Single-molecule nucleosome spacing coordinates chromatin fiber interactions

Nucleosome spacing influences higher-order chromatin fiber organization in vitro but how this relates to cellular chromosome structure remains contentious. To address this, we developed Ligation Analysis of Single-molecule Sequence Interactions (LASSI), a single-molecule epigenomic method that combines proximity ligation with near-nucleotide resolution, long-read adenine methyltransferase footprinting. LASSI measures nucleosome spacing patterns on interacting segments of chromatin genome-wide in cells, quantifying coupled chromosome organization in 1D and 3D. Applying LASSI to mouse embryonic stem cells (mESCs), we discover a genome-wide structural pattern we term fiber homotypy, where chromatin fibers with similar nucleosome spacing patterns interact more frequently in 3D. This pattern persists over long intrachromosomal distances in cis and interchromosomally in trans. Fiber homotypy negatively scales with genomic distance, though differently than contact probability, implying distinct mechanisms. It is further promoted by topologically associated domains (TADs), A/B compartments, and shared histone modification domains, suggesting instructive roles for each of these processes. Coarse-grain molecular dynamics simulations of chromatin fibers informed by LASSI reveal that the intrinsically heterogeneous spacing of nucleosomes along chromatin fibers in cells is a key regulator of homotypy. This variability tunes the free energy landscape of chromatin fiber interactions, promoting the compartmentalization of similar 1D chromatin structures via specific types of fiber-fiber interaction. Further linking compartmentalization and fiber homotypy, we demonstrate that loop extrusion antagonizes this phenomenon. Depletion of the cohesin subunit RAD21 in mESCs increases fiber homotypy genome-wide, while depletion of the cohesin unloader WAPL decreases fiber homotypy, consistent with effects seen on A/B compartmentalization. Our results demonstrate that fiber-fiber interactions driven by shared nucleosome spacing patterns instruct higher-order chromosome organization. Moreover, we show clear structural interdependence across cellular chromatin length-scales, likely tuned by processes ranging from nucleosome positioning to loop extrusion.