A proximal direct repeat motif characterized as a negative regulatory element in the human renin gene.
A proximal direct repeat motif characterized as a negative regulatory element in the human renin gene.
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DOI：
10.1097/00004872-201106001-01122
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发表时间：
2011-06
影响因子：
4
通讯作者：
T. Konoshita;S. Fuchs;Y. Makino;S. Wakahara;I. Miyamori
中科院分区：
文献类型：
--
作者：
T. Konoshita;S. Fuchs;Y. Makino;S. Wakahara;I. Miyamori
肾素基因表达的调节被认为是调节总的肾素-血管紧张素系统的基础。人肾素基因在近端区域（-259/-245 bp）含有直接重复（DR）基序AGGGGTCAC-AGGGCCA，其含有与核受体超家族结合核心基序AGGTCA相似的序列，并且与COUP-TFII共有序列最相似。通过足迹分析、电迁移率变化分析和报告基因分析，对DR基序作为功能性顺式元件在肾皮质和绒毛膜蜕膜细胞中的作用进行了评价。DR基序位点通过足迹分析得到保护，具有清晰的超敏和轻微的超敏区域，与一致的DR一致。EMSA强烈怀疑其中一种结合蛋白是COUP-TFII共识特异性的。用肾素产生细胞的核提取物获得的DNA/蛋白质复合物可以被同源竞争物完全阻断，并且被DR基序的后半突变寡核苷酸强烈阻断，但不被前半突变寡核苷酸阻断。最后，第二半突变构建体的转录活性略有升高，并且在报告基因测定中，第一半突变构建体的转录活性比野生型构建体显著强两倍。这些结果表明，人肾素基因的DR基序位点是一个负调控元件，参与双重转录抑制，核受体超家族成员与该位点结合，在人肾素基因表达中起重要作用，其中一个结合蛋白可能是COUP-TFII。
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.