Perkins A S, Kim J H
Department of Pathology, Yale University School of Medicine, New Haven, Connecticut 06520-8023, USA.
J Biol Chem. 1996 Jan 12;271(2):1104-10. doi: 10.1074/jbc.271.2.1104.
EVI1 is a zinc finger oncoprotein that binds via fingers 1-7 to the sequence GACAAGATAA. The target genes on which EVI1 acts are unknown. This binding motif overlaps with that for the GATA transcription factors, (T/A)GATA(A/G), and GATA-1 can bind to and activate transcription via a GACAAGATAA motif. The possibility has been raised that, when overexpressed in leukemogenesis, EVI1 may function by interfering with the differentiation-promoting action of GATA factors. To explore this, we have assessed the affinity of EVI1 for the GATA binding sites derived from erythroid-specific GATA-1 target genes, and found only low affinity interactions. We examined the contacts between EVI1 and DNA by methylation interference studies, which revealed extensive contacts between EVI1 and its binding site. The importance of the contacts for high affinity binding was shown by in vitro quantitative gel shift studies and in vivo cotransfection studies. To examine what types of sequences from mouse genomic DNA bind to EVI1, we isolated and sequenced five EVI1-binding fragments, and each showed the GACAAGATA site. The data presented contribute to our knowledge of the binding specificity of EVI1, and yield a clearer picture of what sequences can, and cannot, act as targets for EVI1 action.
EVI1是一种锌指癌蛋白，它通过第1至7个锌指与序列GACAAGATAA结合。EVI1作用的靶基因尚不清楚。这种结合基序与GATA转录因子的结合基序（T/A）GATA（A/G）重叠，并且GATA-1可以通过GACAAGATAA基序结合并激活转录。有人提出，在白血病发生过程中过表达时，EVI1可能通过干扰GATA因子的促分化作用发挥功能。为了探究这一点，我们评估了EVI1对源自红系特异性GATA-1靶基因的GATA结合位点的亲和力，结果发现只有低亲和力相互作用。我们通过甲基化干扰研究检测了EVI1与DNA之间的接触，结果显示EVI1与其结合位点之间存在广泛接触。体外定量凝胶迁移研究和体内共转染研究表明了这些接触对于高亲和力结合的重要性。为了检测小鼠基因组DNA中的哪些序列类型与EVI1结合，我们分离并测序了五个EVI1结合片段，每个片段都显示出GACAAGATA位点。所呈现的数据有助于我们了解EVI1的结合特异性，并更清楚地了解哪些序列可以，哪些序列不能作为EVI1作用的靶标。
