Advertisement

Protein arginine methyltransferase 1 in the generation of immune megakaryocytes: A perspective review

Open AccessPublished:September 21, 2022DOI:https://doi.org/10.1016/j.jbc.2022.102517
      Megakaryocytes (Mks) in bone marrow are heterogeneous in terms of polyploidy. They not only produce platelets but also support the self-renewal of hematopoietic stem cells and regulate immune responses. Yet, how the diverse functions are generated from the heterogeneous Mks is not clear at the molecular level. Advances in single-cell RNA seq analysis from several studies have revealed that bone marrow Mks are heterogeneous and can be clustered into 3 to 4 subpopulations: a subgroup that is adjacent to the hematopoietic stem cells, a subgroup expressing genes for platelet biogenesis, and a subgroup expressing immune-responsive genes, the so-called immune Mks that exist in both humans and mice. Immune Mks are predominantly in the low-polyploid (≤8 N nuclei) fraction and also exist in the lung. Protein arginine methyltransferase 1 (PRMT1) expression is positively correlated with the expression of genes involved in immune response pathways and is highly expressed in immune Mks. In addition, we reported that PRMT1 promotes the generation of low-polyploid Mks. From this perspective, we highlighted the data suggesting that PRMT1 is essential for the generation of immune Mks via its substrates RUNX1, RBM15, and DUSP4 that we reported previously. Thus, we suggest that protein arginine methylation may play a critical role in the generation of proinflammatory platelet progeny from immune Mks, which may affect many immune, thrombotic, and inflammatory disorders.

      Keywords

      Abbreviations:

      HSC (hematopoietic stem cell), LPS (lipopolysaccharide), MDS (myelodysplasia syndrome), Mks (megakaryocytes), PRMT (protein arginine methyltransferase), scRNA-seq (Single-cell RNA sequencing), vWF (von Willebrand factor)
      Protein arginine methylation is a common protein modification that only occurs in eukaryotic cells. A family of protein arginine methyltransferases (PRMTs) has at least nine members, which either symmetrically or asymmetrically modify the guanidino nitrogen atoms of the arginine side chain in the context of proteins. The basic molecular functions of these enzymes were comprehensively reviewed (
      • Fulton M.D.
      • Brown T.
      • Zheng Y.G.
      The biological axis of protein arginine methylation and asymmetric dimethylarginine.
      ,
      • Wu Q.
      • Schapira M.
      • Arrowsmith C.H.
      • Barsyte-Lovejoy D.
      Protein arginine methylation: from enigmatic functions to therapeutic targeting.
      ). Among the family members, PRMT1 is the most evolutionarily conserved enzyme existing from yeast to mammals and accounts for most of the asymmetric arginine methylation activity. PRMT1 participates in a broad range of molecular processes such as transcriptional regulation, RNA splicing, protein trafficking, protein translation, and signal transduction (
      • Guccione E.
      • Richard S.
      The regulation, functions and clinical relevance of arginine methylation.
      ). PRMT1 is ubiquitously expressed in all tissues with the highest expression levels observed in the female reproductive system (https://www.gtexportal.org/home/). Straight knockout of PRMT1 is embryonic lethal in mice. PRMT1 has been demonstrated to be critical for cardiovascular fitness (
      • Jeong M.H.
      • Jeong H.J.
      • Ahn B.Y.
      • Pyun J.H.
      • Kwon I.
      • Cho H.
      • et al.
      PRMT1 suppresses ATF4-mediated endoplasmic reticulum response in cardiomyocytes.
      ,
      • Pyun J.H.
      • Kim H.J.
      • Jeong M.H.
      • Ahn B.Y.
      • Vuong T.A.
      • Lee D.I.
      • et al.
      Cardiac specific PRMT1 ablation causes heart failure through CaMKII dysregulation.
      ,
      • Murata K.
      • Lu W.
      • Hashimoto M.
      • Ono N.
      • Muratani M.
      • Nishikata K.
      • et al.
      PRMT1 deficiency in mouse Juvenile heart induces dilated cardiomyopathy and reveals cryptic alternative splicing products.
      ,
      • Pyun J.H.
      • Ahn B.Y.
      • Vuong T.A.
      • Kim S.W.
      • Jo Y.
      • Jeon J.
      • et al.
      Inducible Prmt1 ablation in adult vascular smooth muscle leads to contractile dysfunction and aortic dissection.
      ), pancreatic development (
      • Lee K.
      • Kim H.
      • Lee J.
      • Oh C.M.
      • Song H.
      • Kim H.
      • et al.
      Essential role of protein arginine methyltransferase 1 in pancreas development by regulating protein stability of neurogenin 3.
      ), and brain development (
      • Hashimoto M.
      • Fukamizu A.
      • Nakagawa T.
      • Kizuka Y.
      Roles of protein arginine methyltransferase 1 (PRMT1) in brain development and disease.
      ,
      • Hashimoto M.
      • Murata K.
      • Ishida J.
      • Kanou A.
      • Kasuya Y.
      • Fukamizu A.
      Severe hypomyelination and developmental defects are caused in mice lacking protein arginine methyltransferase 1 (PRMT1) in the central nervous system.
      ). High expression of PRMT1 has been linked to poor overall survival in many types of cancers; conversely, inhibition of PRMT1 activity blocks the proliferation of leukemia and solid tumors such as lung, breast, and ovarian cancers.
      Arginine methylation like phosphorylation is involved in the relay of extracellular and intracellular signals. PRMT1 activity and subcellular localization are regulated by phosphorylation. The yeast homolog of PRMT1 (Hmt1) has been shown to be inactivated through dephosphorylation in response to starvation, although the corresponding phosphorylation site on Hmt1 is not available in mammalian PRMT1 (
      • Messier V.
      • Zenklusen D.
      • Michnick S.W.
      A nutrient-responsive pathway that determines M phase timing through control of B-cyclin mRNA stability.
      ). The casein kinase 1A1 gene (CSNK1A1) is located within the del(5q) in myelodysplasia syndrome (MDS). Haploinsufficiency of CSNK1A1 in Csnk1a1−/+ KO mice causes mild dysplasia of megakaryocytes (Mks) in the bone marrow (
      • Schneider R.K.
      • Adema V.
      • Heckl D.
      • Jaras M.
      • Mallo M.
      • Lord A.M.
      • et al.
      Role of casein kinase 1A1 in the biology and targeted therapy of del(5q) MDS.
      ). Casein kinase 1A phosphorylates PRMT1 and directs it to chromatin (
      • Bao X.
      • Siprashvili Z.
      • Zarnegar B.J.
      • Shenoy R.M.
      • Rios E.J.
      • Nady N.
      • et al.
      CSNK1a1 regulates PRMT1 to maintain the progenitor state in self-renewing somatic tissue.
      ). Thus, haploinsufficiency of CSNK1A1 may affect PRMT1-mediated epigenetic regulation in megakaryopoiesis. There are many potential phosphorylation sites on PRMT1 (
      • Guo A.
      • Gu H.
      • Zhou J.
      • Mulhern D.
      • Wang Y.
      • Lee K.A.
      • et al.
      Immunoaffinity enrichment and mass spectrometry analysis of protein methylation.
      ,
      • Rust H.L.
      • Subramanian V.
      • West G.M.
      • Young D.D.
      • Schultz P.G.
      • Thompson P.R.
      Using unnatural amino acid mutagenesis to probe the regulation of PRMT1.
      ), although systematic studies on how phosphorylation changes PRMT1 activity have not been performed. In turn, PRMT1 methylates and activates various kinases. In leukemia cells, PRMT1 methylates and activates FLT3 kinase and FLT3 ITD kinase, which are commonly found in acute myelogenous leukemia (
      • Zhu Y.
      • He X.
      • Lin Y.C.
      • Dong H.
      • Zhang L.
      • Chen X.
      • et al.
      Targeting PRMT1-mediated FLT3 methylation disrupts maintenance of MLL-rearranged acute lymphoblastic leukemia.
      ). Furthermore, epidermal growth factor receptor is methylated by PRMT1 in triple-negative breast cancer cells, and methylation enhances epidermal growth factor receptor activity (
      • Nakai K.
      • Xia W.
      • Liao H.W.
      • Saito M.
      • Hung M.C.
      • Yamaguchi H.
      The role of PRMT1 in EGFR methylation and signaling in MDA-MB-468 triple-negative breast cancer cells.
      ). The expression of PRMT1 is upregulated by insulin and cytokines in cell lines (
      • Iwasaki H.
      • Yada T.
      Protein arginine methylation regulates insulin signaling in L6 skeletal muscle cells.
      ,
      • Sun Q.
      • Liu L.
      • Roth M.
      • Tian J.
      • He Q.
      • Zhong B.
      • et al.
      PRMT1 upregulated by epithelial proinflammatory cytokines participates in COX2 expression in fibroblasts and chronic antigen-induced pulmonary inflammation.
      ), whereas the expression of PRMT1 is downregulated by nutritional stress (
      • Zhang X.
      • Li L.
      • Li Y.
      • Li Z.
      • Zhai W.
      • Sun Q.
      • et al.
      mTOR regulates PRMT1 expression and mitochondrial mass through STAT1 phosphorylation in hepatic cell.
      ). PRMT1 is required for the activation of SMAD signaling via methylation of the suppressor SMAD6 (
      • Xu J.
      • Wang A.H.
      • Oses-Prieto J.
      • Makhijani K.
      • Katsuno Y.
      • Pei M.
      • et al.
      Arginine methylation initiates BMP-induced Smad signaling.
      ) and for activation of the mTOR pathway (
      • Zhang X.
      • Li L.
      • Li Y.
      • Li Z.
      • Zhai W.
      • Sun Q.
      • et al.
      mTOR regulates PRMT1 expression and mitochondrial mass through STAT1 phosphorylation in hepatic cell.
      ). PRMT1 also interacts with the interferon alpha receptor and promotes the interferon response (
      • Abramovich C.
      • Yakobson B.
      • Chebath J.
      • Revel M.
      A protein-arginine methyltransferase binds to the intracytoplasmic domain of the IFNAR1 chain in the type I interferon receptor.
      ). Further still, the Wnt signaling pathway is activated by PRMT1 through the methylation of GSK3 kinase (
      • Albrecht L.V.
      • Ploper D.
      • Tejeda-Munoz N.
      • De Robertis E.M.
      Arginine methylation is required for canonical Wnt signaling and endolysosomal trafficking.
      ). Taken together, the variation in PRMT1 activity further diversifies the outcomes of phosphorylation-mediated signaling and thus contributes to cell heterogeneity.
      PRMT1 expression levels are varied in blood cells with the lowest in hematopoietic stem cells (HSCs) and the highest in megakaryocyte-erythroid progenitors (
      • Zhang L.
      • Tran N.T.
      • Su H.
      • Wang R.
      • Lu Y.
      • Tang H.
      • et al.
      Cross-talk between PRMT1-mediated methylation and ubiquitylation on RBM15 controls RNA splicing.
      ). PRMT1 knockout in Mx1-cre mice increased the number and polyploidy of Mks in the bone marrow but caused bone marrow failure a few weeks after the induction thereof (
      • Zhu L.
      • He X.
      • Dong H.
      • Sun J.
      • Wang H.
      • Zhu Y.
      • et al.
      Protein arginine methyltransferase 1 is required for maintenance of normal adult hematopoiesis.
      ). Here, we summarize our findings on how PRMT1 regulates megakaryopoiesis at the molecular level and offer our perspective on the novel roles of PRMT1 in the generation and the functions of a group of immune Mks.

