Kurokawa M, Tanaka T, Tanaka K, Hirano N, Ogawa S, Mitani K, Yazaki Y, Hirai H
Third Department of Internal Medicine, Faculty of Medicine, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113, Japan.
J Biol Chem. 1996 Jul 12;271(28):16870-6. doi: 10.1074/jbc.271.28.16870.
The AML1 gene encodes DNA-binding proteins that contain the runt homology domain and is found at the breakpoints of t(8;21), t(3;21), and t(12;21) translocations associated with myelogenous leukemias. AML1 heterodimerizes with PEBP2beta/CBFbeta, resulting in the enhanced affinity with DNA. The runt homology domain is responsible for binding with DNA and heterodimerizing with PEBP2beta/CBFbeta. AML1 is suggested to perform a pivotal role in myeloid cell differentiation, whereas it can cause neoplastic transformation when overexpressed in fibroblasts. In this study, we demonstrated that the reducing reagent, dithiothreitol (DTT), markedly enhances the DNA binding of AML1 expressed in COS7 cells. Oxidation by diamide or modification by N-ethylmaleimide of the free sulfhydryl residues inhibited the interaction of AML1 with DNA. The diamide effect was reversible with excess of DTT, whereas DTT could not restore the DNA binding of AML1 treated with N-ethylmaleimide. Site-directed mutagenesis of the amino acid residue 72, a highly conserved cysteine in the runt homology domain of AML1, to serine almost completely abolished DNA binding without altering the interaction with PEBP2beta/CBFbeta. This substitution also impaired transactivation through the consensus DNA sequence and transformation of fibroblasts induced by AML1b. These data indicate an essential role of the conserved cysteine residue in DNA binding of AML1, and it is possible that the redox state of AML1 could contribute to the regulation of its function.
AML1基因编码包含 runt 同源结构域的 DNA 结合蛋白，在与骨髓性白血病相关的 t(8;21)、t(3;21) 和 t(12;21) 易位的断点处被发现。AML1 与 PEBP2β/CBFβ 形成异二聚体，从而增强与 DNA 的亲和力。runt 同源结构域负责与 DNA 结合并与 PEBP2β/CBFβ 形成异二聚体。AML1 被认为在髓样细胞分化中起关键作用，而当它在成纤维细胞中过度表达时可导致肿瘤转化。在本研究中，我们证明了还原试剂二硫苏糖醇（DTT）显著增强了 COS7 细胞中表达的 AML1 与 DNA 的结合。二酰胺氧化或 N-乙基马来酰亚胺对游离巯基残基的修饰抑制了 AML1 与 DNA 的相互作用。二酰胺的作用可被过量的 DTT 逆转，而 DTT 不能恢复用 N-乙基马来酰亚胺处理的 AML1 与 DNA 的结合。将 AML1 的 runt 同源结构域中高度保守的半胱氨酸残基 72 定点突变为丝氨酸几乎完全消除了与 DNA 的结合，而不改变与 PEBP2β/CBFβ 的相互作用。这种取代也损害了通过共有 DNA 序列的反式激活以及由 AML1b 诱导的成纤维细胞转化。这些数据表明保守的半胱氨酸残基在 AML1 与 DNA 结合中起重要作用，并且 AML1 的氧化还原状态可能有助于其功能的调节。
