The stability of cellular phenotypes in developing organisms depends on error-free transmission of epigenetic and genetic information during mitosis. Methylation of cytosine residues in genomic DNA is a key epigenetic mark that modulates gene expression and prevents genome instability. Here, we report on a genetic test of the relationship between DNA replication and methylation in the context of the developing vertebrate organism instead of cell lines. Our analysis is based on the identification of hypomorphic alleles of dnmt1 , encoding the DNA maintenance methylase Dnmt1, and pole1 , encoding the catalytic subunit of leading-strand DNA polymerase epsilon holoenzyme (Pole). Homozygous dnmt1 mutants exhibit genome-wide DNA hypomethylation, whereas the pole1 mutation is associated with increased DNA methylation levels. In dnmt1 / pole1 double-mutant zebrafish larvae, DNA methylation levels are restored to near normal values, associated with partial rescue of mutant-associated transcriptional changes and phenotypes. Hence, a balancing antagonism between DNA replication and maintenance methylation buffers against replicative errors contributing to the robustness of vertebrate development.

Unconventional T cells, such as innate natural killer T cells (iNKT) cells, are an important part of vertebrate immune defences. iNKT recognise glycolipids through a T cell receptor (TCR) that is composed of a semi-invariant TCR α chain, paired with a restricted set of TCR β chains. Here, we show that splicing of the cognate Trav11-Traj18-Trac pre-mRNA encoding the characteristic Vα14Jα18 variable region of this semi-invariant TCR depends on the presence of Tnpo3. The Tnpo3 gene encodes a nuclear transporter of the β-karyopherin family whose cargo includes various splice regulators. The block of iNKT cell development in the absence of Tnpo3 can be overcome by transgenic provision of a rearranged Trav11-Traj18-Trac cDNA, indicating that Tnpo3 deficiency does not interfere with the development of iNKT cells per se. Our study thus identifies a role for Tnpo3 in regulating the splicing of the pre-mRNA encoding the cognate TCRα chain of iNKT cells.

Bone marrow hematopoietic stem cells (HSCs) hold the unique capability to reconstitute the hematopoietic system. In inbred mice housed in gnotobiotic facilities, the top of the hematopoietic hierarchy is occupied by dormant HSCs. These dormant HSCs reversibly exit quiescence during stress. Whether HSC dormancy also exists in humans remains unclear. In this work, we show by single-cell RNA-seq analysis that human bone marrow HSCs are organized on an axis of dormancy. This dormancy axis is characterized by a gradual upregulation of processes such as translation, ribosomal biosynthesis, cell cycle and ROS. We identify the orphan receptor GPRC5C to enrich for human dormant HSCs. GPRC5C is also essential for HSC function, as demonstrated by genetic loss- and gain-of-function analyses. Through structural modelling and biochemical assays, we uncover that hyaluronic acid, a bone marrow extracellular matrix component, preserves dormancy through GPRC5C. This work identifies the hyaluronic acid–GPRC5C signalling axis as an essential component controlling the state of dormancy in HSCs. Now we are investigating the role of this signaling axis in the context of hematopoietic aging and leukemia. Bone marrow hematopoietic stem cells (HSCs) hold the unique capability to reconstitute the hematopoietic system. In inbred mice housed in gnotobiotic facilities, the top of the hematopoietic hierarchy is occupied by dormant HSCs. These dormant HSCs reversibly exit quiescence during stress. Whether HSC dormancy also exists in humans remains unclear. In this work, we show by single-cell RNA-seq analysis that human bone marrow HSCs are organized on an axis of dormancy. This dormancy axis is characterized by a gradual upregulation of processes such as translation, ribosomal biosynthesis, cell cycle and ROS. We identify the orphan receptor GPRC5C to enrich for human dormant HSCs. GPRC5C is also essential for HSC function, as demonstrated by genetic loss- and gain-of-function analyses. Through structural modelling and biochemical assays, we uncover that hyaluronic acid, a bone marrow extracellular matrix component, preserves dormancy through GPRC5C. This work identifies the hyaluronic acid–GPRC5C signalling axis as an essential component controlling the state of dormancy in HSCs. Now we are investigating the role of this signaling axis in the context of hematopoietic aging and leukemia.

