Protein arginine methylation is a common type of post-translational modification. PRMT1, the major type I protein arginine methyltransferase, catalyzes the formation of asymmetric dimethyl-arginine and is implicated in various cellular processes, including hematopoiesis and tumorigenesis. We have shown that PRMT1 expression is naturally low in hematopoietic stem cells (HSCs). However, the functions of PRMT1 in hematopoietic stem cell self-renewal and differentiation are yet to be revealed. We have found a cyanine-based fluorescent probe (E84) that can specifically label PRMT1 protein. E84 staining dynamically captures intracellular PRMT1 level and was used to separate live HSC populations with differential PRMT1 expression by flow cytometry. Subsequent bone marrow transplantation of E84high or E84low Lin−Sca1+cKit+ (LSK) cells showed that E84low LSK cells were much more advantageous in reconstituting each blood cell lineages, compared to the E84high counterparts, meaning that the stem-ness of HSCs is negatively correlated with endogenous PRMT1. Therefore, inhibition of PRMT1 was expected to enhance the number and differentiation potential of functional HSCs. The treatment of a PRMT1-specific inhibitor (MS023) to mice resulted in an enlarged LT-HSC population in bone marrow and decreased frequency of granulocyte progenitor cells. In vitro colony formation assays further demonstrated that PRMT1 is required for GMP differentiation.
Then we asked whether copious expression of PRMT1 promotes the differentiation of HSC. In this line, we made a LoxP-STOP-LoxP-PRMT1 transgenic mouse model, which induces PRMT1 overexpression upon the expression of Cre recombinase from tissue-specific promoters. We established Mx1-Cre-PRMT1 (Mx1-Tg) mice. Mx1-Tg mice were injected with poly(I:C) for PRMT1 induction and analyzed at four weeks after the last dose. We found that, as predicted, LT-HSC population was reduced and the Pre-GM population was raised. Accordingly, more CFU-Gs but less GEMMs were grown on CFU assays. We further utilized this animal model to compare the blood reconstitution capabilities of bone marrow cells from Mx1-Tg vs. WT mice in the same repopulating conditions. We performed competitive bone marrow transplantation by injecting Mx1-Tg/WT (CD45.2) bone marrow plus supporting cells (CD45.1) to irradiated mice, followed by 5 doses of poly(I:C) induction. Recipient mice were analyzed during a course of approximately 16 weeks. Mx1-Tg cells were outcompeted by WT cells in reconstituting every blood lineages. Taken together, we conclude that PRMT1 promotes HSC differentiation and accelerates HSC exhaustion during the stress caused by bone marrow irradiation.
To understand the mechanism on PRMT1-mediated stress hematopoiesis, we also made Pf4-Cre PRMT1 transgenic mice. When PRMT1 is specifically expressed in MK cells, the number of LT-HSCs was also reduced, implying that PRMT1 affects the self-renewal of LT-HSCs via communication between MK cells and HSCs. Mechanistically, two PRMT1 substrates - RBM15 and DUSP4 - are critical for stem cell self-renewal. We further characterized how PRMT1 activates p38 kinase pathway via directly methylating DUSP4 thus induces ubiquitylation and degradation of DUSP4. The arginine methylation site on DUSP4 has been identified. Moreover, introducing methyl-R mutated DUSP4 back to PRMT1-overexpressing cells partially rescued the loss of HSC differentiation potential. This data adds a new link between arginine methylation and protein phosphorylation mediated by MAP kinases/phosphatases. In addition, we discovered that RBM15 controls alternative RNA splicing and RNA processing in a PRMT1-dosage dependent manner. In this report, we will further address how RBM15 target genes, such as enzymes involved in fatty acid metabolic pathways, affect HSC differentiation. In summary, we report that arginine methylation is a novel regulator for the HSC differentiation via controlling p38-regulated stress pathway and metabolic reprogramming.
Disclosures
No relevant conflicts of interest to declare.
ER-associated degradation preserves hematopoietic stem cell quiescence and self-renewal by restricting mTOR activity