MECHANISMS OF TRANSFORMATION OF MURINE HEMATOPOIETIC CELLS BY ME26 VIRUS

该项目的目标是使用基于逆转录病毒的小鼠模型 系统来理解转录的不适当激活 造血细胞中的因素可导致 白血病。我们一直在研究含有MYB-ETS的ME26病毒， 它通过一种新的机制导致小鼠白血病的高发病率 涉及红系特异基因的不适当激活 造血祖细胞。我们之前已经表明，ME26 病毒蛋白可反式激活红系特异性启动子 转录因子GATA-1，然后与GATA-1协同 转录激活促红细胞生成素受体的蛋白质和 其他红系特有基因。这导致了不受监管的 病毒感染细胞在红系存在时的增殖 激素促红细胞生成素。为了更好地理解 病毒蛋白可以反式激活GATA-1启动子，我们设计并 分析缺失突变体以确定启动子中的哪些序列 对ME26病毒蛋白的激活至关重要。我们的结果 表明GATA-1启动子3‘端序列 包括两个CACCC元件，是ME26反式激活所必需 病毒，而其他上游站点有助于通过 病毒。CACCC位点突变导致ME26病毒消失 激活。含ME26的细胞提取物的相互作用 病毒蛋白和含有两个CACCC元件的GATA-1片段 通过电泳迁移率变化分析进行检测，结果 显示两者之间没有直接的互动。然而，我们可以检测到 转录因子Sp1与该序列结合的普遍存在的转录因子Sp1这些 数据表明，CACCC元素是GATA-1所必需的 启动子被ME26病毒反式激活，病毒蛋白可能 通过与Sp1结合间接反式激活启动子。

The objective of this project is to use a retrovirus-based mouse model system to understand how inappropriate activation of transcription factors in hematopoietic cells can result in the development of leukemia. We have been studying the myb-ets-containing ME26 virus, which causes a high incidence of leukemia in mice by a novel mechanism involving the inappropriate activation of erythroid-specific genes in hematopoietic precursor cells. We have previously shown that the ME26 viral protein can transactivate the promoter of the erythroid-specific transcription factor, GATA-1, and then cooperate with the GATA-1 protein to transcriptionally activate the erythropoietin receptor and other erythroid-specific genes. This results in the unregulated proliferation of virus-infected cells in the presence of the erythroid hormone erythropoietin. In an attempt to better understand how the viral protein can transactivate the GATA-1 promoter, we engineered and analyzed deletion mutants to determine which sequences in the promoter are crucial for activation by the ME26 viral protein. Our results indicate that sequences in the 3' end of the GATA-1 promoter, which include two CACCC elements, are essential for transactivation by ME26 virus, while other upstream sites contribute to full activation by the virus. Mutation of the CACCC sites abolishes ME26 viral transactivation. The interaction of cell extracts containing ME26 viral protein and the GATA-1 fragment containing the two CACCC elements was examined by electrophoretic mobility shift analysis and the results showed no direct interaction between the two. However, we could detect the ubiquitous transcription factor Sp1 bound to this sequence. These data demonstrate that the CACCC element is necessary for GATA-1 promoter transactivation by ME26 virus and that the viral protein may indirectly transactivate the promoter by binding to Sp1.