Fig 1: Polyubiquitylation and instability of DUSP4 promoted by PRMT1-involved R351 methylationFor a Figure360 author presentation of this figure, see https://doi.org/10.1016/j.celrep.2021.109421.(A) Polyubiquitylation of DUSP4, but not its R351K variant, is stimulated by PRMT1. 293T cells were transfected with DUSP4 (wild-type and R351K mutant), 6× His-tagged ubiquitin, and PRMT1 (V1 and V2 isoforms). Cells were treated with MG132 for 6 h prior to harvest. Fractions of pull-downs (upper panel) and inputs (bottom panel) were applied for western blotting (n = 3, representative western blots).(B) PRMT1-dependent stability levels of wild-type and R351K DUSP4 were determined in 293T cells by western blotting (n = 3, representative western blots).(C) Half-life time of wild-type and R351K DUSP4. Normalized protein stability curves are plotted in the right panel (n = 3, representative western blots).(D) Mechanistic description of DUSP4 stability modulated by PRMT1-dependent R351 methylation. R351 methylation of DUSP4 by PRMT1 triggers its polyubiquitylation and thus its degradation; R351K mutation abolishes the methylation and thus suppresses polyubiquitylation and degradation (n = 3, representative western blots).(E) Mk differentiation in the presence of wild-type and R351K DUSP4. Human CD34+ cells infected with lentiviruses expressing DUSP4s (wild-type or R351K mutant) were induced for Mk differentiation. Percentage of CD41a+CD42b+ cells was determined by FACS after 7 days. Representative plots and statistics are shown (n = 3, independent experiments). Data are shown as mean ± SD. Two-tailed paired t test, *p = 0.05.(F) Co-immunoprecipitation of HUWE1 and DUSP4. Flag-tagged DUSP4 was used to immunoprecipitate myc-tagged HUWE1 in co-transfected 293T cells (n = 3, representative western blots after immunoprecipitation).(G) The protein ubiquitylation of DUSP4 is measured in 293T cells transfected with plasmids as shown on top of the gels (n = 3, representative western blots).(H) Mutant and wild-type DUSP4 were expressed together with HUWE1 shRNA in 293T cells for western blotting with respective antibodies (n = 3, representative western blots).(I) Protein ubiquitylation assays with wild-type and mutant DUSP4. DUSP4 wild-type protein and mutant protein were expressed in 293T cells transfected with or without the plasmid combination of HUWE1 and PRMT1 as indicated on the top of the gel for affinity purification with Ni-NTA beads (n = 3, representative western blots).
Fig 2: Identification of DUSP4 for optimal Mk differentiation(A) Schematic of shRNA-based screening assay to identify essential DUSPs for Mk-induced Mk differentiation. Human CD34+ cells infected with lentiviruses expressing shRNAs against DUSPs were cultured for Mk differentiation.(B) Heatmap of the percentages of CD41a+CD42b+ cells upon DUSP knockdown on day 8. Fold changes were normalized to the percentage of double-positive cells with the group treated with control shRNA.(C) Representative flow chart of FACS analysis of Mk differentiation using BM cells (top panel) and CB cells (bottom panel) cultured in TPO-containing medium.(D) Summary of FACS analysis. Statistics are based on the data of three independent experiments (n = 3) with the bone marrow (BM) or cord blood (CB) cells from two donors. Data are shown as mean ± SD. Two-tailed paired t test, *p = 0.05, **p = 0.01.(E) Schematic description of Mk differentiation of human peripheral blood-derived CD34+ cells with DUSP4 knockdown.(F and G) Representative and complete FACS analysis of CD41a and CD42b markers for Mk differentiation with peripheral blood CD34+ cells upon DUSP4 knockdown on day 7. Representative plots (F) and statistics (G) are shown (n = 3, independent experiments). Data are shown as mean ± SD. Two-tailed paired t test, *p = 0.05, **p = 0.01.
Fig 3: Negative correlation between PRMT1 and Mk differentiation revealed by single-cell RNA-seq (scRNA-seq) analysis(A) Experimental design for scRNA-seq analysis. Sample 1: Native CD34+ cells were cells isolated directly from BM. Sample 2: CD34+ cells were cells cultured in TPO and SCF for 8 days before sorting with CD41a and CD42b. Sample 3: The non-CD41a+CD42b+ cells were sorted from sample 2. Sample 4: The CD41a+CD42b+ cells were sorted from sample 2.(B and C) SPRING plots of single-cell transcriptomes. Individual cells are presented according to their origins (B) or transcriptome-associated cell types (C). Ba, basophilic or mast cell; D, dendritic; Er, erythroid; GN, granulocytic neutrophil; Ly, lymphocytic; M, monocytic; Mk, megakaryocytic; MPP, multipotential progenitor.(D) Pathway analysis of the top 400 genes with the strongest negative Spearman correlation to PRMT1 expression level in the TPO/SCF-stimulated CD41a+CD42b+ population. Red bars highlight Mk-relevant biological pathways.(E) Spearman correlation coefficients between PRMT1 and any gene revealed by scRNA-seq in TPO/SCF-stimulated CD41a+CD42b+ cells. Representative genes with significant correlation and functional relevance are annotated.(F) Gene set enrichment analysis (GSEA) of PRMT1-correlated genes in the TPO/SCF-stimulated CD41a+CD42b+ population. GSEA inputs are Spearman correlation coefficients of PRMT1 versus any gene with single-cell resolution.(G) Normalized expression of representative transcripts co-plotted against that of PRMT1 transcript in TPO/SCF-stimulated CD41a+CD42b+ cells with single-cell resolution.
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