Thrombopoiesis regulated by a transcription factor NF-E2

本研究的目的是通过分析异源二聚体转录因子NF-E2.1的功能来阐明血小板生成的调控机制。结构和功能分析的MafG的小MafA缺失突变体，缺乏40个氨基酸的C-末端没有拯救缺陷的前血小板形成的mafG-空小鼠的巨核细胞时，突变MafG从转基因供应。该结果清楚地表明MafG C-末端区域对于巨核细胞中的小Ma功能是关键的。我们通过酵母双杂交筛选与MafG C-末端区域相互作用的蛋白。我们发现NF-E2 p45与全长MafG相互作用，但不与没有C-末端的MafG相互作用。有趣的是，CNC家族转录因子的另一个成员和NF-E2 p45的相关因子Nrf 2确实与MafG相互作用，而不管C-末端区域的存在。这些结果表明MafG C端区域可能参与了肿瘤的发生， 关于我们 MafG的异二聚体配偶体分子的选择。NF-E2 p45的亚细胞定位我们建立了在巨核细胞中表达Flag标记的NF-E2 p45或GFP融合的NF-E2 p45的转基因小鼠系。我们还获得了表达Flag-HA-His标记的NF-E2 p45的稳定的巨核细胞系。通过使用这些小鼠和细胞，我们正在检查NF-E2 p45. 3的亚细胞分布。NF-E2 p45的靶基因我们从小Maf突变巨核细胞和对照巨核细胞的消减筛选中鉴定出一个新的基因克隆325。我们在小鼠中破坏了325基因，发现小鼠是健康的和可生育的，显示正常的血小板计数。我们正计划给予小鼠抗血小板抗体和/或骨髓抑制剂，并观察其对血小板生成的影响。我们还对NF-E2 p45基因敲除小鼠进行了转录组分析，发现参与血小板功能和结构的因子减少，而抗氧化反应基因和解毒酶基因增加，这表明氧化应激参与了巨核细胞生成和血小板生成的过程。少

The goal of this study was to clarify the regulatory mechanisms of thrombopoiesis through analyzing the function of a heterodimeric transcription factor NF-E2.1. Structure and function analysis of small MafA deletion mutant of MafG that lacks 40 amino acids of its C-terminus did not rescue the defective proplatelet formation of megakaryocytes from mafG-null mice when the mutant MafG was supplied from the transgene. This results clearly showed that the MafG C-terminal region is critical to the small Ma function in megakaryocytes. We investigated the proteins interacting to the MafG C-terminal region through the yeast two hybrid screening. We found that NF-E2 p45 interacts with the full length of MafG but not with MafG without the C-terminus. Interestingly, Nrf2, another member of CNC family transcription factor and a related factor of NF-E2 p45, does interact with MafG irrespective of the presence the C-terminal region. These results imply that MafG C-terminal region may contribute to t … More he selection of a heterodimeric partner molecule of MafG.2. Subcellular localization of NF-E2 p45We established transgenic mouse lines expressing Flag-tagged NF-E2 p45 or GFP-fused NF-E2 p45 in megakaryocytes. We also obtained a stable transformant megakaryocytic cell lines expressing Flag-HA-His tagged NF-E2 p45. By using these mice and cells, we are examining the subcellular distribution of NF-E2 p45.3. Target genes of NF-E2 p45We identified a novel gene clone 325 from the subtraction screening of small Maf mutant megakaryocytes and the control megakaryocytes. We disrupted the 325 gene in mice and found that the mice is healthy and fertile displaying the normal platelet count. We are planning to administer anti-platelet antibody and/or myelosupressive reagents to the mice and to see their effects on the thrombopoiesis. We also performed transcriptome analysis of NF-E2 p45-null mice and found that the factors involved in the platelet function and structures are decreased while antioxidant response genes and detoxifying enzyme genes were increased, which suggests the involvement of oxidative stress in the process of megakaryopoiesis and thrombopoiesis. Less