DNA methylation on N(6)-adenine in mammalian embryonic stem cells
Authors: Yifei Liu
Yongquan Lai
Samuel G Mackintosh
Shijia Zhu
James A Swenberg
Gang Fang
Tao P Wu
Lawrence S Hon
Tao Wang
Alan Tackett
Guilin Wang
Mei Zhong
Stephanie D Byrum
Andrew Z Xiao
Matthew G Seetin
Kaixuan Lin
Journal: Nature (2016)
Discipline: Multidisciplinary
Keywords: 5-methylcytosine; DNA methylation; N(6)-methyladenine; Alkbh1; transcriptional silencing
Affiliations: Department of Genetics and Yale Stem Cell Center, Yale School of Medicine, New Haven, Connecticut 06520, USA., Pacific Biosciences, 1380 Willow Road, Menlo Park, California 94025, USA., Environmental Sciences &Engineering, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599, USA., Department of Genetics and Genomic Sciences and Icahn Institute for Genomics and Multiscale Biology, Icahn School of Medicine at Mount Sinai, New York 10029, USA., Department of Biochemistry and Molecular Biology, University of Arkansas for Medical Sciences, Little Rock, Arkansas 72205, USA., Yale Stem Cell Center and Department of Cell Biology, Yale School of Medicine, New Haven, Connecticut 06520, USA., Department of Molecular Biophysics &Biochemistry, Yale Center for Genome Analysis, Yale School of Medicine, New Haven, Connecticut 06520, USA.

Abstract:  It has been widely accepted that 5-methylcytosine is the only form of DNA methylation in mammalian genomes. Here we identify N(6)-methyladenine as another form of DNA modification in mouse embryonic stem cells. Alkbh1 encodes a demethylase for N(6)-methyladenine. An increase of N(6)-methyladenine levels in Alkbh1-deficient cells leads to transcriptional silencing. N(6)-methyladenine deposition is inversely correlated with the evolutionary age of LINE-1 transposons; its deposition is strongly enriched at young (&lt;1.5 million years old) but not old (&gt;6 million years old) L1 elements. The deposition of N(6)-methyladenine correlates with epigenetic silencing of such LINE-1 transposons, together with their neighbouring enhancers and genes, thereby resisting the gene activation signals during embryonic stem cell differentiation. As young full-length LINE-1 transposons are strongly enriched on the X chromosome, genes located on the X chromosome are also silenced. Thus, N(6)-methyladenine developed a new role in epigenetic silencing in mammalian evolution distinct from its role in gene activation in other organisms. Our results demonstrate that N(6)-methyladenine constitutes a crucial component of the epigenetic regulation repertoire in mammalian genomes. 

