Elucidation of novel mechanism of thyroid hormone action

ZAKI-4 是我们最近在人类皮肤成纤维细胞中发现的一种甲状腺激素反应基因。添加 3, 3', 5-三碘甲状腺原氨酸 (T_3)（甲状腺激素的一种活性形式）可增强皮肤成纤维细胞中 ZAKII-4 基因的转录。然而，转录激活被放线菌酮（蛋白质合成的翻译抑制剂）预处理完全阻断。结果表明，T_3需要从头合成蛋白质来激活ZAKI-4基因的转录。 T_3 似乎诱导了一些增强 ZAKI-4 基因转录的因子。由于人们普遍认为T_3通过与其核受体结合并调节靶基因的表达来发挥作用，因此鉴定增强ZAKI-4基因转录的T_3诱导因子可以揭示甲状腺激素作用的新机制。因此，我们设计了当前的研究项目，以利用ZAKI-4基因的5'侧翼区域来阐明ZAKI-4基因的T_… More 3依赖性转录激活机制。我们通过引物延伸方法鉴定了转录起始位点。使用PromoterFinderTM，我们在ZAKI-4的5'侧翼区域克隆了一个DNA片段（约2.8 kb，命名为alphaE2-5）。我们构建了带有 alphaE2-5 的嵌合荧光素酶报告基因，并将该构建体转染到表达 ZAKI-4 的人胶质瘤细胞系 U-251-MG 中。使用 alphaE2-5，观察到荧光素酶活性增加了 5 倍，表明 alphaE2-5 含有介导 ZAKI-4 启动子活性的区域。我们还对启动子区域进行了精确分析，发现-28至-4区域介导启动子活性以及T_3依赖性转录调控。通过使用 U-251MG 的核提取物进行电泳迁移率变动分析 (EMSA)，我们确认提取物含有与 TFZ-1 结合的蛋白质。我们进一步用TFZ-1作为探针通过Southern印迹分析了该蛋白质，并证明该蛋白质的表观分子量为45kDa，并且通过添加10^-7MT_3表达增加。这些结果表明，命名为TMZ45的蛋白质​​是介导T_3对ZAKI-4转录调节的因子。目前我们正在尝试使用酵母一杂交系统克隆 TMZ45 的 cDNA。较少的

ZAKI-4 is a thyroid hormone-responsive gene that we have recently identified in human skin fibroblasts. Addition of 3, 3', 5-triiodothyronine (T_3), an active form of thyroid hormone, enhances the transcription of the ZAKII-4 gene in skin fibroblasts. However, transcriptional activation was completely blocked by the pretreatment with cycloheximide, a translation inhibitor of protein synthesis. The result indicates that de novo protein synthesis is required to activate the transcription of the ZAKI-4 gene by T_3. It appears that T_3 induces some factor(s) which enhances ZAKI-4 gene transcription. Since it has generally been accepted that T_3 exerts its effect through binding to its nuclear receptor and regulating expression of target genes, the identification of the T_3 induced factor(s) that enhance the transcription of the ZAKI-4 gene could uncover a novel mechanism of thyroid hormone action. Therefore, we designed the current research project in order to elucidate the mechanism of T_ … More 3 dependent transcriptional activation of ZAKI-4 gene by using a 5'-flanking region of the ZAKI-4 gene. We identified the transcription start site by the primer extension method. Using PromoterFinderTM, we cloned a DNA fragment (about 2.8 kb, designated as alphaE2-5) in the 5' flanking region of ZAKI-4. We constructed a chimeric luciferase reporter gene with alphaE2-5 and transfected this construct to a ZAKI-4 expressing human glioma cell line, U-251-MG.With alphaE2-5, a 5-fold increase in luciferase activity was observed, indicating that alphaE2-5 contains a region which mediates the promoter activity of ZAKI-4. We have also precisely analyzed the promoter region and found that a region from -28 to -4 mediated the promoter activity as well as the T_3 dependent transcriptional regulation. By electrophoretic mobility shift assay (EMSA) using the nuclear extract from U-251MG, we confirmed that the extract contained a protein which binds to TFZ-1. We further analyzed the protein by South Western blotting with TFZ-1 as a probe and demonstrated that the apparent molecular mass of the protein is 45 kDa and expression increased by the addition of 10^<-7>M T_3. These results suggest that the protein designated as TMZ45 is the factor which mediates transcriptional regulation of ZAKI-4 by T_3. Currently we are trying to clone the cDNA for TMZ45 using the yeast one hybrid system. Less