Functional Contribution of Small Maf Proteins in Hematopoietic Cells

(1) mafG基因的转录调控。通过5′-RACE鉴定出3个独立的启动子。含有mafG启动子的7kbp基因组片段能够在转基因小鼠的脑、晶状体、肺和肾中指导组织特异性报告基因的表达，但不能在造血细胞中指导。我们将对mafG基因下游的另外3kbp片段进行测试。(2) mag -null巨核细胞减法分析。从血小板形成（PPF）缺陷的巨核细胞中提取的mRNA与对照野生型巨核细胞的mRNA相减。从减法中获得的几个克隆被发现与细胞骨架蛋白有关，如肌动蛋白和微管蛋白。还获得了一个新的克隆体，命名为#325，它被发现在从鱼类到人类的各种脊椎动物中都有保存。#325的表达随着未成熟造血细胞向巨核细胞的分化而增加。我们制备了#325过表达转基因小鼠，并在小鼠体内分离了#325基因，以检测该基因在体内的功能。(3) MafG c端区分析。删除MafG c端区域生成MafGAC。该突变分子在培养细胞中过表达时也抑制了野生型MafG的转录，当将MafGAC与p45的协同转录活性与MafG进行比较时，MafGAC的转录活性明显强于野生型MafG。我们将在体内测试MafGAC的功能。

(1) Transcriptional regulation of mafG gene. Three independent promoters were identified by 5'-RACE in mafG gene. A 7 kbp-genomic fragment containing the mafG promoters were able to direct tissue-specific reporter gene expression in brain, lens, lung and kidney, but not in hematopoietic cells of transgenic mouse. We are going to test additional 3 kbp fragment downstream of mafG gene.(2) Subtraction analysis of mafG-null megakaryocytes. mRNA derived from proplatelet formation (PPF)-defective mafG-/-::mafK+/- megakaryocytes were subtracted by mRNA of control wild type megakaryocytes. Several clones obtained from the subtraction were found to be related to cytoskeletal proteins, such as actin and tubulin. One novel clone, named #325, was also obtained, and it was found to be conserved among various vertebrates from fish to human. The expression of #325 increased according to the differentiation of immature hematopoietic cells to megakaryocytes. We are generating #325-overexpressing transgenic mouse and disaipting #325 gene in mouse to examine the in vivo function of the gene.(3) Analysis of MafG C-terminal region. MafG C-terminal region was deleted to generate MafGAC. The mutant molecule also repressed the transcription as well as the wild type MafG when they were overexpressed in cultured cells, When the synergistic transcriptional activity of MafGAC with p45 was compared to that of MafG, MafGAC showed much stronger transcriptional activity than wild type MafG. We are going to test the function of MafGAC in vivo.