We report the isolation of bcl-x, a bcl-2-related gene that can function as a bcl-2-independent regulator of programmed cell death (apoptosis). Alternative splicing results in two distinct bcl-x mRNAs. The protein product of the larger mRNA, bcl-xL, is similar in size and predicted structure to Bcl-2. When stably transfected into an IL-3-dependent cell line, bcl-xL inhibits cell death upon growth factor withdrawal at least as well as bcl-2. Surprisingly, the second mRNA species, bcl-xS, encodes a protein that inhibits the ability of bcl-2 to enhance the survival of growth factor-deprived cells. In vivo, bcl-xS mRNA is expressed at high levels in cells that undergo a high rate of turnover, such as developing lymphocytes. In contrast, bcl-xL is found in tissues containing long-lived postmitotic cells, such as adult brain. Together these data suggest that bcl-x plays an important role in both positive and negative regulation of programmed cell death.

R-spondin proteins strongly potentiate Wnt signalling and function as stem-cell growth factors. Despite the biological and therapeutic significance, the molecular mechanism of R-spondin action remains unclear. Here we show that the cell-surface transmembrane E3 ubiquitin ligase zinc and ring finger 3 (ZNRF3) and its homologue ring finger 43 (RNF43) are negative feedback regulators of Wnt signalling. ZNRF3 is associated with the Wnt receptor complex, and inhibits Wnt signalling by promoting the turnover of frizzled and LRP6. Inhibition of ZNRF3 enhances Wnt/β-catenin signalling and disrupts Wnt/planar cell polarity signalling in vivo. Notably, R-spondin mimics ZNRF3 inhibition by increasing the membrane level of Wnt receptors. Mechanistically, R-spondin interacts with the extracellular domain of ZNRF3 and induces the association between ZNRF3 and LGR4, which results in membrane clearance of ZNRF3. These data suggest that R-spondin enhances Wnt signalling by inhibiting ZNRF3. Our study provides new mechanistic insights into the regulation of Wnt receptor turnover, and reveals ZNRF3 as a tractable target for therapeutic exploration.

Trimethylation on H3K27 (H3K27me3) mediated by Polycomb repressive complex 2 (PRC2) has been linked to embryonic stem cell (ESC) identity and pluripotency. EZH2, the catalytic subunit of PRC2, has been reported as the sole histone methyltransferase that methylates H3K27 and mediates transcriptional silencing. Analysis of Ezh2(-/-) ESCs suggests existence of an additional enzyme(s) catalyzing H3K27 methylation. We have identified EZH1, a homolog of EZH2 that is physically present in a noncanonical PRC2 complex, as an H3K27 methyltransferase in vivo and in vitro. EZH1 colocalizes with the H3K27me3 mark on chromatin and preferentially preserves this mark on development-related genes in Ezh2(-/-) ESCs. Depletion of Ezh1 in cells lacking Ezh2 abolishes residual methylation on H3K27 and derepresses H3K27me3 target genes, demonstrating a role of EZH1 in safeguarding ESC identity. Ezh1 partially complements Ezh2 in executing pluripotency during ESC differentiation, suggesting that cell-fate transitions require epigenetic specificity.

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Succinate acts as an extracellular mediator signaling through the G protein-coupled receptor GPR91. Here we show that dendritic cells had high expression of GPR91. In these cells, succinate triggered intracellular calcium mobilization, induced migratory responses and acted in synergy with Toll-like receptor ligands for the production of proinflammatory cytokines. Succinate also enhanced antigen-specific activation of human and mouse helper T cells. GPR91-deficient mice had less migration of Langerhans cells to draining lymph nodes and impaired tetanus toxoid-specific recall T cell responses. Furthermore, GPR91-deficient allografts elicited weaker transplant rejection than did the corresponding grafts from wild-type mice. Our results suggest that the succinate receptor GPR91 is involved in sensing immunological danger, which establishes a link between immunity and a metabolite of cellular respiration.

LGR4/5 receptors and their cognate RSPO ligands potentiate Wnt/β-catenin signalling and promote proliferation and tissue homeostasis in epithelial stem cell compartments. In the liver, metabolic zonation requires a Wnt/β-catenin signalling gradient, but the instructive mechanism controlling its spatiotemporal regulation is not known. We have now identified the RSPO-LGR4/5-ZNRF3/RNF43 module as a master regulator of Wnt/β-catenin-mediated metabolic liver zonation. Liver-specific LGR4/5 loss of function (LOF) or RSPO blockade disrupted hepatic Wnt/β-catenin signalling and zonation. Conversely, pathway activation in ZNRF3/RNF43 LOF mice or with recombinant RSPO1 protein expanded the hepatic Wnt/β-catenin signalling gradient in a reversible and LGR4/5-dependent manner. Recombinant RSPO1 protein increased liver size and improved liver regeneration, whereas LGR4/5 LOF caused the opposite effects, resulting in hypoplastic livers. Furthermore, we show that LGR4(+) hepatocytes throughout the lobule contribute to liver homeostasis without zonal dominance. Taken together, our results indicate that the RSPO-LGR4/5-ZNRF3/RNF43 module controls metabolic liver zonation and is a hepatic growth/size rheostat during development, homeostasis and regeneration.

In eukaryotes, Suv39h H3K9 trimethyltransferases are required for pericentric heterochromatin formation and function. In early mouse preimplantation embryos, however, paternal pericentric heterochromatin lacks Suv39h-mediated H3K9me3 and downstream marks. Here we demonstrate Ezh2-independent targeting of maternally provided polycomb repressive complex 1 (PRC1) components to paternal heterochromatin. In Suv39h2 maternally deficient zygotes, PRC1 also associates with maternal heterochromatin lacking H3K9me3, thereby revealing hierarchy between repressive pathways. In Rnf2 maternally deficient zygotes, the PRC1 complex is disrupted, and levels of pericentric major satellite transcripts are increased at the paternal but not the maternal genome. We conclude that in early embryos, Suv39h-mediated H3K9me3 constitutes the dominant maternal transgenerational signal for pericentric heterochromatin formation. In absence of this signal, PRC1 functions as the default repressive back-up mechanism. Parental epigenetic asymmetry, also observed along cleavage chromosomes, is resolved by the end of the 8-cell stage--concurrent with blastomere polarization--marking the end of the maternal-to-embryonic transition.

Effective use of conditional Cre recombinase-loxP gene modification requires Cre-expressing mouse strains with defined patterns of expression. To assess the in vivo functionality of Cre-expressing mice, we have engineered an improved reporter strain for monitoring Cre-mediated excisions. The ␤-galactosidase-neomycin phosphotransferase fusion gene (␤geo)-trapped ROSA26 locus was modified by gene targeting such that ␤geo is expressed only after Cremediated excision of loxP-f lanked DNA sequences. ␤geo from the excised ROSA26 allele is expressed ubiquitously in embryos and adult mice. By mating the reporter strain with Cre-expressing transgenic mice, we have shown that the loxP-f lanked ROSA26 allele is accessible to Cre during early embryogenesis, as well as in a specific hematopoietic lineage (T lymphocytes). This improved reporter strain should facilitate monitoring in vivo Cre-mediated excision events in a variety of experimental contexts.