      Multiple routes for the generation of Mks

      Single-cell RNA sequencing (scRNA-seq) technology elucidates the heterogeneity and continuum within HSCs and hematopoietic progenitors of all blood lineages (
      • Giladi A.
      • Paul F.
      • Herzog Y.
      • Lubling Y.
      • Weiner A.
      • Yofe I.
      • et al.
      Single-cell characterization of haematopoietic progenitors and their trajectories in homeostasis and perturbed haematopoiesis.
      ,
      • Nestorowa S.
      • Hamey F.K.
      • Pijuan Sala B.
      • Diamanti E.
      • Shepherd M.
      • Laurenti E.
      • et al.
      A single-cell resolution map of mouse hematopoietic stem and progenitor cell differentiation.
      ,
      • Paul F.
      • Arkin Y.
      • Giladi A.
      • Jaitin D.A.
      • Kenigsberg E.
      • Keren-Shaul H.
      • et al.
      Transcriptional heterogeneity and lineage commitment in myeloid progenitors.
      ,
      • Tabula Muris Consortium
      • Overall Coordination
      • Logistical Coordination
      • Organ Collection and Processing
      • Library Preparation and Sequencing
      • Computational Data Analysis
      • et al.
      Single-cell transcriptomics of 20 mouse organs creates a Tabula Muris.
      ,
      • Tusi B.K.
      • Wolock S.L.
      • Weinreb C.
      • Hwang Y.
      • Hidalgo D.
      • Zilionis R.
      • et al.
      Population snapshots predict early haematopoietic and erythroid hierarchies.
      ,
      • Laurenti E.
      • Doulatov S.
      • Zandi S.
      • Plumb I.
      • Chen J.
      • April C.
      • et al.
      The transcriptional architecture of early human hematopoiesis identifies multilevel control of lymphoid commitment.
      ,
      • van Galen P.
      • Hovestadt V.
      • Wadsworth Ii M.H.
      • Hughes T.K.
      • Griffin G.K.
      • Battaglia S.
      • et al.
      Single-cell RNA-seq reveals AML hierarchies relevant to disease progression and immunity.
      ). HSCs give rise to megakaryocyte-erythroid progenitor cells, which further differentiate into Mks in the bone marrow under normal homeostatic conditions (
      • Sanada C.
      • Xavier-Ferrucio J.
      • Lu Y.C.
      • Min E.
      • Zhang P.X.
      • Zou S.
      • et al.
      Adult human megakaryocyte-erythroid progenitors are in the CD34+CD38mid fraction.
      ,
      • Xavier-Ferrucio J.
      • Krause D.S.
      Concise review: bipotent megakaryocytic-erythroid progenitors: concepts and controversies.
      ). Single-cell sequencing analysis suggests that Mks may be generated from more than one type of progenitor or stem cell (Fig. 1) (
      • Psaila B.
      • Mead A.J.
      Single-cell approaches reveal novel cellular pathways for megakaryocyte and erythroid differentiation.
      ). Both platelets and Mks store the von Willebrand factor (vWF) protein, a complex plasma glycoprotein that modulates platelet adhesion at the site of a vascular injury. A subpopulation of HSCs expressing high levels of vWF protein tends to differentiate into Mks with high efficiency even though these vWF+ HSCs retain their capability to generate other blood lineages in bone marrow transplantation assays (
      • Carrelha J.
      • Meng Y.
      • Kettyle L.M.
      • Luis T.C.
      • Norfo R.
      • Alcolea V.
      • et al.
      Hierarchically related lineage-restricted fates of multipotent haematopoietic stem cells.
      ). Phenotypic HSCs expressing high levels of c-Kit (a tyrosine kinase receptor for stem cell factor) differentiate more efficiently into Mks but lose their self-renewal capability (
      • Shin J.Y.
      • Hu W.
      • Naramura M.
      • Park C.Y.
      High c-Kit expression identifies hematopoietic stem cells with impaired self-renewal and megakaryocytic bias.
      ). Thrombopoietin receptor (also known as MPL or c-MPL) is required for Mk production. In c-Mpl−/− mice, the frequency of a group of multipotent progenitors, namely MPP2, expressing CD41 was reduced, whereas the other multipotent progenitor populations did not change. Subsequently, MPP2 was exclusively responsible for the generation of Mks (
      • Pietras E.M.
      • Reynaud D.
      • Kang Y.A.
      • Carlin D.
      • Calero-Nieto F.J.
      • Leavitt A.D.
      • et al.
      Functionally distinct subsets of lineage-biased multipotent progenitors control blood production in normal and regenerative conditions.
      ). Consistently, CD41+CMP (common myeloid progenitors) have been shown to generate only Mks (
      • Miyawaki K.
      • Iwasaki H.
      • Jiromaru T.
      • Kusumoto H.
      • Yurino A.
      • Sugio T.
      • et al.
      Identification of unipotent megakaryocyte progenitors in human hematopoiesis.
      ). The CD41+CD38CD34+ progenitors are committed for Mk differentiation (
      • Sanada C.
      • Xavier-Ferrucio J.
      • Lu Y.C.
      • Min E.
      • Zhang P.X.
      • Zou S.
      • et al.
      Adult human megakaryocyte-erythroid progenitors are in the CD34+CD38mid fraction.
      ). Challenged with lipopolysaccharide (LPS), quiescent but primed stem-like megakaryocyte progenitors, which express high levels of CD41 within the phenotypic long-term HSCs, quickly expand and undergo maturation and protein synthesis to generate platelets (
      • Haas S.
      • Hansson J.
      • Klimmeck D.
      • Loeffler D.
      • Velten L.
      • Uckelmann H.
      • et al.
      Inflammation-induced emergency megakaryopoiesis driven by hematopoietic stem cell-like megakaryocyte progenitors.
      ). Altogether, these studies suggest that although CD41 is a common marker for Mk cells at different developmental stages, there are many paths or many subgroups of HSCs or hematopoietic progenitors that can generate mature Mks, which may account for Mk heterogeneity and hence platelet heterogeneity.
      Figure thumbnail gr1
      Figure 1PRMT1 expression is higher in low-polyploid immune Mks. Univariate analysis of scRNA data (
      • Sun S.
      • Jin C.
      • Si J.
      • Lei Y.
      • Chen K.
      • Cui Y.
      • et al.
      Single-cell analysis of ploidy and the transcriptome reveals functional and spatial divergency in murine megakaryopoiesis.
      ). A, compare PRMT1 expression in human Mks with different polyploidy. B, PRMT1 expression levels in immune Mks (CD53+CD41+ or LSP1+CD41+) versus platelet generation Mks. C, potential role of PRMT1 in megakaryopoiesis. HSCs are heterogeneous. A group of HSCs expressing vWF is biased toward megakaryocyte differentiation, although they can differentiate into other blood lineages. Several types of progenitor cells are biased toward megakaryocyte differentiation. These progenitor cells (SL-MkPs) express CD41 on the cell surface, along with markers for phenotypic HSCs. Common myeloid progenitor cells expressing CD41 are unipotent for megakaryocyte differentiation. In addition, MEPs retain the binary differentiation capability for both erythroid and megakaryocyte differentiation. MEP cells expressed the highest levels of PRMT1. When progenitors undergo further differentiation into mature megakaryocytes, the activity of PRMT1 determines the direction to immune Mks or platelet-generating Mks and HSC-niche Mks. HSC, hematopoietic stem cell; MEP, megakaryocyte-erythroid progenitor; Mks, megakaryocytes; PRMT, protein arginine methyltransferase; SL-MkP, stem-like megakaryocyte progenitors; vWF, von Willebrand factor.

