Down-regulation of GATA1 uncouples STAT5-induced erythroid differentiation from stem/progenitor cell proliferation

Abstract

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...

Identification of HIF2α as an important STAT5 target gene in human hematopoietic stem cells

“…24 This allowed the identification of STAT5-induced target genes associated with erythroid commitment, as well as target genes that did not associate with erythropoiesis. By comparing this last gene set with the list of target genes that were induced by STAT5 in HSCs, we were able to identify 32 genes that might potentially play a role in the long-term self-renewal phenotype induced by STAT5.…”

“…Next, we aimed to identify genes that would specifically associate with the long-term expansion phenotypes imposed on HSCs by STAT5. By down-modulating GATA1 in STAT5-transduced cord blood CD34 ϩ cells, we have been able to dissect the erythroid differentiation phenotypes from HSC self-renewal phenotypes 24 ( Figure 2D). Lentiviral transduction of GATA1 RNAi vectors completely abrogated the STAT5-induced erythroid commitment, whereas long-term expansion, CAFC formation, and HSC selfrenewal remained intact.…”

“…Using this approach, we were able to identify 117 GATA1-independent STAT5 target genes. 24 By comparing this list of GATA1-independent target genes with the STAT5 target genes in HSCs, we were able to identify 32 genes that would associate with long-term expansion and CAFC formation in the HSC compartment ( Figure 2E and Table 1). Data for HIF2␣, GPR171, PIM1, OSM, TUBB1, and SOX21 are shown in Figure 2F; also shown are potential STAT5-binding sites within the…”

“…12 Although it is clear that STAT5 fulfills essential roles in both normal hematopoiesis and in the development or maintenance of leukemia, little is known about the molecular mechanisms that are involved. By making use of an inducible model in which constitutive STAT5 activity can be introduced in specific compartments of the hematopoietic system, 24,25 our current data show that STAT5 can impose a long-term proliferative advantage on the CD34 ϩ /CD38 Ϫ HSC population, but not on progenitors. Geneexpression profiling in STAT5-transduced HSC and progenitor cells and in STAT5-activated, GATA1-down-modulated cells led to the identification of 32 GATA1-independent STAT5 target genes in the HSC population that could be linked to the STAT5-induced long-term proliferation and self-renewal phenotype.…”

“…For lentiviral transduction, we used a pLVUT lentiviral vector 24 in which the short hairpin against human HIF2␣ (GCTGACTCTTTGCTCTAAT) was cloned into the ClaI EcoR1 restriction site. For the control, a short hairpin RNA targeting firefly luciferase was used.…”