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Many individual genetic risk loci have been associated with multiple common human diseases. However, the molecular basis of this pleiotropy often remains unclear. We present an integrative approach to reveal the molecular mechanism underlying the PROCR locus, associated with lower coronary artery disease (CAD) risk but higher venous thromboembolism (VTE) risk. We identify PROCR-p.Ser219Gly as the likely causal variant at the locus and protein C as a causal factor. Using genetic analyses, human recall-by-genotype and in vitro experimentation, we demonstrate that PROCR-219Gly increases plasma levels of (activated) protein C through endothelial protein C receptor (EPCR) ectodomain shedding in endothelial cells, attenuating leukocyte–endothelial cell adhesion and vascular inflammation. We also associate PROCR-219Gly with an increased pro-thrombotic state via coagulation factor VII, a ligand of EPCR. Our study, which links PROCR-219Gly to CAD through anti-inflammatory mechanisms and to VTE through pro-thrombotic mechanisms, provides a framework to reveal the mechanisms underlying similar cross-phenotype associations.

Funder: National Key R&D Program of China (grant number 2017YFC1600100) National Key Program for Infectious Diseases of China (no. 2012ZX10004215) National Nature Science Foundation of China (no. 82173580) National Nature Science Foundation of China (no. 81872678) Chinese Academy of Medical Sciences (CAMS) Innovation Fund for Medical Sciences (2021-I2M-1-044), Antimicrobial resistance of Shigella sonnei has become a global concern. Here, we report a phylogenetic group of S. sonnei with extensive drug resistance, including a combination of multidrug resistance, coresistance to ceftriaxone and azithromycin (cefRaziR), reduced susceptibility to fluoroquinolones, and even colistin resistance (colR). This distinct clone caused six waterborne shigellosis outbreaks in China from 2015 to 2020. We collect 155 outbreak isolates and 152 sporadic isolates. The cefRaziR isolates, including outbreak strains, are mainly distributed in a distinct clade located in global Lineage III. The outbreak strains form a recently derived monophyletic group that may have emerged circa 2010. The cefRaziR and colR phenotypes are attributed to the acquisition of different plasmids, particularly the IncB/O/K/Z plasmid coharboring the blaCTX-M-14, mphA, aac(3)-IId, dfrA17, aadA5, and sul1 genes and the IncI2 plasmid with an mcr-1 gene. Genetic analyses identify 92 accessory genes and 60 single-nucleotide polymorphisms associated with the cefRaziR phenotype. Surveillance of this clone is required to determine its dissemination and threat to global public health.

Hotspot mutations in the PEST-domain of NOTCH1 and NOTCH2 are recurrently identified in B cell malignancies. To address how NOTCH-mutations contribute to a dismal prognosis, we have generated isogenic primary human tumor cells from patients with Chronic Lymphocytic Leukemia (CLL) and Mantle Cell Lymphoma (MCL), differing only in their expression of the intracellular domain (ICD) of NOTCH1 or NOTCH2. Our data demonstrate that both NOTCH-paralogs facilitate immune-escape of malignant B cells by up-regulating PD-L1, partly dependent on autocrine interferon-γ signaling. In addition, NOTCH-activation causes silencing of the entire HLA-class II locus via epigenetic regulation of the transcriptional co-activator CIITA. Notably, while NOTCH1 and NOTCH2 govern similar transcriptional programs, disease-specific differences in their expression levels can favor paralog-specific selection. Importantly, NOTCH-ICD also strongly down-regulates the expression of CD19, possibly limiting the effectiveness of immune-therapies. These NOTCH-mediated immune escape mechanisms are associated with the expansion of exhausted CD8+ T cells in vivo.

Funder: This work was supported by Cancer Research UK (MdlR (A22257), FB, LMOB, CK, H-CC, VC); Sir Henry Dale Fellowship jointly funded by the Wellcome Trust and the Royal Society (MdlR (WT107609); LMOB); Gates Cambridge Trust (AK); JH is undertaking a PhD funded by the Cambridge School of Clinical Medicine, Frank Edward Elmore Fund and the Medical Research Council’s Doctoral Training Partnership (award reference: 1954837). TA is grateful for the support from the Austrian Science Fund (FWF P33070) and the European Research Council (ERC – STG: 101039320)., Th17 cells are key drivers of autoimmune disease. However, the signaling pathways regulating Th17 polarization are poorly understood. Hedgehog signaling regulates cell fate decisions during embryogenesis and adult tissue patterning. Here we find that cell-autonomous Hedgehog signaling, independent of exogenous ligands, selectively drives the polarization of Th17 cells but not other T helper cell subsets. We show that endogenous Hedgehog ligand, Ihh, signals to activate both canonical and non-canonical Hedgehog pathways through Gli3 and AMPK. We demonstrate that Hedgehog pathway inhibition with either the clinically-approved small molecule inhibitor vismodegib or genetic ablation of Ihh in CD4+ T cells greatly diminishes disease severity in two mouse models of intestinal inflammation. We confirm that Hedgehog pathway expression is upregulated in tissue from human ulcerative colitis patients and correlates with Th17 marker expression. This work implicates Hedgehog signaling in Th17 polarization and intestinal immunopathology and indicates the potential therapeutic use of Hedgehog inhibitors in the treatment of inflammatory bowel disease.

