Massively multiplex single-molecule oligonucleosome footprinting

Abstract

Our understanding of the beads-on-a-string arrangement of nucleosomes has been built largely on high-resolution sequence-agnostic imaging methods and sequence-resolved bulk biochemical techniques. To bridge the divide between these approaches, we present the single-molecule adenine methylated oligonucleosome sequencing assay (SAMOSA). SAMOSA is a high-throughput single-molecule sequencing method that combines adenine methyltransferase footprinting and single-molecule real-time DNA sequencing to natively and nondestructively measure nucleosome positions on individual chromatin fibres. SAMOSA data allows unbiased classification of single-molecular 'states' of nucleosome occupancy on individual chromatin fibres. We leverage this to estimate nucleosome regularity and spacing on single chromatin fibres genome-wide, at predicted transcription factor binding motifs, and across both active and silent human epigenomic domains. Our analyses suggest that chromatin is comprised of a diverse array of both regular and irregular single-molecular oligonucleosome patterns that differ subtly in their relative abundance across epigenomic domains. This irregularity is particularly striking in constitutive heterochromatin, which has typically been viewed as a conformationally static entity. Our proof-of-concept study provides a powerful new methodology for studying nucleosome organization at a previously intractable resolution, and offers up new avenues for modeling and visualizing higher-order chromatin structure.

Segmental duplications and their variation in a complete human genome

“…6 and S21). While more human genomes and diverse tissues need to be interrogated to assess the significance of this observation, it is clear that phased genome assemblies (47) with long-read functional readouts such as methylation (64), transcription, or Fiber-seq (85,86) provide a powerful approach to understanding the regulatory landscape of duplicated and copy number polymorphic genes in the human genome.…”

DiMeLo-seq: a long-read, single-molecule method for mapping protein-DNA interactions genome-wide

“…DiMeLo-seq combines elements of antibody-directed protein-DNA mapping approaches (Skene and Henikoff 2017;Schmid, Durussel, and Laemmli 2004;van Schaik et al 2020) to deposit methylation marks near a specific target protein, then uses long-read sequencing to read out these exogenous methylation marks directly (Abdulhay et al 2020;Stergachis et al 2020;Lee et al 2020;Shipony et al 2020;Wang et al 2019). Taking advantage of the lack of N 6 -methyldeoxyadenosine in human DNA (O'Brown et al 2019), we fused the antibody-binding Protein A to the nonspecific deoxyadenosine methyltransferase Hia5 (Stergachis et al 2020;Drozdz et al 2012) (pA-Hia5) to catalyze the formation of N 6 -methyl-deoxyadenosine (hereafter mA) in the DNA proximal to targeted chromatin-associated proteins (Fig.…”

“…Each of these features provides an opportunity to study genome regulation in unprecedented ways. Recent technologies have begun to take advantage of long-read sequencing to identify accessible regions and CpG methylation on native single molecules, but they cannot target specific protein-DNA interactions (Abdulhay et al 2020;Stergachis et al 2020;Lee et al 2020;Shipony et al 2020;Wang et al 2019). Here we extend these capabilities to map specific regulatory elements and demonstrate the advantages of DiMeLo-Seq by mapping lamina associated domains, CTCF binding sites, histone modifications/variants, and CpG methylation across the genome and through complex repetitive domains.…”

Single-Molecule Techniques to Study Chromatin

“…Recently, using SMRT-seq only, two techniques named Fiber-seq and Single-Molecule-Adenine Methylated Oligonucleosome Sequencing Assay (SAMOSA) were developed using an unspecific N6-Methyladenine (m6A) DNA-methyltransferase for nucleosome detection in higher resolution ( Abdulhay et al, 2020 ; Stergachis et al, 2020 ). Eukaryotic genomes are devoid of m6A but at the same time, adenines are present on double stranded DNA with an average rate of one every two DNA bases, providing unprecedented coverage of methylation sites and therefore higher resolution compared to CpG methylation.…”

“…Similarly, SAMOSA was first applied as a proof of concept on in vitro assembled chromatin confirming its ability to track nucleosome positioning. As a next step, oligonucleosomes of K562 cells were used showing impartial nucleosome mapping on both euchromatin and heterochromatin regions ( Abdulhay et al, 2020 ). Surprisingly, this study revealed elevated heterogeneity in both actively transcribed euchromatin and constitutive heterochromatin regions, the latter being typically viewed as a conformationally more static epigenomic domain.…”