Methyltransferase complex subunit METTL3 maintains genome stability of erythroid cells via MTHFD1-mediated nucleotide biosynthesis
Linlin Zhang, Huizhi Zhao, Shihui Wang, Xueting Wu, Donghao Liu, Hengchao Zhang, Qianqian Yang, Ying Cheng, Xiuyun Wu, Jiangwei Zhao, Shijie Zhang, Huan Zhang, Haojian Zhang, Qiaozhen Kang, Lixiang Chen, Xiuli An, Xiaoli Qu
Linlin Zhang, Huizhi Zhao, Shihui Wang, Xueting Wu, Donghao Liu, Hengchao Zhang, Qianqian Yang, Ying Cheng, Xiuyun Wu, Jiangwei Zhao, Shijie Zhang, Huan Zhang, Haojian Zhang, Qiaozhen Kang, Lixiang Chen, Xiuli An, Xiaoli Qu
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Research Article Cell biology Hematology

Methyltransferase complex subunit METTL3 maintains genome stability of erythroid cells via MTHFD1-mediated nucleotide biosynthesis

Abstract

N6-methyladenosine (m6A) is a prevalent modification of mammalian mRNA. Increasing evidence has documented diverse roles of m6A in normal cell physiology and diseases. However, its functional role in erythropoiesis remains poorly understood. In this study, we found that deletion of Mettl3 using the EpoR-Cre mouse led to microcytic/hypochromic anemia due to defective erythropoiesis along with impaired hemoglobin biosynthesis. Mechanically, Mettl3 deficiency disrupted nucleotide biosynthesis, which induced DNA damage, leading to apoptosis of colony-forming unit–erythroid cells and cell-cycle arrest of erythroblasts. Integrated m6A-seq and RNA-seq analysis along with biochemical studies identified Mthfd1, a key enzyme involved in nucleotide biosynthesis, as a Mettl3 direct target gene. Furthermore, deletion of Mettl3 led to decreased expression of Mthfd1, accompanied by a shortage of nucleotides deoxythymidine monophosphate and inosine monophosphate, in erythroid cells. Additionally, inhibition of METTL3 in human erythroid cells led to similar phenotypic and molecular changes, indicating a conserved role of METTL3 in human and murine erythropoiesis. Our findings have identified an METTL3-m6A-MTHFD1 axis that plays a critical role in erythropoiesis by maintaining genome stability of erythroid cells via regulation of nucleotide biosynthesis. These findings provide important insights into the regulatory mechanisms of erythropoiesis and may have implications for underlying the mechanisms of anemias.

Authors

Linlin Zhang, Huizhi Zhao, Shihui Wang, Xueting Wu, Donghao Liu, Hengchao Zhang, Qianqian Yang, Ying Cheng, Xiuyun Wu, Jiangwei Zhao, Shijie Zhang, Huan Zhang, Haojian Zhang, Qiaozhen Kang, Lixiang Chen, Xiuli An, Xiaoli Qu

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Figure 4

RNA-seq analysis of CFU-E cells and ProEs.

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RNA-seq analysis of CFU-E cells and ProEs. (A) PCA of transcriptomes sho...
(A) PCA of transcriptomes shows the separation between control CFU-E and Mettl3-deficient CFU-EKit-lo cells. (B) Volcano plots of differential expression genes in control CFU-E cells and Mettl3-deficient CFU-EKit-lo cells. (C) Bar plot of DEGs between BM control CFU-E and Mettl3-deficient CFU-EKit-lo cells. (D) GO enrichment analysis of genes upregulated in Mettl3-deficient CFU-EKit-lo cells. (E) Confocal images of control and Mettl3-deficient CFU-EKit-lo cells stained for DNA (Hoechst, blue) and γ-H2AX foci (green), and quantification of γ-H2AX immunofluorescent intensity in Mettl3-deficient mice CFU-EKit-lo cells, normalized to control mice CFU-E cells (n = 5–21/group). (F) Heatmap showing expression levels (TPM) of DNA replication/cell cycle, nucleotide biosynthesis, DNA damage response, and pro-apoptosis–related genes in control CFU-E cells and Mettl3-deficient CFU-EKit-lo cells from RNA-seq data. (G) PCA of transcriptomes revealing distinct clustering between BM control and Mettl3-deficient ProEs. (H) Volcano plots of DEGs in ProEs between control and Mettl3fl/fl EpoRcre/- mice. (I) Bar plot showing numbers of DEGs between BM control and Mettl3-deficient ProEs. (J) GO terms enriched for downregulated genes in Mettl3-deficient ProEs. (K) Heatmap of gene expression for genes involved in DNA replication and DNA damage repair. (L) γ-H2AX immunofluorescence for DNA damage in control and Mettl3-deficient erythroblasts (scale bar: 10 μm), and quantification of γ-H2AX immunofluorescent intensity in Mettl3-deficient mice, normalized to control mice erythroblast cells (n = 11–19/group). (M) Western blot analysis of γ-H2AX expression and corresponding quantification in erythroblast cells (n = 3/group). (N) Heatmap showing TPM of cell-cycle genes in control and Mettl3-deficient ProEs. Data are presented as the mean ± SD. Comparisons between 2 groups were performed using an unpaired 2-tailed Student’s t test. **P < 0.01, ***P < 0.001.