The analysis of cell lineage specific expression of perforin gene

MEF是ETS转录因子Elf亚家族成员。MEF被证明激活GN-CSF和IL-3基因的表达，其中Elf-1，最接近MEF的基因，也反式激活。然而，基因敲除实验表明，MEF是必不可少的穿孔素基因在NK细胞中的表达，而不是在抑制性T细胞，另一方面，Elf-1敲除小鼠没有表现出明确的表型。为了阐明穿孔素基因谱系特异性表达的机制，我们计划了两件事：（1）确定与MEF相关的蛋白质;（2）将其与Elf-1的蛋白质进行比较。为此，我们以MEF为诱饵进行了“串联亲和纯化”（TAP）分析。将转染了MEF表达质粒的细胞用IG G cepharose珠和钙调蛋白regine裂解和纯化两次，然后用SDS-PGE分离与MEW相关的蛋白。在用蛋白质染色可视化后，用LC-MAS光谱法分析每个条带。检测到的蛋白质有：Myb结合蛋白、核仁素、RNA解旋酶、波形蛋白、核磷蛋白。其中，近三分之一的急性髓系白血病患者的核磷蛋白发生突变，提示其在造血细胞中的重要作用。我们现在正在分析MEF和核磷蛋白之间的直接物理和功能相互作用。

MEF is a member of Elf-sub family of ETS transcription factors. MEF was shown to activate the expression of GN-CSF and IL-3 genes, which Elf-1, the closest gene to MEF, also transactivates. However, gene knock-out experiments demonstrated that MEF is indispensable for the expression of perforin gene in NK cells but not in suppressor T-cells, on the other hand, Elf-1 knock-out mice did not show clear phenotype. To elucidate the mechanisms of lineage specific expression of perforin gene, we planned two things : (1)determine the protein(s) that associate with MEF and (2)compare it with those of Elf-1. For this purpose, we performed "tandem affinity purification"(TAP) analysis using MEF as bait Cells trasfected with the expression plasmid for MEF were lysed and purified twice with Ig G cepharose beads and calmodulin regine, then associated proteins with MEW were fractionated by SDS-PGE. After visualized with protein staining, each bands was analysed with LC-MAS spectrometry. Proteins picked up were : Myb binding protein, nucleolin, RNA helicase, vimentin, nucleophosmin. Among those, nucleophosmin was shown to be mutated in one-third of acute myeloid leukemia recently, suggesting its important roles in hematopoietic cells. We are now in the process of the analysis for the direct physical and functional interactions between MEF and nucleophosmin.