      A subgroup of Mks as uncharacterized immune cells

      Evidence of Mks as immune cells existed before scRNA-seq technology was applied (
      • Cunin P.
      • Nigrovic P.A.
      Megakaryocytes as immune cells.
      ). Transplantation of Mks expressing interleukin-1 can induce arthritis (
      • Finkielsztein A.
      • Schlinker A.C.
      • Zhang L.
      • Miller W.M.
      • Datta S.K.
      Human megakaryocyte progenitors derived from hematopoietic stem cells of normal individuals are MHC class II-expressing professional APC that enhance Th17 and Th1/Th17 responses.
      ). Mks activate T cells such as Th17 via major histocompatibility complex (MHC) class II protein expressed on the Mk surface (
      • Finkielsztein A.
      • Schlinker A.C.
      • Zhang L.
      • Miller W.M.
      • Datta S.K.
      Human megakaryocyte progenitors derived from hematopoietic stem cells of normal individuals are MHC class II-expressing professional APC that enhance Th17 and Th1/Th17 responses.
      ). Recently, scRNA-seq technology has demonstrated that heterogeneity also exists in mature blood cells such as neutrophils (
      • Xie X.
      • Shi Q.
      • Wu P.
      • Zhang X.
      • Kambara H.
      • Su J.
      • et al.
      Single-cell transcriptome profiling reveals neutrophil heterogeneity in homeostasis and infection.
      ) and Mks in both humans and mice (
      • Psaila B.
      • Mead A.J.
      Single-cell approaches reveal novel cellular pathways for megakaryocyte and erythroid differentiation.
      ,
      • Liu C.
      • Wu D.
      • Xia M.
      • Li M.
      • Sun Z.
      • Shen B.
      • et al.
      Characterization of cellular heterogeneity and an immune subpopulation of human megakaryocytes.
      ,
      • Pariser D.N.
      • Hilt Z.T.
      • Ture S.K.
      • Blick-Nitko S.K.
      • Looney M.R.
      • Cleary S.J.
      • et al.
      Lung megakaryocytes are immune modulatory cells.
      ,
      • Sun S.
      • Jin C.
      • Si J.
      • Lei Y.
      • Chen K.
      • Cui Y.
      • et al.
      Single-cell analysis of ploidy and the transcriptome reveals functional and spatial divergency in murine megakaryopoiesis.
      ,
      • Wang H.
      • He J.
      • Xu C.
      • Chen X.
      • Yang H.
      • Shi S.
      • et al.
      Decoding human megakaryocyte development.
      ,
      • Yeung A.K.
      • Villacorta-Martin C.
      • Hon S.
      • Rock J.R.
      • Murphy G.J.
      Lung megakaryocytes display distinct transcriptional and phenotypic properties.
      ). Mks residing in the bone marrow have multiple nuclei, which account for their large sizes ranging from 50 to 100 μm. In addition to their sizes, their low number in the bone marrow makes handling fragile Mks challenging in experiments. Therefore, characterization of Mks especially the large polyploid Mks is often missed in regular scRNA-seq analysis. Sun et al. (
      • Sun S.
      • Jin C.
      • Si J.
      • Lei Y.
      • Chen K.
      • Cui Y.
      • et al.
      Single-cell analysis of ploidy and the transcriptome reveals functional and spatial divergency in murine megakaryopoiesis.
      ) utilized a large nozzle and a lower flow rate to isolate Mks based on surface markers, namely CD41, and polyploid status and then applied the scRNA-seq method (Smart-seq2) to collect the transcriptome profiles of individual Mks. For smart-seq2, Mks spanning all degrees of ploidy were collected. In contrast, the automated cell collection procedure employed by 10X Genomics preferentially enriches for low-ploid cells, while high-ploid Mks have limited access to channels of 10X microfluid and may not be sorted because of their fragility and large size (>65 μm in diameter). Moreover, Smart-seq2 detected more genes in a cell, especially low-abundance transcripts, as well as alternatively spliced transcripts than did 10X Genomics Chromium (
      • Wang X.
      • He Y.
      • Zhang Q.
      • Ren X.
      • Zhang Z.
      Direct comparative analyses of 10X Genomics Chromium and smart-seq2.
      ). Mks were clustered into several groups: platelet-generating Mks, immune Mks, and HSC-niche Mks in addition to a group of Mks that expressed high levels of cell cycle genes. Both mice and humans have the immune-Mk subgroups that express immune response genes and transcription factors; PU.1 that is known to promote monocyte differentiation and suppress late megakaryopoiesis (
      • Huang G.
      • Zhang P.
      • Hirai H.
      • Elf S.
      • Yan X.
      • Chen Z.
      • et al.
      PU.1 is a major downstream target of AML1 (RUNX1) in adult mouse hematopoiesis.
      ) and IRF8 that activates proinflammatory gene expression (
      • Giladi A.
      • Paul F.
      • Herzog Y.
      • Lubling Y.
      • Weiner A.
      • Yofe I.
      • et al.
      Single-cell characterization of haematopoietic progenitors and their trajectories in homeostasis and perturbed haematopoiesis.
      ). Therefore, immune Mks may resemble partial functions of monocytes but do not express CD11b, as do typical monocytes. Consistent with the immune phenotype, immune Mks also express high levels of Runt-related transcription factor 1 (RUNX1) (
      • Leon Machado J.A.
      • Steimle V.
      The MHC class II transactivator CIITA: not (quite) the odd-one-out anymore among NLR proteins.
      ), CIITA (
      • Leon Machado J.A.
      • Steimle V.
      The MHC class II transactivator CIITA: not (quite) the odd-one-out anymore among NLR proteins.
      ), STAT1 (
      • Cowland J.B.
      • Muta T.
      • Borregaard N.
      IL-1beta-specific up-regulation of neutrophil gelatinase-associated lipocalin is controlled by IkappaB-zeta.
      ), and NFKBBIZ (
      • Cowland J.B.
      • Muta T.
      • Borregaard N.
      IL-1beta-specific up-regulation of neutrophil gelatinase-associated lipocalin is controlled by IkappaB-zeta.
      ), which regulate immunity. In addition to transcription factors, immune response genes such as HLA-DQA1, LILRB1, CSF2RA, CSF3R, and IL1R2 are differentially and highly expressed in immune Mks. In contrast, platelet-generating Mks express high levels of GATA2, NF-E2, and FLI-1, transcription factors that are well known for inducing Mk maturation.
      After analyzing the differentially expressed genes between different subgroups, Sun et al. utilized the cell membrane proteins CD53 and CD41 to sort the immune Mks for in vitro functional assays. They demonstrated that the CD53+CD41+ population expanded in response to LPS challenge within the first 12 h with an upregulation of a preexisting PU.1 (encoded by the SPI1 gene) and IRF8-associated inflammatory transcription. Isolated primary immune Mks were capable of engulfing and ingesting bacterial particles, as well as activating T cell expansion when cocultured with T cells in vitro in the presence of anti-CD3 and CD28 antibodies. These murine immune Mks express higher levels of MHC class II molecules when stimulated with LPS, implying that immune Mks can present foreign antigens during infection.
      The existence of immune Mks in human adult bone marrow was independently verified by Liu et al. (
      • Liu C.
      • Wu D.
      • Xia M.
      • Li M.
      • Sun Z.
      • Shen B.
      • et al.
      Characterization of cellular heterogeneity and an immune subpopulation of human megakaryocytes.
      ) using Smart-seq2. They identified two surface markers, CD48 and CD148, that define immune Mks. When mice were challenged with LPS or interferon gamma, the CD48+CD148+ Mk population expanded in number and expressed high levels of Toll-like receptor 4 (TLR4), TLR2, S100 calcium-binding protein A8 (S100A8A), and other proteins related to inflammatory response. The existence of immune Mks has also been demonstrated by Pariser et al. (
      • Pariser D.N.
      • Hilt Z.T.
      • Ture S.K.
      • Blick-Nitko S.K.
      • Looney M.R.
      • Cleary S.J.
      • et al.
      Lung megakaryocytes are immune modulatory cells.
      ) using Mks isolated from the lungs. Strikingly, lung Mks express CD53, LSP1, CD48, and CD148 on the surface, similar to immune Mks in the bone marrow (
      • Liu C.
      • Wu D.
      • Xia M.
      • Li M.
      • Sun Z.
      • Shen B.
      • et al.
      Characterization of cellular heterogeneity and an immune subpopulation of human megakaryocytes.
      ,
      • Sun S.
      • Jin C.
      • Si J.
      • Lei Y.
      • Chen K.
      • Cui Y.
      • et al.
      Single-cell analysis of ploidy and the transcriptome reveals functional and spatial divergency in murine megakaryopoiesis.
      ). Whether immune Mks coexpress CD53 and CD48 on the same cells has not yet been investigated using flow cytometry. Unlike bone marrow–derived Mks, lung Mks are generally smaller in size (
      • Lefrancais E.
      • Ortiz-Munoz G.
      • Caudrillier A.
      • Mallavia B.
      • Liu F.
      • Sayah D.M.
      • et al.
      The lung is a site of platelet biogenesis and a reservoir for haematopoietic progenitors.
      ). Lung Mks express MHC class II complexes and activate T cells when primed with ovalbumin (OVA) protein, which is widely used as an antigen for presentation. In addition to antigen presentation, lung Mks express genes involved in inflammatory reactions (
      • Yeung A.K.
      • Villacorta-Martin C.
      • Hon S.
      • Rock J.R.
      • Murphy G.J.
      Lung megakaryocytes display distinct transcriptional and phenotypic properties.
      ,
      • Lefrancais E.
      • Ortiz-Munoz G.
      • Caudrillier A.
      • Mallavia B.
      • Liu F.
      • Sayah D.M.
      • et al.
      The lung is a site of platelet biogenesis and a reservoir for haematopoietic progenitors.
      ). Therefore, adult lung Mks are functionally similar to immune Mks from the bone marrow. Further proof is provided in the same Sun et al. study, in which scRNA analysis of lung-derived Mks and immune Mks from bone marrow were compared. Both groups of Mks had enriched immune-related gene expression signatures. The two types of immune Mks have comparable levels of the characteristic markers, LSP1 and CD53, and transcription factors SPI1/PU.1 and IRF8. By comparing bone marrow–derived Mks with lung-derived Mks from published adult and fetal scRNA data (
      • Pariser D.N.
      • Hilt Z.T.
      • Ture S.K.
      • Blick-Nitko S.K.
      • Looney M.R.
      • Cleary S.J.
      • et al.
      Lung megakaryocytes are immune modulatory cells.
      ,
      • Yeung A.K.
      • Villacorta-Martin C.
      • Hon S.
      • Rock J.R.
      • Murphy G.J.
      Lung megakaryocytes display distinct transcriptional and phenotypic properties.
      ), Sun et al. revealed that immune Mks are conserved and exist in different tissues (bone marrow or lung) and developmental stages (fetal or adult). While adult lung Mks were enriched with transcriptional signatures of immune-related processes, including pathogen recognition, phagocytosis, and antigen presentation, fetal lung and adult bone marrow immune Mks showed differential expression signatures in phagocytosis and antigen presentation, respectively (Table 1). These differences might be explained by the fact that lung-derived Mks are constantly exposed to bacteria, viruses, and other environmental hazards. An alternative explanation is that 5% of Mks have neutrophils inside their cells via emperipolesis, which is stimulated by LPS (
      • Cunin P.
      • Nigrovic P.A.
      Megakaryocytes as immune cells.
      ). Thus, it is possible that the inflammatory signature may partially reflect the genes expressed in neutrophils inside some of the adult lung-derived immune Mks.
      Table 1Differentially expressed genes in immune Mks from different tissues (
      • Sun S.
      • Jin C.
      • Si J.
      • Lei Y.
      • Chen K.
      • Cui Y.
      • et al.
      Single-cell analysis of ploidy and the transcriptome reveals functional and spatial divergency in murine megakaryopoiesis.
      )
      TissueGenes and functions
      Adult bone marrowPlatelet functions: Pf4, Flna, Nfe2, Serpine 2, Vwf, Ppbp

      Antigen presentation: Cd74, H2-Ab1, H2-Eb1, H2-DMa, H2-DMb1

      Anti-bacteria peptides: S100a8, S100a9, Camp
      Adult lungAntigen presentation: Cd74, H2-Ab1, H2-Eb1, H2-DMa, H2-DMb1

      Phagocytosis: C1qa, C1qb, C1qc, C5ar1, Ctsh, Ctsc, Ctsb, Ctsz, Ctsd, Ctsl, Lamp1, Fcgr3

      Chemotaxis: Cxcl16, Cd14, Cxcl2, Ccl2, Ccrl2
      Fetal lungPhagocytosis: C1qa, C1qb, C1qc, C5ar1, Ctsh, Ctsc, Ctsb, Ctsz, Ctsd, Ctsl, Lamp1, Fcgr3
      Another scRNA-seq analysis of Mks further confirmed a subgroup of Mks expressing genes with an immune response signature, including CD53, as described in other groups (
      • Wang J.
      • Xie J.
      • Wang D.
      • Han X.
      • Chen M.
      • Shi G.
      • et al.
      CXCR4(high) megakaryocytes regulate host-defense immunity against bacterial pathogens.
      ). They used CXCR4 as an additional marker to isolate immune Mks for additional immunological assays. They discovered that CXCR4-high Mks migrated to the spleen and liver upon stimulation by bacterial infection. CXCR4high Mks secrete IL-6 and TNFα and contact myeloid cells as an adaptive immune response in addition to an enhanced innate immune response. In an early study, low-polyploid Mks were shown to express MHC class II molecules, whereas high-polyploid Mks expressed MHC class I (
      • Raslova H.
      • Kauffmann A.
      • Sekkai D.
      • Ripoche H.
      • Larbret F.
      • Robert T.
      • et al.
      Interrelation between polyploidization and megakaryocyte differentiation: a gene profiling approach.
      ). Interestingly, since CXCR4high Mks express MHC class II molecules, we suspect that these Mks are not fully mature or able to generate platelets. To date, platelets expressing MHC class II molecules have not been reported.
      Whether CD53+CD41+ Mks can recapitulate all the biological functions of immune Mks, as defined by scRNA-seq analysis, requires further investigation. Both MHC classes I and II are involved in antigen presentation in Mks (
      • Finkielsztein A.
      • Schlinker A.C.
      • Zhang L.
      • Miller W.M.
      • Datta S.K.
      Human megakaryocyte progenitors derived from hematopoietic stem cells of normal individuals are MHC class II-expressing professional APC that enhance Th17 and Th1/Th17 responses.
      ,
      • Zufferey A.
      • Speck E.R.
      • Machlus K.R.
      • Aslam R.
      • Guo L.
      • McVey M.J.
      • et al.
      Mature murine megakaryocytes present antigen-MHC class I molecules to T cells and transfer them to platelets.
      ). MHC class I is expressed in mature Mks (
      • Finkielsztein A.
      • Schlinker A.C.
      • Zhang L.
      • Miller W.M.
      • Datta S.K.
      Human megakaryocyte progenitors derived from hematopoietic stem cells of normal individuals are MHC class II-expressing professional APC that enhance Th17 and Th1/Th17 responses.
      ). Accordingly, MHC class I gene expression was observed in all four groups defined in our scRNA-seq studies. Since none of the MHC markers or PRMT1 were exclusively expressed in a specific group, a low percentage of high-polyploid Mks may still express PRMT1 and participate in immune reactions.

