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 longterm 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.
IntroductionSignal transducer and activator of transcription-5 (STAT5) is a member of a family of transcription factors that is composed of 7 genes (STAT1 to STAT6, with STAT5 being encoded by 2 genes, STAT5A and STATB). The pathways mediated by these transcription factors are involved in many processes in hematopoietic cells, including regulation of proliferation, antiapoptosis, differentiation, and self-renewal. 1,2 STAT transcription factors contain a conserved tyrosine residue in their SH2 domain, which on phosphorylation, gives rise to heterodimerization or homodimerization (or tetramerization 3 ) with another STAT molecule, leading to nuclear translocation. Constitutively activated STAT5 has been found in blast cells of up to 70% of acute myeloid leukemia cases. 4 Besides its function as a transcription factor, tyrosine phosphorylated STAT5 has also been described to interact with other partners to induce phosphoinositide-3 kinase activity, thereby contributing to leukemic transformation. 5 STAT transcription factors can be activated by growth factors via JAK kinases, present at the cytoplasmic domains of growth factor receptors, as well as by intrinsic kinase activity of certain membrane receptors or cytoplasmic tyrosine kinases. 6 STAT5 is expressed in a large variety of hematopoietic cells and involved in the signal transduction of many different growth factors and cytokines. Early acting cytokines, such as Fms-like tyrosine kinase 3 ligand (FLT-3L), thrombopoietin, and stem cell factor, all mediate at le...