Methylation of Dual Specificity Phosphatase 4 Controls Cell Differentiation

Mitogen-activated protein kinases are inactivated by dual specificity phosphatases (DUSPs), whose activities are tightly regulated during cell differentiation. Using knockdown screening and single-cell transcriptional analysis, we determined that DUSP4 is the phosphatase that specifically inactivates p38 kinase for the promotion of megakaryocyte (Mk) differentiation. Mechanistically, PRMT1-mediated methylation of DUSP4 triggers its ubiquitinylation by an E3 ligase HUWE1. Interestingly, the mechanistic axis of the DUSP4 degradation and p38 activation is also associated with a transcriptional signature of immune activation in Mk cells. In the context of thrombocytopenia observed in myelodysplastic syndromes (MDS), we demonstrated that high levels of p38 MAPK and PRMT1 are associated with low platelet counts and adverse prognosis, while pharmacological inhibition of p38 MAPK or PRMT1 stimulates megakaryopoiesis. These findings provide mechanistic insights into the role of the PRMT1-DUSP4-p38 axis on Mk differentiation and present a targeting strategy for treatment of thrombocytopenia associated with MDS. Human bone marrow-derived CD34+ cells were cultured in a prestimulation medium (IMDM + 20% BIT + Basic Cytokine Mix: 100 ng/mL SCF, 20 ng/mL TPO, 100 ng/mL FLT3 Ligand and 50 ng/mL IL-6) for four days and examined for the surface markers CD41a/CD42b before processing for scRNA-seq. For un-stimulated cells (Sample 1 in Figure 4), approximately 100,000 cells were harvested. The rest of CD34+ cells were cultured in a Mkstimulating medium (IMDM + 20% BIT + MK Cytokine Mix: 25 ng/mL SCF, 50 ng/mL TPO), which was replaced with fresh Mk-stimulating medium on Day 4 and Day 7. On Day 8 poststimulation, approximately 100,000 cells were collected as TPO/SCF-stimulated cells (Sample 2 in Figure 4). FACS sorting of the TPO/SCF-stimulated cells allowed the collection of approximately 88,000 TPO/SCF-stimulated, CD41a+CD42b+ cells (Sample 4 in Figure 4) and 100,000 TPO/SCF-stimulated, non-CD41a+CD42b+ cells (Sample 3 in Figure 4). The four sets of cell samples were immediately subject to Drop-Seq-based single cell RNA sequencing as detailed below.