14 15 Background: Topologically associating domains (TADs) are fundamental units of three-16 dimensional (3D) nuclear organization. The regions bordering TADs-TAD boundaries-17 regulate gene expression by restricting interactions of cis-regulatory sequences to their target 18genes. TAD and TAD boundary disruption have been implicated in rare disease pathogenesis; 19 however, we have a limited framework for integrating cross-cell-type TAD maps into the study of 20 genome evolution and interpretation of common trait-associated variants. Here, we investigate 21 an unexplored attribute of 3D genome architecture-the stability of TAD boundaries across cell 22 types-and demonstrate its relevance to understanding how genetic variation in TADs 23 contributes to complex disease.
24Results: By synthesizing TAD maps across 37 diverse cell types with multiple annotations and 25 41 genome-wide association studies (GWAS), we investigate the differences in function and 26 evolutionary constraint between TADs versus TAD boundaries. We demonstrate that TAD 27 boundaries are more evolutionarily constrained than TADs. We find that boundaries are 28 enriched for disease heritability, especially for immunologic, hematologic, and metabolic traits. 29 Next, we stratify boundaries by their stability across cell types and show that more than 30% of 30 boundaries are unique to a specific tissue. Compared to these unique boundaries, cell-type-31 stable boundaries are enriched for CTCF binding, evolutionary constraint, housekeeping genes, 32 and trait heritability, suggesting greater functional importance. 33 Conclusions: Cell-type-stable TAD boundaries have greater sequence-level constraint and 34 complex trait heritability enrichment than cell-type-unique boundaries and TADs overall. Thus, 35 considering TAD and boundary stability across cell types provides valuable context for 36 understanding the genome's functional landscape and enabling 3D-structural aware variant 37 interpretation. 38 39 2 BACKGROUND 40 41 The three-dimensional (3D) conformation of the genome facilitates the regulation of gene 42 expression. 1-4 Using chromosome conformation capture technologies (3C, 4C, 5C, Hi-C), 5-7 43recent studies have demonstrated that modulation of gene expression via 3D chromatin 44 structure is important for many physiologic and pathologic cellular functions, including cell type 45 identity, cellular differentiation, and risk for multiple rare diseases and cancer. [8][9][10][11][12][13] Nonetheless, 46 many fundamental questions remain about the functions of and evolutionary constraints on 3D 47 74 associating" TADs and the "insulatory" boundaries. For example, in cross-species multiple 75 sequence alignments, syntenic break enrichment at TAD boundaries suggests a long-term 76 evolutionary preference for rearrangements that "shuffle" intact TADs, rather than "break" 77 them. 25,26 Additionally, TADs often contain clusters of co-regulated genes-e.g., cytochrome 78 genes and olfactory receptors. 7,20,27 Intra-TAD structural variation...