Bone marrow haematopoietic stem cells (HSCs) are vital for lifelong maintenance of healthy haematopoiesis. In inbred mice housed in gnotobiotic facilities, the top of the haematopoietic hierarchy is occupied by dormant HSCs, which reversibly exit quiescence during stress. Whether HSC dormancy exists in humans remains debatable. Here, using single-cell RNA sequencing, we show a continuous landscape of highly purified human bone marrow HSCs displaying varying degrees of dormancy. We identify the orphan receptor GPRC5C, which enriches for dormant human HSCs. GPRC5C is also essential for HSC function, as demonstrated by genetic loss- and gain-of-function analyses. Through structural modelling and biochemical assays, we show that hyaluronic acid, a bone marrow extracellular matrix component, preserves dormancy through GPRC5C. We identify the hyaluronic acid–GPRC5C signalling axis controlling the state of dormancy in mouse and human HSCs.

Bone marrow (BM) hematopoietic stem cells (HSCs) are vital for the lifelong maintenance of the hematopoietic system. Experimental mice are housed in germ-free facilities, where the top of the hematopoietic hierarchy is occupied by dormant HSCs, which reversibly exit quiescence during stress. The existence of dormant HSC in humans remains debatable. Using single-cell RNA-seq analysis, we show a continuous landscape of highly purified human BM HSCs displaying varying degrees of dormancy. The orphan receptor GPRC5C was identified to enrich for dormant human HSC and is essential for HSC function, as demonstrated by genetic loss- and gain-of-function analyses. Through biochemical assays, we show that hyaluronic acid (HA), a BM extracellular matrix component, preserves dormancy through GPRC5C. We identify the HA–GPRC5C signalling axis controlling the state of dormancy in mouse and human HSCs. Leukemic stem cells (LSCs) share numerous features with healthy HSCs. Therefore we investigated the functionality of GPRC5C in the context of acute myeloid leukemia (AML). High Gprc5c levels accelerated disease development, while the loss of Gprc5c abrogated AML-initiating potential. Mechanistically, Gprc5c increased intracellular branched-chain amino acids (BCAAs) and the expression of BCAA transporters, Slc7a5 and Slc43a1. These results suggest the Gprc5c–Slc7a5/Slc43a1–BCAA cascade as a therapeutic target that can compromise leukemia function in AML. Indeed, targeting SLC7A5 with JPH203 elicited strong anti-leukemic effects in human AML by attenuating oxidative phosphorylation. This anti-leukemic effect was strengthened in the presence of venetoclax and azacitidine.Our results suggest that GPRC5C is functionally critical in both healthy and disease hematopoiesis.

To capture the global gene network regulating the differentiation of immature T cells in an unbiased manner, large-scale forward genetic screens in zebrafish were conducted and combined with genetic interaction analysis. After ENU mutagenesis, genetic lesions associated with failure of T cell development were identified by meiotic recombination mapping, positional cloning, and whole genome sequencing. Recessive genetic variants in 33 genes were identified and confirmed as causative by additional experiments. The mutations affected T cell development but did not perturb the development of an unrelated cell type, growth hormone-expressing somatotrophs, providing an important measure of cell-type specificity of the genetic variants. The structure of the genetic network encompassing the identified components was established by a subsequent genetic interaction analysis, which identified many instances of positive (alleviating) and negative (synthetic) genetic interactions. Several examples of synthetic lethality were subsequently phenocopied using combinations of small molecule inhibitors. These drugs not only interfered with normal T cell development, but also elicited remission in a model of T cell acute lymphoblastic leukaemia. Our findings illustrate how genetic interaction data obtained in the context of entire organisms can be exploited for targeted interference with specific cell types and their malignant derivatives.

