectors; D007987:Gonadotropin-Releasing Hormone / Q000235:genetics* / Q000378:metabolism; D009687:Nuclear Proteins / Q000378:metabolism; D015345:Oligonucleotide Probes; D017354:Point Mutation; D011401:Promoter Regions, Genetic / Q000502:physiology*; D051381:Rats; D015533:Transcriptional Activation / Q000502:physiology; D014212:Tretinoin / Q000378:metabolism / Q000502:physiology*
D009687:Nuclear Proteins; D015345:Oligonucleotide Probes; D007987:Gonadotropin-Releasing Hormone; D014212:Tretinoin