      Immune Mks may be responsible for generating proinflammatory platelets

      Both high- and low-polyploid Mks can produce platelets, although high polyploidy may do so more efficiently (
      • Machlus K.R.
      • Italiano Jr., J.E.
      The incredible journey: from megakaryocyte development to platelet formation.
      ). Therefore, the heterogeneity of Mks determines the heterogeneity of platelets. Neonatal platelets are less active in response to stimulants than adult platelets (
      • Rajasekhar D.
      • Kestin A.S.
      • Bednarek F.J.
      • Ellis P.A.
      • Barnard M.R.
      • Michelson A.D.
      Neonatal platelets are less reactive than adult platelets to physiological agonists in whole blood.
      ), which may be due to the low polyploidy of fetal Mks (
      • de Alarcon P.A.
      • Graeve J.L.
      Analysis of megakaryocyte ploidy in fetal bone marrow biopsies using a new adaptation of the feulgen technique to measure DNA content and estimate megakaryocyte ploidy from biopsy specimens.
      ). Immune Mks were enriched in the fraction of low-polyploid Mks (
      • Sun S.
      • Jin C.
      • Si J.
      • Lei Y.
      • Chen K.
      • Cui Y.
      • et al.
      Single-cell analysis of ploidy and the transcriptome reveals functional and spatial divergency in murine megakaryopoiesis.
      ). Lung Mks, like bone marrow immune Mks, have low polyploidy (
      • Yeung A.K.
      • Villacorta-Martin C.
      • Hon S.
      • Rock J.R.
      • Murphy G.J.
      Lung megakaryocytes display distinct transcriptional and phenotypic properties.
      ). Platelet budding from lung Mks has been visualized in vivo using two-photon microscopy (
      • Lefrancais E.
      • Ortiz-Munoz G.
      • Caudrillier A.
      • Mallavia B.
      • Liu F.
      • Sayah D.M.
      • et al.
      The lung is a site of platelet biogenesis and a reservoir for haematopoietic progenitors.
      ). Given the transcriptome similarity between lung Mks and bone marrow immune Mks, it is possible that immune Mks in bone marrow like lung Mks (
      • Lefrancais E.
      • Ortiz-Munoz G.
      • Caudrillier A.
      • Mallavia B.
      • Liu F.
      • Sayah D.M.
      • et al.
      The lung is a site of platelet biogenesis and a reservoir for haematopoietic progenitors.
      ) can produce platelets. Although immune Mks express genes responsible for inflammation, whether platelets generated from immune Mks are proinflammatory has not been tested experimentally. According to scRNA-seq analysis (
      • Wang H.
      • He J.
      • Xu C.
      • Chen X.
      • Yang H.
      • Shi S.
      • et al.
      Decoding human megakaryocyte development.
      ), human fetal liver and embryo yolk sac cells contain Mks. Further analysis demonstrated that a subpopulation of early embryonic Mks expresses immune response genes (
      • Wang H.
      • He J.
      • Xu C.
      • Chen X.
      • Yang H.
      • Shi S.
      • et al.
      Decoding human megakaryocyte development.
      ), suggesting that there may be two developmental paths of megakaryopoiesis, with one directing toward Mks expressing immune-responsive genes and the other toward platelet production. However, according to the Mk distribution along these developmental paths, it cannot be ruled out that immune Mks are in transit to become platelet-producing Mks. Inflammation can be a prerequisite for platelet production in certain progenitors. Intriguingly, these stem-like megakaryocyte progenitors also express interferon response genes that are critical for platelet generation, as IFNAR−/− mice cannot produce Mk-specific proteins in these phenotypic HSCs (
      • Haas S.
      • Hansson J.
      • Klimmeck D.
      • Loeffler D.
      • Velten L.
      • Uckelmann H.
      • et al.
      Inflammation-induced emergency megakaryopoiesis driven by hematopoietic stem cell-like megakaryocyte progenitors.
      ). These data are consistent with the observation that STAT1 activation in interferon signaling promotes megakaryopoiesis (
      • Huang Z.
      • Richmond T.D.
      • Muntean A.G.
      • Barber D.L.
      • Weiss M.J.
      • Crispino J.D.
      STAT1 promotes megakaryopoiesis downstream of GATA-1 in mice.
      ).
      Many clinical studies have shown that platelets play a role not only in thrombus formation but also in innate immunity, inflammation, and carcinogenesis (
      • Vardon-Bounes F.
      • Ruiz S.
      • Gratacap M.P.
      • Garcia C.
      • Payrastre B.
      • Minville V.
      Platelets are critical key players in sepsis.
      ,
      • Frydman G.H.
      • Tessier S.N.
      • Wong K.H.K.
      • Vanderburg C.R.
      • Fox J.G.
      • Toner M.
      • et al.
      Megakaryocytes contain extranuclear histones and may be a source of platelet-associated histones during sepsis.
      ,
      • Van Bergen M.
      • Marneth A.E.
      • Hoogendijk A.J.
      • Van Alphen F.P.J.
      • Van den Akker E.
      • Laros-Van Gorkom B.A.P.
      • et al.
      Specific proteome changes in platelets from individuals with GATA1-, GFI1B-, and RUNX1-linked bleeding disorders.
      ,
      • Zaid Y.
      • Puhm F.
      • Allaeys I.
      • Naya A.
      • Oudghiri M.
      • Khalki L.
      • et al.
      Platelets can associate with SARS-cov-2 RNA and are hyperactivated in COVID-19.
      ). Platelets from patients with sepsis and COVID-19 have different transcriptome profiles and protein components compared to those from normal human controls (
      • Frydman G.H.
      • Tessier S.N.
      • Wong K.H.K.
      • Vanderburg C.R.
      • Fox J.G.
      • Toner M.
      • et al.
      Megakaryocytes contain extranuclear histones and may be a source of platelet-associated histones during sepsis.
      ,
      • Van Bergen M.
      • Marneth A.E.
      • Hoogendijk A.J.
      • Van Alphen F.P.J.
      • Van den Akker E.
      • Laros-Van Gorkom B.A.P.
      • et al.
      Specific proteome changes in platelets from individuals with GATA1-, GFI1B-, and RUNX1-linked bleeding disorders.
      ,
      • Zaid Y.
      • Puhm F.
      • Allaeys I.
      • Naya A.
      • Oudghiri M.
      • Khalki L.
      • et al.
      Platelets can associate with SARS-cov-2 RNA and are hyperactivated in COVID-19.
      ). Under septic conditions, immune Mks may expand and produce high levels of pathogenic platelets that express high levels of inflammatory response genes. Therefore, determining the molecular mechanism underlying the differentiation of immune Mks is critical for uncovering the genesis of proinflammatory platelets.

