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 6

METTL3 inhibition impaired human erythropoiesis.

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METTL3 inhibition impaired human erythropoiesis. (A) Global m6A levels i...
(A) Global m6A levels in total RNA after DMSO or STM2457 treatment (n = 3/group). (B) Colony-forming ability of progenitor cells on day 6. Scale bar: 100 μm. (C) Proliferation curve of human CD34+ cells treated with DMSO or STM2457 (n = 3/group). (D) Representative flow cytometry profiles and quantification of erythroid cell apoptosis (n = 3/group). (E) Schematic illustration and purity assessment of sorted human CFU-E and GPA+ cells for EdU assay. (F) Flow cytometry quantified the proportion of EdU+ erythroid cells in sorted human CFU-E cells and (G) quantification of the EdU MFI in CFU-E cells during S phase (n = 3/group). (H) The proportion of EdU+ erythroid cells was determined by flow cytometry in sorted human GPA+ cells. (I) Measurement of EdU MFI in GPA+ cells during S phase (n = 3/group). (J) Representative Western blot analysis of γ-H2AX, pATM, pATR, and pCHK1 in erythroid cells treated with DMSO or STM2457 on day 7 (n = 3/group). (K) qRT-PCR measurement of MTHFD1 mRNA levels after METTL3 inhibition (n = 3/group). (L) Western blot analysis of MTHFD1 protein level in METTL3-deficient cells (n = 3/group). (M) Exogenous IMP supplementation partially rescued the proliferation deficit in STM2457-treated erythroid cells (n = 3/group). (N) Analysis of the effect of exogenous IMP on apoptosis in STM2457-treated erythroid cells (n = 3/group). (O) Exogenous IMP supplementation partially rescued the cell-cycle defect in STM2457-treated erythroid cells (n = 3/group). Data are presented as mean ± SD. Comparisons between 2 groups were performed using an unpaired 2-tailed Student’s t test. A 2-way ANOVA with Tukey’s post hoc test was used to calculate statistical significance among multiple groups. *P < 0.05, **P < 0.01, ***P < 0.001.