Chang C P, de Vivo I, Cleary M L
Department of Pathology, Stanford University School of Medicine, California 94305, USA.
Mol Cell Biol. 1997 Jan;17(1):81-8. doi: 10.1128/MCB.17.1.81.
E2a-Pbx1 chimeric oncoproteins result from fusion of the E2A and PBX1 genes at the sites of t(1;19) chromosomal translocations in a subset acute lymphoblastic leukemias. Experimentally, E2a-Pbx1 transforms a variety of cell types, including fibroblasts, myeloid progenitors, and lymphoblasts. Structure-function studies have shown that contributions from both E2a and Pbx1 are necessary for oncogenesis, but the Pbx1 homeodomain is dispensable and the required portion of Pbx1 has not been delineated. In this study, we used deletional and site-directed mutagenesis to identify portions of Pbx1 necessary for oncogenic and transcriptional activities of E2a-Pbx1. These studies defined a motif (named the Hox cooperativity motif [HCM]) carboxy terminal to the Pbx homeodomain that is required for cooperative DNA binding, cellular transcriptional activity, and the oncogenic potential of E2a-Pbx1. The HCM is highly conserved throughout the Pbx/exd subfamily of divergent homeodomain proteins and functions in DNA-binding assays as a potential contact site for Hox dimerization. E2a-Pbx1 proteins with interstitial deletion or single-point mutations in the HCM could neither activate transcription in cellular assays nor transform NIH 3T3 cells. An E2a-Pbx1 mutant containing 50 amino acids of Pbx1b spanning the HCM but lacking the homeodomain was capable of inducing fibroblast transformation. Thus, the HCM is a necessary and sufficient contribution of Pbx1 for oncogenesis induced by E2a-Pbx1 and accounts for its homeodomain-independent transforming properties. Since subtle alterations of the Pbx HCM result in complete abrogation of transforming activity whereas the homeodomain is entirely dispensable, we conclude that interactions mediated by the HCM are more important for transformation by E2a-Pbx1 than interactions with cognate Pbx DNA sites.
E2a-Pbx1嵌合癌蛋白是由E2A基因与PBX1基因在一部分急性淋巴细胞白血病的t(1;19)染色体易位位点处融合产生的。在实验中，E2a-Pbx1可转化多种细胞类型，包括成纤维细胞、髓系祖细胞和淋巴细胞。结构功能研究表明，E2a和Pbx1两者的作用对于肿瘤发生都是必需的，但Pbx1同源结构域是可有可无的，且尚未明确Pbx1所需的部分。在本研究中，我们利用缺失突变和定点诱变来确定Pbx1中对E2a-Pbx1的致癌和转录活性所必需的部分。这些研究确定了一个位于Pbx同源结构域羧基末端的基序（命名为Hox协同基序[HCM]），它是E2a-Pbx1协同DNA结合、细胞转录活性和致癌潜能所必需的。HCM在不同同源结构域蛋白的整个Pbx/exd亚家族中高度保守，并且在DNA结合试验中作为Hox二聚化的潜在接触位点发挥作用。在HCM中具有间隙缺失或单点突变的E2a-Pbx1蛋白在细胞试验中既不能激活转录，也不能转化NIH 3T3细胞。一个包含跨越HCM但缺乏同源结构域的50个Pbx1b氨基酸的E2a-Pbx1突变体能够诱导成纤维细胞转化。因此，HCM是Pbx1对E2a-Pbx1诱导肿瘤发生所必需且充分的贡献，并解释了其不依赖同源结构域的转化特性。由于Pbx HCM的细微改变会导致转化活性完全丧失，而同源结构域则完全是可有可无的，我们得出结论，由HCM介导的相互作用对于E2a-Pbx1的转化比与同源Pbx DNA位点的相互作用更为重要。