DNA methylation is a universal epigenetic mechanism involved in regulation of gene expression and genome stability. The DNA maintenance methylase DNMT1 ensures that DNA methylation patterns are faithfully transmitted to daughter cells during cell division. Because loss of DNMT1 is lethal, a pan-organismic analysis of DNMT1 function is lacking. We identified new recessive dnmt1 alleles in medaka and zebrafish and, guided by the structures of mutant proteins, generated a recessive variant of mouse Dnmt1. Each of the three missense mutations studied here distorts the catalytic pocket and reduces enzymatic activity. Because all three DNMT1 mutant animals are viable, it was possible to examine their phenotypes throughout life. The consequences of genome-wide hypomethylation of DNA of somatic tissues in the Dnmt1 mutants are surprisingly mild but consistently affect the development of the lymphoid lineage. Our findings indicate that developing lymphocytes in vertebrates are sensitive to perturbations of DNA maintenance methylation.

The transcriptome of eukaryotic cells is constantly monitored for errors to avoid the production of undesired protein variants. The evolutionarily conserved nonsense-mediated mRNA decay (NMD) pathway degrades aberrant mRNAs, but also functions in the regulation of transcript abundance in response to changed physiological states. Here, we describe a zebrafish mutant of upf1, encoding the central component of the NMD machinery. Fish homozygous for the upf1(t20450) allele (Y163X) survive until day 10 after fertilization, presenting with impaired T cell development as one of the most conspicuous features of the mutant phenotype. Analysis of differentially expressed genes identified dysregulation of the premRNA splicing pathway, accompanied by perturbed autoregulation of canonical splicing activators (SRSF) and repressors (HNRNP). In upf1-deficient mutants, NMD-susceptible transcripts of ribosomal proteins that are known for their role as noncanonical splicing regulators were greatly increased, most notably, rpl10a. When the levels of NMD-susceptible rpl10a transcripts were artificially increased in zebrafish larvae, T cell development was significantly impaired, suggesting that perturbed autoregulation of rpl10a splicing contributes to failing T cell development in upf1 deficiency. Our results identify an extraribosomal tissue-specific function to rpl10a in the immune system, and thus exemplify the advantages of the zebrafish model to study the effects of upf1-deficiency in the context of a vertebrate organism.

Evidence for transgenerational inheritance of epigenetic information in vertebrates is scarce. Aberrant patterns of DNA methylation in gametes may set the stage for transmission into future generations. Here, we describe a viable hypomorphic allele of dnmt1 in zebrafish that causes widespread demethylation of CpG dinucleotides in sperm and somatic tissues. We find that homozygous mutants are essentially normal, with the exception of drastically impaired lymphopoiesis, affecting both larval and adult phases of T cell development. The phenotype of impaired larval (but not adult) T cell development is transmitted to subsequent generations by genotypically wildtype fish. We further find that about 200 differentially methylated regions in sperm DNA of transmitting and non-transmitting males, including hypermethylated sites associated with runx3 and rptor genes, whose reduced activities are associated with impaired larval T cell development. Our results indicate a particular sensitivity of larval T cell development to transgenerationally inherited epimutations.

In mammals, T cell development critically depends on the IL-7 cytokine signaling pathway. Here we describe the identification of the zebrafish ortholog of mammalian IL-7 based on chromosomal localization, deduced protein sequence, and expression patterns. To examine the biological role of il7 in teleosts, we generated an il7 allele lacking most of its coding exons using CRISPR/Cas9-based mutagenesis. il7-deficient animals are viable and exhibit no obvious signs of immune disorder. With respect to intrathymic T cell development, il7 deficiency is associated with only a mild reduction of thymocyte numbers, contrasting with a more pronounced impairment of T cell development in il7r-deficient fish. Genetic interaction studies between il7 and il7r mutants, and il7 and crlf2(tslpr) mutants suggest the contribution of additional, as-yet unidentified cytokines to intrathymic T cell development. Such activities were also ascertained for other cytokines, such as il2 and il15, collectively indicating that in contrast to the situation in mammals, T cell development in the thymus of teleosts is driven by a degenerate multicomponent network of γc cytokines; this explains why deficiencies of single components have little detrimental effect. In contrast, the dependence on a single cytokine in the mammalian thymus has catastrophic consequences in cases of congenital deficiencies in genes affecting the IL-7 signaling pathway. We speculate that the transition from a degenerate to a nonredundant cytokine network supporting intrathymic T cell development emerged as a consequence of repurposing evolutionarily ancient constitutive cytokine pathways for regulatory functions in the mammalian peripheral immune system.

