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Background High-throughput transcriptomic datasets are often examined to discover new actors and regulators of a biological response. To this end, graphical interfaces have been developed and allow a broad range of users to conduct standard analyses from RNA-seq data, even with little programming experience. Although existing solutions usually provide adequate procedures for normalization, exploration or differential expression, more advanced features, such as gene clustering or regulatory network inference, often miss or do not reflect current state of the art methodologies. Results We developed here a user interface called DIANE (Dashboard for the Inference and Analysis of Networks from Expression data) designed to harness the potential of multi-factorial expression datasets from any organisms through a precise set of methods. DIANE interactive workflow provides normalization, dimensionality reduction, differential expression and ontology enrichment. Gene clustering can be performed and explored via configurable Mixture Models, and Random Forests are used to infer gene regulatory networks. DIANE also includes a novel procedure to assess the statistical significance of regulator-target influence measures based on permutations for Random Forest importance metrics. All along the pipeline, session reports and results can be downloaded to ensure clear and reproducible analyses. Conclusions We demonstrate the value and the benefits of DIANE using a recently published data set describing the transcriptional response of Arabidopsis thaliana under the combination of temperature, drought and salinity perturbations. We show that DIANE can intuitively carry out informative exploration and statistical procedures with RNA-Seq data, perform model based gene expression profiles clustering and go further into gene network reconstruction, providing relevant candidate genes or signalling pathways to explore. DIANE is available as a web service (https://diane.bpmp.inrae.fr), or can be installed and locally launched as a complete R package.

Objectives Transcriptional regulatory modules are usually modelled via a network, in which nodes correspond to genes and edges correspond to regulatory associations between them. In the model yeast Saccharomyces cerevisiae, the topological properties of such a network are well-described (distribution of degrees, hierarchical levels, organization in network motifs, etc.). To go further on this, our aim was to search for additional information resulting from the new combination of classical representations of transcriptional regulatory networks with more realistic models of the spatial organization of S. cerevisiae genome in the nucleus. Results Taking advantage of independent studies with high-quality datasets, i.e. lists of target genes for specific transcription factors and chromosome positions in a three dimensional space representing the nucleus, particular spatial co-localizations of genes that shared common regulatory mechanisms were searched. All transcriptional modules of S. cerevisiae, as described in the latest release of the YEASTRACT database were analyzed and significant biases toward co-localization for a few sets of target genes were observed. To help other researchers to reproduce such analysis with any list of genes of their interest, an interactive web tool called 3D-Scere (https://3d-scere.ijm.fr/) is provided.

International audience; In 46,XY men, testis is determined by a genetic network(s) that both promotes testis formation and represses ovarian development. Disruption of this process results in a lack of testis-determination and affected individuals present with 46,XY gonadal dysgenesis (GD), a part of the spectrum of Disorders/Differences of Sex Development/Determination (DSD). A minority of all cases of GD are associated with pathogenic variants in key players of testis-determination, SRY, SOX9, MAP3K1 and NR5A1. However, most of the cases remain unexplained. Recently, unbiased exome sequencing approaches have revealed new genes and loci that may cause 46,XY GD. We critically evaluate the evidence to support causality of these factors and describe how functional studies are continuing to improve our understanding of genotype-phenotype relationships in genes that are established causes of GD. As genomic data continues to be generated from DSD cohorts, we propose several recommendations to help interpret the data and establish causality.

International audience; Leveraging systems biology approaches, we illustrate how metabolically distinct species of Clostridia protect against or worsen Clostridioides difficile infection in mice by modulating the pathogen's colonization, growth, and virulence to impact host survival. Gnotobiotic mice colonized with the amino acid fermenter Paraclostridium bifermentans survive infection with reduced disease severity, while mice colonized with the butyrate-producer, Clostridium sardiniense, succumb more rapidly. Systematic in vivo analyses revealed how each commensal alters the gut-nutrient environment to modulate the pathogen's metabolism, gene regulatory networks, and toxin production. Oral administration of P. bifermentans rescues conventional, clindamycin-treated mice from lethal C. difficile infection in a manner similar to that of monocolonized animals, thereby supporting the therapeutic potential of this commensal species. Our findings lay the foundation for mechanistically informed therapies to counter C. difficile disease using systems biology approaches to define host-commensal-pathogen interactions in vivo.

Genetic studies of metabolites have identified thousands of variants, many of which are associated with downstream metabolic and obesogenic disorders. However, these studies have relied on univariate analyses, reducing power and limiting context-specific understanding. Here we aim to provide an integrated perspective of the genetic basis of metabolites by leveraging the Finnish Metabolic Syndrome In Men (METSIM) cohort, a unique genetic resource which contains metabolic measurements, mostly lipids, across distinct time points as well as information on statin usage. We increase effective sample size by an average of two-fold by applying the Covariates for Multi-phenotype Studies (CMS) approach, identifying 588 significant SNP-metabolite associations, including 228 new associations. Our analysis pinpoints a small number of master metabolic regulator genes, balancing the relative proportion of dozens of metabolite levels. We further identify associations to changes in metabolic levels across time as well as genetic interactions with statin at both the master metabolic regulator and genome-wide level., Genome-wide association studies of metabolites have revealed hundreds of genetic associations using univariate analyses. Here, the authors use a multivariate approach to perform association analyses for 158 serum metabolites, followed by fine mapping and GxE interaction tests with statin use and age.

