Fisher R C, Olson M C, Pongubala J M, Perkel J M, Atchison M L, Scott E W, Simon M C
Institute for Human Gene Therapy, University of Pennsylvania, Philadelphia, Pennsylvania 19104, USA.
Mol Cell Biol. 1998 Jul;18(7):4347-57. doi: 10.1128/MCB.18.7.4347.
Gene targeting of transcription factor PU.1 results in an early block to fetal hematopoiesis, with no detectable lymphoid or myeloid cells produced in mouse embryos. Furthermore, PU.1(-/-) embryonic stem (ES) cells fail to differentiate into Mac-1(+) and F4/80(+) macrophages in vitro. We have previously shown that a PU.1 transgene under the control of its own promoter restores the ability of PU. 1(-/-) ES cells to differentiate into macrophages. In this study, we take advantage of our PU.1(-/-) ES cell rescue system to genetically test which previously identified PU.1 functional domains are necessary for the development of mature macrophages. PU.1 functional domains include multiple N-terminal acidic and glutamine-rich transactivation domains, a PEST domain, several serine phosphorylation sites, and a C-terminal Ets DNA binding domain, all delineated and characterized by using standard biochemical and transactivational assays. By using the production of mature macrophages as a functional readout in our assay system, we have established that the glutamine-rich transactivation domain, a portion of the PEST domain, and the DNA binding domain are required for myelopoiesis. Deletion of three acidic domains, which exhibit potent transactivation potential in vitro, had no effect on the ability of PU.1 to promote macrophage development. Furthermore, mutagenesis of four independent sites of serine phosphorylation also had no effect on myelopoiesis. Collectively, our results indicate that PU.1 interacts with important regulatory proteins during macrophage development via the glutamine-rich and PEST domains. The PU.1(-/-) ES cell rescue system represents a powerful, in vitro strategy to functionally map domains of PU.1 essential for normal hematopoiesis and the generation of mature macrophages.
转录因子PU.1的基因靶向导致胎儿造血早期受阻，小鼠胚胎中未产生可检测到的淋巴细胞或髓细胞。此外，PU.1(-/-)胚胎干细胞（ES细胞）在体外无法分化为Mac-1(+)和F4/80(+)巨噬细胞。我们之前已经表明，在其自身启动子控制下的PU.1转基因可恢复PU.1(-/-)ES细胞分化为巨噬细胞的能力。在本研究中，我们利用我们的PU.1(-/-)ES细胞拯救系统进行基因测试，以确定哪些先前鉴定的PU.1功能域对于成熟巨噬细胞的发育是必需的。PU.1功能域包括多个N端酸性和富含谷氨酰胺的反式激活域、一个PEST域、几个丝氨酸磷酸化位点以及一个C端Ets DNA结合域，所有这些都是通过使用标准生化和反式激活测定法来描绘和表征的。通过在我们的测定系统中使用成熟巨噬细胞的产生作为功能读数，我们已经确定富含谷氨酰胺的反式激活域、PEST域的一部分以及DNA结合域是髓系造血所必需的。删除在体外表现出强大反式激活潜力的三个酸性域，对PU.1促进巨噬细胞发育的能力没有影响。此外，四个独立丝氨酸磷酸化位点的诱变对髓系造血也没有影响。总的来说，我们的结果表明，PU.1在巨噬细胞发育过程中通过富含谷氨酰胺的域和PEST域与重要的调节蛋白相互作用。PU.1(-/-)ES细胞拯救系统代表了一种强大的体外策略，用于在功能上绘制对正常造血和成熟巨噬细胞生成至关重要的PU.1域。