Since the generation of cell-type specific knockout models, the importance of inter-cellular communication between neural, vascular, and microglial cells during neural development has been increasingly appreciated. However, the extent of communication between these major cell populations remains to be systematically mapped. Here, we describe EMBRACE (embryonic brain cell extraction using FACS), a method to simultaneously isolate neural, mural, endothelial, and microglial cells to more than 94% purity in ∼4 h. Utilizing EMBRACE we isolate, transcriptionally analyze, and build a cell-cell communication map of the developing mouse brain. We identify 1,710 unique ligand-receptor interactions between neural, endothelial, mural, and microglial cells in silico and experimentally confirm the APOE-LDLR, APOE-LRP1, VTN-KDR, and LAMA4-ITGB1 interactions in the E14.5 brain. We provide our data via the searchable "Brain interactome explorer", available at https://mpi-ie.shinyapps.io/braininteractomeexplorer/. Together, this study provides a comprehensive map that reveals the richness of communication within the developing brain.

SUMMARY:Due to the rapidly increasing scale and diversity of epigenomic data, modular and scalable analysis workflows are of wide interest. Here we present snakePipes, a workflow package for processing and downstream analysis of data from common epigenomic assays: ChIP-seq, RNA-seq, Bisulfite-seq, ATAC-seq, Hi-C and single-cell RNA-seq. snakePipes enables users to assemble variants of each workflow and to easily install and upgrade the underlying tools, via its simple command-line wrappers and yaml files.AVAILABILITY AND IMPLEMENTATION:snakePipes can be installed via conda: `conda install -c mpi-ie -c bioconda -c conda-forge snakePipes'. Source code (https://github.com/maxplanck-ie/snakepipes) and documentation (https://snakepipes.readthedocs.io/en/latest/) are available online.SUPPLEMENTARY INFORMATION:Supplementary data are available at Bioinformatics online.

Solid pseudopapillary neoplasms (SPN) of the pancreas are rare, low-grade malignant neoplasms that metastasise to the liver or peritoneum in 10-15% of cases. They almost invariably present somatic activating mutations of CTNNB1. No comprehensive molecular characterisation of metastatic disease has been conducted to date. We performed whole-exome sequencing and copy-number variation (CNV) analysis of 10 primary SPN and comparative sequencing of five matched primary/metastatic tumour specimens by high-coverage targeted sequencing of 409 genes. In addition to CTNNB1-activating mutations, we found inactivating mutations of epigenetic regulators (KDM6A, TET1, BAP1) associated with metastatic disease. Most of these alterations were shared between primary and metastatic lesions, suggesting that they occurred before dissemination. Differently from mutations, the majority of CNVs were not shared among lesions from the same patients and affected genes involved in metabolic and pro-proliferative pathways. Immunostaining of 27 SPNs showed that loss or reduction of KDM6A and BAP1 expression was significantly enriched in metastatic SPNs. Consistent with an increased transcriptional response to hypoxia in pancreatic adenocarcinomas bearing KDM6A inactivation, we showed that mutation or reduced KDM6A expression in SPNs is associated with increased expression of the HIF1α-regulated protein GLUT1 at both primary and metastatic sites. Our results suggest that BAP1 and KDM6A function is a barrier to the development of metastasis in a subset of SPNs, which might open novel avenues for the treatment of this disease. © 2018 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.

Cytidine deaminases of the AID/APOBEC family catalyze C-to-U nucleotide transitions in mRNA or DNA. Members of the APOBEC3 branch are involved in antiviral defense, whereas AID contributes to diversification of antibody repertoires in jawed vertebrates via somatic hypermutation, gene conversion, and class switch recombination. In the extant jawless vertebrate, the lamprey, two members of the AID/APOBEC family are implicated in the generation of somatic diversity of the variable lymphocyte receptors (VLRs). Expression studies linked CDA1 and CDA2 genes to the assembly of VLRA/C genes in T-like cells and the VLRB genes in B-like cells, respectively. Here, we identify and characterize several CDA1-like genes in the larvae of different lamprey species and demonstrate that these encode active cytidine deaminases. Structural comparisons of the CDA1 variants highlighted substantial differences in surface charge; this observation is supported by our finding that the enzymes require different conditions and substrates for optimal activity in vitro. Strikingly, we also found that the number of CDA-like genes present in individuals of the same species is variable. Nevertheless, irrespective of the number of different CDA1-like genes present, all lamprey larvae have at least one functional CDA1-related gene encoding an enzyme with predicted structural and chemical features generally comparable to jawed vertebrate AID. Our findings suggest that, similar to APOBEC3 branch expansion in jawed vertebrates, the AID/APOBEC family has undergone substantial diversification in lamprey, possibly indicative of multiple distinct biological roles.

