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Enabled by long-read sequencing technologies, particularly Single Molecule, Real-Time sequencing, N6-methyladenine (6mA) footprinting is a transformative methodology for revealing the heterogenous and dynamic distribution of nucleosomes and other DNA-binding proteins. Here, we present ipdTrimming, a novel 6mA-calling pipeline that outperforms existing tools in both computational efficiency and accuracy. Utilizing this optimized experimental and computational framework, we are able to map nucleosome positioning and transcription factor occupancy in nuclear DNA and establish high-resolution, long-range binding events in mitochondrial DNA. Our study highlights the potential of 6mA footprinting to capture coordinated nucleoprotein binding and to unravel epigenetic heterogeneity. 

Although DNAN⁶-adenine methylation (6mA) is best known in prokaryotes, its presence in eukaryotes has recently generated great interest. Biochemical and genetic evidence supports that AMT1, an MT-A70 family methyltransferase (MTase), is crucial for 6mA deposition in unicellular eukaryotes. Nonetheless, the 6mA transmission mechanism remains to be elucidated. Taking advantage of single-molecule real-time circular consensus sequencing (SMRT CCS), here we provide definitive evidence for semiconservative transmission of 6mA inTetrahymena thermophila. In wild-type (WT) cells, 6mA occurs at the self-complementary ApT dinucleotide, mostly in full methylation (full-6mApT); after DNA replication, hemi-methylation (hemi-6mApT) is transiently present on the parental strand, opposite to the daughter strand readily labeled by 5-bromo-2′-deoxyuridine (BrdU). In ΔAMT1cells, 6mA predominantly occurs as hemi-6mApT. Hemi-to-full conversion in WT cells is fast, robust, and processive, whereas de novo methylation in ΔAMT1cells is slow and sporadic. InTetrahymena, regularly spaced 6mA clusters coincide with the linker DNA of nucleosomes arrayed in the gene body. Importantly, in vitro methylation of human chromatin by the reconstituted AMT1 complex recapitulates preferential targeting of hemi-6mApT sites in linker DNA, supporting AMT1's intrinsic and autonomous role in maintenance methylation. We conclude that 6mA is transmitted by a semiconservative mechanism: full-6mApT is split by DNA replication into hemi-6mApT, which is restored to full-6mApT by AMT1-dependent maintenance methylation. Our study dissects AMT1-dependent maintenance methylation and AMT1-independent de novo methylation, reveals a 6mA transmission pathway with a striking similarity to 5-methylcytosine (5mC) transmission at the CpG dinucleotide, and establishes 6mA as a bona fide eukaryotic epigenetic mark.