Yamada Kazuya, Kawata Hiroko, Matsuura Kaoru, Shou Zhangfei, Hirano Satoko, Mizutani Tetsuya, Yazawa Takashi, Yoshino Miki, Sekiguchi Toshio, Kajitani Takashi, Miyamoto Kaoru
Department of Biochemistry, Fukui Medical University, Japan.
Biochem Biophys Res Commun. 2002 Sep 20;297(2):368-74. doi: 10.1016/s0006-291x(02)02203-9.
Zinc-fingers and homeoboxes 1 (ZHX1) is a protein that interacts with the activation domain of the A subunit of nuclear factor-Y. The function of ZHX1, as a transcription factor, was characterized and their domains were mapped. To determine the nuclear localization signal, expression vectors, in which various truncated forms of ZHX1 were fused to the C-terminal of green fluorescence protein (GFP), were transfected into human embryonic kidney (HEK) 293 cells. All GFP-ZHX1 fusion proteins including an arginine-rich region that corresponds to the amino acid sequence between 734 and 768 were localized in the nuclei. A dimerization domain of the ZHX1 was also mapped using protein-protein interaction assays. The homeodomain (HD) 1 consisting of the amino acid sequence between 272 and 432 of ZHX1 was necessary and sufficient for dimerization. Lastly, the transcriptional activity of ZHX1 was examined using a mammalian one-hybrid system. ZHX1, fused to the C-terminal of the GAL4 DNA-binding domain, was co-transfected with luciferase reporter plasmids with or without five copies of the GAL4-binding site into HEK293 cells. The luciferase activity was decreased in both concentration- and GAL4-binding site-dependent manner. The acidic region corresponding to the amino acid sequence between 831 and 873 was a repressor domain and dimerization was prerequisited for full repressor activity.
锌指蛋白与同源框蛋白1（ZHX1）是一种与核因子-Y A亚基的激活结构域相互作用的蛋白质。对ZHX1作为转录因子的功能进行了表征，并对其结构域进行了定位。为了确定核定位信号，将各种截短形式的ZHX1与绿色荧光蛋白（GFP）的C末端融合的表达载体转染到人胚肾（HEK）293细胞中。所有包含对应于734至768氨基酸序列的富含精氨酸区域的GFP-ZHX1融合蛋白都定位于细胞核中。还使用蛋白质-蛋白质相互作用分析对ZHX1的二聚化结构域进行了定位。由ZHX1的272至432氨基酸序列组成的同源结构域（HD）1对于二聚化是必要且充分的。最后，使用哺乳动物单杂交系统检测了ZHX1的转录活性。将与GAL4 DNA结合结构域的C末端融合的ZHX1与带有或不带有五个GAL4结合位点拷贝的荧光素酶报告质粒共转染到HEK293细胞中。荧光素酶活性以浓度和GAL4结合位点依赖性方式降低。对应于831至873氨基酸序列的酸性区域是一个抑制结构域，二聚化是完全抑制活性的先决条件。
