Single-molecule long-read sequencing reveals the chromatin basis of gene expression

Abstract

Genome-wide chromatin accessibility and nucleosome occupancy profiles have been widely investigated, while the longrange dynamics remain poorly studied at the single-cell level. Here, we present a new experimental approach, methyltransferase treatment followed by single-molecule long-read sequencing (MeSMLR-seq), for long-range mapping of nucleosomes and chromatin accessibility at single DNA molecules and thus achieve comprehensive-coverage characterization of the corresponding heterogeneity. MeSMLR-seq offers direct measurements of both nucleosome-occupied and nucleosome-evicted regions on a single DNA molecule, which is challenging for many existing methods. We applied MeSMLR-seq to haploid yeast, where single DNA molecules represent single cells, and thus we could investigate the combinatorics of many (up to 356) nucleosomes at long range in single cells. We illustrated the differential organization principles of nucleosomes surrounding the transcription start site for silent and actively transcribed genes, at the single-cell level and in the long-range scale. The heterogeneous patterns of chromatin status spanning multiple genes were phased. Together with single-cell RNAseq data, we quantitatively revealed how chromatin accessibility correlated with gene transcription positively in a highly heterogeneous scenario. Moreover, we quantified the openness of promoters and investigated the coupled chromatin changes of adjacent genes at single DNA molecules during transcription reprogramming. In addition, we revealed the coupled changes of chromatin accessibility for two neighboring glucose transporter genes in response to changes in glucose concentration.

The DNA methylation landscape in cancer

“…Variations on scBS-Seq have seen the ability to measure DNA methylation and transcription in parallel from single cells (scM&T-Seq, [134]) and single cell nucleosome occupancy and methylation sequencing (scNOMe-Seq, [135]). Wang et al [136] have recently devised a new approach (methyltransferase treatment followed by single-molecule long-read sequencing (MeSMLR-Seq) based upon the original NOMe-seq protocol, which adopts single molecule long read sequencing (Oxford Nanopore Technologies) to enable in excess of 53 kB read lengths (up to 356 nucleosomes). MeSMLR-Seq offers one of the first epigenetic applications of long-read sequencing and great potential to uncover biological insight over short-read sequencing options; however, there is currently little cost or convenience benefit in adopting these approaches in their current form.…”

On the role of transcription in positioning nucleosomes

“…It is worth mentioning that the yeast results shown in Fig 1A deviate from the simulated profiles in that the second peak is higher than the first. However, this same trend was not observed in recent single-molecule studies that simultaneously measure the position of multiple nucleosomes from the same DNA [ 45 , 46 ]. Additional experiments are needed to evaluate the robustness and mechanism of this particular feature further.…”

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].…”