      Protein arginine methylation for the generation of immune Mks

      Based on our published data on PRMT1-mediated megakaryopoiesis and its direct effects on the protein concentrations of the transcription factor PU.1, the RNA-binding motif protein 15, RBM15, and dual specificity phosphatase 4 (DUSP4), we argue that protein arginine methylation plays a critical role in the generation of immune Mks. Our scRNA-seq analysis of human cord blood cells stimulated with thrombopoietin and stem cell factor for MK differentiation revealed that the expression of PRMT1 was positively correlated with the expression of proinflammatory genes in individual Mks (
      • Su H.
      • Jiang M.
      • Senevirathne C.
      • Aluri S.
      • Zhang T.
      • Guo H.
      • et al.
      Methylation of dual-specificity phosphatase 4 controls cell differentiation.
      ). We previously demonstrated that PRMT1 expression plays a critical role in megakaryocyte differentiation (
      • Zhang L.
      • Tran N.T.
      • Su H.
      • Wang R.
      • Lu Y.
      • Tang H.
      • et al.
      Cross-talk between PRMT1-mediated methylation and ubiquitylation on RBM15 controls RNA splicing.
      ,
      • Zhao X.
      • Jankovic V.
      • Gural A.
      • Huang G.
      • Pardanani A.
      • Menendez S.
      • et al.
      Methylation of RUNX1 by PRMT1 abrogates SIN3A binding and potentiates its transcriptional activity.
      ). Constitutively high expression of PRMT1 blocks Mks from further developing into high-polyploid cells. In addition, our unpublished data from mice conditionally overexpressing PRMT1 under the platelet factor 4 (PF4) promoter, which drives the expression of PRMT1 mainly in Mk-committed cells, demonstrated that, in 6-week-old mice, overexpression of PRMT1 leads to decreased polyploidization with no effect on platelet count. Conversely, inhibition of asymmetric methyltransferase activity with the potent inhibitor, MS023 at 50 mg/kg, increases platelet count and polyploidy in mice (
      • Su H.
      • Jiang M.
      • Senevirathne C.
      • Aluri S.
      • Zhang T.
      • Guo H.
      • et al.
      Methylation of dual-specificity phosphatase 4 controls cell differentiation.
      ). Given that the majority of immune Mks have low polyploidy (≤8N), we further investigated PRMT1 expression levels using the scRNA-seq data reported by Sun et al. (
      • Sun S.
      • Jin C.
      • Si J.
      • Lei Y.
      • Chen K.
      • Cui Y.
      • et al.
      Single-cell analysis of ploidy and the transcriptome reveals functional and spatial divergency in murine megakaryopoiesis.
      ). We found that PRMT1 was highly expressed in immune Mks and in the low polyploidy fraction (Fig. 1), which is consistent with our findings that high expression of PRMT1 blocks Mk differentiation into high-polyploid cells. PRMT1 is largely expressed in the bone marrow blood cells of patients with MDS. Given that thrombocytopenia is an ominous symptom of MDS, our findings support the use of PRMT1 inhibition to combat this symptom in affected patients (
      • Su H.
      • Jiang M.
      • Senevirathne C.
      • Aluri S.
      • Zhang T.
      • Guo H.
      • et al.
      Methylation of dual-specificity phosphatase 4 controls cell differentiation.
      ).
      Genes regulated by RUNX1, RBM15, and DUSP4 in the presence of different levels of PRMT1 expression may determine the direction of Mk differentiation into immune or platelet-generating Mks (Fig. 2). Mechanistically, we have identified that methylation of RUNX1 by PRMT1 disrupts its binding to a transcriptional repressor complex, the mSIN3 complex, thus activating PU.1 transcription (
      • Zhao X.
      • Jankovic V.
      • Gural A.
      • Huang G.
      • Pardanani A.
      • Menendez S.
      • et al.
      Methylation of RUNX1 by PRMT1 abrogates SIN3A binding and potentiates its transcriptional activity.
      ). PU.1 is a master transcription factor that drives monocyte-granulocyte differentiation and blocks Mk differentiation (
      • Huang G.
      • Zhang P.
      • Hirai H.
      • Elf S.
      • Yan X.
      • Chen Z.
      • et al.
      PU.1 is a major downstream target of AML1 (RUNX1) in adult mouse hematopoiesis.
      ). Consistently, we discovered that PU.1 is repressed by RUNX1 when PRMT1 is downregulated during Mk differentiation. RUNX1 is also required for the maturation of Mks and polyploidization, as a transcriptional repressor of the MYH10 gene, which encodes nonmuscle myosin IIB heavy chain protein for blocking polyploidization (
      • Lordier L.
      • Bluteau D.
      • Jalil A.
      • Legrand C.
      • Pan J.
      • Rameau P.
      • et al.
      RUNX1-induced silencing of non-muscle myosin heavy chain IIB contributes to megakaryocyte polyploidization.
      ).
      Figure thumbnail gr2
      Figure 2The generation of immune Mks is dependent of PRMT1 dose according to our perspectives. Many signals have been shown to activate PRMT1 via increasing its protein levels or phosphorylation. According to our studies, once PRMT1 is upregulated, PRMT1 can methylate DUSP4, RUNX1, and RBM15. Methylation of DUSP4 and RBM15 leads to their degradation; thus, p38 kinase is maintained in activation status, and the mRNAs of proinflammatory genes are stable because of RBM15 degradation. PRMT1 upregulation also converts RUNX1 into a transcriptional activator of PU.1 via methylation. PU.1 activates the transcription of innate immune response genes. Collectively, these results indicate that PRMT1 upregulates the generation of immune Mks. In contrast, PRMT1 downregulation upregulates DUSP4 and RBM15 and converts RUNX1 to a transcriptional repressor. RBM15 is required for the generation of full-length GATA1 and c-Mpl mRNAs via RNA splicing. Given that RBM15 transcription is dependent on RUNX1 activation, transient upregulation of PRMT1 may be required for the production of RBM15. Downregulation of PRMT1 is required for the generation of platelet-generating Mks. Thus, the dynamic regulation of PRMT1 is required for optimal differentiation. Mks, megakaryocytes; PRMT, protein arginine methyltransferase.
      The gene encoding RBM15 is part of a recurrent chromosome translocation, t(1;22), in acute megakaryoblastic leukemia (
      • Ma Z.
      • Morris S.W.
      • Valentine V.
      • Li M.
      • Herbrick J.A.
      • Cui X.
      • et al.
      Fusion of two novel genes, RBM15 and MKL1, in the t(1;22)(p13;q13) of acute megakaryoblastic leukemia.
      ,
      • Mercher T.
      • Coniat M.B.
      • Monni R.
      • Mauchauffe M.
      • Nguyen Khac F.
      • Gressin L.
      • et al.
      Involvement of a human gene related to the Drosophila spen gene in the recurrent t(1;22) translocation of acute megakaryocytic leukemia.
      ). Downregulation of RBM15 promotes the generation of Mk progenitors, while RBM15 is required for Mk maturation into high-polyploid cells (
      • Niu C.
      • Zhang J.
      • Breslin P.
      • Onciu M.
      • Ma Z.
      • Morris S.W.
      c-Myc is a target of RNA-binding motif protein 15 in the regulation of adult hematopoietic stem cell and megakaryocyte development.
      ). Furthermore, methylation of RBM15 by PRMT1 triggers its degradation via CNOT4-mediated ubiquitination, whereas overexpression of RBM15 can rescue the PRMT1-mediated block of Mk differentiation (
      • Zhang L.
      • Tran N.T.
      • Su H.
      • Wang R.
      • Lu Y.
      • Tang H.
      • et al.
      Cross-talk between PRMT1-mediated methylation and ubiquitylation on RBM15 controls RNA splicing.
      ). RBM15 may control the stability of mRNAs via binding to the 3′UTR; thus, methylation of RBM15 by PRMT1 stabilizes the mRNAs involved in inflammatory responses, such as IL-16, IL12RB2, IL1A, and IL18BP. Additionally, RBM15 controls RNA splicing to produce full-length mRNAs of many genes, including GATA1, RUNX1, TAL1, and c-MPL, which are required for Mk maturation and polyploidization (
      • Zhang L.
      • Tran N.T.
      • Su H.
      • Wang R.
      • Lu Y.
      • Tang H.
      • et al.
      Cross-talk between PRMT1-mediated methylation and ubiquitylation on RBM15 controls RNA splicing.
      ,
      • Niu C.
      • Zhang J.
      • Breslin P.
      • Onciu M.
      • Ma Z.
      • Morris S.W.
      c-Myc is a target of RNA-binding motif protein 15 in the regulation of adult hematopoietic stem cell and megakaryocyte development.
      ,
      • Jin S.
      • Su H.
      • Tran N.T.
      • Song J.
      • Lu S.S.
      • Li Y.
      • et al.
      Splicing factor SF3B1K700E mutant dysregulates erythroid differentiation via aberrant alternative splicing of transcription factor TAL1.
      ,
      • Xiao N.
      • Laha S.
      • Das S.P.
      • Morlock K.
      • Jesneck J.L.
      • Raffel G.D.
      Ott1 (Rbm15) regulates thrombopoietin response in hematopoietic stem cells through alternative splicing of c-Mpl.
      ). Given that PRMT1 promotes the alternative splicing of c-MPL RNAs that encode for truncated c-MPL proteins, which respond poorly to thrombopoietin, Mks expressing high levels of PRMT1 may be less efficient in producing platelets. We previously reported that RUNX1 activates the transcription of RBM15 (
      • Tran N.T.
      • Su H.
      • Khodadadi-Jamayran A.
      • Lin S.
      • Zhang L.
      • Zhou D.
      • et al.
      The AS-RBM15 lncRNA enhances RBM15 protein translation during megakaryocyte differentiation.
      ). Paradoxically, RBM15 protein is degraded by PRMT1-mediated ubiquitylation. As a result, along with the activation of PU.1, Mks expressing PRMT1 remain immune Mks. Because RBM15 is required for MK polyploidization, downregulation of PRMT1 is necessary for extending the stability of RBM15 protein. Nevertheless, activation of PRMT1 is still required for the initiation of megakaryopoiesis via RUNX1-mediated transcriptional activation of RBM15. Thus, the timing and amplitude of transcriptional activation by PRMT1 and RUNX1 determine the protein levels of RBM15, which may further determine the heterogeneity of mature Mks (Fig. 2). Furthermore, Myc is required for generation of Mk progenitors, whereas its downregulation is required for platelet generation (
      • Takayama N.
      • Nishimura S.
      • Nakamura S.
      • Shimizu T.
      • Ohnishi R.
      • Endo H.
      • et al.
      Transient activation of c-MYC expression is critical for efficient platelet generation from human induced pluripotent stem cells.
      ). Since PRMT1 has a consensus E-box, Myc may activate PRMT1 during megakaryopoiesis.
      Recently, we discovered that DUSP4 is a critical downstream target of PRMT1 in megakaryopoiesis (
      • Su H.
      • Jiang M.
      • Senevirathne C.
      • Aluri S.
      • Zhang T.
      • Guo H.
      • et al.
      Methylation of dual-specificity phosphatase 4 controls cell differentiation.
      ). Methylation of DUSP4 by PRMT1 triggers methylation-dependent recruitment of the E3 ligase, HUWE1, which triggers the degradation of methylated DUSP4. Ectopic expression of DUSP4 rescues PRMT1-mediated blockage and promotes polyploidization of mature Mks. Consistently, knockdown of HUWE1 promotes MK differentiation. We found that DUSP4 specifically dephosphorylates p38 mitogen-activated protein (MAP) kinase in Mks. Activation of MAPK1, a MAP kinase, is required for megakaryopoiesis (
      • Mazharian A.
      • Watson S.P.
      • Severin S.
      Critical role for ERK1/2 in bone marrow and fetal liver-derived primary megakaryocyte differentiation, motility, and proplatelet formation.
      ), whereas activation of p38 MAP kinase blocks megakaryopoiesis (
      • Desterke C.
      • Bilhou-Nabera C.
      • Guerton B.
      • Martinaud C.
      • Tonetti C.
      • Clay D.
      • et al.
      FLT3-mediated p38-MAPK activation participates in the control of megakaryopoiesis in primary myelofibrosis.
      ). Given that p38 MAP kinase is often activated in inflammatory reactions (
      • Wang Q.
      • Reszka-Blanco N.
      • Cheng L.
      • Li G.
      • Zhang L.
      • Su L.
      p38 MAPK is critical for nuclear translocation of IRF-7 during CpG-induced type I IFN expression in human plasmacytoid dendritic cells.
      ), it is likely that PRMT1-regulated DUSP4 degradation indirectly activates the p38 MAP kinase pathway to promote inflammation. Consistently, the activation of p38 kinase has been revealed in Mks isolated from primary fibrosis (
      • Desterke C.
      • Bilhou-Nabera C.
      • Guerton B.
      • Martinaud C.
      • Tonetti C.
      • Clay D.
      • et al.
      FLT3-mediated p38-MAPK activation participates in the control of megakaryopoiesis in primary myelofibrosis.
      ) and MDS (
      • Su H.
      • Jiang M.
      • Senevirathne C.
      • Aluri S.
      • Zhang T.
      • Guo H.
      • et al.
      Methylation of dual-specificity phosphatase 4 controls cell differentiation.
      ,
      • Navas T.A.
      • Mohindru M.
      • Estes M.
      • Ma J.Y.
      • Sokol L.
      • Pahanish P.
      • et al.
      Inhibition of overactivated p38 MAPK can restore hematopoiesis in myelodysplastic syndrome progenitors.
      ), which leads to defective megakaryopoiesis. Observations in DUSP4 KO mice have demonstrated the critical role of DUSP4 in the inflammatory response to infection (
      • Parveen S.
      • Chowdhury A.R.
      • Jawed J.J.
      • Majumdar S.B.
      • Saha B.
      • Majumdar S.
      Immunomodulation of dual specificity phosphatase 4 during visceral leishmaniasis.
      ,
      • Al-Mutairi M.S.
      • Cadalbert L.C.
      • McGachy H.A.
      • Shweash M.
      • Schroeder J.
      • Kurnik M.
      • et al.
      MAP kinase phosphatase-2 plays a critical role in response to infection by Leishmania mexicana.
      ). DUSP4 is a nuclear phosphatase which binds to other substrates, such as chromatin-bound proteins (
      • Hsiao W.Y.
      • Lin Y.C.
      • Liao F.H.
      • Chan Y.C.
      • Huang C.Y.
      Dual-specificity phosphatase 4 regulates STAT5 protein stability and helper T cell polarization.
      ). Accordingly, PRMT1 can also participate in a p38 kinase–independent pathway, via methylation of DUSP4. For example, DUSP4 may affect epigenetic regulation by modulating phosphorylation of chromatin-bound proteins such as phosphorylated H3S10 (
      • Su H.
      • Tran N.T.
      • Guo H.
      • Luo M.
      • Zhao X.
      PRMT1-mediated methylation of DUSP4 determines Megakaryocyte-erythroid lineage choice by regulating p38 singlaing.
      ). This requires further investigation.

