Search

Characterization of gene regulatory networks is fundamental to understanding homeostatic development. This process can be simplified by analyzing relatively simple genomes such as the genome of Drosophila melanogaster. In this work we have developed a computational framework in Drosophila to explore for the presence of gene regulatory circuits between two large groups of transcriptional regulators: the epigenetic group of the Polycomb/trithorax (PcG/trxG) proteins and the microRNAs (miRNAs). We have searched genome-wide for miRNA targets in PcG/trxG transcripts as well as for Polycomb Response Elements (PREs) in miRNA genes. Our results show that 10% of the analyzed miRNAs could be controlling PcG/trxG gene expression, while 40% of those miRNAs are putatively controlled by the selected set of PcG/trxG proteins. The integration of these analyses has resulted in the predicted existence of 3 classes of miRNA-PcG/trxG crosstalk interactions that define potential regulatory circuits. In the first class, miRNA-PcG circuits are defined by miRNAs that reciprocally crosstalk with PcG. In the second, miRNA-trxG circuits are defined by miRNAs that reciprocally crosstalk with trxG. In the third class, miRNA-PcG/trxG shared circuits are defined by miRNAs that crosstalk with both PcG and trxG regulators. These putative regulatory circuits may uncover a novel mechanism in Drosophila for the control of PcG/trxG and miRNAs levels of expression. The computational framework developed here for Drosophila melanogaster can serve as a model case for similar analyses in other species. Moreover, our work provides, for the first time, a new and useful resource for the Drosophila community to consult prior to experimental studies investigating the epigenetic regulatory networks of miRNA-PcG/trxG mediated gene expression.

CHKA expression in HPNE cells and pancreas cancer cells

Acute treatment with MN58b: effect on ABCB gene expression

Supplementary Table 1. Metrics of the RNA-Seq experiment; Supplementary Table 2. Clinical and pathological characteristics of patients whose tumors were analyzed for CHKA expression; Supplementary Table 3. IC50 of MN58b for the PDAC cell lines used in the study; Supplementary Table 4. IC50 for gemcitabine, oxaliplatin, and 5-FU in control (ShNt, non-targetting) and CHKA-knockdown (Sh-1 and Sh-2) cells; Supplementary Table 5. Effect of modulation of ABCB transporter activity on the sensitivity (IC50) to treatment with MN58b of IMIM-PC-2 and IMIM-PC-2R cells.

Analysis of antibody specifity

RNA-Seq analysis of IMIM-PC-2 and IMIM-PC-2-R cells

Detailed results of combination treatments

Among malignant mesotheliomas (MM), the sarcomatoid subtype is associated with higher chemoresistance and worst survival. Due to its low incidence, there has been little progress in the knowledge of the molecular mechanisms associated with sarcomatoid MM, which might help to define novel therapeutic targets. In this work, we show that loss of PTEN expression is frequent in human sarcomatoid MM and PTEN expression levels are lower in sarcomatoid MM than in the biphasic and epithelioid subtypes. Combined Pten and Trp53 deletion in mouse mesothelium led to nonepithelioid MM development. In Pten;Trp53-null mice developing MM, the Gαi2-coupled receptor subunit activated MEK/ERK and PI3K, resulting in aggressive, immune-suppressed tumors. Combined inhibition of MEK and p110β/PI3K reduced mouse tumor cell growth in vitro. Therapeutic inhibition of MEK and p110β/PI3K using selumetinib (AZD6244, ARRY-142886) and AZD8186, two drugs that are currently in clinical trials, increased the survival of Pten;Trp53-null mice without major toxicity. This drug combination effectively reduced the proliferation of primary cultures of human pleural (Pl) MM, implicating nonepithelioid histology and high vimentin, AKT1/2, and Gαi2 expression levels as predictive markers of response to combined MEK and p110β/PI3K inhibition. Our findings provide a rationale for the use of selumetinib and AZD8186 in patients with MM with sarcomatoid features. This constitutes a novel targeted therapy for a poor prognosis and frequently chemoresistant group of patients with MM, for whom therapeutic options are currently lacking.Significance:Mesothelioma is highly aggressive; its sarcomatoid variants have worse prognosis. Building on a genetic mouse model, a novel combination therapy is uncovered that is relevant to human tumors.

This file contains Supplementary Material and Methods. Supplementary Table S1 summarizes the antibodies used for IHC and WB; Supplementary Table S2 shows the primers and the shRNAs sequences used; Supplementary Table S3 shows the Selumetinib and AZD8186 concentration ranges used for in vitro experiments; Supplementary Table S4. Summary of patient and tumor characteristics corresponding to the TMA from each institution; Supplementary Table S5. Drug sensitivity values for Selumetinib/AZD8186 responder cells. Supplementary Figure S1. Histology of the metastases of Pten;Trp53-null mouse tumors. Representative H&E staining of kidney, spleen and pancreas metastases of Pten;Trp53-null tumors. Scale bar 200µm (right panels) and 50µm (left panels). Supplementary Figure S2. Signaling analysis of Pten;Trp53-null and Pik3ca*;Trp53-null mouse tumors. Supplementary Figure S3. Characterization of cell lines derived from Pten;Trp53-null and Pik3ca*;Trp53-null tumors. Supplementary Figure S4. Effect of MEK and p110b inhibitors on signaling in Pten;Trp53-null and Pik3ca*;Trp53-null cells. Supplementary Figure S5. A fraction of Selumetinib/AZD8186 treated mice had no macroscopically detectable tumors at autopsy. Supplementary Figure S6. Analysis of synergy between Selumetinib and AZD8186 in the extended panel of primary human PMM cells. Supplementary Figure S7. Comparison of the response of human Pl-MM cultures to the Selumetinib/AZD8186 combination compared to cisplatin. Viability of primary human PMM cells after 72h exposure to serial dilutions of cisplatin or constant ratio combination of Selumetinib/AZD8186. Compound concentration range: 0.025-20µM for Selumetinib and AZD8186 and 1.25-40 µM for cisplatin. The combination ratios for Selumetinib and AZD8186 in each specific culture are specified in Supplementary table S3. Mean+ SEM (n=2-3).

The consumption of highly processed foods, along with other dietary and lifestyle poor habits, has an impact on health by increasing the risk of several non-communicable pathologies, such as diabetes. Gut microbiome composition, in specific, can be modulated by nutrients, deriving in different metabolic outcomes that have an influence on this high disease susceptibility and making it a possible therapeutic target for these comorbidities. In this work, gut microbiome of 60 and 46 individuals from 2 different studies focused, among other aspects, on diet-microbiome interactions, was characterised. By means of differential abundance analyses and supervised machine learning techniques based on random forest, gradient boosting and support vector machines, a set of microbial genera that could be potential biomarkers for the differentiation of individuals with poorer dietary patterns was discovered, after comparing coincidences in these taxa among classifiers and testing them for significant differences. Among these,Dialister,PhoceaandPseudoflavonifractorwere suggested to have a role in the way highly processed foods affect health negatively, along withPrevotellaceae NK3B31 groupand an undetermined genus fromMuribaculaceaein the opposite sense. Furthermore, all the identified genera in this study had already been linked to type 2 diabetes, among whichBacteroidesandPseudoflavonifractorproved to be differentially abundant in groups of individuals with different levels of biomarkers for this disease. Nevertheless, further research via longitudinal studies and experimental validation of these genera should be carried out to confirm the association of these taxa with diet and diabetes.

