Single-molecule sequencing to uncover high-resolution epigenome patterns

Nearly all essential nuclear processes act on DNA packaged into series of nucleosomes termed chromatin fibers. However, our understanding of how these processes (e.g. DNA replication, RNA transcription, chromatin extrusion, nucleosome remodeling) actually occur on such fibers remains unresolved. Our current 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 fibers. SAMOSA data allows unbiased classification of single-molecular ’states’ of nucleosome occupancy on individual chromatin fibers. We leverage this to estimate nucleosome regularity and spacing on single chromatin fibers 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. ATP-dependent chromatin remodelers are one of the major regulators of patterns within the epigenome. As a follow-up from our proof-of-concept study, we developed SAMOSA-ChAAT, a massively multiplex single-molecule footprinting platform to map the primary structure of individual, precisely-reconstituted chromatin templates subjected to virtually any chromatin-associated reaction. As proof-of-concept, we apply SAMOSA-ChAAT to study ATP-dependent chromatin remodeling by the essential imitation switch (ISWI) ATPase SNF2h, whose mechanism-of-action remains contentious. Using our approach, we discover that SNF2h operates as a density-dependent, length-sensing nucleosome sliding enzyme, whose ability to decrease or increase DNA accessibility depends on single-fiber nucleosome density. We validate our in vitro findings with single-fiber accessibility measurements in vivo, finding that the regulatory ‘mode’ of SNF2h-containing complexes (i.e. ‘opening’ vs. ‘closing’ chromatin) is dictated by the underlying nucleosome-density of individual chromatin fibers: at canonically-defined heterochromatin, SNF2h generates evenly-spaced nucleosome arrays of multiple nucleosome repeat lengths; at SNF2h-dependent accessible sites, the enzyme slides nucleosomes to increase accessibility of motifs for the essential transcription factor CTCF. Our approach and data demonstrate, for the first time, how chromatin remodelers can effectively sense nucleosome density to induce diametrically-opposed regulatory effects within the nucleus. More generally, our novel approach promises molecularly-precise views of any of the essential processes shaping nuclear physiology