      Perspectives

      We have developed a novel vital fluorescent dye to label intracellular PRMT1 so that we can isolate live cells according to PRMT1 expression levels for functional studies (
      • Su H.
      • Sun C.W.
      • Liu S.M.
      • He X.
      • Hu H.
      • Pawlik K.M.
      • et al.
      Defining the epigenetic status of blood cells using a cyanine-based fluorescent probe for PRMT1.
      ). How immune Mks expressing high levels of PRMT1 may be expanded under disease conditions in vivo requires further investigation. Targeting PRMT1 has been shown to generate neoantigens in cancer cells, accounting for its high efficacy in cancer treatment in combination with immune checkpoint blockers (
      • Ding L.
      • Odunsi K.
      RNA splicing and immune-checkpoint inhibition.
      ,
      • Fong J.Y.
      • Pignata L.
      • Goy P.A.
      • Kawabata K.C.
      • Lee S.C.
      • Koh C.M.
      • et al.
      Therapeutic targeting of RNA splicing catalysis through inhibition of protein arginine methylation.
      ). Platelets are known to promote cancer proliferation and metastasis (
      • Stone R.L.
      • Nick A.M.
      • McNeish I.A.
      • Balkwill F.
      • Han H.D.
      • Bottsford-Miller J.
      • et al.
      Paraneoplastic thrombocytosis in ovarian cancer.
      ), and whether platelets generated in cancer patients are different from those generated in healthy individuals has been explored (
      • Roweth H.G.
      • Battinelli E.M.
      Lessons to learn from tumor-educated platelets.
      ). However, whether platelets from cancer patients are generated by immune Mks is not yet known. Here, we argue that targeting PRMT1, which blocks the generation of immune Mks and their pathogenic platelet progenies, may create a hostile tumor microenvironment for cancer cells to adapt to. Recently, inhibition of PRMT1 in combination with PD-1 immune checkpoint blockers has shown promising results in the treatment of melanoma (
      • Fedoriw A.
      • Shi L.
      • O'Brien S.
      • Smitheman K.N.
      • Wang Y.
      • Hou J.
      • et al.
      Inhibiting type I arginine methyltransferase activity promotes T cell-mediated antitumor immune responses.
      ). Understanding PRMT1’s role in generating immune Mks will help to elucidate the molecular mechanisms underlying immune therapy.
      Platelets carry granules that contain pre-mRNAs from Mks (
      • Davizon-Castillo P.
      • Rowley J.W.
      • Rondina M.T.
      Megakaryocyte and platelet transcriptomics for discoveries in human health and disease.
      ). Upregulation of PRMT1 in immune Mks activates RUNX1-mediated transcription, RBM15-mediated RNA processing, and DUSP4-controlled signaling events, which could induce Mks to produce platelets with already spliced mRNAs encoding for proinflammatory cytokines and changes in surface proteins that alter the communication between platelets and immune cells, such as monocytes, macrophages, and T cells (
      • Morrell C.N.
      • Aggrey A.A.
      • Chapman L.M.
      • Modjeski K.L.
      Emerging roles for platelets as immune and inflammatory cells.
      ). Platelets from patients with severe COVID-19 have activated p38 kinase (
      • Manne B.K.
      • Denorme F.
      • Middleton E.A.
      • Portier I.
      • Rowley J.W.
      • Stubben C.J.
      • et al.
      Platelet gene expression and function in COVID-19 patients. Blood 136(11):1317-1329. 1. Infection by Leishmania mexicana.
      ). Mks have been detected from pulmonary and cardiac systems including brain capillaries in COVID-19 patients (
      • Battina H.L.
      • Alentado V.J.
      • Srour E.F.
      • Moliterno A.R.
      • Kacena M.A.
      Interaction of the inflammatory response and megakaryocytes in COVID-19 infection.
      ). We speculated that immune Mks migrate to lung and cardiac tissues in large amount especially in severe COVID-19 patients and that COVID-19 platelets may be generated from immune Mks, expressing higher levels of PRMT1 in the lungs, which lead to severe thrombosis. The number of interferon-activated Mks increases in severe COVID-19 cases according to multiomics analysis (
      • Bernardes J.P.
      • Mishra N.
      • Tran F.
      • Bahmer T.
      • Best L.
      • Blase J.I.
      • et al.
      Longitudinal multi-omics analyses identify responses of megakaryocytes, erythroid cells, and plasmablasts as hallmarks of severe COVID-19.
      ). The contribution of Mks to cytokine storms was implied in another scRNA analysis of COVID-19 immune cells (
      • Ren X.
      • Wen W.
      • Fan X.
      • Hou W.
      • Su B.
      • Cai P.
      • et al.
      COVID-19 immune features revealed by a large-scale single-cell transcriptome atlas.
      ). Thus, it is plausible to predict that targeting PRMT1 could be used to alleviate thrombo-inflammation in patients with severe COVID-19. In addition, platelets are etiological factors for cardiovascular diseases, including atherosclerosis (
      • Burger P.C.
      • Wagner D.D.
      Platelet P-selectin facilitates atherosclerotic lesion development.
      ). Pathogenic platelets from PRMT1-controlled immune Mks may establish and accelerate atherosclerosis progression. Apart from platelets, immune Mks may communicate independently with tumor cells and other immune cells during pathogenesis.
      Taken together, we posit that PRMT1 plays a critical role in the generation of immune Mks. Intensive research is urgently needed to study the genesis of both immune Mks and their pathogenic platelet progenies and to determine their roles in innate and adaptive immune responses to infection, cancers, and cardiovascular diseases.

      Dataset

      Sun, S., Jin, C., Si, J., Lei, Y., Chen, K., Cui, Y., Liu, Z., Liu, J., Zhao, M., Zhang, X., Tang, F., Rondina, M. T., Li, Y., and Wang, Q. F. (2021) Single-cell analysis of ploidy and the transcriptome reveals functional and spatial divergency in murine megakaryopoiesis. Genome Sequence Archive for Human, HRA000114.

      Conflict of interest

      X. L. Z. is a consultant for Alexion, Takeda, Sanofi-Genzyme, and BioMedica. X. L. Z. is also a cofounder of Clotsolution. All other authors declare no conflict of interest.

      Acknowledgments

      We would like to thank Dr Diane Krause at Yale for critical comments and Drs Minkui Luo and Amit Verma for critical input on megakaryocyte differentiation.

      Author contributions

      X. Z. and Y. L. conceptualization; X. Z., X. L. Z., Q.-F. W., and Y. L. writing–original draft; Z. C., Y. C., and X. L. Z. writing–review and editing; Z. C. and Y. L. data curation.

      Funding and additional information

      This study was partially supported by Beijing Natural Science Foundation JQ21024 for Y. L., National Natural Science Foundation of China 82130008 for Q.-F. W., NIDDK grant DK110574 for X. Z.