Several Candida species can undergo a heritable and reversible transition from a ‘white’ state to a mating proficient ‘opaque’ state. This ability relies on highly interconnected transcriptional networks that control cell-type-specific gene expression programs over multiple generations. Candida albicans, the most prominent pathogenic Candida species, provides a well-studied paradigm for the white-opaque transition. In this species, a network of at least eight transcriptional regulators controls the balance between white and opaque states that have distinct morphologies, transcriptional profiles, and physiological properties. Given the reversible nature and the high frequency of white-opaque transitions, it is widely assumed that this switch is governed by epigenetic mechanisms that occur independently of any changes in DNA sequence. However, a direct genomic comparison between white and opaque cells has yet to be performed. Here, we present a whole-genome comparative analysis of C. albicans white and opaque cells. This analysis revealed rare genetic changes between cell states, none of which are linked to white-opaque switching. This result is consistent with epigenetic mechanisms controlling cell state differentiation in C. albicans and provides direct evidence against a role for genetic variation in mediating the switch.

Developmental genes are often regulated by multiple elements with overlapping activity. Yet, in most cases, the relative function of those elements and their contribution to endogenous gene expression remain poorly characterized. An example of this phenomenon is that distinct sets of enhancers have been proposed to direct Fgf8 in the limb apical ectodermal ridge and the midbrain-hindbrain boundary. Using in vivo CRISPR/Cas9 genome engineering, we functionally dissect this complex regulatory ensemble and demonstrate two distinct regulatory logics. In the apical ectodermal ridge, the control of Fgf8 expression appears distributed between different enhancers. In contrast, we find that in the midbrain-hindbrain boundary, one of the three active enhancers is essential while the other two are dispensable. We further dissect the essential midbrain-hindbrain boundary enhancer to reveal that it is also composed by a mixture of essential and dispensable modules. Cross-species transgenic analysis of this enhancer suggests that its composition may have changed in the vertebrate lineage., Developmental genes are often regulated by multiple elements, yet their relative contribution to gene expression remains poorly understood. Here the authors apply in vivo CRISPR/Cas9 genome engineering to find two distinct regulatory logics directing Fgf8 in the limb apical ectodermal ridge and the midbrain-hindbrain boundary.

>Hepatitis B virus (HBV) is a global health concern. Viral and host factors orchestrate the natural history of HBV infection, but the impact of host factors that influence the clinical course of the disease remains poorly understood. The aim of this study was to identify host factors crucial to the HBV life cycle by conducting a meta-analysis utilizing public microarray datasets. Methods: An integrative meta-analysis of expression data from two microarray datasets of HBVinfected liver tissues and healthy uninfected livers was conducted to identify gene expression signatures and overlapping biological processes modulating infection/disease. Results: Using integrative meta-analysis of expression data (INMEX), we identified across two datasets a total of 841 genes differentially expressed during HBV infection, including 473 upregulated and 368 downregulated genes. In addition, through functional enrichment and pathway analysis, we observed that Jak-STAT, TLR, and NF-κB are the most relevant signaling pathways in chronic HBV infection. The network-based meta-analysis identified NEDD8, SKP2, JUN, and HIF1A as the most highly ranked hub genes. Conclusion: Thus, these results may provide valuable information about novel potential host factors modulating chronic HBV infection. Such factors may serve as potential targets for the development of novel therapeutics such as activin receptor-like kinase inhibitors.

Clostridium tetani produces a potent neurotoxin, the tetanus toxin (TeNT), which is responsible for an often-fatal neurological disease (tetanus) characterized by spastic paralysis. Prevention is efficiently acquired by vaccination with the TeNT toxoid, which is obtained by C. tetani fermentation and subsequent purification and chemical inactivation. C. tetani synthesizes TeNT in a regulated manner. Indeed, the TeNT gene (tent) is mainly expressed in the late exponential and early stationary growth phases. The gene tetR (tetanus regulatory gene), located immediately upstream of tent, encodes an alternative sigma factor which was previously identified as a positive regulator of tent. In addition, the genome of C. tetani encodes more than 127 putative regulators, including 30 two-component systems (TCSs). Here, we investigated the impact of 12 regulators on TeNT synthesis which were selected based on their homology with related regulatory elements involved in toxin production in other clostridial species. Among nine TCSs tested, three of them impact TeNT production, including two positive regulators that indirectly stimulate tent and tetR transcription. One negative regulator was identified that interacts with both tent and tetR promoters. Two other TCSs showed a moderate effect: one binds to the tent promoter and weakly increases the extracellular TeNT level, and another one has a weak inverse effect. In addition, CodY (control of dciA (decoyinine induced operon) Y) but not Spo0A (sporulation stage 0) or the DNA repair protein Mfd (mutation frequency decline) positively controls TeNT synthesis by interacting with the tent promoter. Moreover, we found that inorganic phosphate and carbonate are among the environmental factors that control TeNT production. Our data show that TeNT synthesis is under the control of a complex network of regulators that are largely distinct from those involved in the control of toxin production in Clostridium botulinum or Clostridium difficile.