Transmission of epigenetic information between generations occurs in nematodes, flies and plants, mediated by specialised small RNA pathways, modified histones and DNA methylation. Similar processes in mammals can also affect phenotype through intergenerational or trans-generational mechanisms. Here we generate a luciferase knock-in reporter mouse for the imprinted Dlk1 locus to visualise and track epigenetic fidelity across generations. Exposure to high-fat diet in pregnancy provokes sustained re-expression of the normally silent maternal Dlk1 in offspring (loss of imprinting) and increased DNA methylation at the somatic differentially methylated region (sDMR). In the next generation heterogeneous Dlk1 mis-expression is seen exclusively among animals born to F1-exposed females. Oocytes from these females show altered gene and microRNA expression without changes in DNA methylation, and correct imprinting is restored in subsequent generations. Our results illustrate how diet impacts the foetal epigenome, disturbing canonical and non-canonical imprinting mechanisms to modulate the properties of successive generations of offspring.

Funder: Wellcome Trust, Many individual genetic risk loci have been associated with multiple common human diseases. However, the molecular basis of this pleiotropy often remains unclear. We present an integrative approach to reveal the molecular mechanism underlying the PROCR locus, associated with lower coronary artery disease (CAD) risk but higher venous thromboembolism (VTE) risk. We identify PROCR-p.Ser219Gly as the likely causal variant at the locus and protein C as a causal factor. Using genetic analyses, human recall-by-genotype and in vitro experimentation, we demonstrate that PROCR-219Gly increases plasma levels of (activated) protein C through endothelial protein C receptor (EPCR) ectodomain shedding in endothelial cells, attenuating leukocyte-endothelial cell adhesion and vascular inflammation. We also associate PROCR-219Gly with an increased pro-thrombotic state via coagulation factor VII, a ligand of EPCR. Our study, which links PROCR-219Gly to CAD through anti-inflammatory mechanisms and to VTE through pro-thrombotic mechanisms, provides a framework to reveal the mechanisms underlying similar cross-phenotype associations.

Funder: Herchel Smith Funds, The establishment of cell identity during embryonic development involves the activation of specific gene expression programmes and is underpinned by epigenetic factors including DNA methylation and histone post-translational modifications. G-quadruplexes are four-stranded DNA secondary structures (G4s) that have been implicated in transcriptional regulation and cancer. Here, we show that G4s are key genomic structural features linked to cellular differentiation. We find that G4s are highly abundant in human embryonic stem cells and are lost during lineage specification. G4s are prevalent in enhancers and promoters. G4s that are found in common between embryonic and downstream lineages are tightly linked to transcriptional stabilisation of genes involved in essential cellular functions as well as transitions in the histone post-translational modification landscape. Furthermore, the application of small molecules that stabilise G4s causes a delay in stem cell differentiation, keeping cells in a more pluripotent-like state. Collectively, our data highlight G4s as important epigenetic features that are coupled to stem cell pluripotency and differentiation.

Funder: Center for Trophoblast Research, The mechanism behind transgenerational epigenetic inheritance is unclear, particularly through the maternal grandparental line. We previously showed that disruption of folate metabolism in mice by the Mtrr hypomorphic mutation results in transgenerational epigenetic inheritance of congenital malformations. Either maternal grandparent can initiate this phenomenon, which persists for at least four wildtype generations. Here, we use genome-wide approaches to reveal genetic stability in the Mtrr model and genome-wide differential DNA methylation in the germline of Mtrr mutant maternal grandfathers. We observe that, while epigenetic reprogramming occurs, wildtype grandprogeny and great grandprogeny exhibit transcriptional changes that correlate with germline methylation defects. One region encompasses the Hira gene, which is misexpressed in embryos for at least three wildtype generations in a manner that distinguishes Hira transcript expression as a biomarker of maternal phenotypic inheritance.

