Targeted ShRNA Screening Identified Critical Role of Pleckstrin-2 in Erythropoiesis

Abstract

Abstract 3199Mammalian erythropoiesis is globally regulated by erythropoietin (Epo). Epo binds to its receptor on the cell surface of erythroid precursor; induces a series of downstream pathways that promote cell differentiation and inhibit apoptosis. Recent genome wide transcriptional profile study demonstrated that over 500 genes are up-regulated during erythropoiesis. Many of these genes encode erythroid specific proteins that play well-known functions in red cells. However, the functions of the most other genes in the erythroid cells are still unknown. To identify novel genes in erythropoiesis, we infected mouse fetal liver erythroblasts with lentiviruses containing mammalian shRNA knockdown library that selectively includes the most highly upregulated 100 genes with unknown functions in erythroid cells. The infected cells were cultured in two different conditions for the characterization of early and late stage erythropoiesis using a high throughput flow cytometry based analysis. With these methods, we identified 33 novel genes that regulate cell differentiation or apoptosis in early stage erythropoisis; 20 genes play important roles in late stage erythropoiesis including enucleation. Significantly, there is an overlap of 16 genes that function in both early and late stage erythropoiesis. We focused on pleckstrin-2, which is specifically and abundantly expressed in erythroid cells, to further characterize its detailed functions in red cell development. We found that knockdown of pleckstrin-2 leads to dramatic apoptosis in early stage erythropoiesis. Knockdown of pleckstrin-2 in late stage erythropoiesis blocks enucleation with no apparent effects on cell differentiation, proliferation or apoptosis. We further discovered that pleckstrin-2 deficiency in early and late erythroblasts disrupts normal actin cytoskeleton as evidenced by super-resolution immunofluorescence microscope. To elucidate the detailed mechanisms of the functions of pleckstrin-2 in different stages of erythropoiesis, we performed proteomic studies and identified candidate proteins that interact with pleckstrin-2 that may contribute to the phenotypes of apoptosis and enucleation defects. In summary, our study identified pleckstrin-2 as a critical regulator of mammalian erythropoiesis and proved the significance of large-scale shRNA screening in the discovery of novel genes in development. Disclosures:No relevant conflicts of interest to declare.