Inhibition of S-Adenosylmethionine Synthesis Promotes Erythropoiesis Via Epigenetic Modifications
Authors: Kato Hiroki , Long Nguyen Chi , Ishii Yusho , Matsumoto Mitsuyo , Rhee Catherine , Saigusa Daisuke , Funayama Ryo , Okae Hiroaki , Fujiwara Tohru , Muto Akihiko , Harigae Hideo , Scadden David T. , Igarashi Kazuhiko 
Published: November 2021

Abstract
Erythroid differentiation involves global gene expression repression, chromatin condensation and enucleation, mitochondria removal and other marked cellular changes. Given the necessity for these dynamic alterations, it is hardly surprising that epigenetic modifications possess important roles for erythropoiesis. S-adenosylmethionine (SAM), a principle methyl donor for DNA and histone methylations, would be involved in this process. Yet little is known about the specific roles for SAM synthesis in erythropoiesis.
SAM is synthesized from methionine and ATP via the enzymatic activity of Mat2a and we evaluated the in vivo role of SAM synthesis by treating wild type mice (C57BL/6) with a selective Mat2a inhibitor (cycloleucine). As expected, the Mat2a inhibitor administration (henceforth Mat2ai) reduced SAM in bone marrow (BM) cells (SAM; 3.17±0.43 and 0.93±0.10 area ratio for ctrl and Mat2ai, p &lt; 0.01, n = 4 mice). Interestingly, Mat2ai increased erythropoiesis in BM (Ter119 + cell; 46.3±3.1 and 116.4±14.2×10 6 cells for ctrl and Mat2ai, p &lt; 0.01, n = 8 mice) and in blood (hemoglobin concentrations in peripheral blood; 13.7±0.18 and 16.3±0.26 g/dl for ctrl and Mat2ai, p &lt; 0.01, n = 8 mice). However, serum erythropoietin concentration decreased (erythropoietin; 254.2±34.1 and 42.7±5.70 pg/ml for ctrl and Mat2ai, p &lt; 0.01, n = 10 mice). Therefore, Mat2ai promoted erythropoiesis in vivo without increasing erythropoietin. To reveal the point where the erythroid differentiation was affected, immature and mature erythroblast subsets in BM were assessed. Although immature erythroblasts were not changed by Mat2ai (24.1±2.80 and 23.8±3.86×10 6 cells for ctrl and Mat2ai, p = 0.95, n = 8 mice), mature erythroblasts in BM increased following Mat2ai (18.9±2.48 and 81.2±9.73×10 6 cells for ctrl and Mat2ai, p &lt; 0.01, n = 8 mice). Therefore, Mat2ai promoted erythroid maturation from immature erythroblast in BM. To reveal the mechanistic insight of this promotion of erythroid maturation by Mat2ai, we performed RNA sequencing of immature erythroblast in BM. This analysis revealed that most genes were down-regulated by Mat2ai (differentially expressed genes by Mat2ai; DOWN 2578 genes, UP 72 genes). In line with this notion, transposase-accessible chromatin sequencing (ATAC-seq) of immature erythroblasts revealed that chromatin accessibility was reduced. While DNA methylation analysis (whole genome bisulfite sequence) of immature erythroblasts revealed slightly reduced global DNA methylation (approximately 2%), there were no clear correlations between changes in promotor (or gene-body) DNA methylation and transcription. This result suggests that DNA methylation changes possess limited roles for the erythroid maturation promoted by Mat2ai. On the other hand, we found that an active histone methylation mark (H3K4me3) was selectively reduced by Mat2ai and that the changes of gene expression and H3K4me3 enrichment (revealed by chromatin immunoprecipitation followed by sequencing) correlated (r = 0.66). Therefore, the loss of H3K4me3, but not the DNA methylation, might contribute to the global gene expression repression for erythroid maturation induced by Mat2ai. Finally, in vitro human erythroid differentiation analysis using CD34 + cord blood cells further revealed that therapeutic and genetic inhibition of SAM synthesis induced erythroid maturation, which was cancelled by extracellular administration of SAM. Therefore, SAM synthesis inhibition is a non-erythropoietin trigger for erythroid maturation and this process occurs in human cells.
Collectively, we found that SAM synthesis inhibition promoted erythroid maturation in both mouse and human. Histone methylation alteration induced by SAM synthesis inhibition might contribute to this phenomenon. These findings may pave the way to develop a new therapeutic strategy for anemia in erythropoietin independent manner.

Disclosures
Harigae: Kyowakirin: Other: Subsidies or Donations; Astellas Pharma: Other: Subsidies or Donations; Ono pharma: Honoraria, Other: Subsidies or Donations; Janssen Pharma: Honoraria; Chugai Pharma: Honoraria; Novartis Pharma: Honoraria, Research Funding; Bristol Myers Squibb: Honoraria. Scadden: Magenta Therapeutics: Current holder of individual stocks in a privately-held company, Membership on an entity's Board of Directors or advisory committees; Clear Creek Bio: Current holder of individual stocks in a privately-held company, Membership on an entity's Board of Directors or advisory committees; LifeVaultBio: Current holder of individual stocks in a privately-held company, Membership on an entity's Board of Directors or advisory committees; Agios Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees; Editas Medicines: Membership on an entity's Board of Directors or advisory committees; Fate Therapeutics: Current holder of individual stocks in a privately-held company; Clear Creek Bio: Current holder of individual stocks in a privately-held company, Membership on an entity's Board of Directors or advisory committees; Dainippon Sumitomo Pharma: Other: Sponsored research; FOG Pharma:: Consultancy; Garuda Therapeutics: Current holder of individual stocks in a privately-held company, Membership on an entity's Board of Directors or advisory committees; VCanBio: Consultancy; Inzen Therapeutics: Membership on an entity's Board of Directors or advisory committees.