In mammalian cells, chromosomal replication starts at thousands of origins at which replisomes are assembled. Replicative stress triggers additional initiation events from ‘dormant’ origins whose genomic distribution and regulation are not well understood. In this study, we have analyzed origin activity in mouse embryonic stem cells in the absence or presence of mild replicative stress induced by aphidicolin, a DNA polymerase inhibitor, or by deregulation of origin licensing factor CDC6. In both cases, we observe that the majority of stress-responsive origins are also active in a small fraction of the cell population in a normal S phase, and stress increases their frequency of activation. In a search for the molecular determinants of origin efficiency, we compared the genetic and epigenetic features of origins displaying different levels of activation, and integrated their genomic positions in three-dimensional chromatin interaction networks derived from high-depth Hi-C and promoter-capture Hi-C data. We report that origin efficiency is directly proportional to the proximity to transcriptional start sites and to the number of contacts established between origin-containing chromatin fragments, supporting the organization of origins in higher-level DNA replication factories.

Obesity has an impact on health by increasing the risk of various diseases. However, these risks might also depend on the metabolic health status, as it seems that metabolically healthy obese subjects are under a reduced risk of suffering comorbidities such as colorectal cancer. The gut microbiome has an effect on obesity and metabolic disorders through several integration pathways, making it a potential therapeutic target for these diseases. In this study, we characterized the gut microbiota of 356 obese and non-obese European individuals with different comorbidities associated with obesity. Using approaches based on supervised machine learning and network biology, we found a set of biomarkers of interest for differentiating metabolically healthy from unhealthy subjects. Then, we performed a linear discriminant analysis of effect size on a population of 1593 colorectal cancer, adenoma and control subjects assembled by the COST Action ML4Microbiome to investigate their role in colorectal cancer risk. Four of our biomarkers appeared in both approaches, suggesting their possible role in colorectal cancer development, prognosis and follow up:Clostridium leptum, Gordonibacter pamelaeae, Eggerthella lentaandCollinsella intestinalis. Further research via longitudinal studies or experimental validation of these microbial species would be necessary to confirm this association.

BackgroundThere is a lack of knowledge for the general public about gut microbiome implications on health and the ability of diet and lifestyle to change it, impacting the well-being and preventing the development of NCDs. Participatory actions with innovative digital methodologies such as photovoice motivate citizens to self-implement healthier habits and improve their health status.MethodsWe conducted a participatory action research initiative #PictureYourMicrobes (https://bit.ly/3wKuRYt), which consisted of a photovoice project accompanied by self-tracking citizen science activities (self-reported nutritional questionnaires and stool sample collection for microbial profiling). Learning outcomes related to recognizing the importance of nutrition over gut microbiome’s health and including prebiotics and probiotic foods in the diet were defined and evaluated through pre-and post-knowledge questionnaires.ResultsWe recruited 70 volunteers to participate and set up six photovoice groups with homogeneous age and educational levels. Each group met online for four sessions composed of guided group discussions, where facilitators helped participants integrate their microbial profiling results with diet and nutritional habits. Participants took 156 photographs they thought best reflected how daily habits influence gut microbiota health. After contextualising and critically analysing the photographs and their reflections, they selected 64 photos sorted into four categories for a photobook (https://bit.ly/3LrUHEF) and exhibitions. They developed policy recommendations as a form of community-based solutions to improve gut microbiome health. Finally, we demonstrate that participants improved their knowledge about microbiota and its relationship with health through learning outcomes evaluation.ConclusionsParticipatory action research, such as photovoice, contributed to the awareness and adoption of new healthy habits, including the consumption of probiotics and prebiotic foods for a healthy gut microbiome. Moreover, it significantly impacted participants and provided a societal impact on other people through photobooks, pictures exhibitions, and digital media.Key questionsWhat is already known on this topic?Recent reports have shown that the gut microbiome (the complex of microorganisms that inhabit the gut) is altered in individuals with non-communicable diseases (NCDs).Citizens are unaware of the importance of the gut microbiome over health and the capability of diet and lifestyle modifications to modulate it and prevent the development of NCDs.What this study adds?The knowledge of the “own microbiome” allows people to reflect on their nutritional decisions and raise awareness about taking care of their microbiome and health.The use of participative action methodologies and co-creation activities in communication and dissemination allow participants to share their experiences and learnings with other citizens.Participative action methodologies used to teach complex health-related issues allows the transmission of knowledge from scientists to citizens.How might this study affect research, practice, or policy?Photovoice activity contributed to the awareness and adoption of new healthy habits, and participants became conscious of their nutritional habits through their reflections.Participatory action research activities allowed participants to propose solutions to maintain a healthy life and prevent NCDs critically and informally.Combining the results of these methodologies with social networks, digital photo books, and physical exhibitions amplifies the outcomes.Citizens developed policy recommendations as a form of community-based solutions to enhance gut microbiome health.

Introduction: Celiac disease (CeD) is an autoimmune disease affecting the small intestine due to gluten consumption. The treatment involves adherence to a gluten-free diet (GFD); however, up to 30% of patients show symptoms after one year on a GFD, presenting non-responsive CeD (NR-CeD). Evidence suggests that the microbiome plays a role in CeD progression and symptoms manifestations. Here we study the association of microbiome profiles with the persistence of symptoms in NR-CeD. Methods: We performed shotgun metagenomics to stool samples of NR-CeD patients (n=40). We integrate the results with public data from healthy controls (n=37) and patients with CeD undergoing GFD (n=52). We performed an analysis combining the datasets, calculated diversity indexes, and performed a Linear discriminant analysis integrated with effect size (LEfSe) to find microbial biomarkers of each condition. Results: We found that the microbiome of NR-CeD was less diverse than healthy controls and R-CeD (Table). After LEfSe analysis (Figure) we found that the microbiome from healthy controls was dominated by bacteria from the phylum Firmicutes and Actinobacteria (i.e: f_Ruminocacceae, g_Bifidobacterium,). The R-CeD microbiome was characterised by bacteria from the phylum Proteobacteria, Verrucomicrobia, and Firmicutes (c_Clostridia, c_Negativicutes). NR-CeD microbiome was enriched in bacteria from the phylum Bacteroidetes (o_Bacteroidales), and Firmicutes (c_Clostridia). Conclusion: CeD patients under prolonged treatment shift their microbiome differently than healthy controls and NR-CeD, suggesting specific microbial changes associated with symptoms prevalence. Moreover, we found microbial biomarkers associated with NR-CeD, mainly genus related to active CeD in the literature (Bacteroides spp.)

