Abstract
N6-methyldeoxyadenine (6mA) is a noncanonical DNA base modification present at low levels in plant and animal genomes1,2,3,4, but its prevalence and association with genome function in other eukaryotic lineages remains poorly understood. Here we report that abundant 6mA is associated with transcriptionally active genes in early-diverging fungal lineages5. Using single-molecule long-read sequencing of 16 diverse fungal genomes, we observed that up to 2.8% of all adenines were methylated in early-diverging fungi, far exceeding levels observed in other eukaryotes and more derived fungi. 6mA occurred symmetrically at ApT dinucleotides and was concentrated in dense methylated adenine clusters surrounding the transcriptional start sites of expressed genes; its distribution was inversely correlated with that of 5-methylcytosine. Our results show a striking contrast in the genomic distributions of 6mA and 5-methylcytosine and reinforce a distinct role for 6mA as a gene-expression-associated epigenomic mark in eukaryotes.
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Acknowledgements
We thank J.K. Henske, C. Swift, S.P. Gilmore and K.V. Solomon for preparing DNA and/or RNA for P. finnis and A. robustus; T. Porter for DNA and RNA preparation for Catenaria anguillulae; P. Liu for preparation of DNA and RNA for R. globosum and L. transversale; and D. Carter-House for preparation of genomic DNA of H. vesiculosa and R. globosum for bisulfite sequencing. For bisulfite sequencing of H. vesiculosa and R. globosum, we thank N.A. Rohr for library preparation and the Georgia Advanced Computing Resource Center (GACRC) for computational resources. Work conducted by the US Department of Energy Joint Genome Institute, a DOE Office of Science User Facility, is supported by the Office of Science of the US Department of Energy under Contract No. DE-AC02-05CH11231. This work was partially supported by funding from the National Science Foundation (DEB-1441715 to JES, DEB-1441604 to J.W.S. and DEB-1354625 to T.Y.J. and I.V.G.); Any opinions, findings, conclusions or recommendations expressed in this material are those of the authors and do not necessarily reflect the views of the National Science Foundation. This work was further supported by the Office of Science (BER), US Department of Energy (DE-SC0010352) and the Institute for Collaborative Biotechnologies through grant W911NF-09- 0001. R.J.S. is supported by funding from the Office of the Vice President of Research at UGA as well as the Pew Charitable Trusts.
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S.J.M., R.O.D. and I.V.G. designed the study. S.J.M. and R.O.D. collected and analyzed data under the supervision of G.K.-R. and I.V.G. R.C.K. optimized the protocol for 6mA IP-sequencing and PacBio library preparation. R.C.K. and C.D. sequenced genomes, including IP-sequencing. R.C.K., C.D., A.J.B. and R.J.S. conducted bisulfite sequencing. S.J.M., R.O.D. and A.J.B. analyzed bisulfite sequencing data. K.B.L., R.L. and T.R.N. conducted LC-mass spectrometry analysis. B.P.B. analyzed mass spectrometry data. K.L., B.B.A. and A.C. assembled genomes. S.J.M., S.H., A.K., S.R.A. and A.S. annotated genomes. A.L. and E.L. assembled transcriptomes. S.J.M., W.S. and G.K.-R. analyzed transcriptomes. A.G., D.C., J.M., T.Y.J., M.A.O'M., J.E.S., J.W.S. and I.V.G. coordinated genome projects. S.J.M. wrote the manuscript with significant input from A.V. and I.V.G.; and I.V.G. coordinated the project.
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Integrated supplementary information
Supplementary Figure 1 Coverage, quality and reproducibility of 6mA marks across fungi.
Line graph showing per-strand coverage of 6mA marks in genomes of a) Dikarya (shown in greyscale to distinguish between different lineages), and b) early-diverging fungi. 6mA marks below a minimum coverage cutoff of 15x and above a maximum coverage (determined independently for each genome) were removed from downstream analyses. Coverage ranges for each lineage are shown in the figure legend. c) Modification quality value (mQV) distribution for each lineage and filtering cutoff used (black bar, 25 mQV). d) Box plots showing distribution of methylation ratios for methylated adenines in each genome analyzed (black bar within boxes shows median ratio). Methylation ratio refers to the proportion of molecules mapped to a given site which are methylated.
