Analysis of hematopoietic lineage plasticity by using inducible transcription factors

为了研究不同造血系分化的分子机制和可能的谱系转换或跨分化，我们构建了髓系转录因子C/EBPα和PU.1的诱导形式，并检测了它们在体外和体内的作用。将全长C/EBPa和PU1与雌激素受体的配体结合区(C/EBPα-ER和PU.1-ER)融合在一起，使其能被4-羟基三苯氧胺(4-HT)激活。我们进行了亚克隆。C/eBPα-ER和PU1-ER分别构建成逆转录病毒载体pMX-IRES-GFP和感染病毒。有趣的是，4-羟色胺处理BaF_3/CEBPα-ER，BaF_3/PU_3/PU_1-ER细胞发生生长停滞和细胞凋亡，表明C/E_BPα-ER和PU_1-ER正常工作。接下来，我们将C1EBP、α-ER和PU1-ER亚克隆到H-2K启动子载体中，制成转基因小鼠。获得了8株C/EBPa-ER和5株PU.1-ER的转基因整合。RT-PCR分析表明，在8株C/EBpα-ER转基因株中，有5株表达α-ER蛋白，Western印迹显示只有1株表达C/EBPDNA-ER蛋白。C/EBP型α-ER在胸腺和脾中高表达，在骨髓和外周血中中等表达。凝胶漂移分析表明，4-羟色胺处理后，C/eBPα-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/EBPα活性如何影响不同发育阶段的造血细胞的分化。

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.