Senescence is a state of stable proliferative arrest, generally accompanied by the senescence-associated secretory phenotype, which modulates tissue homeostasis. Enhancer-promoter interactions, facilitated by chromatin loops, play a key role in gene regulation but their relevance in senescence remains elusive. Here, we use Hi-C to show that oncogenic RAS-induced senescence in human diploid fibroblasts is accompanied by extensive enhancer-promoter rewiring, which is closely connected with dynamic cohesin binding to the genome. We find de novo cohesin peaks often at the 3′ end of a subset of active genes. RAS-induced de novo cohesin peaks are transcription-dependent and enriched for senescence-associated genes, exemplified by IL1B, where de novo cohesin binding is involved in new loop formation. Similar IL1B induction with de novo cohesin appearance and new loop formation are observed in terminally differentiated macrophages, but not TNFα-treated cells. These results suggest that RAS-induced senescence represents a cell fate determination-like process characterised by a unique gene expression profile and 3D genome folding signature, mediated in part through cohesin redistribution on chromatin., Senescence is a state of stable proliferative arrest. Here, the authors perform Hi-C analysis on oncogenic RAS-induced senescence in human fibroblasts and characterize the changes in the 3D genome folding associated with the senescence-specific gene expression profile, which are mediated in part through cohesin redistribution on chromatin.

Little is known about the impact of pretreatment drug resistance (PDR) on the efficacy of second generation integrase inhibitors. We sequenced pretreatment plasma specimens from the ADVANCE trial (NCT03122262). Our primary outcome was 96-week virologic success, defined as a sustained viral load, Here the authors combine next generation sequencing on plasma from participants of the ADVANCE clinical trial with virological and follow-up data to investigate the impact of pre-treatment drug resistance (PDR) to non-nucleoside reverse transcriptase inhibitors (NNRTIs) on the efficacy of second-generation integrase inhibitors and find an association between NNRTI resistance prior to treatment and long-term treatment.

Long non-coding RNAs (lncRNAs) are important regulatory molecules that are implicated in cellular physiology and pathology. In this work, we dissect the functional role of the HOXB-AS3 lncRNA in patients with NPM1-mutated (NPM1mut) acute myeloid leukemia (AML). We show that HOXB-AS3 regulates the proliferative capacity of NPM1mut AML blasts in vitro and in vivo. HOXB-AS3 is shown to interact with the ErbB3-binding protein 1 (EBP1) and guide EBP1 to the ribosomal DNA locus. Via this mechanism, HOXB-AS3 regulates ribosomal RNA transcription and de novo protein synthesis. We propose that in the context of NPM1 mutations, HOXB-AS3 overexpression acts as a compensatory mechanism, which allows adequate protein production in leukemic blasts., LncRNAs at the HOX gene locus are increased in expression in NPM1 mutant acute myeloid leukemia. In this study, the authors show that one such lncRNA, HOX3B-AS3, has a role in regulating gene transcription and protein synthesis in leukemia cells.

Funder: U.S. Department of Health & Human Services | NIH | NCI | Division of Cancer Epidemiology and Genetics, National Cancer Institute (National Cancer Institute Division of Cancer Epidemiology and Genetics), Long non-coding RNAs (lncRNAs) are important regulatory molecules that are implicated in cellular physiology and pathology. In this work, we dissect the functional role of the HOXB-AS3 lncRNA in patients with NPM1-mutated (NPM1mut) acute myeloid leukemia (AML). We show that HOXB-AS3 regulates the proliferative capacity of NPM1mut AML blasts in vitro and in vivo. HOXB-AS3 is shown to interact with the ErbB3-binding protein 1 (EBP1) and guide EBP1 to the ribosomal DNA locus. Via this mechanism, HOXB-AS3 regulates ribosomal RNA transcription and de novo protein synthesis. We propose that in the context of NPM1 mutations, HOXB-AS3 overexpression acts as a compensatory mechanism, which allows adequate protein production in leukemic blasts.

Funder: U.S. Department of Health & Human Services | NIH | NCI | Division of Cancer Epidemiology and Genetics, National Cancer Institute (National Cancer Institute Division of Cancer Epidemiology and Genetics), Long non-coding RNAs (lncRNAs) are important regulatory molecules that are implicated in cellular physiology and pathology. In this work, we dissect the functional role of the HOXB-AS3 lncRNA in patients with NPM1-mutated (NPM1mut) acute myeloid leukemia (AML). We show that HOXB-AS3 regulates the proliferative capacity of NPM1mut AML blasts in vitro and in vivo. HOXB-AS3 is shown to interact with the ErbB3-binding protein 1 (EBP1) and guide EBP1 to the ribosomal DNA locus. Via this mechanism, HOXB-AS3 regulates ribosomal RNA transcription and de novo protein synthesis. We propose that in the context of NPM1 mutations, HOXB-AS3 overexpression acts as a compensatory mechanism, which allows adequate protein production in leukemic blasts.