N-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:
 The theory that 6mA occurs on mammalian mtDNA has already been rigorously tested and it was shown to be absent ( 0.0001% 6mdA dA; 1 ppm), see Table 1 https: www.ncbi.nlm.nih.gov pubmed 16684535 Perhaps the authors are picking up some form of contaminating circular DNA, either from sample or reagent contamination? Given the numerous inherent flaws in current methodologies for 6mA measurements in eukaryotes, Iu2019m highly skeptical that these results could be valid. Two recent publications have described multiple experimental and methodological artifacts inherent to 6mA detection methodologies. These include both sample and reagent contamination from bacterial DNA, RNA contamination (which renders all 6mA antibody-based methods problematic), nonspecificity of 6mA antibodies (including binding to unmodified adenines), and flaws in sequencing methodologies (e.g. SMRTseq). Notably, two recent reports indicate that there is not sufficient evidence to conclude that 6mA occurs, or is u201cregulatedu201d in mammalian DNA. https: advances.sciencemag.org content 6 12 eaay3335 https: www.ncbi.nlm.nih.gov pubmed 31159718 Finally, in another recent paper where METTL4 was shown to be a mammalian snRNA m6Am methyltransferase, the authors also measured mtDNA 6mA levels in WT and in METTL4 KO cells and saw no difference, nor did they see appreciable levels of 6mA at all (in either nuclear or mtDNA), despite a highly sensitive UHPLC-ms ms method ( 1 ppm). If you take a close look at their Fig S2, you can see that there is no discernible 6mA peak in either nuclear genomic DNA or mtDNA from either METTL4 KO or WT human cell lines. https: www.ncbi.nlm.nih.gov pubmed 31913360 , 
 These results most likely constitute a technical or experimental artifact. Prior reports, using clean mitochondrial purification methods and highly sensitive UHPLC-ms ms techniques found no evidence for presence of 6mA in mammalian mitochondrial DNA. From the results of this study 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] , 
 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). ,