ABSTRACT
Epigenetic modifications of histones are associated with development and pathogenesis of disease. Existing approaches cannot provide insights into long-range interactions and represent the average chromatin state. Here we describe BIND&MODIFY, a method using long-read sequencing for profiling histone modifications and transcription factors on individual DNA fibers. We use recombinant fused protein A-M.EcoGII to tether methyltransferase M.EcoGII to protein binding sites to label neighboring regions by methylation. Aggregated BIND&MODIFY signal matches bulk ChIP-seq and CUT&TAG. BIND&MODIFY can simultaneously measure histone modification status, transcription factor binding, and CpG 5mC methylation at single-molecule resolution and also quantifies correlation between local and distal elements.
Subject(s)
Eukaryota , Histones , Eukaryota/genetics , Histones/metabolism , Chromatin , Methylation , DNA/metabolism , DNA MethylationABSTRACT
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.
