YAP, but Not RSPO-LGR4/5, Signaling in Biliary Epithelial Cells Promotes a Ductular Reaction in Response to Liver Injury.

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 BEC-like organoids, suggesting that RSPO-LGR4/5-mediated WNT/ß-catenin activity is important for a DR. We addressed the roles of this and other signaling pathways in a DR by performing a focused CRISPR-based 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/ß-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.

Formation of new chromatin domains determines pathogenicity of genomic duplications.

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.

A gradient of ROR2 protein stability and membrane localization confers brachydactyly type B or Robinow syndrome phenotypes.

Mutations in ROR2 cause dominant brachydactyly type B (BDB1) or recessive Robinow syndrome (RRS), each characterized by a distinct combination of phenotypic features. We here report a novel nonsense mutation in ROR2 (c.1324C>T; p.R441X) causing intracellular protein truncation in a patient exhibiting features of RRS in conjunction with severe recessive brachydactyly. The mutation is located at the same position as a previously described frame shift mutation causing dominant BDB1. To investigate the apparent discrepancy in phenotypic outcome, we analysed ROR2 protein stability and distribution in stably transfected cell lines expressing exact copies of several human RRS and BDB1 intracellular mutations. RRS mutant proteins were less abundant and retained intracellularly, although BDB1 mutants were stable and predominantly located at the cell membrane. The p.R441X mutation showed an intermediate pattern with membrane localization but also high endoplasmic reticulum retention. Furthermore, we observed a correlation between the severity of BDB1, the location of the mutation, and the amount of membrane-associated ROR2. Membrane protein fraction quantification revealed a gradient of distribution and stability correlating with the clinical phenotypes. This gradual model was confirmed by crossing mouse models for RRS and BDB1, yielding double heterozygous animals that exhibited an intermediate phenotype. We propose a model in which the RRS versus the BDB1 phenotype is determined by the relative degree of protein retention/degradation and the amount of mutant protein reaching the plasma membrane.