Long-Range Single-Molecule Mapping Reveals the Multikilobase-Scale Organization of Accessible Chromatin in Eukaryotes

Gene regulation in most eukaryotes is carried out through the action of sequence-specific regulatory factors at specific locations in the genome. The association of these proteins with regulatory elements is accompanied by reduced nucleosomal occupancy, and, accordingly, higher sensitivity to enzymatic digestion of DNA. This property, the basis of assays such as DNAse-seq and ATAC-seq, has long been a highly useful proxy for the identification of regulatory elements, such as promoters, enhancers and insulators, but important fundamental questions are left unanswered by such approaches. Regulatory elements are often located at significant genomic distances from each other, yet they act in concert to influence gene expression. However, enzymatic digestion techniques followed by short-read sequencing technologies can only measure the state of each such element individually. The state of chromatin at medium-range scales has until now been impossible to characterize, and the extent and patterns of coordination of regulatory activity along the genome are not understood. We address these questions by developing a novel method for mapping chromatin accessibility on the multikilobase scale. Our approach is based on using a combination of treating intact chromatin with 5-methylcytosine (5mC) and N6 -methyladenosine (m6A) methyltransferases followed by a nanopore sequencing-based readout of methylated bases and the corresponding accessibility state of individual DNA molecules. It allows the assessment of patterns of coaccessibility between distally located regulatory elements, the quantification of the distribution of chromatin states within a population of individual chromatin molecules at varying length scales, and the measurement of chromatin accessibility in previously “invisible” to short reads repetitive regions of the genome. We present in detail the insights derived from its the application to the genome of the yeast Saccharomyces cerevisiae, and discuss the future scope and prospects of the technology for studying more complex eukaryotic genomes.