Mettl3-Mediated m6a Modification Is Essential for the Maintenance of Genomic Stability of Erythroid Cells

Erythropoiesis is the process by which hematopoietic stem cells proliferate and undergo multiple stages of differentiation to produce mature erythrocytes. This is a sophisticated process orchestrated by various mechanisms, including epigenetic mechanisms. N6-methyladenosine (m⁶A) is an abundant modification of mRNA. An increasingly important role of m⁶A in normal cell physiology and disease has been documented. Yet our know knowledge on the role of m⁶A in erythropoiesis is very limited. A recent study showed that knocking down of Mettl3, the only catalytic enzyme for RNA methylation, in CD34⁺ cells significantly impaired in vitro human erythropoiesis. However, the role of Mettl3 during erythropoiesis in vivo remains unknown.

In the present study, we deleted Mettl3 in erythroid cells by crossing Mettl3^fl/fl mice with Epor-tdTomato-cre mice while Mettl3^fl/flEpor^cre/cre mice were embryonic lethal, Mettl3^fl/flEpor^cre/- mice were viable but exhibited severe anemia as demonstrated by decreased RBC counts, hemoglobin and hematocrit along with a 6-fold increase in blood EPO levels. Analyses of bone marrow (BM) erythropoiesis revealed significantly impaired erythropoiesis as demonstrated by reductions in the number of erythroid colonies and erythroblasts of Mettl3^fl/flEpor^cre/- mice compared to control mice. In addition, We found decreased cell cycle of Mettl3-deficient CFU-E and erythroblasts along with increased apoptosis.

To investigate the molecular mechanisms for the impaired cell cycle and increased apoptosis, we compared the transcriptomes between control and Mettl3-deficient CFU-E and proerythroblasts. We found that in both CFU-E and proerythroblasts about 2000 genes were differentially expressed. Gene ontology (GO) analysis of DEGs revealed that pathways involved in DNA metabolism and DNA replication genes were downregulated while pathway involved in DNA damage response was upregulated, indicating DNA damage which was further demonstrated by drastically elevated levels of γ-H2AX, a marker for DNA damage. These findings indicate that Mettl3 plays a critical role in maintaining genome stability of erythroid cells. Additionally, pathway involved in mitotic cell cycle process was downregulated and pathway involved in programmed cell death was upregulated in Mettl3-deficient cells compared to control cells, which could be a consequence of unrepairable DNA damage.

To further define the molecular mechanisms for the role of Mettl3 in maintaining genome stability of erythroid cells, we performed SLIM-seq (Super-low input m⁶A-seq) of CFU-E cells. Analysis of the SLIM-seq data revealed that 1,523 genes were m⁶A-modified. GO analysis revealed that these m⁶A-modified genes are involved in processes such as mRNA metabolism, cell cycle progression, and DNA metabolic processes. Intersecting the m⁶A-modified genes with the significantly downregulated genes following Mettl3 deletion identified 129 overlapping genes as potential targets of Mettl3. Functional enrichment analysis of these genes revealed significant enrichment in the "purine-containing compound biosynthetic process" pathway, which is involved in nucleotide biosynthesis and metabolism. The top three ranked genes were Pgm2, Mthfd1, and Adss. Given that Mthfd1 was the most downregulated gene in both CFU-E and proerythroblasts upon Mettl3 deletion, we next focused on Mthfd1. We found that the half-life of Mthfd1 mRNA was shortened in Mettl3-deficent cells. Furthermore, a dual luciferase assay showed that Mettl3 directly regulates the 3' untranslated regions of Mthfd1 mRNAs, indicating that the expression of Mthfd1 is directly regulated by m⁶A.

Finally, knocking down METTL3 in human CD34+ cells led to similar phenotypic and molecular changes including marked inhibition of cell growth, impaired erythroid colony formation, severe apoptosis, increased γ-H2AX levels and significant downregulation of MTHFD1 expression. Furthermore, MTHFD1 knockdown in human CD34+ cells phenocopied the changes of METTL3-knockdown cells. These results indicate conserved role of METLL3 in human and murine.

In summary, we have uncovered a previously unrecognized role for METTL3 in erythropoiesis and identified the underlying molecular mechanisms. Our findings provide insights into erythrocyte biology which may have implications in understanding of the anemia associated with METTL3 inhibitor therapy.