Cho S, Chung J J, Choe Y, Choi H S, Han Kim D, Rhee K, Kim K
School of Biological Sciences and Research Center for Cell Differentiation, Seoul National University, Seoul, 151-742, South Korea.
Brain Res Mol Brain Res. 2001 Mar 5;87(2):204-13. doi: 10.1016/s0169-328x(01)00021-3.
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.
我们之前证明，全反式维甲酸（all-trans-RA）可调节大鼠下丘脑片段和GT1-1神经元细胞中促性腺激素释放激素（GnRH）的释放及基因表达。大鼠GnRH基因的启动子分析显示，全反式维甲酸对GnRH转录的增强作用是由位于大鼠GnRH启动子-1640 / -1438内的顺式元件介导的。在本研究中，我们试图在大鼠GnRH基因的全反式维甲酸反应区域内定位功能性维甲酸反应元件（RAREs）。序列分析表明，在该启动子序列中存在三个假定的AGGTCA相关序列重复（-1637 / -1617、-1579 / -1562和-1494 / -1470）。其中，只有-1494 / -1470序列能够在电泳迁移率变动分析中竞争GT1-1核提取物与共有RARE（AGGTCA的直接重复序列，间隔5个碱基对，DR-5）的特异性结合，反之亦然。此外，与共有RARE一样，-1494 / -1470序列插入SV40启动子上游区域时可赋予对全反式维甲酸的反应性。用全反式或9-顺式维甲酸处理GT1-1细胞可增加GT1-1核提取物与共有RARE以及与-1494 / -1470序列的特异性结合，而不影响与cAMP反应元件（CRE）的特异性结合。两种维甲酸均诱导GT1-1细胞中RARβ基因表达。-1494 / -1470序列（5'-TCTTAGGACTCTGTGTGACCTAAGA）类似于TGACCT（AGGTCA的互补序列）的直接重复序列，间隔5个碱基对（即反向的DR-5）。将-1494 / -1470序列的第二个核心识别基序突变为与共有RARE差异更大的序列（从TGACCT变为TTACAT）可消除对全反式维甲酸的反应性，而将第一个核心识别基序突变为更类似TGACCT的序列（从AGGACT变为TGAACT）则可增加对全反式维甲酸的反应性。这些结果表明，-1494 / -1470序列确实是修饰型DR-5类型的一个弱但功能性的RARE。综上所述，这些数据表明全反式维甲酸通过GnRH启动子远端区域存在的功能性RARE增强GnRH转录。