Retracted: No

PubPeer comments:
 This job was Great! Authors just open another door for Epigenetics! , 
 The findings of this study appear to be irreproducible according to a recent paper ( http: onlinelibrary.wiley.com doi 10.1002 anie.201700424 full ) showing that m6A cannot be detected by highly sensitive and accurate mass-spec techniques applied on DNA harvested from mouse ESCs expanded in both serum LIF and naive ground state 2i LIF. It is likely that the findings constitute an artifact of the sequencing method used. , 
 Merry Christmas to all the scientists who have been, are, or will be working on mammalian N6-mdA! For mammalian N6-mdA, till today, its existence and biological function have been confirmed in several independent model systems in 2017. (Dr. Peng Jin s Nature Comm paper showed N6-mdA in mouse brain. And, You will see several papers out soon!) For the paper, which Parasabella Crassichaetae mentioned above, several points in the result are suspicious: 1. The ES cells authors used in the work showed ultra-high level 8-oxo-dG, which means the ES cells authors cultured has ultra-high ROS stress. This is unnormal. 2. The authors used the  2i condition to culture ES cells. It is well-known that the  2i could cause whole genome 5mC hypomethylation. And, we just confirmed  2i indeed could demethylate N6-mdA, too. And, very recently, two Nature papers published just told us,  2i will abolish the ES cell pluripotency. 3. For the MS approach, the sensitivity is not the only factor to judge the method worked or not. In one word, the authors do not have enough solid data to support their conclusion. They cannot claim  No m6-dA or m4-dC in mouse ESC . , 
 #3 I agree with peer #2 and am also suspicious of these findings. The paper (http: onlinelibrary.wiley.com doi 10.1002 anie.201700424 full) by Schiffers et al. 2017 is not the first paper indicating the N6-mdA is absent from mouse genomic DNA. An independent study in 2006 (http: onlinelibrary.wiley.com doi 10.1016 j.febslet.2006.04.074 abstract) did not detect any N6-mdA in mouse tissues, using a highly sensitive method capable of detecting less than 1 N6-mdA site per million dA! Importantly, in the recent study by Schiffers et al. 2017, the authors were meticulous and made sure to include a negative control digestion reaction, to determine the background level of N6-mdA in the digestion reaction alone. DNA degradase Plus (the enzymes used by Wu et al. for preparation of DNA samples for MS) has nucleic acid, including low levels of N6-mdA methylated DNA. Unfortunately, the authors did not properly control for this issue in the paper. It is essential to present N6-mdA measurements for the DNA Degradase Plus alone, which the authors did not show. I suspect that the reported N6-mdA signal in mouse ES cells might be explained by the presence of small amounts of N6-mdA in the DNA Degradase reaction mix. Finally, SMRTseq and methylated DNA IP sequencing methodologies are prone to false positives (especially at repetitive DNA elements), so it is not surprising to falsely identify N6-mdA sites using these approaches (http: journals.plos.org ploscompbiol article?id=10.1371 journal.pcbi.1002935 and https: www.biorxiv.org content early 2017 11 24 224279). In conclusion, I too am skeptical about the existence and epigenetic relevance of N6-mdA in metazoa. , 
 Agree with peer 2 and 4! The real 6mA level has to be reconsider in terms of the existing levels in the degradase enzymes and the field should set a standard for that as a control. , 
 Authors should check another recent well executed study showing that previous reports of 6mA in higher organisms are artifactual: No evidence for DNA N6-methyladenine in mammals https: advances.sciencemag.org content 6 12 eaay3335 , 
 No MS data, and no control data points. Only check T-cell, then claim  No 6mA in Mammals ?! , 
 This paper also could not reproduce the findings that 6mA occurs in eukaryotic genomic DNA https: bmcgenomics.biomedcentral.com articles 10.1186 s12864-019-5754-6 , 
 https: www.ncbi.nlm.nih.gov pubmed 28371147 https: www.ncbi.nlm.nih.gov pmc articles PMC7080441 , 
 #9 Recent work clearly indicates that 6mA detection and sequencing methods are severely flawed when used outside of bacteria and certain unicellular eukaryotes. The new studies show that there is no evidence for 6mA in mammals or other metazoa, and therefore the conclusions of this Nature paper can be considered invalidated. The authors should directly acknowledge their mistake and formally address this issue. https: www.ncbi.nlm.nih.gov pmc articles PMC7080441 https: www.ncbi.nlm.nih.gov pubmed 28371147 https: bmcgenomics.biomedcentral.com articles 10.1186 s12864-019-5754-6 , 
 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 young full-length LINE-1 elements (L1s). 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). For L1 elements, although 6mASCOPE appeared to suggest a higher 6mA A level in the young full-length L1s than in older L1s, a further comparison with a WGA negative control did not support 6mA enrichment in young L1 elements (fig. S16), highlighting the importance of using negative controls to capture possible uncharacterized biases , 
 Multiple other labs have failed to validate the presence of 6mA in mammals after controlling for methodological flaws and experimental artifacts in detection quantification methods like UHPLC-ms ms. https: www.ncbi.nlm.nih.gov pmc articles PMC7080441 https: www.ncbi.nlm.nih.gov pubmed 28371147 https: bmcgenomics.biomedcentral.com articles 10.1186 s12864-019-5754-6 And more recently - https: www.science.org doi 10.1126 science.abe7489 ,