Analysis of hematopoietic lineage plasticity by using inducible transcription factors

利用诱导转录因子分析造血谱系可塑性

• 批准号: 14370298
• 负责人: NAKAJIMA Hideaki
• 金额: $ 8.9万
• 依托单位: The University of Tokyo
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (B)
• 财政年份: 2002
• 资助国家: 日本
• 起止时间: 2002 至 2003
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-14370298/
• 关键词: differentiation; transcription factor; C; EBPα; PU.1; transigenic mouse; hematopoiesis; EBPε

In order to investigate the molecular mechanism of differentiation and possible lineage switch or trans-differentiation in various hematopoietic lineages, we generated inducible form of myeloid transcription factors C/EBP α and PU.1 and examined their effect in vitro or in vivo. Full length C/EBP a and PU.1 were fused in frame with ligand binding domain of estrogen receptor (C/EBP α -ER and PU.1-ER) so that they can be activated by 4-hydroxy tamoxifen (4-HT). We subcloned. C/EBP α -ER and PU.1-ER into retrovirus vector, pMX-IRES-GFP and infected virus to BaF3 cells. Interestingly, 4-HT treatment of BaF3/CEBP α -ER, BaF3/PU.1l-ER cells induced growth arrest and apoptosis, indicating that C/EBP α -ER and PU.1-ER are working properly. Next we subcloned Cl EBP α -ER and PU.1-ER in H-2K promoter vector made transgenic mice. We obtained transgene integration in 8 lines for C/ EBP a -ER and 5 lines for PU.1-ER. RT-PCR analysis showed that mRNA is expressed in 5 out of 8 C/EBP α -ER transgenics and only one line expressed C/EBP α -ER protein by western blot. C/EBP α -ER was expressed highly in thymus and spleen, moderately in bone marrow and peripheral blood. Gel-shift assay showed that C/ EBP α -ER protein bound to conserved C/EBP binding sequence in response to 4-HT. With these mice in our hand, we are now planning to investigate how ectopically induced C/ EBP α activity affects differentiation of hematopoietic cells at various developmental stages.

为了研究分化的分子机制以及在各种造血谱系中可能的谱系转换或转分化，我们产生了可诱导形式的髓样转录因子C/EBP α和PU. 1，并在体外或体内检测了它们的作用。将全长C/EBP α和PU. 1与雌激素受体的配体结合域（C/EBP α -ER和PU. 1-ER）进行框内融合，使其能被4-羟基三苯氧胺（4-HT）激活。我们进行了亚克隆。将C/EBP α -ER和PU. 1-ER分别克隆入逆转录病毒载体pMX-IRES-GFP中，感染BaF 3细胞。有趣的是，4-HT处理BaF 3/CEBP α -ER和BaF 3/PU. 1 l-ER细胞可诱导生长停滞和凋亡，表明C/EBP α -ER和PU. 1-ER正常工作。然后将C1-EBP α -ER和PU.1-ER分别亚克隆到H-2K启动子载体中，制备转基因小鼠。我们在8个株系中获得了C/EBPa-ER的转基因整合，在5个株系中获得了PU.1-ER的转基因整合。RT-PCR检测结果显示，8株转基因细胞中有5株表达mRNA，Western blot检测结果显示，只有1株表达C/EBP α -ER蛋白。C/EBP α -ER在胸腺和脾脏中呈高表达，在骨髓和外周血中呈中等表达。凝胶迁移实验表明，C/ EBP α -ER蛋白与保守的C/EBP结合序列结合。有了这些小鼠，我们现在计划研究异位诱导的C/ EBP α活性如何影响造血细胞在不同发育阶段的分化。

项目成果

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Carpino N.：“p70 的鉴定、cDNA 克隆和靶向删除，p70 是一种新型、普遍表达的含有 SH3 结构域的蛋白质。”Mol.Cell.Biol.. 22. 7491-7500 (2002)

Kumagai H.: "Identification and characterization of a new pair of immunoglobulin-like receptors LMIR1 and 2 derived from murine bone marrow-derived mast cells."Biochem.Biophys.Res.Commun.. 307. 719-729 (2003)

Kumagai H.：“来自鼠骨髓源性肥大细胞的一对新的免疫球蛋白样受体 LMIR1 和 2 的鉴定和表征。”Biochem.Biophys.Res.Commun.. 307. 719-729 (2003)

Truong BT.: "CCAAT/Enhancer binding proteins repress the leukemic phenotype of acute myeloid leukemia."Blood. 101. 1141-1148 (2003)

Truong BT.：“CCAAT/增强子结合蛋白抑制急性髓性白血病的白血病表型。”血液。

Watanabe N., Nakajima H., Suzuki H., Oda A., Matsubara Y., Moroi M., Terauchi Y., Kadowaki T., Suzuki H., Koyasu S., Ikeda Y., Hanada M.: "Functional phenotype of phosphoinositide 3-kinase p85alpha-null platelets characterized by an impaired response to G

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Kitamura T., Koshino Y., Shibata F., Oki T., Nakaiima H., Nosaka T., Kumagai H.: "Retrovirus-mediated gene transfer and expression cloning : powerful tools in functional genomics."Exp.Hematol.. 31. 1007-1014 (2003)

Kitamura T.、Koshino Y.、Shibata F.、Oki T.、Nakaiima H.、Nosaka T.、Kumagai H.：“逆转录病毒介导的基因转移和表达克隆：功能基因组学的强大工具。”Exp.Hematol..