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

Abstract

Active regulatory elements in eukaryotes are typically characterized by an open, nucleosomedepleted chromatin structure; mapping areas of open chromatin has accordingly emerged as a widely used tool in the arsenal of modern functional genomics. However, existing approaches for profiling chromatin accessibility are limited by their reliance on DNA fragmentation and short read sequencing, which leaves them unable to provide information about the state of chromatin on larger scales or reveal coordination between the chromatin state of individual distal regulatory elements. To address these limitations, we have developed a method for profiling accessibility of individual chromatin fibers at multi-kilobase length scale (SMAC-seq, or Single-Molecule long-read Accessible Chromatin mapping sequencing assay), enabling the simultaneous, highresolution, single-molecule assessment of the chromatin state of distal genomic elements. Our strategy is based on combining the preferential methylation of open chromatin regions by DNA methyltransferases (CpG and GpC 5-methylcytosine (5mC) and N 6 -methyladenosine (m 6 A) enzymes) and the ability of long-read single-molecule nanopore sequencing to directly read out the methylation state of individual DNA bases. Applying SMAC-seq to the budding yeast Saccharomyces cerevisiae, we demonstrate that aggregate SMAC-seq signals match bulk-level accessibility measurements, observe single-molecule protection footprints of nucleosomes and transcription factors, and quantify the correlation between the chromatin states of distal genomic elements.

Chromatin accessibility profiling methods

“…As genomes of many eukaryotes contain abundant endogenous CpG methylation, and bisulfite sequencing measures methylation on cytosines, exogenous enzymes are required that methylate other dinucleotide contexts. The approach has limited spatial resolution, as it relies on GpC nucleotides that are rare in mammalian genomes, only found once every 20-30 bp, and it is common to find much larger stretches of sequence having no informative positions at all 103 . However, in species such as yeast and Drosophila, which lack endogenous methylation, a combination of both a GpC and a CpG methyltransferase can be used, which increases assay resolution down to ∼10 bp.…”

“…For instance, non-specific methyl ation, such as m 6 A deposited via EcoGII, or other modifications (Tet-assisted pyridine borane sequencing (lrTAPS) 115 ) can be combined with nano pore or PacBio sequencing to obtain a fine-scale read-out of chromatin accessibility at the single-molecule level. This can be done either on total genomic DNA -with the single-molecule long-read accessible chromatin mapping sequencing (SMAC-seq) assay 103 or mapping chromatin fibres onto a DNA template using methyltransferases (Fiber-seq) 116 -or in combination with a phasing MNase digestion step (single-molecule adenine methylated oligonucleosome sequencing assay (SAMOSA) 117 ). The large number of informative positions allows for fine-scale footprinting almost everywhere in the genome.…”

Recent advances in the detection of base modifications using the Nanopore sequencer

“…[67]. nanoNOMe, MeSMLR-seq, and SMAC-seq are methods that combine the accessibility of one or more methyltransferases and the direct detection of methylated regions by the Nanopore sequencer instead of requiring BS-seq [68][69][70]. These methods can detect open chromatin patterns of single long DNA molecules (Fig.…”

“…Although nanoNOMe and MeSMLR-seq employ a GpC methyltransferase similar to NOMe-seq, SMAC-seq employs a m6A methyltransferase (EcoGII) and CpG-specific methyltransferase (M.SssI) in addition to the GpC methyltransferase to increase the resolution [68]. Shipony et al and Wang et al applied SMAC-seq and MeSMLR-seq to S. cerevisiae, respectively [68,70]. In SMAC-seq, Tombo is used for methylation detection [68].…”

“…Shipony et al and Wang et al applied SMAC-seq and MeSMLR-seq to S. cerevisiae, respectively [68,70]. In SMAC-seq, Tombo is used for methylation detection [68]. Lee et al applied nanoNOMe to four human cell lines and detected DNA methylation using Nanopolish [69].…”