Mammalian DNA folds into 3D structures that facilitate and regulate genetic processes such as transcription, DNA repair, and epigenetics. Several insights derive from chromosome capture methods, such as Hi-C, which allow researchers to construct contact maps depicting 3D interactions among all DNA segment pairs. These maps show a complex cross-scale organization spanning megabase-pair compartments to short-ranged DNA loops. To better understand the organizing principles, several groups analyzed Hi-C data assuming a Russian-doll-like nested hierarchy where DNA regions of similar sizes merge into larger and larger structures. Apart from being a simple and appealing description, this model explains, e.g., the omnipresent chequerboard pattern seen in Hi-C maps, known as A/B compartments, and foreshadows the co-localization of some functionally similar DNA regions. However, while successful, this model is incompatible with the two competing mechanisms that seem to shape a significant part of the chromosomes' 3D organization: loop extrusion and phase separation. This paper aims to map out the chromosome's actual folding hierarchy from empirical data. To this end, we take advantage of Hi-C experiments and treat the measured DNA-DNA interactions as a weighted network. From such a network, we extract 3D communities using the generalized Louvain algorithm. This algorithm has a resolution parameter that allows us to scan seamlessly through the community size spectrum, from A/B compartments to topologically associated domains (TADs). By constructing a hierarchical tree connecting these communities, we find that chromosomes are more complex than a perfect hierarchy. Analyzing how communities nest relative to a simple folding model, we found that chromosomes exhibit a significant portion of nested and non-nested community pairs alongside considerable randomness. In addition, by examining nesting and chromatin types, we discovered that nested parts are often associated with active chromatin. These results highlight that cross-scale relationships will be essential components in models aiming to reach a deep understanding of the causal mechanisms of chromosome folding. Author summaryThe 3D organization of mammalian DNA affects genetic processes, such as transcription, DNA repair, and epigenetics. To unravel the complexity of the 3D structure, researchers developed numerous experimental methods, the most advanced being Hi-C. This method enables scientists to create "contact maps" illustrating the 3D interactions among all pairs of DNA segments across the genome. These maps unveiled a multi-scale organization, ranging from megabase-pair compartments to short-range DNA loops. Common explanations for this organization rest on nested hierarchies, where DNA regions of similar sizes coalesce into larger structures. However, such a model is incompatible with competing molecular mechanisms, primarily loop extrusion and phase separation, that shape the chromosomes' 3D organization at different scales.Our study aims to map out the actual chromosome folding relationships using Hi-C data sets. Treating the data as a weighted network of DNA-DNA interactions, we identified 3D communities across different network scales using the Generalized Louvain method, a standard community detection algorithm. By building a tree linking these communities, we discovered that chromosome organization is more intricate than a perfect hierarchy suggests. Instead, we found that chromosomes exhibit a mix of nested and non-nested community pairs alongside considerable randomness. The nested parts often associate with active chromatin. These results highlight that cross-scale relationships are critical for understanding the causal mechanisms of chromosome folding.
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