Protein Arginine Methyltransferase 1 Expands and Mobilizes Immune Megakaryocytes and Generates Hyperactive Platelets in Stress Conditions

Protein arginine methyltransferase 1 (PRMT1) inhibits the differentiation of megakaryocytes (Mk) into highly polyploid cells. Through single-cell RNA sequencing (scRNA-seq) analysis, we found that PRMT1 expression is linked to the expression of genes associated with inflammatory response in Mks cultured in vitro with TPO and SCF (Su et al. 2021). Furthermore, we observed elevated levels of PRMT1 expression in a subset of megakaryocytes exhibiting a gene expression signature related to immune reactions, using Mks isolated from mouse bone marrow using published scRNA-seq data (Zhao, et al. 2022).

In previous studies, we reported that PRMT1 is upregulated in human CD34⁺ stem/progenitor cells from myeloid dysplasia syndrome patients (Su et al. 2021). Additionally, we noted increased PRMT1 expression in long-term hematopoietic stem cells in the bone marrow of mice injected with lipopolysaccharide. To further our research, we generated a transgenic PRMT1 mouse (Pf4-PRMT1) that conditionally expresses human PRMT1 when the stop cassette is removed by cre recombinase expressed from the platelet factor 4 (Pf4) promoter. These mice exhibit a normal lifespan but an expanded population of granulocyte-monocyte progenitors. While the frequency of Cd41⁺ckit⁺ Mk progenitors is only slightly increased, the mice produce hyperactive platelets with a proteomic profile resembling platelets isolated from ovarian cancer mice.

We then examined the subpopulations of Mks from the bone marrow, peripheral blood, spleen, and liver. We found that immune Mks, defined by CD41⁺CD42⁺CD53⁺ (Sun et al 2021), are expanded in the PF4-PRMT1 mice. Intriguingly, the immune megakaryocytes are more abundant in peripheral blood and spleen, suggesting that PRMT1 promotes the migration of immune megakaryocytes out of the bone marrow. Additionally, PRMT1 facilitates the immune Mks to expand and migrate to peripheral blood and spleen in response to LPS challenge. In line with this, we observed that the megakaryocytes from the Pf4-PRMT1 transgenic mice migrate farther away from the centers in colony formation assays. In vitro liquid culture with TPO, SCF, and IL6 of bone marrow cells expanded the population of immune megakaryocytes in a PRMT1-dependent manner. We then tested immune Mks isolated from ovarian cancer mice in mobility assays.

Subsequently, we conducted phagocytosis assays with in vitro cultured bone marrow cells. We found that immune megakaryocytes efficiently phagocytose DQ-Ova when PRMT1 is upregulated. Furthermore, PRMT1 upregulates MHC class II on the CD53⁺ immune Mks. In previous studies, we reported that PRMT1 methylates DUSP4, leading to its degradation and that DUSP4 promotes Mk differentiation (Su et al. 2021). Therefore, we analyzed the frequency of immune megakaryocytes in DUSP4 knockout mice and noted an expansion of immune megakaryocytes, indicating PRMT1-DUSP4 axis is involved in generating immune Mks and their immune responses. At the molecular levels, we identified PRMT1 targets in scRNA-seq analysis of Mks isolated from the transgenic mice. We found that PRMT1 upregulates immune response pathways in immune Mks. Interestingly, these genes mostly upregulated in immune Mks,such as Ifi27l2a, HIST1H2A, ATPap1, and RNAset2b, and RHD12, are immune response genes that have not been extensively studied. Previously, we reported that PRMT1 targets RUNX1 and RBM15 in megakaryopoiesis. Some of the RUNX1 targets overlap with the upregulated genes in the scRNA-seq assays. The candidate genes involved in Mk migration will be discussed in the presentation.

In summary, our research on PRMT1 and immune Mks in stress conditions paves the way for future studies and potential breakthroughs in the field of hematology and immunology.