Neuroendocrine tumours (NETs) may arise throughout the body and are a highly heterogeneous, relatively rare class of neoplasms difficult to study also for the lack of disease models. Despite this, knowledge on their molecular alterations has expanded in the latest years, also building from genetic syndromes causing their onset. Pancreatic NETs (PanNETs) have been among the most studied, and research so far has outlined a series of recurring features, as inactivation of MEN1, VHL, TSC1/2 genes and hyperactivation of the PI3K/mTOR pathway. Next-generation sequencing has added new information by showing the key role of alternative lengthening of telomeres, driven in a fraction of PanNETs by inactivation of ATRX/DAXX. Despite this accumulation of knowledge, single studies often relied on few cases or were limited to the DNA, RNA, protein or epigenetic level with lack of integrative analysis. The International Cancer Genome Consortium aimed at removing these barriers through a strict process of data and samples collection, to produce whole-genome integrated analyses for many tumour types. The results of this effort on PanNETs have been recently published and, while confirming previous observations provide a first snapshot of how heterogeneous is the combination of genetic alterations that drive this tumour type, yet converging into four pathways whose alteration has been enriched by newly discovered mechanisms. While calling for further integration of genetic and epigenetic analyses, these data allow to reconcile previous findings in a defined frame and may provide clinical research with markers for patients stratification and to guide targeted therapy decisions.

Next-generation sequencing (NGS) was applied to 148 lung neuroendocrine tumours (LNETs) comprising the four World Health Organization classification categories: 53 typical carcinoid (TCs), 35 atypical carcinoid (ACs), 27 large-cell neuroendocrine carcinomas, and 33 small-cell lung carcinomas. A discovery screen was conducted on 46 samples by the use of whole-exome sequencing and high-coverage targeted sequencing of 418 genes. Eighty-eight recurrently mutated genes from both the discovery screen and current literature were verified in the 46 cases of the discovery screen, and validated on additional 102 LNETs by targeted NGS; their prevalence was then evaluated on the whole series. Thirteen of these 88 genes were also evaluated for copy number alterations (CNAs). Carcinoids and carcinomas shared most of the altered genes but with different prevalence rates. When mutations and copy number changes were combined, MEN1 alterations were almost exclusive to carcinoids, whereas alterations of TP53 and RB1 cell cycle regulation genes and PI3K/AKT/mTOR pathway genes were significantly enriched in carcinomas. Conversely, mutations in chromatin-remodelling genes, including those encoding histone modifiers and members of SWI-SNF complexes, were found at similar rates in carcinoids (45.5%) and carcinomas (55.0%), suggesting a major role in LNET pathogenesis. One AC and one TC showed a hypermutated profile associated with a POLQ damaging mutation. There were fewer CNAs in carcinoids than in carcinomas; however ACs showed a hybrid pattern, whereby gains of TERT, SDHA, RICTOR, PIK3CA, MYCL and SRC were found at rates similar to those in carcinomas, whereas the MEN1 loss rate mirrored that of TCs. Multivariate survival analysis revealed RB1 mutation (p=0.0005) and TERT copy gain (p=0.016) as independent predictors of poorer prognosis. MEN1 mutation was associated with poor prognosis in AC (p=0.0045), whereas KMT2D mutation correlated with longer survival in SCLC (p=0.0022). In conclusion, molecular profiling may complement histology for better diagnostic definition and prognostic stratification of LNETs. (C) 2016 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.

