Transcriptional regulation of the CLC-K1 promoter by myc-associated zinc finger protein and kidney-enriched Kruppel-like factor, a novel zinc finger repressor
Transcriptional regulation of the CLC-K1 promoter by myc-associated zinc finger protein and kidney-enriched Kruppel-like factor, a novel zinc finger repressor
复制标题
DOI：
10.1128/mcb.20.19.7319-7331.2000
复制
发表时间：
2000-10-01
影响因子：
5.3
通讯作者：
Marumo, F
中科院分区：
文献类型：
--
作者：
Uchida, S;Tanaka, Y;Marumo, F
CLC-K1 和 CLC-K2（两种肾脏特异性 CLC 氯离子通道）的表达在组织特异性的基础上受到转录调节。先前的研究表明，转录起始位点附近的 GA 元件对于 CLC-K1 和 CLC-K2 基因启动子的基础和细胞特异性活性非常重要。为了鉴定 GA 结合蛋白，通过酵母单杂交系统筛选了人肾 cDNA 文库。克隆了 Cys2-His2 锌指基因的新成员 KKLF（“肾脏富集 Kruppel 样因子”）和先前分离的 MAZ（“myc 相关锌指蛋白”）。 KKLF被发现在肝脏、肾脏、心脏和骨骼肌中大量表达，免疫组织化学显示KKLF蛋白在心脏和骨骼肌间质细胞、星状细胞和肝脏成纤维细胞中的核定位。在肾脏中，KKLF 蛋白定位于间质细胞、系膜细胞和肾单位段，其中 CLC-K1 和 CLC-K2 不表达。凝胶迁移率变化测定揭示了重组 KKLF 和 MAZ 蛋白与 CLC-K1 GA 元件的序列特异性结合，精细突变测定阐明了 KKLF 结合位点的共有序列是 GGGGNGGNG。在瞬时转染实验中，MAZ 对 CLC-K1-荧光素酶报告基因的转录具有强烈的激活作用。另一方面，KKLF 与 MAZ 共表达似乎阻断了 MAZ 的激活作用。这些结果表明，一组新型锌指蛋白可能有助于通过其 GA 顺式元件调节 CLC-K1 和 CLC-K2 通道基因的严格组织和肾单位片段特异性表达。
The expression of CLC-K1 and CLC-K2, two kidney-specific CLC chloride channels, is transcriptionally regulated on a tissue-specific basis. Previous studies have shown that a GA element near their transcriptional start sites is important for basal and cell-specific activities of the CLC-K1 and CLC-K2 gene promoters. To identify the GA-binding proteins, the human kidney cDNA library was screened by a yeast one-hybrid system. A novel member of the Cys2-His2 zinc finger gene designated KKLF (for "kidney-enriched Kruppel-like factor") and the previously isolated MAZ (for "myc-associated zinc finger protein") were cloned. KKLF was found to be abundantly expressed in the liver, kidneys, heart, and skeletal muscle, and immunohistochemistry revealed the nuclear localization of KKLF protein in interstitial cells in heart and skeletal muscle, stellate cells, and fibroblasts in the liver. In the kidneys, KKLF protein was localized in interstitial cells, mesangial cells, and nephron segments, where CLC-K1 and CLC-K2 were not expressed. A gel mobility shift assay revealed sequence-specific binding of recombinant KKLF and MAZ proteins to the CLC-K1 GA element, and the fine-mutation assay clarified that the consensus sequence for the KKLF binding site was GGGGNGGNG. In a transient-transfection experiment, MAZ had a strong activating effect on transcription of the CLC-K1-luciferase reporter gene. On the other hand, KKLF coexpression with MAZ appeared to block the activating effect of MAZ. These results suggest that a novel set of zinc finger proteins may help regulate the strict tissue- and nephron segment-specific expression of the CLC-K1 and CLC-K2 channel genes through their GA cis element.