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Complex genetic networks control hematopoietic stem cell differentiation into progenitors that give rise to billions of erythrocytes daily. Previously, we described a role for the master regulator of erythropoiesis, GATA-1, in inducing genes encoding components of the autophagy machinery. In this context, the Forkhead transcription factor, Foxo3, amplified GATA-1-mediated transcriptional activation. To determine the scope of the GATA-1/Foxo3 cooperativity, and to develop functional insights, we analyzed the GATA-1/Foxo3-dependent transcriptome in erythroid cells. GATA-1/Foxo3 repressed expression of Exosc8, a pivotal component of the exosome complex, which mediates RNA surveillance and epigenetic regulation. Strikingly, downregulating Exosc8, or additional exosome complex components, in primary erythroid precursor cells induced erythroid cell maturation. Our results demonstrate a new mode of controlling erythropoiesis in which multiple components of the exosome complex are endogenous suppressors of the erythroid developmental program.

Abstract 367 Developmental processes such as hematopoiesis are regulated by complex genetic networks, which can be deconstructed into regulatory modules termed network motifs. Although considerable progress has been made in defining genetic networks that control hematopoiesis, many questions remain regarding how such networks are established and maintained. We provide evidence for an important network motif involving the master regulator of erythropoiesis GATA-1 and the forkhead transcription factor FoxO3, which instigates autophagy as an important component of the erythroid cell genetic network. Autophagy mediates organelle remodeling, including the consumption of mitochondria, as a critical developmental process and as a cellular quality control mechanism. Whereas autophagy has been analyzed extensively under stress conditions, mechanisms that instigate and regulate autophagy in specialized cell and tissue types are much less understood. In a genetic complementation assay in G1E-ER-GATA-1 cells, ER-GATA-1 directly activated transcription of essential autophagy genes, including the gene encoding Microtubule Associated Protein 1 Light Chain 3B (LC3B). GATA-1-mediated LC3B induction was associated with increased LC3B-positive autophagosomes, based on immunofluorescence studies. We developed transmission electron microscopy/immunogold labeling assays, which uniquely allow one to mechanistically dissect how a single regulatory factor (GATA-1) orchestrates organelle remodeling as a crucial step in terminal maturation. This analysis provided evidence that GATA-1 induction of LC3B is linked to increased autophagosome/autophagolysosome numbers per cell and to increased autophagosome/autophagolysosome size. As these organelles mediate mitochondrial clearance as a crucial step in erythropoiesis, we investigated the underlying molecular mechanisms. We demonstrated that GATA-1 directly activates autophagy genes and induces FoxO3, another direct activator of autophagy genes. This dual regulatory mechanism constitutes a type 1 coherent feed-forward loop, a network motif that can buffer input signal noise (GATA-1 level/activity), thus preventing a premature output (autophagy). In principle, a sustained input signal translates into an irreversible autophagy output. GATA-1 induced FoxO3 protein 6.7 fold (p < 0.001) and FoxO3 chromatin occupancy (3 fold, p < 0.0001). FoxO3 occupied chromatin adjacent to the GATA-1 occupancy site at select GATA-1 target gene loci. siRNA-mediated knockdown of FoxO3 in G1E-ER-GATA-1 cells reduced the capacity of GATA-1 to regulate select autophagy genes. A similar impact of FoxO3 loss on these genes was detected with bone marrow-derived erythroblasts from FoxO3-nullizygous mice. Our results establish a novel genetic mechanism that instigates cell type-specific autophagy as an important step in erythroid cell maturation. These studies support a model in which GATA-1-dependent autophagy is a crucial driving force in erythropoiesis, and specific molecular steps in organelle remodeling are inextricably linked to the erythroid cell genetic network. Perturbation of this mechanism may lead to the accumulation of functionally defective intermediates in autophagosome/autophagolysosome assembly with ensuing erythroid cell maturation defects and pathologies. Disclosures: No relevant conflicts of interest to declare.