GSE60087
Epo-Induced Erythroid Maturation Is Dependent on Plc?1 Signaling
Erythropoiesis is a tightly regulated process. Development of red blood cells occurs through differentiation of hematopoietic stem cells into more committed progenitors and finally into erythrocytes. Binding of erythropoietin to its receptor (EpoR) is strictly required for erythropoiesis as it promotes survival and late maturation of erythroid progenitors. In vivo and in vitro studies have highlighted the requirement of EpoR signaling through Jak2 tyrosine-kinase and Stat5a/b as a central pathway. Here, we demonstrate that phospholipase C gamma 1 (Plc?1) is activated downstream of EpoR/Jak2 independently of Stat5. Plc?1-deficient proerythroblasts and erythroid progenitors exhibited strong impairment in differentiation and colony-forming potential. In vivo, suppression of Plc?1 in immunophenotypically defined hematopoietic stem cells (Lin-Sca1+KIT+CD48-CD150+) severely reduced erythroid development. To identify Plc?1 effector molecules involved in regulation of erythroid differentiation we assessed for changes on the level of transcription and DNA methylation after inactivation of  Plc?1. The single common downstream effector was H2AFY2, which encodes for the histone variant macroH2A2 (mH2A2). Suppression of macroH2A2 expression recapitulated the effects of Plc?1 knockdown on erythroid maturation. Taken  together, our findings identify Plc?1 and its downstream target macroH2A2, as a “non-canonical” signaling pathway essential for erythroid differentiation. Overall design: MCIP-seq was used to interrogate methylation changes during Epo-induced differentiation of murine I/11 erythroblastic cell line upon knock-down of Plcy1 as compared to a mock control. Two different shRNA constructs that target Plcg1 were investigated at two different time points.