N6-Deoxyadenosine Methylation in Mammalian Mitochondrial DNA
Authors: Xiaolong Cui
Tong Wu
Xiaoyang Dou
Ziyang Hao
Pei-Hua Peng
Yueh-Te Lin
Chuan He
Kou-Juey Wu
Li-Sheng Zhang
Lulu Hu
Pingping Zhu
Yawei Gao
Caiping Tan
Allen Zhu
Jianzhao Liu
Hui-Lung Sun
Kai-Wen Hsu
Journal: Molecular Cell (2020)
Discipline: MOLECULAR BIOLOGY & GENETICS
Keywords: METTL4; N(6)-methyldeoxyadenosine (6mA); TFAM; methyltransferase; mitochondrial DNA methylation
Affiliations: Department of Chemistry, Department of Biochemistry and Molecular Biology, Institute for Biophysical Dynamics, Howard Hughes Medical Institute, The University of Chicago, 929 East 57th Street, Chicago, IL 60637, USA., Department of Chemistry, Department of Biochemistry and Molecular Biology, Institute for Biophysical Dynamics, Howard Hughes Medical Institute, The University of Chicago, 929 East 57th Street, Chicago, IL 60637, USA; School of Life Science, Zhengzhou University, Zhengzhou 450001, China., Department of Chemistry, Department of Biochemistry and Molecular Biology, Institute for Biophysical Dynamics, Howard Hughes Medical Institute, The University of Chicago, 929 East 57th Street, Chicago, IL 60637, USA; MOE Key Laboratory of Bioinorganic and Synthetic Chemistry, School of Chemistry and Chemical Engineering, Sun Yat-sen University, Guangzhou 510275, China., Cancer Genome Research Center, Chang Gung Memorial Hospital at Linkou, Taoyuan 333, Taiwan., Department of Chemistry, Department of Biochemistry and Molecular Biology, Institute for Biophysical Dynamics, Howard Hughes Medical Institute, The University of Chicago, 929 East 57th Street, Chicago, IL 60637, USA; Clinical and Translational Research Center of Shanghai First Maternity and Infant Hospital, Shanghai Key Laboratory of Signaling and Disease Research, School of Life Sciences and Technology, Tongji University, Shanghai 200092, China., Department of Chemistry, Department of Biochemistry and Molecular Biology, Institute for Biophysical Dynamics, Howard Hughes Medical Institute, The University of Chicago, 929 East 57th Street, Chicago, IL 60637, USA. Electronic address: .

Abstract:  N6-Methyldeoxyadenosine (6mA) has recently been shown to exist and play regulatory roles in eukaryotic genomic DNA (gDNA). However, the biological functions of 6mA in mammals have yet to be adequately explored, largely due to its low abundance in most mammalian genomes. Here, we report that mammalian mitochondrial DNA (mtDNA) is enriched for 6mA. The level of 6mA in HepG2 mtDNA is at least 1,300-fold higher than that in gDNA under normal growth conditions, corresponding to approximately four 6mA modifications on each mtDNA molecule. METTL4, a putative mammalian methyltransferase, can mediate mtDNA 6mA methylation, which contributes to attenuated mtDNA transcription and a reduced mtDNA copy number. Mechanistically, the presence of 6mA could repress DNA binding and bending by mitochondrial transcription factor (TFAM). Under hypoxia, the 6mA level in mtDNA could be further elevated, suggesting regulatory roles for 6mA in mitochondrial stress response. Our study reveals DNA 6mA as a regulatory mark in mammalian mtDNA. 

Retracted: No

PubPeer comments:
 Prior work, using clean mitochondrial purification methods and highly sensitive UHPLC-ms ms techniques found no evidence for the presence of 6mA in mammalian mitochondrial DNA. https: www.ncbi.nlm.nih.gov pubmed 16684535:  ...Since it has been suggested that mitochondria could be the degenerate descendants of endosymbiotic bacteria, and since bacterial DNA is known to contain 6mA, mitochondrial DNA from human placenta was also included in this study. In contrast to bacterial DNA, which contains 6mA, no 6mA was detected in mitochondrial DNA [ 1 ppm] In addition, it was recently found that METTL4 functions as a mammalian snRNA m6Am methyltransferase https: www.ncbi.nlm.nih.gov pubmed 31913360. In that study from Dr. Yang Shi s lab, the authors also measured mtDNA 6mA levels in WT, METTL4 KO and METTL4 overexpressing cells and did not detect any 6mA at all (in either nuclear or mtDNA), despite the fact that they used a highly sensitive UHPLC-ms ms method (capable of detecting 1 ppm). If you examine the chromatograms shown in their Supplemental data, you can see that there is no discernible 6mA peak in either nuclear genomic DNA or mtDNA from any of the samples (regardless of METTL4 status! I therefore urge the authors to rigorously re-evaluate the validity of their results and conclusions. , 
 Readers should become aware of a recent paper Critical assessment of DNA adenine methylation in eukaryotes using quantitative deconvolution , that could not reproduce the finding that 6mA modification is enriched in mitochondrial DNA. From the text: Previous studies have reported enrichment of 6mA in mitochondrial DNA (mtDNA) (12, 13, 21, 38) and in young full-length LINE-1 elements (L1s) (10, 11, 21). For mtDNA, 6mASCOPE did not find 6mA enrichment in the 7205 CCS reads from the HEK293 sample that mapped to mtDNA, in comparison to a negative control (targeted mitochondrial genome amplification, 10u20135.72; CI, 10u20136.00 to 10u20134.90; fig. S15). ,