B cells have the unique property to somatically alter their immunoglobulin (IG) genes by V(D)J recombination, somatic hypermutation (SHM) and class-switch recombination (CSR). Aberrant targeting of these mechanisms is implicated in lymphomagenesis, but the mutational processes are poorly understood. By performing whole genome and transcriptome sequencing of 181 germinal center derived B-cell lymphomas (gcBCL) we identified distinct mutational signatures linked to SHM and CSR. We show that not only SHM, but presumably also CSR causes off-target mutations in non-IG genes. Kataegis clusters with high mutational density mainly affected early replicating regions and were enriched for SHM- and CSR-mediated off-target mutations. Moreover, they often co-occurred in loci physically interacting in the nucleus, suggesting that mutation hotspots promote increased mutation targeting of spatially co-localized loci (termed hypermutation by proxy). Only around 1% of somatic small variants were in protein coding sequences, but in about half of the driver genes, a contribution of B-cell specific mutational processes to their mutations was found. The B-cell-specific mutational processes contribute to both lymphoma initiation and intratumoral heterogeneity. Overall, we demonstrate that mutational processes involved in the development of gcBCL are more complex than previously appreciated, and that B cell-specific mutational processes contribute via diverse mechanisms to lymphomagenesis. This study has been supported by the German Ministry of Science and Education (BMBF) in the framework of the ICGC MMML-Seq project (01KU1002A-J) the MMML-MYC-SYS project (036166B) and the project ICGC DE-MINING (01KU1505E), the European Union in the framework of the BLUEPRINT Project (HEALTH-F5-2011-282510) and the KinderKrebsInitiative Buchholz/Holm-Seppensen. This work was supported by the BMBF-funded Heidelberg Center for Human Bioinformatics (HD-HuB) within the German Network for Bioinformatics Infrastructure (de.NBI) (#031A537A, #031A537C). Former grant support of MMML by the Deutsche Krebshilfe (2003–2011) is gratefully acknowledged. We acknowledge COSMIC and use of Cancer Gene Census. Part of the work was performed in association with SFB1074 (particularly subproject B1) funded by DFG. We wish to thank Barbara Hutter, Ivo Buchhalter, Zuguang Gu, and Natalie Jäger for skillful technical assistance. We thank the High-Throughput Sequencing Unit of the Genome and Proteome Core Facility and the Omics IT and Data Management Core Facility of the German Cancer Research Center (DKFZ, Heidelberg) as well as the Institute of Clinical Molecular Biology (IKMB, Christian-Albrechts-University Kiel) for excellent technical support and expertise. DH is a member of the Hartmut Hoffmann-Berling International Graduate School of Molecular and Cellular Biology (HBIGS) and of the MD/PhD-program of the University of Heidelberg. KK and UHT are funded by the Helmholtz International Graduate School for Cancer Research at the German Cancer Research Center. SHB, HK, and SH acknowledge support by LIFE (Leipzig Research Center for Civilization Diseases), Leipzig University. LIFE is funded by the European Union, the European Regional Development Fund (ERDF), the European Social Fund (ESF), and the Free State of Saxony. This work has been carried out with the help of the Interdisciplinary Bank of Biomaterials and Data of the University Hospital of Würzburg and the Julius Maximilian University of Würzburg (idbw). Peer Reviewed "Article signat per 70 autors/es:Daniel Hübschmann, Kortine Kleinheinz, Rabea Wagener, Stephan H. Bernhart, Cristina López, Umut H. Toprak, Stephanie Sungalee, Naveed Ishaque, Helene Kretzmer, Markus Kreuz, Sebastian M. Waszak, Nagarajan Paramasivam, Ole Ammerpohl, Sietse M. Aukema, Renée Beekman, Anke K. Bergmann, Matthias Bieg, Hans Binder, Arndt Borkhardt, Christoph Borst, Benedikt Brors, Philipp Bruns, Enrique Carrillo de Santa Pau, Alexander Claviez, Gero Doose, Andrea Haake, Dennis Karsch, Siegfried Haas, Martin-Leo Hansmann, Jessica I. Hoell, Volker Hovestadt, Bingding Huang, Michael Hummel, Christina Jäger-Schmidt, Jules N. A. Kerssemakers, Jan O. Korbel, Dieter Kube, Chris Lawerenz, Dido Lenze, Joost H. A. Martens, German Ott, Bernhard Radlwimmer, Eva Reisinger, Julia Richter, Daniel Rico, Philip Rosenstiel, Andreas Rosenwald, Markus Schillhabel, Stephan Stilgenbauer, Peter F. Stadler, José I. Martín-Subero, Monika Szczepanowski, Gregor Warsow, Marc A. Weniger, Marc Zapatka, Alfonso Valencia, Hendrik G. Stunnenberg, Peter Lichter, Peter Möller, Markus Loeffler, Roland Eils, Wolfram Klapper, Steve Hoffmann, Lorenz Trümper, ICGC MMML-Seq consortium, ICGC DE-Mining consortium, BLUEPRINT consortium, Ralf Küppers, Matthias Schlesner & Reiner Siebert"

Consumption of high-energy-yielding diets, rich in fructose and lipids, is a factor contributing to the current increase in non-alcoholic fatty liver disease prevalence. Gut microbiota composition and short-chain fatty acids (SCFAs) production alterations derived from unhealthy diets are considered putative underlying mechanisms. This study aimed to determine relationships between changes in gut microbiota composition and SCFA levels by comparing rats featuring diet-induced steatohepatitis with control counterparts fed a standard diet. A high-fat high-fructose (HFHF) feeding induced higher body, liver and mesenteric adipose tissue weights, increased liver triglyceride content and serum transaminase, glucose, non-HDL-c and MCP-1 levels. Greater liver malondialdehyde levels and glutathione peroxidase activity were also observed after feeding the hypercaloric diet. Regarding gut microbiota composition, a lowered diversity and increased abundances of bacteria from the Clostridium sensu stricto 1, Blautia, Eubacterium coprostanoligenes group, Flavonifractor, and UBA1819 genera were found in rats featuring diet-induced steatohepatitis, as well as higher isobutyric, valeric and isovaleric acids concentrations. These results suggest that hepatic alterations produced by a hypercaloric HFHF diet may be related to changes in overall gut microbiota composition and abundance of specific bacteria. The shift in SCFA levels produced by this unbalanced diet cannot be discarded as potential mediators of the reported hepatic and metabolic alterations.

Background: Dysbiosis of the microbiome has been related to the Celiac disease (CeD) progress, an autoimmune disease characterised by gluten intolerance developed in genetically susceptible individuals under certain environmental factors. The microbiome contributes to CeD pathophysiology modulating the immune response by the action of short-chain fatty acids (SCFA), affecting gut barrier integrity allowing the entrance of gluten derived proteins, and degrading immunogenic peptides of gluten through endoprolyl peptidase enzymes. Results: We reviewed state of the art in taxonomic composition for CeD and compiled the larger dataset of 16S prokaryotic ribosomal RNA (rRNA) gene high-throughput sequencing for consensus profiling. We present for the first time an integrative analysis of metataxonomic data from CeD patients, including samples from different body sites (saliva, pharynx, duodenum, and stool). We found the presence of coordinated changes through the gastrointestinal tract characterised by an increase in Actinobacteria species in the upper tract (pharynx and duodenum), and an increase in Proteobacteria in the lower tract (duodenum and stool), as well as site-specific changes evidencing a dysbiosis in CeD patients' microbiota. Moreover, we described the effect of adherence to a gluten-free diet (GFD) evidenced by an increase in beneficial bacteria and a decrease in some Betaproteobacteriales but not fully restoring CeD-related dysbiosis. Conclusions: We illustrate that the gut microbiota acts as an enhancer of immune response in CeD through the production of lipopolysaccharides and other bacterial components that activate the immune response and by decrease SCFA producers bacteria. Furthermore, microbial changes observed through the gastrointestinal tract of CeD patients may help manage the disease and follow-up GFD treatment.

BackgroundDysbiosis of the microbiome has been related to the Celiac disease (CeD) progress, an autoimmune disease characterised by gluten intolerance developed in genetically susceptible individuals under certain environmental factors. The microbiome contributes to CeD pathophysiology modulating the immune response by the action of short-chain fatty acids (SCFA), affecting gut barrier integrity allowing the entrance of gluten derived proteins, and degrading immunogenic peptides of gluten through endoprolyl peptidase enzymes. We reviewed state of the art in taxonomic composition for CeD and compiled the larger dataset of 16S prokaryotic ribosomal RNA (rRNA) gene high-throughput sequencing for consensus profiling. We present for the first time an integrative analysis of metataxonomic data from CeD patients, including samples from different body sites (saliva, pharynx, duodenum, and stool). We found the presence of coordinated changes through the gastrointestinal tract characterised by an increase in Actinobacteria species in the upper tract (pharynx and duodenum), and an increase in Proteobacteria in the lower tract (duodenum and stool), as well as site- specific changes evidencing a dysbiosis in CeD patients’ microbiota. Moreover, we described the effect of adherence to a gluten-free diet (GFD) evidenced by an increase in beneficial bacteria and a decrease in some Betaproteobacteriales but not fully restoring CeD-related dysbiosis. We illustrate that the gut microbiota acts as an enhancer of immune response in CeD through the production of lipopolysaccharides and other bacterial components that activate the immune response and by decrease SCFA producers bacteria. Furthermore, microbial changes observed through the gastrointestinal tract of CeD patients may help manage the disease and follow-up GFD treatment.