Lymphocytes represent basic components of vertebrate adaptive immune systems, suggesting the utility of non-mammalian models to define the molecular basis of their development and differentiation. Our forward genetic screens in zebrafish for recessive mutations affecting early T cell development revealed several major genetic pathways. The identification of lineage-specific transcription factors and specific components of cytokine signaling and DNA replication and/or repair pathways known from studies of immunocompromisedmammals provided an evolutionary cross-validation of the screen design. Unexpectedly, however, genes encoding proteins required for pre-mRNA processing were enriched in the collection of mutants identified here. In both zebrafish and mice, deficiency of the splice regulator TNPO3 impairs intrathymic T cell differentiation, illustrating the evolutionarily conserved and cell-type-specific functions of certain pre-mRNA-processing factors for T cell development.

Background: Currently, no reliable blood-based assay for early detection of pancreatic ductal adenocarcinoma (PDAC) is available. Cell-free DNA (cfDNA) quantitation in patients' plasma has been recently applied in monitoring several cancer types. This study evaluates the diagnostic potential of cfDNA in PDAC patients.Methods: Plasma cfDNA levels and integrity ratio were assayed using quantitative real-time PCR of Alu-repeat amplicons in patients with pancreatic ductal adenocarcinoma (n=50), pancreatic neuroendocrine tumor (n=23), and chronic pancreatitis (n=20), as well as in healthy volunteers without evidence of pancreatic disease (n=23).Results: The total load of cfDNA, obtained by Alu83 quantitation, was the highest in PDAC patients than in any of the other patient groups (Welch t test; p<0.001) and was an average predictor of PDAC disease (AUC=0.664; CI, 0.56-0.77). A nonlinear association between Alu83 levels and subjects' age was detected (Spearman's rho=0.35; p<0.001) in the overall population, as well as within the PDAC patients' group (Spearman's rho=0.47; p<0.001). Necrosis-derived cfDNA fragments, quantitated with the Alu244 amplicon, were barely detectable in any of the samples and, in that respect, comparable between the different subject groups. CfDNA integrity estimation (Alu244/Alu83 ratio) was significantly affected by the limited detectability of plasma Alu244 levels.Conclusion: The lack of detectable levels of necrosis-derived cfDNA in pancreatic pathologies considerably affects the clinical use of such biomarker in PDAC patients. Different methods of analysis should be applied in the evaluation of the cfDNA diagnostic value in pancreas pathology.

Background Detection of molecular tumor heterogeneity has become of paramount importance with the advent of targeted therapies. Analysis for detection should be comprehensive, timely and based on routinely available tumor samples. Aim To evaluate the diagnostic potential of targeted multigene next-generation sequencing (TM-NGS) in characterizing gastrointestinal cancer molecular heterogeneity. Methods 35 gastrointestinal tract tumors, five of each intestinal type gastric carcinomas, pancreatic ductal adenocarcinomas, pancreatic intraductal papillary mucinous neoplasms, ampulla of Vater carcinomas, hepatocellular carcinomas, cholangiocarcinomas, pancreatic solid pseudopapillary tumors were assessed for mutations in 46 cancer-associated genes, using Ion Torrent semiconductor-based TM-NGS. One ampulla of Vater carcinoma cell line and one hepatic carcinosarcoma served to assess assay sensitivity. TP53, PIK3CA, KRAS, and BRAF mutations were validated by conventional Sanger sequencing. Results TM-NGS yielded overlapping results on matched fresh-frozen and formalin-fixed paraffin-embedded (FFPE) tissues, with a mutation detection limit of 1% for fresh-frozen high molecular weight DNA and 2% for FFPE partially degraded DNA. At least one somatic mutation was observed in all tumors tested; multiple alterations were detected in 20/35 (57%) tumors. Seven cancers displayed significant differences in allelic frequencies for distinct mutations, indicating the presence of intratumor molecular heterogeneity; this was confirmed on selected samples by immunohistochemistry of p53 and Smad4, showing concordance with mutational analysis. Conclusions TM-NGS is able to detect and quantitate multiple gene alterations from limited amounts of DNA, moving one step closer to a next-generation histopathologic diagnosis that integrates morphologic, immunophenotypic, and multigene mutational analysis on routinely processed tissues, essential for personalized cancer therapy.