Single-nucleotide-resolution sequencing of human N6-methyldeoxyadenosine reveals strand-asymmetric clusters associated with SSBP1 on the mitochondrial genome

Abstract

N6-methyldeoxyadenosine (6mA) is a well-characterized DNA modification in prokaryotes but reports on its presence and function in mammals have been controversial. To address this issue, we established the capacity of 6mA-Crosslinking-Exonuclease-sequencing (6mACE-seq) to detect genome-wide 6mA at single-nucleotide-resolution, demonstrating this by accurately mapping 6mA in synthesized DNA and bacterial genomes. Using 6mACE-seq, we generated a human-genome-wide 6mA map that accurately reproduced known 6mA enrichment at active retrotransposons and revealed mitochondrial chromosome-wide 6mA clusters asymmetrically enriched on the heavy-strand. We identified a novel putative 6mA-binding protein in single-stranded DNA-binding protein 1 (SSBP1), a mitochondrial DNA (mtDNA) replication factor known to coat the heavy-strand, linking 6mA with the regulation of mtDNA replication. Finally, we characterized AlkB homologue 1 (ALKBH1) as a mitochondrial protein with 6mA demethylase activity and showed that its loss decreases mitochondrial oxidative phosphorylation. Our results show that 6mA clusters play a previously unappreciated role in regulating human mitochondrial function, despite 6mA being an uncommon DNA modification in the human genome.

Figure 1.

6mACE-seq maps 6mA site in synthetic 6mA-dsDNA. (A) Flowchart documenting the procedure for 6mACE-seq. (B) Counts of 6mACE-seq read start-sites mapped to a 200 bp synthetic dsDNA (PG482/PG483, Supplementary Table S1) with a single 6mA at position 51 of the plus strand. Red and blue represent 6mACE-seq reads that map respectively to the + or − strand. (C) Significance (-log₁₀P-val) at varying coverage was calculated at each position of the dsDNA from (B), and repeated over 100 simulated trials. Average significance +/− SD was then plotted for the plus strand from positions 40 to 60.

Figure 2.

6mACE-seq maps 6mA sites in multiple bacterial genomes. (A) Counts of 6mACE-seq read start-sites at five representative fully-methylated G6mATC sites within the UTI89 E. coli genome. The format follows that of (Figure 1B). (B) Venn diagram showing overlap in 6mA sites identified by 6mACE-seq and all G6mATC sites within the UTI89 E. coli genome. (C) Weblogo representations of consensus 6mA motifs constructed from significant (FDR<0.01) 6mA sites identified using 6mACE-seq performed on genomes of dam+ UTI89 E. coli (top panel), dam+ S. typhimurium (middle panel) and dam- S. typhimurium (bottom panel).

Figure 3.

6mACE-seq identifies 6mA enrichment in young and active human LINEs and SINEs. (A) Plot of genome-wide location of 6mA sites by chromosome. Each dot represents a single 6mA site and the colour intensity corresponds to the statistical significance (-log₁₀P-val) of the 6mA site determined from 6mACE-seq reads. (B) Consensus 6mA motif constructed using all significant (FDR<0.01) 6mA sites across the human genome (C) Pie-chart displaying proportion of 6mA sites mapped to various repetitive genomic elements. (D and E) Enrichment of 6mA sites in various LINE (D) or SINE (E) subfamilies, quantified as the ratio of Observed proportion/Expected proportion of 6mA sites in each transposon subfamily. Age of each LINE1 subfamily and SINE subfamily are plotted accordingly. * represents P-value<10^-19 calculated using a 2-tailed test for population proportion. (F) Counts of 6mACE-seq read start-sites in a representative AluYm1 SINE element. The format follows that of (Figure 1B) with additional horizontal red bars denoting statistically significant 6mA sites (FDR<0.01) mapped to the positive strand.

Figure 4.

6mA is enriched in mtDNA and exhibit strand-asymmetric clustering patterns. (A) UHLPC-MS/MS quantification of 6mA/dA in whole-cell DNA versus purified mtDNA. Data represents average of at least three biological replicates +/− SD. Samples were digested with degradase plus. * represents T-test P < 0.001. (B) Weblogo representation of consensus 6mA motif constructed using all significant (FDR<0.01) 6mA sites across the human mitochondrial genome. (C) Map of mitochondrial 6mA. Inner layer: counts of 6mACE-seq read start-sites following the same format as (Figure 1B). Middle layer: open reading frames of mitochondrial chromosome. Outer layer: significant (FDR<0.01) 6mA sites (hollow circles) determined using raw 6mACE-seq reads. Red and blue represent 6mA sites mapped to the mtDNA + or − strands respectively. (D) Counts of 6mACE-seq read start-sites within chrM:5339–5520. The format follows that of (Figure 1B) with horizontal blue bars denoting statistically significant 6mA sites (FDR<0.01) mapped to the negative heavy strand. Printed genomic sequence corresponds to the negative heavy strand.

Figure 5.

Dense 6mA destabilizes dsDNA structure to potentially promote binding of mitochondrial SSBP1. (A) SILAC-based pulldown from HEK293T mitochondrial extract using dsDNA bait (Supplementary Table S1). Data are plotted as ratio of 6mA/dA binding (forward pulldown) on X-axis and ratio of dA/6mA binding (label-swapped reverse pulldown) on Y-axis. Red circles represent known N⁶-methyladenosine RNA-binding proteins (YTH family and HNRNP family proteins). Blue circles represent all other SILAC-detected candidates. (B) Fluorescence anisotropy curve of fluorescent-tagged dsDNA probes (Supplementary Table S1) with (blue) or without (red) five 6mAs, incubated with varying concentrations of recombinant SSBP1. Data represents average of three technical replicates +/− SD. (C) Melting curve of dsDNA containing 5, 1 or 0 6mA sites (Supplementary Table S1), plotted as the negative first derivative of fluorescence against temperature. Data represents average of three technical replicates.

Figure 6.

ALKBH1 is a mitochondrial protein with 6mA demethylase activity and its loss disrupts mitochondrial function. (A) Western blotting of ALKBH1 in ALKBH1-WT and ALKBH1-KO whole-cell (W) or mitochondrial (M) lysate. Actin and HSP60 are used as cytoplasmic/non-mitochondrial and mitochondrial loading markers. (B) UHPLC-MS/MS quantification of resultant 6mA/dA within 6mA-containing ssDNA (Supplementary Table S1) after incubation without or with recombinant ALKBH1. Data represents average of three biological replicates +/− SD. * T-test P < 0.0001. Samples were digested with 20× diluted NPAP (see ‘Materials and Methods’ section). (C) Plot of oxygen consumption rate, OCR (pmol/min) against time during the Seahorse Mito-Stress test assay of ALKBH1-WT versus two separate ALKBH1-KO clones. Time points for administering of all three drugs used in this assay are as denoted. Data represents average of at least three biological replicates +/− SD. (D and E) ATP production (D) and SRC (E) calculated from OCR measurements in (C). Data represents average of at least three biological replicates +/− SD. ** T-test P < 0.00001. Sample legend follows that of (C).