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首页> 外文期刊>Molecular and Cellular Biology >E2A-HLF-mediated cell transformation requires both the trans-activation domains of E2A and the leucine zipper dimerization domain of HLF.
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E2A-HLF-mediated cell transformation requires both the trans-activation domains of E2A and the leucine zipper dimerization domain of HLF.

机译：E2A-HLF介导的细胞转化需要E2A的反式激活域和HLF的亮氨酸拉链二聚化域。

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The E2A-HLF fusion gene, formed by the t(17;19)(q22;p13) translocation in childhood acute pro-B-cell leukemia, encodes a hybrid protein that contains the paired trans-activation domains of E2A (E12/E47) linked to the basic region/leucine zipper DNA-binding and dimerization domain of hepatic leukemia factor (HLF). To assess the transforming potential of this novel gene, we introduced it into NIH 3T3 murine fibroblasts by using an expression vector that also contained the neomycin resistance gene. Cells selected for resistance to the neomycin analog G418 formed aberrant colonies in monolayer cultures, marked by increased cell density and altered morphology. Transfected cells also grew readily in soft agar, producing colonies whose sizes correlated with E2A-HLF expression levels. Subclones expanded from colonies with high levels of the protein reproducibly formed tumors in nude mice and grew to higher plateau-phase cell densities in reduced-serum conditions than did parental NIH 3T3 cells. By contrast, NIH 3T3 cells expressing mutant E2A-HLF proteins that lacked either of the bipartite E2A trans-activation domains or the HLF leucine zipper domain failed to show oncogenic properties, including anchorage-independent cell growth. Thus, both of the E2A trans-activation motifs and the HLF leucine zipper dimerization domain are essential for the transforming potential of the chimeric E2A-HLF protein, suggesting a model in which aberrant regulation of the expression pattern of downstream target genes contributes to leukemogenesis.
机译：由儿童急性前B细胞白血病中的t（17; 19）（q22; p13）易位形成的E2A-HLF融合基因编码包含E2A（E12 / E47）的成对反式激活结构域的杂合蛋白）与肝白血病因子（HLF）的基本区域/亮氨酸拉链DNA结合和二聚结构域连接。为了评估该新基因的转化潜力，我们通过使用还包含新霉素抗性基因的表达载体将其引入到NIH 3T3鼠成纤维细胞中。选择对新霉素类似物G418具有抗性的细胞在单层培养物中形成异常集落，其特征在于细胞密度增加和形态改变。转染的细胞也容易在软琼脂中生长，产生集落，其大小与E2A-HLF表达水平相关。亚克隆从具有高水平蛋白质的集落中扩增出来，可在裸鼠中形成肿瘤，并且在降低的血清条件下比亲代NIH 3T3细胞生长到更高的高原期细胞密度。相比之下，表达突变E2A-HLF蛋白的NIH 3T3细胞缺少两部分E2A反式激活结构域或HLF亮氨酸拉链结构域，它们均未显示致癌特性，包括不依赖锚定的细胞生长。因此，E2A反式激活基序和HLF亮氨酸拉链二聚化结构域对于嵌合E2A-HLF蛋白的转化潜力都是必不可少的，这提示了下游靶基因表达模式的异常调节有助于白血病发生的模型。

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