Supplementary Figure 2 Validation of SMRT-detected 6mA using mass spectrometry and IP sequencing.
a) Percent methylated adenines as detected by SMRT-analysis (SMRT-6mA) and by Mass-Spectrometry (MS-6mA). As a measure of high confidence SMRT-detected sites, percent of methylated adenines at ApT sites (SMRT-6mApT) is also included. b) 6mA overlap between SMRT and IP-seq methods within a 100kb region of H. vesiculosa scaffold 1. Red tiles: MACs detected through SMRT-analysis. Black tiles: methylated regions identified through IP-sequencing. Significant peaks were detected using macs231, Q-value ≤ 0.01. Read coverage tracks for both the control (inner circle) and pulldown (outer circle) are also shown. c) Comparison of 6mA-IP and SMRT analysis results across all lineages examined. a refers to percent of 6mA bases identified by SMRT analysis prior to filtering.
Supplementary Figure 3 Surrounding nucleotide context and relative genomic occurrence of 6mA.
a) Occurrence of 6mA at 4mers in early-diverged fungi. TAT/ATA trinucleotides are underscored in red. b) Percent of total ApT containing trinucleotides within MACs that are methylated.
Supplementary Figure 4 Expression, thymine and TAT-trinucleotide frequencies flanking and across MACs.
Frequency of TAT trinucleotides (top), thymine bases (middle) and expression (bottom) are plotted upstream, downstream and across MACs. Frequency is calculated as: # occurrences ÷ total # MACs. As MACs vary in length, all MACs ≥ 100 bp were selected, fragmented into 100 sections from start to end, then average frequency is calculated within fragment. MACs are oriented by gene direction.
Supplementary Figure 5 6mA is associated with active genes.
a) Expression and methylation level of all methylated genes, sorted by expression level. Genes are sorted by FPKM value (blue → black), with 6mA levels shown immediately below (white → dark green). While methylated genes rarely lack expression (FPKM < 1.0), the level of 6mA has no influence over the magnitude of expression. If the two were related, we would expect that as expression level increased, we would see a similar pattern in amount of 6mA present, which is not the case. b) FPKM levels of unmethylated genes, sorted by expression level.
Supplementary Figure 6 MAC overlaps with various genomic features.
a) Percent of gene models containing MACs and proximity to their transcriptional start sites. While some MACs directly overlap with the TSS, many are located slightly downstream. b) Fixed window overlaps of MACs with micro RNAs. c) Fixed window overlaps of MACs with tRNAs.
Supplementary Figure 7 6mA presence or absence is related to gene function.
a) Methylation presence/absence at all genes containing common pfam17 domains (present in at least 8 genes) and their deviation from expected. Pfams showing significant (p ≤ 0.05) departures from the expected were identified using Fisher’s exact test followed by FDR correction (significant = red, non-significant = blue). Overall percentage of significant pfams for each genome are shown in parenthesis next to lineage names. b) log2 fold change in methylation presence/absence at genes containing common pfam17 domains across all lineages (present in at least 8 genes across all genomes, significant in at least one lineage). Lineages showing significant departure from the expected are denoted with a * (adjusted p-value ≤ 0.05), or ** (adjusted p-value ≤ 0.01). Green = enriched in unmethylated gene set, purple = enriched in methylated gene set. Constitutively expressed housekeeping proteins, such as mitochondrial Rho proteins (blue arrow) are very frequently methylated, while some genes, such as Leucine-Rich-Repeat containing proteins (orange arrow) show variability across lineages.
Supplementary Figure 8 6mA and 5mC enrichment by region.
Overall percent cytosines methylated per genome (a), context (b) and distribution of both epigenomic marks, 5mC and 6mA, across the genome (c and d, respectively).
Supplementary information
Supplementary Text and Figures
Supplementary Figures 1–8, Supplementary Tables 1 and 2, and Supplementary Notes 1 and 2. (PDF 2294 kb)
Supplementary Dataset 1
Pfam presence across fungi, putative methyltransferases and lineages surveyed. Worksheet 1) Spreadsheet of all pfams showing significant differences (adjusted pval ≤ 0.01) in presence/absence across earlydiverging fungi vs Dikarya. EDF = early-diverging fungi. Worksheet 2) Spreadsheet of methyltransferases showing significant differences (adjusted pval ≤ 0.01) in presence/absence across early-diverging fungi vs Dikarya. EDF = early-diverging fungi. Worksheet 3) Spreadsheet of all lineages included in gene conservation and pfam analyses. (XLSX 389 kb)
Supplementary Dataset 2
Results of Fisher's exact test per lineage. Results of Fisher's exact test examining methylation presence/absence at all common pfams (present in at least 8 genes) for each genome. Results for each lineage are shown on separate worksheets. (XLSX 133 kb)
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Mondo, S., Dannebaum, R., Kuo, R. et al. Widespread adenine N6-methylation of active genes in fungi. Nat Genet 49, 964–968 (2017). https://doi.org/10.1038/ng.3859
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DOI: https://doi.org/10.1038/ng.3859
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