Fears S, Gavin M, Zhang D E, Hetherington C, Ben-David Y, Rowley J D, Nucifora G
University of Chicago, IL 60637, USA.
Proc Natl Acad Sci U S A. 1997 Mar 4;94(5):1949-54. doi: 10.1073/pnas.94.5.1949.
The ETV6/CBFA2 (TEL/AML1) fusion gene occurs as a result of the chromosome translocation t(12;21)(p13;q22) in up to 30% of children diagnosed with B cell precursor (cd10+, cd19+) acute lymphoblastic leukemia. Leukemic cells that have acquired the t(12;21) usually demonstrate loss of the remaining normal ETV6 (TEL) allele. Using reporter gene assays we have functionally characterized both the normal ETV6 and ETV6/CBFA2 fusion proteins in the regulation of the MCSFR proximal promoter. Neither ETV6 or ETV6/CBFA2 has any significant, detectable effect on the promoter by itself. However, both ETV6 and ETV6/CBFA2 inhibit the activation of the promoter by CBFA2B(AML1B) and C/EBPa. We have shown that a 29-bp region of the MCSFR promoter containing the binding sites for CBFA2B and C/EBPa is sufficient for the inhibition by ETV6 and ETV6/CBFA2. Mutational analysis of the MCSFR promoter revealed that binding of both CBFA2B and C/EBPa to their respective sites is necessary for the inhibition by ETV6 and ETV6/CBFA2. Deletion of the helix-loop-helix (HLH) region from the cDNAs of ETV6 and ETV6/CBFA2 decreased but did not completely abrogate the ability of either construct to inhibit promoter activation. We also found that the ETS DNA binding region of ETV6 is necessary for inhibition of the promoter. Addition of ETS1 and FLI1, two ETS family members that have homology in the 5' HLH region, but not Spi1, an ETS family member without the 5' HLH region, also inhibited reporter gene expression. Our data show that the inhibition mediated by ETV6 and ETV6/CBFA2, in the context of the MCSFR promoter, depend on interactions with other proteins, not just CBFA2B. Our results also indicate that the transactivation characteristics of ETV6/CBFA2 are a combination of positive and negative regulatory properties.
在高达30%被诊断为B细胞前体（CD10+、CD19+）急性淋巴细胞白血病的儿童中，ETV6/CBFA2（TEL/AML1）融合基因是由染色体易位t(12;21)(p13;q22)产生的。获得t(12;21)的白血病细胞通常显示剩余正常ETV6（TEL）等位基因的缺失。我们使用报告基因检测在调节MCSFR近端启动子时对正常ETV6和ETV6/CBFA2融合蛋白进行了功能表征。单独的ETV6或ETV6/CBFA2对启动子均无任何显著的、可检测到的影响。然而，ETV6和ETV6/CBFA2均抑制CBFA2B(AML1B)和C/EBPa对启动子的激活。我们已表明，MCSFR启动子中包含CBFA2B和C/EBPa结合位点的一个29 bp区域足以被ETV6和ETV6/CBFA2抑制。MCSFR启动子的突变分析显示，CBFA2B和C/EBPa与其各自位点的结合对于ETV6和ETV6/CBFA2的抑制作用是必需的。从ETV6和ETV6/CBFA2的cDNA中缺失螺旋-环-螺旋（HLH）区域会降低但并未完全消除任一构建体抑制启动子激活的能力。我们还发现ETV6的ETS DNA结合区域对于启动子的抑制是必需的。添加ETS1和FLI1这两个在5' HLH区域具有同源性的ETS家族成员，但不添加没有5' HLH区域的ETS家族成员Spi1，也会抑制报告基因表达。我们的数据表明，在MCSFR启动子的背景下，由ETV6和ETV6/CBFA2介导的抑制作用取决于与其他蛋白质的相互作用，而不仅仅是与CBFA2B的相互作用。我们的结果还表明，ETV6/CBFA2的反式激活特性是正负调节特性的组合。
