Long-range single-molecule mapping of chromatin modification in eukaryotes

Abstract

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.

Nanopore sequencing technology, bioinformatics and applications

“…For example, the movement of DNA replication forks on single DNA molecules has been measured by detection of nucleotide analogs (for example, 5-bromodeoxyuridine (5-BrdU)) using ONT sequencing [177][178][179] , and the 3D chromatin organization in human cells has been analyzed by integrating a chromatin conformation capture technique and ONT sequencing to capture multiple loci in close spatial proximity by single reads 180 . Two other experimental assays, DiMeLo-seq 181 and BIND&MODIFY 182 , use ONT sequencing to map histone modifications (H3K9me3 and H3K27me3), a histone variant (CENP-A) and other specific protein-DNA interactions (for example, CTCF binding profile). They both construct a fusion protein of the adenosine methyltransferase and protein A to convert specific protein-DNA interactions to an artificial 6mA profile, which is subsequently detected by ONT sequencing.…”

Spatial chromatin accessibility sequencing resolves high-order spatial interactions of epigenomic markers

“…Recently, there has been an increasing interest in applying methylation labeling and nanopore sequencing for analysis of chromatin accessibility at the single-molecule level [5][6][7][8][9][10] . In this study, we have developed SCA-seq to study chromatin spatial density by updating the 2D chromatin accessibility map to the three-dimensional space (Fig.…”

“…To understand the heterogeneity of chromatin accessibility in vivo, scientists have been interested in chromatin structure at a single-molecule resolution level in recent years. They developed approaches such as methyltransferase treatment followed by single-molecule long-read sequencing 5 , single-molecule adenine methylated oligonucleosome sequencing assay 6 , nanopore sequencing of nucleosome occupancy and methylome 7 , single-molecule long-read accessible chromatin mapping sequencing 8 9 , and fiber-seq 10 . Subsequently, decondensed genomes were methylated using methyltransferases and then directly sequenced using third-generation sequencing platforms (Nanopore, Pacbio).…”

Single-molecule simultaneous profiling of DNA methylation and DNA-protein interactions with Nanopore-DamID

“…Each molecule must have GATC methylation on both ends to be amplified, reducing resolution and rendering DamID incompatible with bisulfite sequencing.Oxford Nanopore Technologies (ONT) long-read sequencing can directly detect both cytosine and adenine methylation, including in regions refractory to short-read analysis [17][18][19] . Exogenous promiscuous adenine methylases have recently been used to profile chromatin accessibility 20 and, by fusion to protein-A, regions of transcription factor occupancy bound by a cognate antibody 21,22 .However, direct detection of adenine methylation requires high genome-wide coverage, which can be prohibitively expensive in mammals, and most eukaryotes. Here, using LaminB1 and CTCF as examples, we develop a Nanopore-based DamID which enables rapid, cost-effective single-molecule profiling of transcription factor-cytosine methylation interactions.Nanopore sequencing can be used to selectively analyse adaptor-ligated DNA without purification or amplification, for example to enrich fragments cleaved by Cas9 23 .…”

“…Overall, Nanopore-DamID selectively sequences Dam-labelled native DNA, enabling codetection of DNA-protein contacts and DNA methylation. This approach could be combined with other adenine methylase-based approaches [20][21][22] to increase sequencing depth over methylated regions.…”