Konoshita Tadashi, Fuchs Sébastien, Makino Yasukazu, Wakahara Shigeyuki, Miyamori Isamu
Third Department of Internal Medicine, Fukui University School of Medicine, 23-3, Shimoaizuki, Matsuoka, 910-1193, Japan.
J Cell Biochem. 2007 Nov 1;102(4):1043-50. doi: 10.1002/jcb.21341.
The regulation of renin gene expression is thought to be fundamental to regulation of the total renin-angiotensin system. The human renin gene contains a direct repeat (DR) motif AGGGGTCAC-AGGGCCA in the proximal region (-259/-245 bp), which contains similar sequence for nuclear receptor superfamily binding core motif, AGGTCA, and is the most similar to COUP-TFII consensus. The DR motif was evaluated as a functional cis-element with renal cortex and chorio-decidual cells by footprint assay, electromobility shift assay (EMSA) and reporter assay. The DR motif site was protected by footprint analysis with a clear hypersensitive and a minor hypersensitive region in good accordance with the DR of the consensus. One of the binding proteins was strongly suspected to be COUP-TFII-consensus-specific by EMSA. The DNA/protein complexes obtained with nuclear extract of renin producing cells could be completely blocked by homologous competitor and strongly blocked by the second-half mutant oligonucleotide of the DR motif but not by the first-half mutant oligonucleotide. Finally, the transcriptional activity of second-half mutant construct is slightly elevated and that first-half mutant construct is significantly stronger by twofold compared with wild type construct in reporter assay. These findings suggest that the DR motif site of the human renin gene functions as a negative regulatory element involved in a twofold repression of transcription and that member(s) of nucleic receptor superfamily bind the site and play important roles in the human renin gene expression with a possibility that one of the binding protein is COUP-TFII.
肾素基因表达的调控被认为是整个肾素 - 血管紧张素系统调控的基础。人类肾素基因在近端区域（-259 / -245 bp）含有一个直接重复（DR）基序AGGGGTCAC - AGGGCCA，其包含与核受体超家族结合核心基序AGGTCA相似的序列，并且与COUP - TFII共有序列最为相似。通过足迹分析、电泳迁移率变动分析（EMSA）和报告基因分析，将DR基序评估为肾皮质和绒毛膜蜕膜细胞中的功能性顺式元件。通过足迹分析保护了DR基序位点，有一个明显的超敏区域和一个较小的超敏区域，与共有序列的DR高度一致。通过EMSA强烈怀疑其中一种结合蛋白是COUP - TFII共有序列特异性的。用肾素产生细胞的核提取物获得的DNA /蛋白质复合物可被同源竞争者完全阻断，并被DR基序的后半部分突变寡核苷酸强烈阻断，但不被前半部分突变寡核苷酸阻断。最后，在报告基因分析中，与野生型构建体相比，后半部分突变构建体的转录活性略有升高，而前半部分突变构建体的转录活性则显著增强两倍。这些发现表明，人类肾素基因的DR基序位点作为一个负调控元件，参与转录的双重抑制，并且核受体超家族的成员结合该位点并在人类肾素基因表达中发挥重要作用，其中一种结合蛋白有可能是COUP - TFII。