Resveratrol and its 2-methoxy derivative pterostilbene are two phenolic compounds that occur in foodstuffs and feature hepato-protective effects. This study is devoted to analysing and comparing the metabolic effects of pterostilbene and resveratrol on gut microbiota composition in rats displaying NAFLD induced by a diet rich in saturated fat and fructose. The associations among changes induced by both phenolic compounds in liver status and those induced in gut microbiota composition were also analysed. For this purpose, fifty Wistar rats were distributed in five experimental groups: a group of animals fed a standard diet (CC group) and four additional groups fed a high-fat high-fructose diet alone (HFHF group) or supplemented with 15 or 30 mg/kg bw/d of pterostilbene (PT15 and PT30 groups, respectively) or 30 mg/kg bw/d of resveratrol (RSV30 group). The dramatic changes induced by high-fat high-fructose feeding in the gut microbiota were poorly ameliorated by pterostilbene or resveratrol. These results suggest that the specific changes in microbiota composition induced by pterostilbene (increased abundances of Akkermansia and Erysipelatoclostridium, and lowered abundance of Clostridum sensu stricto 1) may not entirely explain the putative preventive effects on steatohepatitis. This research was funded by Ministerio de Economía y Competitividad-Fondo Europeo de Desarrollo Regional (grant number AGL-2015-65719-R MINECO/FEDER, UE), Instituto de Salud Carlos III CIBERobn (grant number CB12/03/30007); University of the Basque Country (grant number GIU 18/173).

This study was supported by the COST Action CA18131 "Statistical and machine learning techniques in human microbiome studies." IM-I was supported by the "MS type I" program (CP16/00163) from the Instituto de Salud Carlos III and co-funded by Fondo Europeo de Desarrollo Regional-FEDER. MN was grateful for the additional support by the project "Information and Communication Technologies for a Single Digital Market in Science, Education and Security" of the Scientific Research Center, NIS-3317 and National roadmaps for research infrastructures (RIs) grant number NIS-3318. LL was supported by Academy of Finland (decision 295741). IE was supported by H2020-EU.4.b. project "Integration of knowledge and biobank resources in comprehensive translational approach for personalized prevention and treatment of metabolic disorders (INTEGROMED)" (grant agreement ID 857572). MD was supported by the Luxembourg National Research Fund (FNR) CORE grant (C18/BM/12585940). The human microbiome has emerged as a central research topic in human biology and biomedicine. Current microbiome studies generate high-throughput omics data across different body sites, populations, and life stages. Many of the challenges in microbiome research are similar to other high-throughput studies, the quantitative analyses need to address the heterogeneity of data, specific statistical properties, and the remarkable variation in microbiome composition across individuals and body sites. This has led to a broad spectrum of statistical and machine learning challenges that range from study design, data processing, and standardization to analysis, modeling, cross-study comparison, prediction, data science ecosystems, and reproducible reporting. Nevertheless, although many statistics and machine learning approaches and tools have been developed, new techniques are needed to deal with emerging applications and the vast heterogeneity of microbiome data. We review and discuss emerging applications of statistical and machine learning techniques in human microbiome studies and introduce the COST Action CA18131 "ML4Microbiome" that brings together microbiome researchers and machine learning experts to address current challenges such as standardization of analysis pipelines for reproducibility of data analysis results, benchmarking, improvement, or development of existing and new tools and ontologies. European Cooperation in Science and Technology (COST) CA18131 European Commission CP16/00163 project "Information and Communication Technologies for a Single Digital Market in Science, Education and Security" of the Scientific Research Center NIS-3317 National roadmaps for research infrastructures (RIs) NIS-3318 Academy of Finland 295741 H2020-EU.4.b. project "Integration of knowledge and biobank resources in comprehensive translational approach for personalized prevention and treatment of metabolic disorders (INTEGROMED)" 857572 Luxembourg National Research Fund (FNR) CORE grant C18/BM/12585940

The number of microbiome-related studies has notably increased the availability of data on human microbiome composition and function. These studies provide the essential material to deeply explore host-microbiome associations and their relation to the development and progression of various complex diseases. Improved data-analytical tools are needed to exploit all information from these biological datasets, taking into account the peculiarities of microbiome data, i.e., compositional, heterogeneous and sparse nature of these datasets. The possibility of predicting host-phenotypes based on taxonomy-informed feature selection to establish an association between microbiome and predict disease states is beneficial for personalized medicine. In this regard, machine learning (ML) provides new insights into the development of models that can be used to predict outputs, such as classification and prediction in microbiology, infer host phenotypes to predict diseases and use microbial communities to stratify patients by their characterization of state-specific microbial signatures. Here we review the state-of-the-art ML methods and respective software applied in human microbiome studies, performed as part of the COST Action ML4Microbiome activities. This scoping review focuses on the application of ML in microbiome studies related to association and clinical use for diagnostics, prognostics, and therapeutics. Although the data presented here is more related to the bacterial community, many algorithms could be applied in general, regardless of the feature type. This literature and software review covering this broad topic is aligned with the scoping review methodology. The manual identification of data sources has been complemented with: (1) automated publication search through digital libraries of the three major publishers using natural language processing (NLP) Toolkit, and (2) an automated identification of relevant software repositories on GitHub and ranking of the related research papers relying on learning to rank approach. This study was supported by COST Action CA18131 “Statistical and machine learning techniques in human microbiome studies”. Estonian Research Council grant PRG548 (JT). Spanish State Research Agency Juan de la Cierva Grant IJC2019-042188-I (LM-Z). EO was founded and OA was supported by Estonian Research Council grant PUT 1371 and EMBO Installation grant 3573. AG was supported by Statutory Research project of the Department of Computer Networks and Systems.

