Massively multiplex single-molecule oligonucleosome footprinting

Abstract

15Our understanding of the beads-on-a-string arrangement of nucleosomes has been built largely on high-16resolution sequence-agnostic imaging methods and sequence-resolved bulk biochemical techniques. To bridge 17 the divide between these approaches, we present the single-molecule adenine methylated oligonucleosome 18 sequencing assay (SAMOSA). SAMOSA is a high-throughput single-molecule sequencing method that 19 combines adenine methyltransferase footprinting and single-molecule real-time DNA sequencing to natively 20and nondestructively measure nucleosome positions on individual chromatin fibres. SAMOSA data allows 21unbiased classification of single-molecular 'states' of nucleosome occupancy on individual chromatin fibres. 22We leverage this to estimate nucleosome regularity and spacing on single chromatin fibres genome-wide, at 23 predicted transcription factor binding motifs, and across both active and silent human epigenomic domains. 24Our analyses suggest that chromatin is comprised of a diverse array of both regular and irregular single-25 molecular oligonucleosome patterns that differ subtly in their relative abundance across epigenomic domains. 26This irregularity is particularly striking in constitutive heterochromatin, which has typically been viewed as a 27 conformationally static entity. Our proof-of-concept study provides a powerful new methodology for studying 28 nucleosome organization at a previously intractable resolution, and offers up new avenues for modeling and 29 visualizing higher-order chromatin structure. 30 31 1-sentence summary: High-throughput single-molecule real-time footprinting of chromatin arrays reveals 32 heterogeneous patterns of oligonucleosome occupancy. 33 34

Single-fiber nucleosome density shapes the regulatory output of a mammalian chromatin remodeling enzyme

“…In previous work, we observed that epigenomic domains were highly heterogeneous with respect to chromatin fiber usage (Abdulhay et al, 2020). In light of this heterogeneity, we sought to determine whether SNF2h loss would consistently impact fiber usage patterns across the epigenome, in line with 'clamping' activity in vivo.…”

Chromatin accessibility profiling methods

“…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.…”

Simultaneous profiling of chromatin accessibility and methylation on human cell lines with nanopore sequencing

“…More recently, this technology was applied to exogenous labeling of chromatin accessibility in S. Cerevisiae , a unicellular eukaryotic model organism without endogenous methylation 9 , 10 . Others have demonstrated this application on the PacBio platform even in human cells, though they did not capture endogenous methylation 11 , 12 .…”