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The epigenetic modifications of histones are essential marks related to the development and disease pathogenesis, including human cancers. Mapping histone modification has emerged as the widely used tool for studying epigenetic regulation. However, existing approaches limited by fragmentation and short-read sequencing cannot provide information about the long-range chromatin states and represent the average chromatin status in samples. We leveraged the advantage of long read sequencing to develop a method “BIND&MODIFY” for profiling the histone modification of individual DNA fiber. Our approach is based on the recombinant fused protein A-EcoGII, which tethers the methyltransferase EcoGII to the protein binding sites and locally labels the neighboring DNA regions through artificial methylations. We demonstrate that the aggregated BIND&MODIFY signal matches the bulk-level ChIP-seq and CUT&TAG, observe the single-molecule heterogenous histone modification status, and quantify the correlation between distal elements. This method could be an essential tool in the future third-generation sequencing ages.

Background Although extrachromosomal DNA (ecDNA) has been intensively studied for several decades, the mechanisms underlying its tumorigenic effects have been revealed only recently. In most conventional sequencing studies, the high-throughput short-read sequencing largely ignores the epigenetic status of most ecDNA regions except for the junctional areas. Methods Here, we developed a method of sequencing enzyme-accessible chromatin in circular DNA (CCDA-seq) based on the use of methylase to label open chromatin without fragmentation and exonuclease to enrich ecDNA sequencing depth, followed by long-read nanopore sequencing. Results Using CCDA-seq, we observed significantly different patterns in nucleosome/regulator binding to ecDNA at a single-molecule resolution. Conclusions These results deepen the understanding of ecDNA regulatory mechanisms.