Cohesin exists in two variants containing STAG1 or STAG2. STAG2 is one of the most mutated genes in cancer and a major bladder tumor suppressor. Little is known about how its inactivation contributes to tumorigenesis. Here, we analyze the genomic distribution of STAG1 and STAG2 and perform STAG2 loss-of-function experiments using RT112 bladder cancer cells; we then analyze the genomic effects by integrating gene expression and chromatin interaction data. Functional compartmentalization exists between the cohesin complexes: cohesin-STAG2 displays a distinctive genomic distribution and mediates short and mid-ranged interactions that engage genes at higher frequency than those established by cohesin-STAG1. STAG2 knockdown results in down-regulation of the luminal urothelial signature and up-regulation of the basal transcriptional program, mirroring differences between STAG2-high and STAG2-low human bladder tumors. This is accompanied by rewiring of DNA contacts within topological domains, while compartments and domain boundaries remain refractive. Contacts lost upon depletion of STAG2 are assortative, preferentially occur within silent chromatin domains, and are associated with de-repression of lineage-specifying genes. Our findings indicate that STAG2 participates in the DNA looping that keeps the basal transcriptional program silent and thus sustains the luminal program. This mechanism may contribute to the tumor suppressor function of STAG2 in the urothelium. Fundación Científica de la Asociación Española Contra el Cáncer (to F.X.R., E.L., in part); V.P. is supported by INSERM, the Fondation Toulouse Cancer Santé and Pierre Fabre Research Institute as part of the Chair of Bioinformatics in Oncology of the CRCT; Bioinfo4women programme at the Barcelona Supercomputing Center; European Union's H2020 Framework Programme through the ERC [609989 to M.A.M.-R., in part]; Spanish Ministerio de Ciencia, Innovación y Universidades [BFU2017-85926-P to M.A.M.-R.]; C.N.I.O. is supported by Ministerio de Ciencia, Innovación y Universidades as a Centro de Excelencia Severo Ochoa [SEV-2015-0510]; C.R.G. acknowledges support from ‘Centro de Excelencia Severo Ochoa 2013–2017’ [SEV-2012-0208]; Spanish ministry of Science and Innovation to the EMBL partnership and the CERCA Programme/Generalitat de Catalunya (to C.R.G.); C.R.G. also acknowledges support of the Spanish Ministry of Science and Innovation through the Instituto de Salud Carlos III, the Generalitat de Catalunya through Departament de Salut and Departament d’Empresa i Coneixement; Spanish Ministry of Science and Innovation with funds from the European Regional Development Fund (ERDF) corresponding to the 2014–2020 Smart Growth Operating Program (to C.N.A.G.). Funding for open access charge: Own funds. Peer Reviewed "Article signat per 15 autors/es: Laia Richart, Eleonora Lapi, Vera Pancaldi, Mirabai Cuenca-Ardura, Enrique Carrillo-de-Santa Pau, Miguel Madrid-Mencía, Hélène Neyret-Kahn, François Radvanyi, Juan Antonio Rodríguez, Yasmina Cuartero, François Serra, François Le Dily, Alfonso Valencia, Marc A Marti-Renom, Francisco X Real "

Cohesin exists in two variants, containing either STAG1 or STAG2. STAG2 is one of the most commonly mutated genes in human cancer, and a major bladder cancer tumor suppressor. Little is known about how its inactivation contributes to tumor development. Here, we analyze the genomic distribution of STAG1 and STAG2 and perform STAG2 loss-of-function experiments using RT112 bladder cancer cells; we then analyze the resulting genomic effects by integrating gene expression and chromatin interaction data. Cohesin-STAG2 is required for DNA contacts within topological domains, but not for compartment maintenance of domain boundary integrity. Cohesin-STAG2-mediated interactions are short-ranged and engage promoters and gene bodies with higher frequency than those mediated by cohesin-STAG1. STAG2 knockdown resulted in a modest but consistent down-regulation of the luminal urothelial differentiation signature, mirroring differences between STAG2-high and STAG2-low bladder tumors. Both lost and gained contacts were enriched among STAG1/STAG2 common sites as well as STAG2-enriched sites. Contacts lost upon depletion of STAG2 were significantly assortative, indicating their proximity at the 3D level, and were associated with changes in gene expression. Overall, our findings indicate that, in urothelial cells, STAG2 is required for the establishment and/or maintenance of DNA looping that, in turn, sustains the luminal differentiation program. This mechanism may contribute to the tumor suppressor function of STAG2 in bladder cancer.

Among malignant mesotheliomas (MM), the sarcomatoid subtype is associated with higher chemoresistance and worst survival. Due to its low incidence, there has been little progress in the knowledge of the molecular mechanisms associated with sarcomatoid MM, which might help to define novel therapeutic targets. In this work, we show that loss of PTEN expression is frequent in human sarcomatoid MM and PTEN expression levels are lower in sarcomatoid MM than in the biphasic and epithelioid subtypes. Combined Pten and Trp53 deletion in mouse mesothelium led to nonepithelioid MM development. In Pten;Trp53-null mice developing MM, the Gαi2-coupled receptor subunit activated MEK/ERK and PI3K, resulting in aggressive, immune-suppressed tumors. Combined inhibition of MEK and p110β/PI3K reduced mouse tumor cell growth in vitro. Therapeutic inhibition of MEK and p110β/PI3K using selumetinib (AZD6244, ARRY-142886) and AZD8186, two drugs that are currently in clinical trials, increased the survival of Pten;Trp53-null mice without major toxicity. This drug combination effectively reduced the proliferation of primary cultures of human pleural (Pl) MM, implicating nonepithelioid histology and high vimentin, AKT1/2, and Gαi2 expression levels as predictive markers of response to combined MEK and p110β/PI3K inhibition. Our findings provide a rationale for the use of selumetinib and AZD8186 in patients with MM with sarcomatoid features. This constitutes a novel targeted therapy for a poor prognosis and frequently chemoresistant group of patients with MM, for whom therapeutic options are currently lacking., [Significance] Mesothelioma is highly aggressive; its sarcomatoid variants have worse prognosis. Building on a genetic mouse model, a novel combination therapy is uncovered that is relevant to human tumors., This work was supported, in part, by grants from Asociación Española Contra el Cáncer (F.X. Real), Spanish Ministry of Economy and Competitivity, Plan Estatal de I+D+I 2013-2016, ISCIII (FIS PI15/00045 to A. Carnero), RTICC (Instituto de Salud Carlos III, grants RD12/0036/0034 to F.X. Real and A. Carnero, respectively), and CIBERONC (CB16/12/00453 and CD16/12/00275 to F.X. Real and A. Carnero, respectively), cofunded by FEDER from Regional Development European Funds (European Union) and Inserm (Institut national de la santé et de la recherche médicale). M. Marqués was supported by a Sara Borrell Fellowship from Instituto de Salud Carlos III. CNIO is supported by Ministerio de Ciencia, Innovación y Universidades as a Centro de Excelencia Severo Ochoa SEV-2015-0510.

Funding: this work was funded by the European Union’s Seventh Framework Program through the Blueprint Consortium (grant agreement 282510). MJSDs work was supported by the Leicester Experimental Cancer Medicine Centre (C325/A15575 Cancer Research UK/UK Department of Health). PG’s work was supported by a grant from the Fondation pour la Recherche Medicale, grant number “DEQ20160334875”. RS’ work was supported by the DFG in the framework of the SFB1074.

Simple Summary Pediatric acute lymphoblastic leukemia is the most common malignancy in children. Based on the genetic characteristics of the tumor, patients are risk-stratified and treated with different treatment intensities. However, in a proportion of cases, known as B-other, no genetic alterations relevant for risk stratification are found with routine diagnostic procedures. In this study, we performed RNA sequencing, a comprehensive and cutting-edge method, of 185 children with B-other leukemia and analyzed gene fusions, expression profiles and mutations. Furthermore, we validated our findings using commonly used diagnostic techniques. Our results identified a subgroup of cases clustering with known leukemia subtypes, e.g., DUX4-positive, and subgroups characterized by mutations in PAX5 and IKZF1, resulting in more cases being assigned to a defined subgroup. Moreover, we identified new fusion partners of ETV6, IKZF1 and PAX5. Our data demonstrate the applicability and technical considerations for the use of RNA sequencing to personalize genetic diagnostics in pediatric leukemia. Abstract B-cell precursor acute lymphoblastic leukemia (BCP-ALL) is the most common cancer in children, and significant progress has been made in diagnostics and the treatment of this disease based on the subtypes of BCP-ALL. However, in a large proportion of cases (B-other), recurrent BCP-ALL-associated genomic alterations remain unidentifiable by current diagnostic procedures. In this study, we performed RNA sequencing and analyzed gene fusions, expression profiles, and mutations in diagnostic samples of 185 children with BCP-ALL. Gene expression clustering showed that a subset of B-other samples partially clusters with some of the known subgroups, particularly DUX4-positive. Mutation analysis coupled with gene expression profiling revealed the presence of distinctive BCP-ALL subgroups, characterized by the presence of mutations in known ALL driver genes, e.g., PAX5 and IKZF1. Moreover, we identified novel fusion partners of lymphoid lineage transcriptional factors ETV6, IKZF1 and PAX5. In addition, we report on low blast count detection thresholds and show that the use of EDTA tubes for sample collection does not have adverse effects on sequencing and downstream analysis. Taken together, our findings demonstrate the applicability of whole-transcriptome sequencing for personalized diagnostics in pediatric ALL, including tentative classification of the B-other cases that are difficult to diagnose using conventional methods.