      References

        • Fulton M.D.
        • Brown T.
        • Zheng Y.G.
        The biological axis of protein arginine methylation and asymmetric dimethylarginine.
        Int. J. Mol. Sci. 2019; 20: 3322
        • Wu Q.
        • Schapira M.
        • Arrowsmith C.H.
        • Barsyte-Lovejoy D.
        Protein arginine methylation: from enigmatic functions to therapeutic targeting.
        Nat. Rev. Drug Discov. 2021; 20: 509-530
        • Guccione E.
        • Richard S.
        The regulation, functions and clinical relevance of arginine methylation.
        Nat. Rev. Mol. Cell Biol. 2019; 20: 642-657
        • Jeong M.H.
        • Jeong H.J.
        • Ahn B.Y.
        • Pyun J.H.
        • Kwon I.
        • Cho H.
        • et al.
        PRMT1 suppresses ATF4-mediated endoplasmic reticulum response in cardiomyocytes.
        Cell Death Dis. 2019; 10: 903
        • Pyun J.H.
        • Kim H.J.
        • Jeong M.H.
        • Ahn B.Y.
        • Vuong T.A.
        • Lee D.I.
        • et al.
        Cardiac specific PRMT1 ablation causes heart failure through CaMKII dysregulation.
        Nat. Commun. 2018; 9: 5107
        • Murata K.
        • Lu W.
        • Hashimoto M.
        • Ono N.
        • Muratani M.
        • Nishikata K.
        • et al.
        PRMT1 deficiency in mouse Juvenile heart induces dilated cardiomyopathy and reveals cryptic alternative splicing products.
        iScience. 2018; 8: 200-213
        • Pyun J.H.
        • Ahn B.Y.
        • Vuong T.A.
        • Kim S.W.
        • Jo Y.
        • Jeon J.
        • et al.
        Inducible Prmt1 ablation in adult vascular smooth muscle leads to contractile dysfunction and aortic dissection.
        Exp. Mol. Med. 2021; 53: 1569-1579
        • Lee K.
        • Kim H.
        • Lee J.
        • Oh C.M.
        • Song H.
        • Kim H.
        • et al.
        Essential role of protein arginine methyltransferase 1 in pancreas development by regulating protein stability of neurogenin 3.
        Diabetes Metab. J. 2019; 43: 649-658
        • Hashimoto M.
        • Fukamizu A.
        • Nakagawa T.
        • Kizuka Y.
        Roles of protein arginine methyltransferase 1 (PRMT1) in brain development and disease.
        Biochim. Biophys. Acta Gen. Subj. 2021; 1865: 129776
        • Hashimoto M.
        • Murata K.
        • Ishida J.
        • Kanou A.
        • Kasuya Y.
        • Fukamizu A.
        Severe hypomyelination and developmental defects are caused in mice lacking protein arginine methyltransferase 1 (PRMT1) in the central nervous system.
        J. Biol. Chem. 2016; 291: 2237-2245
        • Messier V.
        • Zenklusen D.
        • Michnick S.W.
        A nutrient-responsive pathway that determines M phase timing through control of B-cyclin mRNA stability.
        Cell. 2013; 153: 1080-1093
        • Schneider R.K.
        • Adema V.
        • Heckl D.
        • Jaras M.
        • Mallo M.
        • Lord A.M.
        • et al.
        Role of casein kinase 1A1 in the biology and targeted therapy of del(5q) MDS.
        Cancer Cell. 2014; 26: 509-520
        • Bao X.
        • Siprashvili Z.
        • Zarnegar B.J.
        • Shenoy R.M.
        • Rios E.J.
        • Nady N.
        • et al.
        CSNK1a1 regulates PRMT1 to maintain the progenitor state in self-renewing somatic tissue.
        Dev. Cell. 2017; 43: 227-239.e5
        • Guo A.
        • Gu H.
        • Zhou J.
        • Mulhern D.
        • Wang Y.
        • Lee K.A.
        • et al.
        Immunoaffinity enrichment and mass spectrometry analysis of protein methylation.
        Mol. Cell. Proteomics. 2014; 13: 372-387
        • Rust H.L.
        • Subramanian V.
        • West G.M.
        • Young D.D.
        • Schultz P.G.
        • Thompson P.R.
        Using unnatural amino acid mutagenesis to probe the regulation of PRMT1.
        ACS Chem. Biol. 2014; 9: 649-655
        • Zhu Y.
        • He X.
        • Lin Y.C.
        • Dong H.
        • Zhang L.
        • Chen X.
        • et al.
        Targeting PRMT1-mediated FLT3 methylation disrupts maintenance of MLL-rearranged acute lymphoblastic leukemia.
        Blood. 2019; 134: 1257-1268
        • Nakai K.
        • Xia W.
        • Liao H.W.
        • Saito M.
        • Hung M.C.
        • Yamaguchi H.
        The role of PRMT1 in EGFR methylation and signaling in MDA-MB-468 triple-negative breast cancer cells.
        Breast Cancer. 2018; 25: 74-80
        • Iwasaki H.
        • Yada T.
        Protein arginine methylation regulates insulin signaling in L6 skeletal muscle cells.
        Biochem. Biophys. Res. Commun. 2007; 364: 1015-1021
        • Sun Q.
        • Liu L.
        • Roth M.
        • Tian J.
        • He Q.
        • Zhong B.
        • et al.
        PRMT1 upregulated by epithelial proinflammatory cytokines participates in COX2 expression in fibroblasts and chronic antigen-induced pulmonary inflammation.
        J. Immunol. 2015; 195: 298-306
        • Zhang X.
        • Li L.
        • Li Y.
        • Li Z.
        • Zhai W.
        • Sun Q.
        • et al.
        mTOR regulates PRMT1 expression and mitochondrial mass through STAT1 phosphorylation in hepatic cell.
        Biochim. Biophys. Acta Mol. Cell Res. 2021; 1868: 119017
        • Xu J.
        • Wang A.H.
        • Oses-Prieto J.
        • Makhijani K.
        • Katsuno Y.
        • Pei M.
        • et al.
        Arginine methylation initiates BMP-induced Smad signaling.
        Mol. Cell. 2013; 51: 5-19
        • Abramovich C.
        • Yakobson B.
        • Chebath J.
        • Revel M.
        A protein-arginine methyltransferase binds to the intracytoplasmic domain of the IFNAR1 chain in the type I interferon receptor.
        EMBO J. 1997; 16: 260-266
        • Albrecht L.V.
        • Ploper D.
        • Tejeda-Munoz N.
        • De Robertis E.M.
        Arginine methylation is required for canonical Wnt signaling and endolysosomal trafficking.
        Proc. Natl. Acad. Sci. U. S. A. 2018; 115: E5317-E5325
        • Zhang L.
        • Tran N.T.
        • Su H.
        • Wang R.
        • Lu Y.
        • Tang H.
        • et al.
        Cross-talk between PRMT1-mediated methylation and ubiquitylation on RBM15 controls RNA splicing.
        Elife. 2015; 4e07938
        • Zhu L.
        • He X.
        • Dong H.
        • Sun J.
        • Wang H.
        • Zhu Y.
        • et al.
        Protein arginine methyltransferase 1 is required for maintenance of normal adult hematopoiesis.
        Int. J. Biol. Sci. 2019; 15: 2763-2773
        • Giladi A.
        • Paul F.
        • Herzog Y.
        • Lubling Y.
        • Weiner A.
        • Yofe I.
        • et al.
        Single-cell characterization of haematopoietic progenitors and their trajectories in homeostasis and perturbed haematopoiesis.
        Nat. Cell Biol. 2018; 20: 836-846
        • Nestorowa S.
        • Hamey F.K.
        • Pijuan Sala B.
        • Diamanti E.
        • Shepherd M.
        • Laurenti E.
        • et al.
        A single-cell resolution map of mouse hematopoietic stem and progenitor cell differentiation.
        Blood. 2016; 128: e20-e31
        • Paul F.
        • Arkin Y.
        • Giladi A.
        • Jaitin D.A.
        • Kenigsberg E.
        • Keren-Shaul H.
        • et al.
        Transcriptional heterogeneity and lineage commitment in myeloid progenitors.
        Cell. 2015; 163: 1663-1677
        • Tabula Muris Consortium
        • Overall Coordination
        • Logistical Coordination
        • Organ Collection and Processing
        • Library Preparation and Sequencing
        • Computational Data Analysis
        • et al.
        Single-cell transcriptomics of 20 mouse organs creates a Tabula Muris.
        Nature. 2018; 562: 367-372
        • Tusi B.K.
        • Wolock S.L.
        • Weinreb C.
        • Hwang Y.
        • Hidalgo D.
        • Zilionis R.
        • et al.
        Population snapshots predict early haematopoietic and erythroid hierarchies.
        Nature. 2018; 555: 54-60
        • Laurenti E.
        • Doulatov S.
        • Zandi S.
        • Plumb I.
        • Chen J.
        • April C.
        • et al.
        The transcriptional architecture of early human hematopoiesis identifies multilevel control of lymphoid commitment.
        Nat. Immunol. 2013; 14: 756-763
        • van Galen P.
        • Hovestadt V.
        • Wadsworth Ii M.H.
        • Hughes T.K.
        • Griffin G.K.
        • Battaglia S.
        • et al.
        Single-cell RNA-seq reveals AML hierarchies relevant to disease progression and immunity.
        Cell. 2019; 176: 1265-1281
        • Sanada C.
        • Xavier-Ferrucio J.
        • Lu Y.C.
        • Min E.
        • Zhang P.X.
        • Zou S.
        • et al.
        Adult human megakaryocyte-erythroid progenitors are in the CD34+CD38mid fraction.
        Blood. 2016; 128: 923-933
        • Xavier-Ferrucio J.
        • Krause D.S.
        Concise review: bipotent megakaryocytic-erythroid progenitors: concepts and controversies.
        Stem Cells. 2018; 36: 1138-1145
        • Psaila B.
        • Mead A.J.
        Single-cell approaches reveal novel cellular pathways for megakaryocyte and erythroid differentiation.
        Blood. 2019; 133: 1427-1435
        • Carrelha J.
        • Meng Y.
        • Kettyle L.M.
        • Luis T.C.
        • Norfo R.
        • Alcolea V.
        • et al.
        Hierarchically related lineage-restricted fates of multipotent haematopoietic stem cells.
        Nature. 2018; 554: 106-111
        • Shin J.Y.
        • Hu W.
        • Naramura M.
        • Park C.Y.
        High c-Kit expression identifies hematopoietic stem cells with impaired self-renewal and megakaryocytic bias.
        J. Exp. Med. 2014; 211: 217-231
        • Pietras E.M.
        • Reynaud D.
        • Kang Y.A.
        • Carlin D.
        • Calero-Nieto F.J.
        • Leavitt A.D.
        • et al.
        Functionally distinct subsets of lineage-biased multipotent progenitors control blood production in normal and regenerative conditions.
        Cell Stem Cell. 2015; 17: 35-46
        • Miyawaki K.
        • Iwasaki H.
        • Jiromaru T.
        • Kusumoto H.
        • Yurino A.
        • Sugio T.
        • et al.
        Identification of unipotent megakaryocyte progenitors in human hematopoiesis.
        Blood. 2017; 129: 3332-3343
        • Haas S.
        • Hansson J.
        • Klimmeck D.
        • Loeffler D.
        • Velten L.
        • Uckelmann H.
        • et al.
        Inflammation-induced emergency megakaryopoiesis driven by hematopoietic stem cell-like megakaryocyte progenitors.
        Cell Stem Cell. 2015; 17: 422-434
        • Cunin P.
        • Nigrovic P.A.
        Megakaryocytes as immune cells.
        J. Leukoc. Biol. 2019; 105: 1111-1121
        • Finkielsztein A.
        • Schlinker A.C.
        • Zhang L.
        • Miller W.M.
        • Datta S.K.
        Human megakaryocyte progenitors derived from hematopoietic stem cells of normal individuals are MHC class II-expressing professional APC that enhance Th17 and Th1/Th17 responses.
        Immunol. Lett. 2015; 163: 84-95
        • Xie X.
        • Shi Q.
        • Wu P.
        • Zhang X.
        • Kambara H.
        • Su J.
        • et al.
        Single-cell transcriptome profiling reveals neutrophil heterogeneity in homeostasis and infection.
        Nat. Immunol. 2020; 21: 1119-1133
        • Liu C.
        • Wu D.
        • Xia M.
        • Li M.
        • Sun Z.
        • Shen B.
        • et al.
        Characterization of cellular heterogeneity and an immune subpopulation of human megakaryocytes.
        Adv. Sci. (Weinh.). 2021; 8e2100921
        • Pariser D.N.
        • Hilt Z.T.
        • Ture S.K.
        • Blick-Nitko S.K.
        • Looney M.R.
        • Cleary S.J.
        • et al.
        Lung megakaryocytes are immune modulatory cells.
        J. Clin. Invest. 2021; 131e137377
        • Sun S.
        • Jin C.
        • Si J.
        • Lei Y.
        • Chen K.
        • Cui Y.
        • et al.
        Single-cell analysis of ploidy and the transcriptome reveals functional and spatial divergency in murine megakaryopoiesis.
        Blood. 2021; 138: 1211-1224
        • Wang H.
        • He J.
        • Xu C.
        • Chen X.
        • Yang H.
        • Shi S.
        • et al.
        Decoding human megakaryocyte development.
        Cell Stem Cell. 2021; 28: 535-549
        • Yeung A.K.
        • Villacorta-Martin C.
        • Hon S.
        • Rock J.R.
