Department of Hematology, University Medical Center Groningen, University of Groningen, Groningen, The Netherlands.
Blood. 2010 Jun 3;115(22):4367-76. doi: 10.1182/blood-2009-10-250894. Epub 2010 Mar 25.
Previously, we have shown that overexpression of an activated mutant of signal transducer and activator of transcription-5 (STAT5) induces erythropoiesis, impaired myelopoiesis, and an increase in long-term proliferation of human hematopoietic stem/progenitor cells. Because GATA1 is a key transcription factor involved in erythropoiesis, the involvement of GATA1 in STAT5-induced phenotypes was studied by shRNA-mediated knockdown of GATA1. CD34(+) cord blood cells were double transduced with a conditionally active STAT5 mutant and a lentiviral vector expressing a short hairpin against GATA1. Erythropoiesis was completely abolished in the absence of GATA1, indicating that STAT5-induced erythropoiesis is GATA1-dependent. Furthermore, the impaired myelopoiesis in STAT5-transduced cells was restored by GATA1 knockdown. Interestingly, early cobblestone formation was only modestly affected, and long-term growth of STAT5-positive cells was increased in the absence of GATA1, whereby high progenitor numbers were maintained. Thus, GATA1 down-regulation allowed the dissection of STAT5-induced differentiation phenotypes from the effects on long-term expansion of stem/progenitor cells. Gene expression profiling allowed the identification of GATA1-dependent and GATA1-independent STAT5 target genes, and these studies revealed that several proliferation-related genes were up-regulated by STAT5 independent of GATA1, whereas several erythroid differentiation-related genes were found to be GATA1 as well as STAT5 dependent.
先前，我们已经证明，信号转导和转录激活因子 5（STAT5）的激活突变体的过表达可诱导红细胞生成、抑制骨髓生成，并增加人类造血干/祖细胞的长期增殖。由于 GATA1 是参与红细胞生成的关键转录因子，因此通过 shRNA 介导的 GATA1 敲低研究了 GATA1 在 STAT5 诱导表型中的参与。CD34+脐带血细胞与条件性激活 STAT5 突变体和表达针对 GATA1 的短发夹 RNA 的慢病毒载体进行双重转导。在不存在 GATA1 的情况下，完全消除了红细胞生成，表明 STAT5 诱导的红细胞生成依赖于 GATA1。此外，GATA1 敲低恢复了 STAT5 转导细胞中的骨髓生成受损。有趣的是，早期鹅卵石形成仅受到适度影响，并且在不存在 GATA1 的情况下，STAT5 阳性细胞的长期生长增加，从而维持了高祖细胞数量。因此，下调 GATA1 允许从对干细胞/祖细胞长期扩增的影响中分离 STAT5 诱导的分化表型。基因表达谱分析允许鉴定依赖于 GATA1 和独立于 GATA1 的 STAT5 靶基因，这些研究表明，几个与增殖相关的基因独立于 GATA1 被 STAT5 上调，而几个与红细胞分化相关的基因被发现依赖于 GATA1 和 STAT5。