Hematopoiesis is one of the best characterized biological systems but the connection between chromatin changes and lineage differentiation is not yet well understood. We have developed a bioinformatic workflow to generate a chromatin space that allows to classify 42 human healthy blood epigenomes from the BLUEPRINT, NIH ROADMAP and ENCODE consortia by their cell type. This approach let us to distinguish different cells types based on their epigenomic profiles, thus recapitulating important aspects of human hematopoiesis. The analysis of the orthogonal dimension of the chromatin space identify 32,662 chromatin determinant regions (CDRs), genomic regions with different epigenetic characteristics between the cell types. Functional analysis revealed that these regions are linked with cell identities. The inclusion of leukemia epigenomes in the healthy hematological chromatin sample space gives us insights on the healthy cell types that are more epigenetically similar to the disease samples. Further analysis of tumoral epigenetic alterations in hematopoietic CDRs points to sets of genes that are tightly regulated in leukemic transformations and commonly mutated in other tumors. Our method provides an analytical approach to study the relationship between epigenomic changes and cell lineage differentiation. Method availability: https://github.com/david-juan/ChromDet. European Union’s Seventh Framework Programme [FP7/2007–2013, 282510 (BLUEPRINT)]; Spanish Ministry of Economy, Industry and Competitiveness and European Regional Development Fund [Project Retos BFU2015–71241-R]. Funding for open access charge: Project Retos BFU2015–71241-R (to A.V.).

To better understand why human neonates show a poor response to intracellular pathogens, we compared gene expression and histone modification profiles of neonatal naive CD8+ T cells with that of their adult counterparts. We found that neonatal lymphocytes have a distinct epigenomic landscape associated with a lower expression of genes involved in T cell receptor (TCR) signaling and cytotoxicity and a higher expression of genes involved in the cell cycle and innate immunity. Functional studies corroborated that neonatal CD8+ T cells are less cytotoxic, transcribe antimicrobial peptides, and produce reactive oxygen species. Altogether, our results show that neonatal CD8+ T cells have a specific genetic program biased toward the innate immune response. These findings will contribute to better diagnosis and management of the neonatal immune response. This project was specifically supported by a joint EcosNord-Anuies-SEP-Con-acyt project (M11S01). Work in the M.A.S. laboratory is supported by grantsfrom Consejo Nacional de Ciencia y Tecnologı ́a(CONACYT; CB-2011-01168182) and Programa de Mejoramiento del Profesorado (PROMEPSI-UAEM/13/342). Work in the S.S. laboratory is supported by recurrent fundingfrom the Inserm and Aix-Marseille University and by specific grants from theEuropean Union’s FP7 Program (agreement 282510-BLUEPRINT), the Associ-ation pour la Recherche contre le Cancer (ARC) (project SFI20111203756), andthe Aix-Marseille initiative d’excelence (A*MIDEX) project ANR-11-IDEX-0001-02. We thank Centro Estatal de la Transfusio ́n Sanguı ́nea in Cuernavaca for thedonation of leukocyte concentrates and the mothers and babies of HospitalGeneral Parres in Cuernavaca for the donation of cord blood. This study makesuse of data generated by the Blueprint and Roadmap consortia. A full list of theinvestigators who contributed to the generation of the data is availablefromwww.blueprint-epigenome.euandhttp://www.roadmapepigenomics.org/. Funding for the Blueprint project was provided by the European Union’sSeventh Framework Program (FP7/2007-2013) under grant agreement282510 – BLUEPRINT. The Roadmap consortium is financed by the NIH. Weare grateful to Professor C.I. Pogson for critical reading of the manuscript. Sí

c-MYC oncogene is deregulated in most human tumours. Histone marks associated with transcriptionally active genes define high-affinity c-MYC targets. The mechanisms involved in their recognition by c-MYC are unknown. Here we report that c-MYC interacts with BPTF, a core subunit of the NURF chromatin-remodelling complex. BPTF is required for the activation of the full c-MYC transcriptional programme in fibroblasts. BPTF knockdown leads to decreased c-MYC recruitment to DNA and changes in chromatin accessibility. In Bptf-null MEFs, BPTF is necessary for c-MYC-driven proliferation, G1–S progression and replication stress, but not for c-MYC-driven apoptosis. Bioinformatics analyses unveil that BPTF levels correlate positively with c-MYC-driven transcriptional signatures. In vivo, Bptf inactivation in pre-neoplastic pancreatic acinar cells significantly delays tumour development and extends survival. Our findings uncover BPTF as a crucial c-MYC co-factor required for its biological activity and suggest that the BPTF-c-MYC axis is a potential therapeutic target in cancer., c-MYC genomic distribution is dictated by the epigenetic context but the mechanisms are unknown. Here, the authors show that c-MYC requires the chromatin reader BPTF to activate its transcriptional program and promote tumour development in vivo, suggesting that BPTF is a potential target for cancer therapy.

ObjectiveTo estimate the prevalence of depression in patients diagnosed with type 2 diabetes mellitus (T2DM), and to identify sociodemographic, clinical and psychological factors associated with depression in this population. Additionally, we examine the annual incidence rate of depression among patients with T2DM.MethodsWe performed a large prospective cohort study of patients with T2DM from the Madrid Diabetes Study. The first recruitment drive included 3443 patients. The second recruitment drive included 727 new patients. Data have been collected since 2007 (baseline visit) and annually during the follow-up period (since 2008).ResultsDepression was prevalent in 20.03% of patients (n=592; 95% CI 18.6% to 21.5%) and was associated with previous personal history of depression (OR 6.482; 95% CI 5.138 to 8.178), mental health status below mean (OR 1.423; 95% CI 1.452 to 2.577), neuropathy (OR 1.951; 95% CI 1.423 to 2.674), fair or poor self-reported health status (OR 1.509; 95% CI 1.209 to 1.882), treatment with oral antidiabetic agents plus insulin (OR 1.802; 95% CI 1.364 to 2.380), female gender (OR 1.333; 95% CI 1.009 to 1.761) and blood cholesterol level (OR 1.005; 95% CI 1.002 to 1.009). The variables inversely associated with depression were: being in employment (OR 0.595; 95% CI 0.397 to 0.894), low physical activity (OR 0.552; 95% CI 0.408 to 0.746), systolic blood pressure (OR 0.982; 95% CI 0.971 to 0.992) and social support (OR 0.978; 95% CI 0.963 to 0.993). In patients without depression at baseline, the incidence of depression after 1 year of follow-up was 1.20% (95% CI 1.11% to 2.81%).ConclusionsDepression is very prevalent among patients with T2DM and is associated with several key diabetes-related outcomes. Our results suggest that previous mental status, self-reported health status, gender and several diabetes-related complications are associated with differences in the degree of depression. These findings should alert practitioners to the importance of detecting depression in patients with T2DM.

