Cross–Cell-Line Conservation-Resolved Interactions Reveal a Stepwise Cascade of CTCF Loop Formation

The three-dimensional organization of the genome is shaped by CTCF-mediated chromatin loops, which vary widely in strength, conservation, and cell-type specificity. A longstanding model proposes that highly conserved loops form autonomously and establish a structural scaffold that supports the subsequent formation of less conserved, cell-type-specific interactions. However, genome-wide evidence for an ordered dependency among loops of different conservation levels has remained limited. Here, we tested this hierarchical model using high-resolution CTCF ChIA-PET data from eight human cell lines. We developed a two-stage predictive framework in which high-confidence loops were first identified using sequence features, chromatin context, and cross-cell-line conservation, and then augmented with neighboring-interaction features that quantify the influence of pre-existing loops at nearby CTCF anchors. Incorporation of neighboring-interaction information resulted in only modest overall improvements in predictive performance, consistent with the largely autonomous formation of highly conserved loops. To directly assess hierarchical dependencies, we stratified loops into eight conservation classes based on their recurrence across cell lines and systematically evaluated how neighboring interactions from each class contributed to loop prediction in others. This conservation-resolved analysis revealed a strongly ordered pattern: loops within a given conservation class were most strongly influenced by neighboring loops from the immediately more conserved class, with weaker effects from less conserved neighbors and minimal contributions from more distant classes. In contrast, the most highly conserved loops exhibited little dependence on local loop context. Together, these results provide quantitative genome-wide support for a hierarchical, directional model of CTCF loop establishment, in which conserved loops form independently and subsequently facilitate the emergence of progressively less conserved interactions. More broadly, the neighboring-interaction framework presented here offers a generalizable approach for inferring the order and directionality of chromatin loop formation, with potential applications to developmental processes and disease-associated genome reorganization.