Frank R, Zhang J, Uchida H, Meyers S, Hiebert S W, Nimer S D
Laboratory of Molecular Aspects of Hematopoiesis, Sloan-Kettering Institute, New York, NY 10021, USA.
Oncogene. 1995 Dec 21;11(12):2667-74.
The t(8;21) translocation, commonly found in acute myelogenous leukemia (AML), generates a fusion protein containing N-terminal AML1 and C-terminal ETO amino acids. The human AML1 gene encodes several related proteins that specifically bind to the sequence TGT/cGGT, located in the promoter regions of a variety of hematopoietic growth factor genes. To examine the abilities of the AML1B protein (which contains 479 amino acids), a shorter AML1A isoform (which contains amino acids 1-250), and the AML1/ETO fusion protein (which contains AML1A amino acids 1-177) to stimulate transcription from the GM-CSF promoter, we performed co-transfection experiments in T cells using a human GM-CSF promoter-CAT reporter gene plasmid and expression vectors that contain the cDNAs for one of the above proteins. Our data demonstrate that AML1B, but not AML1A or AML1/ETO transactivates the GM-CSF promoter, requiring the TGTGGT sequence contained between base pairs -68 and -53. Furthermore, we show that AML1/ETO, but not AML1A, inhibits the ability of AML1B to stimulate CAT expression. Electrophoretic mobility shift assays demonstrated the specific binding of AML1 proteins to the GM-CSF promoter TGTGGT sequence, which does not require GM-CSF sequences immediately upstream of this binding site. Our data support a role for AML1B as a transcriptional activator and establish that the AML1/ETO fusion protein can act as a dominant negative protein on the human GM-CSF promoter. Although AML1/ETO does not stimulate the transcription of GM-CSF, it may function by inhibiting the normal activity of AML1B in AML cells with the t(8;21) translocation.
常见于急性髓性白血病（AML）的t(8;21)易位会产生一种融合蛋白，该蛋白包含N端的AML1和C端的ETO氨基酸。人类AML1基因编码几种相关蛋白，这些蛋白能特异性结合位于多种造血生长因子基因启动子区域的TGT/cGGT序列。为了检测AML1B蛋白（含479个氨基酸）、较短的AML1A异构体（含1 - 250个氨基酸）以及AML1/ETO融合蛋白（含AML1A的1 - 177个氨基酸）刺激GM - CSF启动子转录的能力，我们在T细胞中进行了共转染实验，使用的是人GM - CSF启动子 - CAT报告基因质粒以及包含上述一种蛋白cDNA的表达载体。我们的数据表明，AML1B能反式激活GM - CSF启动子，而AML1A或AML1/ETO则不能，这需要位于 - 68至 - 53碱基对之间的TGTGGT序列。此外，我们发现AML1/ETO能抑制AML1B刺激CAT表达的能力，而AML1A则不能。电泳迁移率变动分析表明AML1蛋白能特异性结合GM - CSF启动子的TGTGGT序列，该结合不需要此结合位点紧邻的GM - CSF序列。我们的数据支持AML1B作为转录激活因子的作用，并证实AML1/ETO融合蛋白可在人GM - CSF启动子上作为显性负性蛋白发挥作用。虽然AML1/ETO不刺激GM - CSF的转录，但它可能通过抑制t(8;21)易位的AML细胞中AML1B的正常活性发挥作用。