Neutrophils are short-lived blood cells that play a critical role in host defense against infections. To better comprehend neutrophil functions and their regulation, we provide a complete epigenetic overview, assessing important functional features of their differentiation stages from bone marrow-residing progenitors to mature circulating cells. Integration of chromatin modifications, methylation, and transcriptome dynamics reveals an enforced regulation of differentiation, for cellular functions such as release of proteases, respiratory burst, cell cycle regulation, and apoptosis. We observe an early establishment of the cytotoxic capability, while the signaling components that activate these antimicrobial mechanisms are transcribed at later stages, outside the bone marrow, thus preventing toxic effects in the bone marrow niche. Altogether, these data reveal how the developmental dynamics of the chromatin landscape orchestrate the daily production of a large number of neutrophils required for innate host defense and provide a comprehensive overview of differentiating human neutrophils. The work presented here was supported by the Landsteiner Foundation forBlood Transfusion Research (LSBR-1121 to T.W.K.) and by the Dutch CancerFoundation (KUN 2011-4 937 to J.H.A.M.). The work was funded by a grantfrom the European Commission 7th Framework Program (FP72007-2013,grant 282510, BLUEPRINT). M.F. ssupported sh Heart Foundation(BHF) Cambridge Centre of Excellence (RE/13/6/30180). Additional support wasrovided by the Wellcome Trust (WT108749/Z/15/Z to P.F.) and by the Eu-ropean Molecular Biology Laboratory (E.L., A.D.,L.C., P.F.). Research in theOuwehand laboratory is also supported by grants from Bristol-Myers Squibb,the British Heart Foundation, the Medical Research Council, the National Insti-tutefor Health Research (NIHR) (W.H.O. is senior investigator), and NHSBlood and Transplant (NHSB T). Sí

Neutrophils and monocytes provide a first line of defense against infections as part of the innate immune system. Here we report the integrated analysis of transcriptomic and epigenetic landscapes for circulating monocytes and neutrophils with the aim to enable downstream interpretation and functional validation of key regulatory elements in health and disease. We collected RNA-seq data, ChIP-seq of six histone modifications and of DNA methylation by bisulfite sequencing at base pair resolution from up to 6 individuals per cell type. Chromatin segmentation analyses suggested that monocytes have a higher number of cell-specific enhancer regions (4-fold) compared to neutrophils. This highly plastic epigenome is likely indicative of the greater differentiation potential of monocytes into macrophages, dendritic cells and osteoclasts. In contrast, most of the neutrophil-specific features tend to be characterized by repressed chromatin, reflective of their status as terminally differentiated cells. Enhancers were the regions where most of differences in DNA methylation between cells were observed, with monocyte-specific enhancers being generally hypomethylated. Monocytes show a substantially higher gene expression levels than neutrophils, in line with epigenomic analysis revealing that gene more active elements in monocytes. Our analyses suggest that the overexpression of c-Myc in monocytes and its binding to monocyte-specific enhancers could be an important contributor to these differences. Altogether, our study provides a comprehensive epigenetic chart of chromatin states in primary human neutrophils and monocytes, thus providing a valuable resource for studying the regulation of the human innate immune system.

Background and aims Gata6 is required to complete and maintain acinar differentiation in the mouse pancreas. Pancreas-specific Gata6 ablation during development causes extensive and persistent acinar-ductal metaplasia, which is considered an initial step of mutant KRas -driven carcinogenesis. Therefore, the Gata6 -null pancreas might represent a tumour-prone environment. We investigated whether Gata6 plays a role during pancreatic tumorigenesis. Design We analysed genetically engineered mouse models and human pancreatic ductal adenocarcinoma (PDAC) cell lines, using a combination of histopathological studies, genome-wide expression and chromatin immunoprecipitation experiments to understand the role of Gata6 in the initiation and progression of KRas G12V -driven tumours Results We show that Gata6 maintains the acinar differentiation programme, both directly and indirectly, and it concomitantly suppresses ectopic programmes in the pancreas. Gata6 ablation renders acinar cells more sensitive to KRas G12V , thereby accelerating tumour development. Gata6 expression is spontaneously lost in a mouse model of KRas G12V -driven PDAC, in association with altered cell differentiation. Using a combination of ChIP-Seq and RNA-Seq, we show that Gata6 exerts its tumour-suppressive effect through the promotion of cell differentiation, the suppression of inflammatory pathways, and the direct repression of cancer-related pathways. Among them is the epidermal growth factor receptor (EGFR) pathway, the activity of which is upregulated in the normal and preneoplastic Gata6 -null pancreas. Accordingly, GATA6-silencing in human PDAC cells leads to an upregulation of EGFR. Conclusions We propose that, in the pancreas, Gata6 acts as a tumour suppressor by enforcing acinar cell differentiation, by directly and indirectly repressing ectopic differentiation programmes, and by regulating crucial cancer-related gene expression pathways.

Item does not contain fulltext The International Human Epigenome Consortium (IHEC) coordinates the generation of a catalog of high-resolution reference epigenomes of major primary human cell types. The studies now presented (see the Cell Press IHEC web portal at http://www.cell.com/consortium/IHEC) highlight the coordinated achievements of IHEC teams to gather and interpret comprehensive epigenomic datasets to gain insights in the epigenetic control of cell states relevant for human health and disease. PAPERCLIP.

Tissue-specific differentiation and inflammatory programmes are thought to independently contribute to disease. The orphan nuclear receptor NR5A2 is a key regulator of pancreas differentiation and SNPs in or near the human gene are associated with risk of pancreatic cancer. In mice, Nr5a2 heterozygosity sensitizes the pancreas to damage, impairs regeneration, and cooperates with mutant KRas in tumor progression. Through global transcriptomic analysis, we uncover a basal pre-inflammatory state in the pancreas of Nr5a2 heterozygous mice that is reminiscent of pancreatitis-induced inflammation and is conserved in histologically normal human pancreata with reduced NR5A2 mRNA expression. In Nr5a2+/− mice, Nr5a2 undergoes a dramatic transcriptional switch relocating from tissue-specific to inflammatory loci thereby promoting AP-1-dependent gene transcription. Importantly, deletion of c-Jun in the pancreas of these mice rescues the pre-inflammatory phenotype and the defective regenerative response to damage. These findings provide compelling evidence that the same transcriptional networks supporting homeostasis in normal tissue can be subverted to foster inflammation upon genetic or environmental constraints.

