Targeted shRNA screening identified critical roles of pleckstrin-2 in erythropoiesis

Abstract

ABSTRACTliver erythroblast in vitro culture technology together with a targeted array-based high throughput screening system and discovered more than 30 genes that have novel functions in the early and late stages of terminal erythropoiesis. Among these genes, we identified pleckstrin-2 (plek2), which was previously reported to be involved in T-cell cytoskeleton reorganization, 23 play critical roles in terminal erythropoiesis. We demonstrated that plek2 is important in the regulation of actin cytoskeleton, cell differentiation and apoptosis. We also found that plek2 blocks apoptosis in the early stage of terminal erythropoiesis through preventing cofilin's mitochondrial entry. Furthermore, we dissected these functions of plek2 in the early and late stages of terminal erythropoiesis reflecting different intracellular levels of reactive oxygen species (ROS). Methods MaterialsRabbit polyclonal antibody against plek2 was purchased from Proteintech. Rabbit polyclonal antibodies against cofilin and phospho-cofilin were purchased from Cell Signaling Technology. Monoclonal antibody against Hsc70 was purchased from Santa Cruz Biotechnology. All the antibodies for flow cytometric analysis were purchased from eBioscience. Alexa Fluor 488 phalloidin and Alexa Fluor 544 for immunofluorescence were purchased from Invitrogen Molecular Probes. The Hairpin-pLKO.1 lentiviruses were from the Broad Institute (Cambridge, MA, USA). Purification and culture of fetal liver cells for targeted screeningPurification of mouse fetal liver erythroblast progenitors (CFU-E; TER119-negative cells) was based on previously described procedures. 10 Details of procedures including viral transduction and the screening protocol are available in the Online Supplementary Appendix. Retroviral shRNA constructionRetroviral shRNA oligonucleotides against plek2 were designed using an automated shRNA selection web server (http://jura.wi.mit.edu/bioc/siRNAext/home.php). 24 Cloning of shRNA into MSCV-U3-H1 was performed as previously described. 25 The shRNA nucleotide sequences used in the experiments are as follows. Non-targeting shRNA: 5'-GGACTAGTCTCCACGGTGA -3'. Plek2 shRNA: 5' -CAACTCAGTATCTCTGGGA -3'. Flow cytometric analysis and immunoprecipitationFlow cytometric analysis of differentiation and enucleation of cultured mouse fetal erythroblasts was as previously described. Immunofluorescence stain and microscopic analysisDetails of the protocol for immunofluorescence stain and microscopic analysis are available in the Online Supplementary Appendix. Quantitative analysis of intracellular reactive oxygen speciesThe intracellular ROS levels were determined using CM-H2DCFDA (Molecular Probes). Briefly, erythroid cells were incubated with 10 mM CM-H2DCFDA or DMSO control. After incubation for 30 min at 37°C, the cells were harvested, washed, and resuspended in PBS. The levels of fluorescence were immediately detected using flow cytometric analysis. Results Targeted shRNA screening identified genes that play novel functions in the early and late stages of ter...

Loss of pleckstrin-2 reverts lethality and vascular occlusions in JAK2V617F-positive myeloproliferative neoplasms

“…We previously discovered that Plek2 plays important roles in erythroblast survival and enucleation in the early and late stages of terminal erythropoiesis, respectively (17). In that report, we also found that the Plek2 level is V617F driver mutation of JAK2 is the leading cause of the Philadelphia-chromosome-negative myeloproliferative neoplasms (MPNs).…”

“…This interaction prevents cofilin's localization in mitochondria and thereby protects erythroid progenitors from cell death in the oxidative environment found in the early stage of terminal erythropoiesis (17). We were surprised that Plek2-knockout mice did not show significant anemia at a young age.…”

“…Our previous study showed that Plek2 is critical for both the early and late stages of terminal erythropoiesis in vitro in cultured mouse fetal liver erythroid cells (17). However, the role of Plek2 in vivo is unknown.…”

“…Cell culture. Purification of mouse fetal liver erythroblast precursors (CFU-E; Ter119-negative cells) and in vitro culture were performed as previously described (17). Briefly, fetal liver cells were isolated from E13.5 C57BL/6 embryos and mechanically dissociated by pipetting in phosphate-buffered saline (PBS) containing 10% FBS (Gemini).…”

KLF1-null neonates display hydrops fetalis and a deranged erythroid transcriptome

“…These include genes involved in EpoR signaling, enucleation, 61,62 and stress erythropoiesis 63 (Table 2). PLEK2 (Pleckstrin-2) and DYRK3 both have proven roles in terminal erythroid differentiation, 63,64 and both are expressed at ,10% of wildtype levels in the proband. There is KLF1 occupancy of the DYRK3 gene promoter ( Figure 5E).…”