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甲状腺激素反应基因。添加甲状腺激素的活性形式3，3 '，5-三碘甲状腺原氨酸（T_3）可增强皮肤成纤维细胞中ZAKII-4基因的转录。然而，转录激活完全阻断预处理放线菌酮，蛋白质合成的翻译抑制剂。结果表明，T_3激活ZAKI-4基因的转录需要从头蛋白质合成。T_3可能诱导了某些因子增强ZAKI-4基因的转录。T_3是通过与核受体结合，调节靶基因的表达而发挥作用的，因此，T_3诱导因子对ZAKI-4基因转录的促进作用可能为甲状腺激素的作用机制提供新的线索。因此，我们设计了本研究项目，以阐明T_ 关于我们 通过使用ZAKI-4基因的5 ′-侧翼区的ZAKI-4基因的3依赖性转录激活。我们通过引物延伸法鉴定了转录起始位点。利用PromoterFinderTM技术，我们在ZAKI-4的5'侧翼区克隆了一个约2.8kb的DNA片段，命名为alphaE 2 -5。我们构建了一个嵌合荧光素酶报告基因与alphaE 2 -5，并将此构建体转染到ZAKI-4表达的人胶质瘤细胞系，U-251-MG。与alphaE 2 -5，荧光素酶活性增加5倍，观察到，alphaE 2 -5含有一个区域，介导的ZAKI-4的启动子活性。我们还对启动子区进行了精确的分析，发现-28 ~-4区域介导了启动子的活性以及T_3依赖的转录调控。通过使用U-251 MG的核提取物的电泳迁移率变动分析（EMSA），我们证实了提取物含有与TFZ-1结合的蛋白质。以TFZ-1为探针，用Southwestern印迹法进一步分析了该蛋白，结果表明该蛋白的表观分子量为45 kDa，加入10 μ M T_3后表达量增加<-7>。这些结果表明TMZ 45蛋白是T_3介导ZAKI-4转录调控的因子。目前，我们正在尝试用酵母单杂交系统克隆TMZ 45的cDNA。少

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