        • Murphy G.J.
        Lung megakaryocytes display distinct transcriptional and phenotypic properties.
        Blood Adv. 2020; 4: 6204-6217
        • Wang X.
        • He Y.
        • Zhang Q.
        • Ren X.
        • Zhang Z.
        Direct comparative analyses of 10X Genomics Chromium and smart-seq2.
        Genomics Proteomics Bioinformatics. 2021; 19: 253-266
        • Huang G.
        • Zhang P.
        • Hirai H.
        • Elf S.
        • Yan X.
        • Chen Z.
        • et al.
        PU.1 is a major downstream target of AML1 (RUNX1) in adult mouse hematopoiesis.
        Nat. Genet. 2008; 40: 51-60
        • Leon Machado J.A.
        • Steimle V.
        The MHC class II transactivator CIITA: not (quite) the odd-one-out anymore among NLR proteins.
        Int. J. Mol. Sci. 2021; 22: 1074-1083
        • Cowland J.B.
        • Muta T.
        • Borregaard N.
        IL-1beta-specific up-regulation of neutrophil gelatinase-associated lipocalin is controlled by IkappaB-zeta.
        J. Immunol. 2006; 176: 5559-5566
        • Lefrancais E.
        • Ortiz-Munoz G.
        • Caudrillier A.
        • Mallavia B.
        • Liu F.
        • Sayah D.M.
        • et al.
        The lung is a site of platelet biogenesis and a reservoir for haematopoietic progenitors.
        Nature. 2017; 544: 105-109
        • Wang J.
        • Xie J.
        • Wang D.
        • Han X.
        • Chen M.
        • Shi G.
        • et al.
        CXCR4(high) megakaryocytes regulate host-defense immunity against bacterial pathogens.
        Elife. 2022; 11e78662
        • Raslova H.
        • Kauffmann A.
        • Sekkai D.
        • Ripoche H.
        • Larbret F.
        • Robert T.
        • et al.
        Interrelation between polyploidization and megakaryocyte differentiation: a gene profiling approach.
        Blood. 2007; 109: 3225-3234
        • Zufferey A.
        • Speck E.R.
        • Machlus K.R.
        • Aslam R.
        • Guo L.
        • McVey M.J.
        • et al.
        Mature murine megakaryocytes present antigen-MHC class I molecules to T cells and transfer them to platelets.
        Blood Adv. 2017; 1: 1773-1785
        • Machlus K.R.
        • Italiano Jr., J.E.
        The incredible journey: from megakaryocyte development to platelet formation.
        J. Cell Biol. 2013; 201: 785-796
        • Rajasekhar D.
        • Kestin A.S.
        • Bednarek F.J.
        • Ellis P.A.
        • Barnard M.R.
        • Michelson A.D.
        Neonatal platelets are less reactive than adult platelets to physiological agonists in whole blood.
        Thromb. Haemost. 1994; 72: 957-963
        • de Alarcon P.A.
        • Graeve J.L.
        Analysis of megakaryocyte ploidy in fetal bone marrow biopsies using a new adaptation of the feulgen technique to measure DNA content and estimate megakaryocyte ploidy from biopsy specimens.
        Pediatr. Res. 1996; 39: 166-170
        • Huang Z.
        • Richmond T.D.
        • Muntean A.G.
        • Barber D.L.
        • Weiss M.J.
        • Crispino J.D.
        STAT1 promotes megakaryopoiesis downstream of GATA-1 in mice.
        J. Clin. Invest. 2007; 117: 3890-3899
        • Vardon-Bounes F.
        • Ruiz S.
        • Gratacap M.P.
        • Garcia C.
        • Payrastre B.
        • Minville V.
        Platelets are critical key players in sepsis.
        Int. J. Mol. Sci. 2019; 20: 3494-3507
        • Frydman G.H.
        • Tessier S.N.
        • Wong K.H.K.
        • Vanderburg C.R.
        • Fox J.G.
        • Toner M.
        • et al.
        Megakaryocytes contain extranuclear histones and may be a source of platelet-associated histones during sepsis.
        Sci. Rep. 2020; 10: 4621
        • Van Bergen M.
        • Marneth A.E.
        • Hoogendijk A.J.
        • Van Alphen F.P.J.
        • Van den Akker E.
        • Laros-Van Gorkom B.A.P.
        • et al.
        Specific proteome changes in platelets from individuals with GATA1-, GFI1B-, and RUNX1-linked bleeding disorders.
        Blood. 2021; 138: 86-90
        • Zaid Y.
        • Puhm F.
        • Allaeys I.
        • Naya A.
        • Oudghiri M.
        • Khalki L.
        • et al.
        Platelets can associate with SARS-cov-2 RNA and are hyperactivated in COVID-19.
        Circ. Res. 2020; 127: 1404-1418
        • Su H.
        • Jiang M.
        • Senevirathne C.
        • Aluri S.
        • Zhang T.
        • Guo H.
        • et al.
        Methylation of dual-specificity phosphatase 4 controls cell differentiation.
        Cell Rep. 2021; 36109421
        • Zhao X.
        • Jankovic V.
        • Gural A.
        • Huang G.
        • Pardanani A.
        • Menendez S.
        • et al.
        Methylation of RUNX1 by PRMT1 abrogates SIN3A binding and potentiates its transcriptional activity.
        Genes Dev. 2008; 22: 640-653
        • Lordier L.
        • Bluteau D.
        • Jalil A.
        • Legrand C.
        • Pan J.
        • Rameau P.
        • et al.
        RUNX1-induced silencing of non-muscle myosin heavy chain IIB contributes to megakaryocyte polyploidization.
        Nat. Commun. 2012; 3: 717
        • Ma Z.
        • Morris S.W.
        • Valentine V.
        • Li M.
        • Herbrick J.A.
        • Cui X.
        • et al.
        Fusion of two novel genes, RBM15 and MKL1, in the t(1;22)(p13;q13) of acute megakaryoblastic leukemia.
        Nat. Genet. 2001; 28: 220-221
        • Mercher T.
        • Coniat M.B.
        • Monni R.
        • Mauchauffe M.
        • Nguyen Khac F.
        • Gressin L.
        • et al.
        Involvement of a human gene related to the Drosophila spen gene in the recurrent t(1;22) translocation of acute megakaryocytic leukemia.
        Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 5776-5779
        • Niu C.
        • Zhang J.
        • Breslin P.
        • Onciu M.
        • Ma Z.
        • Morris S.W.
        c-Myc is a target of RNA-binding motif protein 15 in the regulation of adult hematopoietic stem cell and megakaryocyte development.
        Blood. 2009; 114: 2087-2096
        • Jin S.
        • Su H.
        • Tran N.T.
        • Song J.
        • Lu S.S.
        • Li Y.
        • et al.
        Splicing factor SF3B1K700E mutant dysregulates erythroid differentiation via aberrant alternative splicing of transcription factor TAL1.
        PLoS One. 2017; 12e0175523
        • Xiao N.
        • Laha S.
        • Das S.P.
        • Morlock K.
        • Jesneck J.L.
        • Raffel G.D.
        Ott1 (Rbm15) regulates thrombopoietin response in hematopoietic stem cells through alternative splicing of c-Mpl.
        Blood. 2015; 125: 941-948
        • Tran N.T.
        • Su H.
        • Khodadadi-Jamayran A.
        • Lin S.
        • Zhang L.
        • Zhou D.
        • et al.
        The AS-RBM15 lncRNA enhances RBM15 protein translation during megakaryocyte differentiation.
        EMBO Rep. 2016; 17: 887-900
        • Takayama N.
        • Nishimura S.
        • Nakamura S.
        • Shimizu T.
        • Ohnishi R.
        • Endo H.
        • et al.
        Transient activation of c-MYC expression is critical for efficient platelet generation from human induced pluripotent stem cells.
        J. Exp. Med. 2010; 207: 2817-2830
        • Mazharian A.
        • Watson S.P.
        • Severin S.
        Critical role for ERK1/2 in bone marrow and fetal liver-derived primary megakaryocyte differentiation, motility, and proplatelet formation.
        Exp. Hematol. 2009; 37: 1238-1249
        • Desterke C.
        • Bilhou-Nabera C.
        • Guerton B.
        • Martinaud C.
        • Tonetti C.
        • Clay D.
        • et al.
        FLT3-mediated p38-MAPK activation participates in the control of megakaryopoiesis in primary myelofibrosis.
        Cancer Res. 2011; 71: 2901-2915
        • Wang Q.
        • Reszka-Blanco N.
        • Cheng L.
        • Li G.
        • Zhang L.
        • Su L.
        p38 MAPK is critical for nuclear translocation of IRF-7 during CpG-induced type I IFN expression in human plasmacytoid dendritic cells.
        J. Immunol. 2018; 200: 109.6
        • Navas T.A.
        • Mohindru M.
        • Estes M.
        • Ma J.Y.
        • Sokol L.
        • Pahanish P.
        • et al.
        Inhibition of overactivated p38 MAPK can restore hematopoiesis in myelodysplastic syndrome progenitors.
        Blood. 2006; 108: 4170-4177
        • Parveen S.
        • Chowdhury A.R.
        • Jawed J.J.
        • Majumdar S.B.
        • Saha B.
        • Majumdar S.
        Immunomodulation of dual specificity phosphatase 4 during visceral leishmaniasis.
        Microbes Infect. 2018; 20: 111-121
        • Al-Mutairi M.S.
        • Cadalbert L.C.
        • McGachy H.A.
        • Shweash M.
        • Schroeder J.
        • Kurnik M.
        • et al.
        MAP kinase phosphatase-2 plays a critical role in response to infection by Leishmania mexicana.
        PLoS Pathog. 2010; 6e1001192
        • Hsiao W.Y.
        • Lin Y.C.
        • Liao F.H.
        • Chan Y.C.
        • Huang C.Y.
        Dual-specificity phosphatase 4 regulates STAT5 protein stability and helper T cell polarization.
        PLoS One. 2015; 10e0145880
        • Su H.
        • Tran N.T.
        • Guo H.
        • Luo M.
        • Zhao X.
        PRMT1-mediated methylation of DUSP4 determines Megakaryocyte-erythroid lineage choice by regulating p38 singlaing.
        Blood. 2015; 126: 2387
        • Su H.
        • Sun C.W.
        • Liu S.M.
        • He X.
        • Hu H.
        • Pawlik K.M.
        • et al.
        Defining the epigenetic status of blood cells using a cyanine-based fluorescent probe for PRMT1.
        Blood Adv. 2018; 2: 2829-2836
        • Ding L.
        • Odunsi K.
        RNA splicing and immune-checkpoint inhibition.
        N. Engl. J. Med. 2021; 385: 1807-1809
        • Fong J.Y.
        • Pignata L.
        • Goy P.A.
        • Kawabata K.C.
        • Lee S.C.
        • Koh C.M.
        • et al.
        Therapeutic targeting of RNA splicing catalysis through inhibition of protein arginine methylation.
        Cancer Cell. 2019; 36: 194-209
        • Stone R.L.
        • Nick A.M.
        • McNeish I.A.
        • Balkwill F.
        • Han H.D.
        • Bottsford-Miller J.
        • et al.
        Paraneoplastic thrombocytosis in ovarian cancer.
        N. Engl. J. Med. 2012; 366: 610-618
        • Roweth H.G.
        • Battinelli E.M.
        Lessons to learn from tumor-educated platelets.
        Blood. 2021; 137: 3174-3180
        • Fedoriw A.
        • Shi L.
        • O'Brien S.
        • Smitheman K.N.
        • Wang Y.
        • Hou J.
        • et al.
        Inhibiting type I arginine methyltransferase activity promotes T cell-mediated antitumor immune responses.
        Cancer Immunol. Res. 2022; 10: 420-436
        • Davizon-Castillo P.
        • Rowley J.W.
        • Rondina M.T.
        Megakaryocyte and platelet transcriptomics for discoveries in human health and disease.
        Arterioscler. Thromb. Vasc. Biol. 2020; 40: 1432-1440
        • Morrell C.N.
        • Aggrey A.A.
        • Chapman L.M.
        • Modjeski K.L.
        Emerging roles for platelets as immune and inflammatory cells.
        Blood. 2014; 123: 2759-2767
        • Manne B.K.
        • Denorme F.
        • Middleton E.A.
        • Portier I.
        • Rowley J.W.
        • Stubben C.J.
        • et al.
        Platelet gene expression and function in COVID-19 patients. Blood 136(11):1317-1329. 1. Infection by Leishmania mexicana.
        PLoS Pathog. 2020; 6e1001192
        • Battina H.L.
        • Alentado V.J.
        • Srour E.F.
        • Moliterno A.R.
        • Kacena M.A.
        Interaction of the inflammatory response and megakaryocytes in COVID-19 infection.
        Exp. Hematol. 2021; 104: 32-39
        • Bernardes J.P.
        • Mishra N.
        • Tran F.
        • Bahmer T.
        • Best L.
        • Blase J.I.
        • et al.
        Longitudinal multi-omics analyses identify responses of megakaryocytes, erythroid cells, and plasmablasts as hallmarks of severe COVID-19.
        Immunity. 2020; 53: 1296-1314.e9
        • Ren X.
        • Wen W.
        • Fan X.
        • Hou W.
        • Su B.
        • Cai P.
        • et al.
        COVID-19 immune features revealed by a large-scale single-cell transcriptome atlas.
        Cell. 2021; 184: 1895-1913.e19
        • Burger P.C.
        • Wagner D.D.
        Platelet P-selectin facilitates atherosclerotic lesion development.
        Blood. 2003; 101: 2661-2666