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 human epigenomic domains. Our analyses suggest that chromatin is comprised 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.
Highlights
The nucleosome is the atomic unit of chromatin
We hypothesized that high-accuracy PacBio single-molecule real-time sequencing could detect m6dA deposited on chromatin templates to natively measure nucleosome positioning
We observed higher average interpulse duration (IPD) in samples exposed to methyltransferase, consistent with a rolling circle polymerase ‘pausing’ at methylated adenine residues in template DNA (Figure 1—figure supplement 2)
Summary
Nucleosomes passively and actively template the majority of nuclear interactions essential to life by determining target site access for transcription factors (Spitz and Furlong, 2012), bookmarking active and repressed chromosomal compartments via post-translational modifications (Zhou et al, 2011), and safeguarding the genome from mutational agents (Papamichos-Chronakis and Peterson, 2013). Complementary biochemical methods using nucleolytic cleavage have successfully mapped the subunit architecture of chromatin structure at high resolution. These cleavage-based approaches can be stratified into those that focus primarily on chromatin accessibility (Klemm et al, 2019) (i.e. measuring ‘competent’ active chromatin [Weintraub and Groudine, 1976]), and those that survey nucleosomal structure uniformly across active and inactive genomic compartments. Understanding links between chromatin and gene regulation requires sensitive methods in all three
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