Kilobase-scale compartments enabled by CRUSH reveal regulatory programs across cell types, single-cells, and ancient mammoths

Achyuth Kalluchi, Ben Nolan, Timothy E. Reznicek, Hannah L. Harris, Devika Udupa, Christopher T. Cummings, M. Jordan Rowley
2026 · doi:10.64898/2026.04.25.719469
preprint OA: closed CC-BY-4.0
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Abstract

How chromatin is spatially organized in the nucleus has long been studied through the lens of large-scale A/B compartments, but whether these sizes reflect true biological units or analytical artifacts has remained unclear. We find that limits imposed by the conventional eigenvector-based compartment calling have required extreme sequencing depth and coarse resolution, obscuring regulatory-scale organization. We developed CRUSH to iteratively refine compartments to 1 kb resolution without the need for extreme sequencing depth, we show that kilobase-scale A/B segregation (micro-compartments) is evident across cell types. Across these maps, we demonstrate that RNA polymerase II pausing contributes to a sub-genic compartment signature at the transcription start site and that active enhancers almost universally occupy the A compartment. We then show that fine-scale compartment maps can resolve cancer subtype-specific regulatory programs, single-cell tissue identity, and cold-adaptation regulomes in a 52,000-year-old woolly mammoth. These findings establish chromatin compartmentalization as a gene-scale regulatory feature with broad implications for development, disease, and genome evolution. Graphical Abstract CRUSH uses iterative resolution walking to identify and correct A/B compartment measurements. This allows identification of A/B compartments at kilobase resolution with low sequencing depth and consistency across methodologies. Kilobase-scale compartments reveal sub-genic compartment organization, compartment identities in single-cells, and a distinct cold-adapted regulome in mammoths.
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Abstract How chromatin is spatially organized in the nucleus has long been studied through the lens of large-scale A/B compartments, but whether these sizes reflect true biological units or analytical artifacts has remained unclear. We find that limits imposed by the conventional eigenvector-based compartment calling have required extreme sequencing depth and coarse resolution, obscuring regulatory-scale organization. We developed CRUSH to iteratively refine compartments to 1 kb resolution without the need for extreme sequencing depth, we show that kilobase-scale A/B segregation (micro-compartments) is evident across cell types. Across these maps, we demonstrate that RNA polymerase II pausing contributes to a sub-genic compartment signature at the transcription start site and that active enhancers almost universally occupy the A compartment. We then show that fine-scale compartment maps can resolve cancer subtype-specific regulatory programs, single-cell tissue identity, and cold-adaptation regulomes in a 52,000-year-old woolly mammoth. These findings establish chromatin compartmentalization as a gene-scale regulatory feature with broad implications for development, disease, and genome evolution. Graphical AbstractCRUSH uses iterative resolution walking to identify and correct A/B compartment measurements. This allows identification of A/B compartments at kilobase resolution with low sequencing depth and consistency across methodologies. Kilobase-scale compartments reveal sub-genic compartment organization, compartment identities in single-cells, and a distinct cold-adapted regulome in mammoths. Competing Interest Statement The authors have declared no competing interest.

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[{'doi': None, 'name': None, 'awards': ['R35GM147467']}, {'doi': None, 'name': 'Child Health Research Institute, Omaha, NE', 'awards': []}]

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License: CC-BY-4.0