Penner C G, Davie J R
Department of Biochemistry and Molecular Biology, Faculty of Medicine, University of Manitoba, Winnipeg, Canada.
Biochem J. 1992 May 1;283 ( Pt 3)(Pt 3):905-11. doi: 10.1042/bj2830905.
We identified the factor(s) that bind to the chicken erythroid-cell-specific histone H5 enhancer region which is located on the 3' end of the gene. In DNAase I footprinting and u.v. cross-linking experiments with nuclear extracts from adult chicken immature erythrocytes, we determined that the trans-acting factor GATA-1 was the predominating protein interacting with the histone H5 enhancer. GATA-2 and GATA-3 were not detected. In contrast, gel-mobility-shift assays and competition experiments demonstrated that several specific complexes formed with the histone H5 enhancer region. Gel-mobility-shift assays with 23 bp oligonucleotides containing the GATA-binding site (AGATAA) of the histone H5 enhancer or of the beta-globin enhancer showed that the GATA sequence was sufficient for the formation of at least five complexes. Diagonal mobility-shift assays demonstrated that multisubunit complexes were forming with the GATA-1 protein. Our interpretation of the results is that GATA-1 interacts with a protein of approx. 105 kDa which, in turn, can associate with protein or protein complexes of approx. 26 kDa, 146 kDa and a protein(s) of molecular mass greater than 450 kDa. The different multisubunit complexes formed via the trans-acting factor GATA-1 may impart different transcriptional responses to the promoter and enhancer elements of the histone H5 and globin genes.
我们鉴定了与鸡红细胞特异性组蛋白H5增强子区域结合的因子，该区域位于该基因的3'端。在对成年鸡未成熟红细胞核提取物进行的DNA酶I足迹分析和紫外线交联实验中，我们确定反式作用因子GATA-1是与组蛋白H5增强子相互作用的主要蛋白质。未检测到GATA-2和GATA-3。相比之下，凝胶迁移率变动分析和竞争实验表明，有几种特异性复合物与组蛋白H5增强子区域形成。用含有组蛋白H5增强子或β-珠蛋白增强子的GATA结合位点（AGATAA）的23 bp寡核苷酸进行凝胶迁移率变动分析表明，GATA序列足以形成至少五种复合物。对角线迁移率变动分析表明，多亚基复合物正在与GATA-1蛋白形成。我们对结果的解释是，GATA-1与一种约105 kDa的蛋白质相互作用，而该蛋白质又可与约26 kDa、146 kDa的蛋白质或蛋白质复合物以及分子量大于450 kDa的一种或多种蛋白质结合。通过反式作用因子GATA-1形成的不同多亚基复合物可能会赋予组蛋白H5和珠蛋白基因的启动子和增强子元件不同的转录反应。
