Phospholipase Cγ1 is crucially required for EpoR/JAK2 controlled erythropoietic differentiation.. Mus musculus

Erythropoiesis is a multi-step process in which the development of red blood cells occurs through expansion and differentiation of hematopoietic stem cells (HSCs) into more committed progenitors. In vivo and in vitro studies have pointed out the major role of proximal erythropoietin receptor (EpoR) signaling through JAK2 tyrosine-kinase as a central regulator of erythropoiesis 1,2. However, little is known on more distant signalling pathways driving EPO/JAK2-induced differentiation of erythroid progenitors. Here, we demonstrate that phospholipase Cγ 1 (PLCγ1) is activated downstream of EpoR/JAK2 and is crucially required for differentiation of erythroid progenitor cells. In the absence of PLCγ1, erythroid progenitors exhibited dramatically impaired differentiation and colony-forming potential. To identify PLCγ1 effector molecules involved in regulation of erythroid differentiation we performed global gene expression and methylome analysis. In PLCγ1-deficient erythroid progenitors, profound changes in the landscape of DNA methylation and gene expression were observed. Taken together, our findings identify PLCγ1 as a central regulator in erythroid development and highlight its physiological relevance in development and maintenance of normal hematopoiesis. Overall design: RNA was isolated from 5x10^5 I/11 cells transfected with either scrambled or PLCg1 shRNA using TRIzol (Life Technologies). RNA was amplified using the Ovation Pico WTA system (Nugen) and labeled using the encore Biotin Module (Nugen). 5 ug of amplified and labeled DNA was hybridized to Affymetrix GeneChip Mouse Genome 430plus 2.0 microarrays according to the manufacturer’s recommendations (Affymetrix).