MECHANISMS OF TRANSFORMATION OF MURINE HEMATOPOIETIC CELLS BY ME26 VIRUS

ME26病毒转化小鼠造血细胞的机制

• 批准号: 2463694
• 负责人: S K RUSCETTI
• 金额: --
• 依托单位: DIVISION OF BASIC SCIENCES - NCI
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/2463694
• 关键词: Retroviridae; chimeric proteins; erythropoietin; gel mobility shift assay; gene deletion mutation; gene expression; genetic promoter element; growth factor receptors; hematopoietic stem cells; laboratory mouse; murine leukemia virus; neoplasm /cancer genetics; neoplastic transformation; oncogenes; oncoproteins; tissue /cell culture; transcription factor; viral leukemogenesis; virus protein

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.

本项目的目的是使用逆转录病毒为基础的小鼠模型 系统来了解转录的不适当激活 造血细胞中的因子可导致 白血病 我们一直在研究含有myb-ets的ME 26病毒， 它通过一种新的机制导致小鼠白血病的高发病率 涉及红细胞特异性基因的不适当激活， 造血前体细胞 我们之前已经证明，ME 26 病毒蛋白可以反式激活红细胞特异性 转录因子加塔-1，然后与加塔-1协同作用 转录激活促红细胞生成素受体的蛋白质， 其他红细胞特异性基因。 这导致了不受监管的 在红细胞存在下病毒感染细胞的增殖 促红细胞生成素激素 为了更好地理解 病毒蛋白可以反式激活加塔-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 对于ME 26病毒蛋白的激活至关重要。 我们的结果 表示加塔-1启动子3 ′端序列， 包括两个CACCC元件，是ME反式激活所必需26 病毒，而其他上游网站有助于完全激活的 病毒 CACCC位点的突变消除了ME 26病毒 反式激活 含有ME 26的细胞提取物的相互作用 病毒蛋白和含有两个CACCC元件的加塔-1片段 通过电泳迁移率变动分析检测， 两者之间没有直接的互动。 然而，我们可以检测到 普遍存在的转录因子Sp1与该序列结合。 这些 数据表明，CACCC元件对于加塔-1是必需的 ME 26病毒的启动子反式激活，并且病毒蛋白可以 通过与Sp1结合间接反式激活启动子。