BACKGROUND: A healthy immune system requires immune cells that adapt rapidly to environmental challenges. This phenotypic plasticity can be mediated by transcriptional and epigenetic variability. RESULTS: We apply a novel analytical approach to measure and compare transcriptional and epigenetic variability genome-wide across CD14+CD16- monocytes, CD66b+CD16+ neutrophils, and CD4+CD45RA+ naïve T cells from the same 125 healthy individuals. We discover substantially increased variability in neutrophils compared to monocytes and T cells. In neutrophils, genes with hypervariable expression are found to be implicated in key immune pathways and are associated with cellular properties and environmental exposure. We also observe increased sex-specific gene expression differences in neutrophils. Neutrophil-specific DNA methylation hypervariable sites are enriched at dynamic chromatin regions and active enhancers. CONCLUSIONS: Our data highlight the importance of transcriptional and epigenetic variability for the key role of neutrophils as the first responders to inflammatory stimuli. We provide a resource to enable further functional studies into the plasticity of immune cells, which can be accessed from: http://blueprint-dev.bioinfo.cnio.es/WP10/hypervariability . We would like to thank K. Pearce and M. Kristiansen (UCL Genomics) for processing the Illumina Infinium HumanMethylation450 BeadChips; D. Balzereit, S. Dökel, A. Kovacsovics, and M. Linser (Max Planck Institute for Molecular Genetics) for help with generating the RNA-seq data; B. Phipson (Murdoch Childrens Research Institute), H.C. Bravo (University of Maryland), and P. Guilhamon (UCL Cancer Institute) for advice on statistical analyses; C. Bock (CeMM Research Center for Molecular Medicine of the Austrian Academy of Sciences) for useful discussions; A. Orozco (University of Costa Rica) for technical support; V. Naranbhai, B. Fairfax, and J. Knight (University of Oxford) for providing access to the neutrophil gene expression data set for replication; and L. Phipps for proofreading the manuscript. We gratefully acknowledge the participation of all NIHR Cambridge BioResource volunteers, and thank the Cambridge BioResource staff for their help with volunteer recruitment. We thank members of the Cambridge BioResource SAB and Management Committee for their support of our study and the NIHR Cambridge Biomedical Research Centre (BRC) for funding. BLUEPRINT Consortium: Cornelis A. Albers (Radboud University), Vyacheslav Amstislavskiy (Max Planck Institute for Molecular Genetics), Sofie Ashford (University of Cambridge), Lorenzo Bomba (Wellcome Trust Sanger Institute), David Bujold (McGill University), Frances Burden (University of Cambridge), Stephan Busche (McGill University), Maxime Caron (McGill University), Shu-Huang Chen (McGill University), Warren A. Cheung (McGill University), Laura Clarke (European Bioinformatics Institute), Irina Colgiu (Wellcome Trust Sanger Institute), Avik Datta (European Bioinformatics Institute), Oliver Delaneau (University of Geneva), Heather Elding (Wellcome Trust Sanger Institute), Samantha Farrow (University of Cambridge), Diego Garrido-Martín (Centre for Genomic Regulation), Bing Ge (McGill University), Roderic Guigo (Centre for Genomic Regulation), Valentina Iotchkova (European Bioinformatics Institute), Kousik Kundu (Wellcome Trust Sanger Institute), Tony Kwan (McGill University), John J. Lambourne (University of Cambridge), Ernesto Lowy (European Bioinformatics Institute), Daniel Mead (Wellcome Trust Sanger Institute), Farzin Pourfarzad (Sanquin Research and Landsteiner Laboratory), Adriana Redensek (McGill University), Karola Rehnstrom (University of Cambridge), Augusto Rendon (University of Cambridge), David Richardson (European Bioinformatics Institute), Thomas Risch (Max Planck Institute for Molecular Genetics), Sophia Rowlston (University of Cambridge), Xiaojian Shao (McGill University), Marie-Michelle Simon (McGill University), Marc Sultan (Max Planck Institute for Molecular Genetics), Klaudia Walter (Wellcome Trust Sanger Institute), Steven P. Wilder (European Bioinformatics Institute), Ying Yan (Wellcome Trust Sanger Institute), Stylianos E. Antonarakis (University of Geneva), Guillaume Bourque (McGill University), Emmanouil T. Dermitzakis (University of Geneva), Paul Flicek (European Bioinformatics Institute), Hans Lehrach (Max Planck Institute for Molecular Genetics), Joost H. A. Martens (Radboud University), Marie-Laure Yaspo (Max Planck Institute for Molecular Genetics), Willem H. Ouwehand (University of Cambridge). Sí

BACKGROUND: Hotspot mutations in the promoter of the gene coding for telomerase reverse transcriptase (TERT) have been described and proposed to activate gene expression. OBJECTIVES: To investigate TERT mutation frequency, spectrum, association with expression and clinical outcome, and potential for detection of recurrences in urine in patients with urothelial bladder cancer (UBC). DESIGN, SETTING, AND PARTICIPANTS: A set of 111 UBCs of different stages was used to assess TERT promoter mutations by Sanger sequencing and TERT messenger RNA (mRNA) expression by reverse transcription-quantitative polymerase chain reaction. The two most frequent mutations were investigated, using a SNaPshot assay, in an independent set of 184 non-muscle-invasive and 173 muscle-invasive UBC (median follow-up: 53 mo and 21 mo, respectively). Voided urine from patients with suspicion of incident UBC (n=174), or under surveillance after diagnosis of non-muscle-invasive UBC (n=194), was tested using a SNaPshot assay. OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS: Association of mutation status with age, sex, tobacco, stage, grade, fibroblast growth factor receptor 3 (FGFR3) mutation, progression-free survival, disease-specific survival, and overall survival. RESULTS AND LIMITATIONS: In the two series, 78 of 111 (70%) and 283 of 357 (79%) tumors harbored TERT mutations, C228T being the most frequent substitution (83% for both series). TERT mutations were not associated with clinical or pathologic parameters, but were more frequent among FGFR3 mutant tumors (p=0.0002). There was no association between TERT mutations and mRNA expression (p=0.3). Mutations were not associated with clinical outcome. In urine, TERT mutations had 90% specificity in subjects with hematuria but no bladder tumor, and 73% in recurrence-free UBC patients. The sensitivity was 62% in incident and 42% in recurrent UBC. A limitation of the study is its retrospective nature. CONCLUSIONS: Somatic TERT promoter mutations are an early, highly prevalent genetic event in UBC and are not associated with TERT mRNA levels or disease outcomes. A SNaPshot assay in urine may help to detect UBC recurrences. Hotspot mutations in the promoter of the gene coding for telomerase reverse transcriptase (TERT) have been described and proposed to activate gene expression.

Francisco Real and colleagues report exome sequencing in urothelial bladder tumors. They show that STAG2, a subunit of the cohesin complex, is recurrently mutated and provide evidence that STAG2 loss does not lead to increases in aneuploidy. Urothelial bladder cancer (UBC) is heterogeneous at the clinical, pathological and genetic levels. Tumor invasiveness (T) and grade (G) are the main factors associated with outcome and determine patient management1. A discovery exome sequencing screen (n = 17), followed by a prevalence screen (n = 60), identified new genes mutated in this tumor coding for proteins involved in chromatin modification (MLL2, ASXL2 and BPTF), cell division (STAG2, SMC1A and SMC1B) and DNA repair (ATM, ERCC2 and FANCA). STAG2, a subunit of cohesin, was significantly and commonly mutated or lost in UBC, mainly in tumors of low stage or grade, and its loss was associated with improved outcome. Loss of expression was often observed in chromosomally stable tumors, and STAG2 knockdown in bladder cancer cells did not increase aneuploidy. STAG2 reintroduction in non-expressing cells led to reduced colony formation. Our findings indicate that STAG2 is a new UBC tumor suppressor acting through mechanisms that are different from its role in preventing aneuploidy.

Immunodeficiency is one of the most important causes of mortality associated with Wolf-Hirschhorn syndrome (WHS), a severe rare disease originated by a deletion in chromosome 4p. The WHS candidate 1 (WHSC1) gene has been proposed as one of the main genes responsible for many of the alterations in WHS, but its mechanism of action is still unknown. Here, we present in vivo genetic evidence showing that Whsc1 plays an important role at several points of hematopoietic development. Particularly, our results demonstrate that both differentiation and function of Whsc1-deficient B cells are impaired at several key developmental stages due to profound molecular defects affecting B cell lineage specification, commitment, fitness, and proliferation, demonstrating a causal role for WHSC1 in the immunodeficiency of WHS patients., FEDER (Fondo de Investigaciones Sanitarias/Instituto de Salud Carlos III) (PI13/00160 and PI14/00025), Fundación Inocente Inocente, and the ARIMMORA EU/FP7 project and by donations from the Asociación Española del Síndrome de Wolf Hirschhorn (AESWH), the Fundación Síndrome de Wolf-Hirschhorn or 4p- (FSWH4p), FECYT Precipita, and Fundación Ramón Areces