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.

Zinc finger protein Zscan4 is selectively expressed in mouse two-cell (2C) embryos undergoing zygotic genome activation (ZGA) and in a rare subpopulation of embryonic stem cells with 2C-like features. Here, we show that Zscan4 specifically recognizes a subset of (CA)n microsatellites, repeat sequences prone to genomic instability. Zscan4-associated microsatellite regions are characterized by low nuclease sensitivity and high histone occupancy. In vitro, Zscan4 binds nucleosomes and protects them from disassembly upon torsional strain. Furthermore, Zscan4 depletion leads to elevated DNA damage in 2C mouse embryos in a transcription-dependent manner. Together, our results identify Zscan4 as a DNA sequence–dependent microsatellite binding factor and suggest a developmentally regulated mechanism, which protects fragile genomic regions from DNA damage at a time of embryogenesis associated with high transcriptional burden and genomic stress.

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

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ATP-dependent chromatin remodelers regulate the DNA accessibility required of virtually all nuclear processes. Biochemical studies have provided insight into remodeler action at the nucleosome level, but how these findings translate to activity on chromatin fibers in vitro and in vivo remains poorly understood. Here, we present a massively multiplex single-molecule platform allowing high-resolution mapping of nucleosomes on fibers assembled on mammalian genomic sequences. We apply this method to distinguish between competing models for chromatin remodeling by the essential ISWI ATPase SNF2h: linker-length-dependent dynamic positioning versus fixed-linker-length static clamping. Our single-fiber data demonstrate that SNF2h operates as a density-dependent, length-sensing chromatin remodeler whose ability to decrease or increase DNA accessibility depends on single-fiber nucleosome density. In vivo, this activity manifests as different regulatory modes across epigenomic domains: at canonically-defined heterochromatin, SNF2h generates evenly-spaced nucleosome arrays of multiple nucleosome repeat lengths; at SNF2h-dependent accessible sites, SNF2h slides nucleosomes to increase accessibility of motifs for the essential transcription factor CTCF. Overall, our generalizable approach provides molecularly-precise views of the processes that shape nuclear physiology. Concurrently, our data illustrate how a mammalian chromatin remodeling enzyme can effectively sense nucleosome density to induce diametrically-opposed regulatory effects within the nucleus.