Imaging and chromosome conformation capture studies have revealed several layers of chromosome organization, including segregation into megabase-large active and inactive compartments, and partitioning into sub-megabase domains (TADs). Yet, it remains unclear how these layers of organization form, interact with one another and impact genome functions. Here, we show that deletion of the cohesin-loading factor Nipbl, in mouse liver, leads to a dramatic reorganization of chromosomal folding. TADs and associated peaks vanish globally, even in the absence of transcriptional changes. In contrast, compartmental segregation is preserved and even reinforced. Strikingly, the disappearance of TADs unmasks a finer compartment structure that accurately reflects the underlying epigenetic landscape. These observations demonstrate that the 3D organization of the genome results from the interplay of two independent mechanisms: 1) cohesin-independent segregation of the genome into fine-scale compartments, defined by chromatin state; 2) cohesin-dependent formation of TADs, possibly by loop extrusion, which contributes to guide distant enhancers to their target genes.

Chromosome conformation capture methods have identified subchromosomal structures of higher-order chromatin interactions called topologically associated domains (TADs) that are separated from each other by boundary regions. By subdividing the genome into discrete regulatory units, TADs restrict the contacts that enhancers establish with their target genes. However, the mechanisms that underlie partitioning of the genome into TADs remain poorly understood. Here we show by chromosome conformation capture (capture Hi-C and 4C-seq methods) that genomic duplications in patient cells and genetically modified mice can result in the formation of new chromatin domains (neo-TADs) and that this process determines their molecular pathology. Duplications of non-coding DNA within the mouse Sox9 TAD (intra-TAD) that cause female to male sex reversal in humans, showed increased contact of the duplicated regions within the TAD, but no change in the overall TAD structure. In contrast, overlapping duplications that extended over the next boundary into the neighbouring TAD (inter-TAD), resulted in the formation of a new chromatin domain (neo-TAD) that was isolated from the rest of the genome. As a consequence of this insulation, inter-TAD duplications had no phenotypic effect. However, incorporation of the next flanking gene, Kcnj2, in the neo-TAD resulted in ectopic contacts of Kcnj2 with the duplicated part of the Sox9 regulatory region, consecutive misexpression of Kcnj2, and a limb malformation phenotype. Our findings provide evidence that TADs are genomic regulatory units with a high degree of internal stability that can be sculptured by structural genomic variations. This process is important for the interpretation of copy number variations, as these variations are routinely detected in diagnostic tests for genetic disease and cancer. This finding also has relevance in an evolutionary setting because copy-number differences are thought to have a crucial role in the evolution of genome complexity.

Long-range regulatory interactions play an important role in shaping gene-expression programs. However, the genomic features that organize these activities are still poorly characterized. We conducted a large operational analysis to chart the distribution of gene regulatory activities along the mouse genome, using hundreds of insertions of a regulatory sensor. We found that enhancers distribute their activities along broad regions and not in a gene-centric manner, defining large regulatory domains. Remarkably, these domains correlate strongly with the recently described TADs, which partition the genome into distinct self-interacting blocks. Different features, including specific repeats and CTCF-binding sites, correlate with the transition zones separating regulatory domains, and may help to further organize promiscuously distributed regulatory influences within large domains. These findings support a model of genomic organization where TADs confine regulatory activities to specific but large regulatory domains, contributing to the establishment of specific gene expression profiles.

Biliary epithelial cells (BECs) form bile ducts in the liver and are facultative liver stem cells that establish a ductular reaction (DR) to support liver regeneration following injury. Liver damage induces periportal LGR5+ putative liver stem cells that can form BEClike organoids, suggesting that RSPO-LGR4/5-mediated WNT/b-catenin activity is important for a DR. We addressed the roles of this and other signaling pathways in a DR by performing a focused CRISPRbased loss-of-function screen in BEC-like organoids, followed by in vivo validation and single-cell RNA sequencing. We found that BECs lack and do not require LGR4/5-mediated WNT/b-catenin signaling during a DR, whereas YAP and mTORC1 signaling are required for this process. Upregulation of AXIN2 and LGR5 is required in hepatocytes to enable their regenerative capacity in response to injury. Together, these data highlight heterogeneity within the BEC pool, delineate signaling pathways involved in a DR, and clarify the identity and roles of injury-induced periportal LGR5+ cells.