Background Efficient treatments against metastatic melanoma dissemination are still lacking. Here, we report that low-cytotoxic concentrations of 5-aza-2′-deoxycytidine, a DNA demethylating agent, prevent in vitro 3D invasiveness of metastatic melanoma cells and reduce lung metastasis formation in vivo. Results We unravelled that this beneficial effect is in part due to MIR-199A2 re-expression by promoter demethylation. Alone, this miR showed an anti-invasive and anti-metastatic effect. Throughout integration of micro-RNA target prediction databases with transcriptomic analysis after 5-aza-2′-deoxycytidine treatments, we found that miR-199a-3p downregulates set of genes significantly involved in invasion/migration processes. In addition, analysis of data from melanoma patients showed a stage- and tissue type-dependent modulation of MIR-199A2 expression by DNA methylation. Conclusions Thus, our data suggest that epigenetic- and/or miR-based therapeutic strategies can be relevant to limit metastatic dissemination of melanoma. Electronic supplementary material The online version of this article (10.1186/s13148-018-0600-2) contains supplementary material, which is available to authorized users.

International audience; Mature spermatozoa contain a whole repertoire of the various classes of cellular RNAs, both coding and non-coding. It was hypothesized that after fertilization they might impact development, a claim supported by experimental evidence in various systems. Despite the current increasing interest in the transgenerational maintenance of epigenetic traits and their possible determination by RNAs, little remains known about conservation in sperm and across generations and the specificities and mechanisms involved in transgenerational maintenance. We identified two distinct fractions of RNAs in mature mouse sperm, one readily extracted in the aqueous phase of the classical TRIzol procedure and a distinct fraction hybridized with homologous DNA in DNA-RNA complexes recovered from the interface, purified after DNase hydrolysis and analyzed by RNA-seq methodology. This DNA-associated RNA (D RNA) was found to represent as much as half of the cell contents in differentiated sperm, in which a major part of the cytoplasmic material has been discarded. Stable complexes were purified free of proteins and identified as hybrids (R-loops) on the basis of their sensitivity to RNase H hydrolysis. Further analysis by RNA-seq identified transcripts from all the coding and non-coding regions of the genome, thus revealing an extensive wave of transcription, prior to or concomitant with the terminal compaction of the chromatin.

Cardiac fibrosis is a final common pathology in inherited and acquired heart diseases that causes cardiac electrical and pump failure. Here, we use systems genetics to identify a pro-fibrotic gene network in the diseased heart and show that this network is regulated by the E3 ubiquitin ligase WWP2, specifically by the WWP2-N terminal isoform. Importantly, the WWP2-regulated pro-fibrotic gene network is conserved across different cardiac diseases characterized by fibrosis: human and murine dilated cardiomyopathy and repaired tetralogy of Fallot. Transgenic mice lacking the N-terminal region of the WWP2 protein show improved cardiac function and reduced myocardial fibrosis in response to pressure overload or myocardial infarction. In primary cardiac fibroblasts, WWP2 positively regulates the expression of pro-fibrotic markers and extracellular matrix genes. TGFβ1 stimulation promotes nuclear translocation of the WWP2 isoforms containing the N-terminal region and their interaction with SMAD2. WWP2 mediates the TGFβ1-induced nucleocytoplasmic shuttling and transcriptional activity of SMAD2., Pathological cardiac fibrosis is a hallmark of diseases leading to heart failure. Here, the authors used systems genetics to identify a pro-fibrotic gene network regulated by WWP2, a E3 ubiquitin ligase, which orchestrates the nucleocytoplasmic shuttling and transcriptional activity of SMAD2 in the diseased heart.

In developing organisms, spatially prescribed cell identities are thought to be determined by the expression levels of multiple genes. Quantitative tests of this idea, however, require a theoretical framework capable of exposing the rules and precision of cell specification over developmental time. Using the gap gene network in the early fly embryo as an example, we use such a framework to show how expression levels of the four gap genes can be jointly decoded into an optimal specification of position with 1% accuracy. The decoder correctly predicts, with no free parameters, the dynamics of pair-rule expression patterns at different developmental time points and in various mutant backgrounds. Precise cellular identities are thus available at the earliest stages of development, contrasting the prevailing view of positional information being slowly refined across successive layers of the patterning network. Our results suggest that developmental enhancers closely approximate a mathematically optimal decoding strategy.

Understanding the control of large-scale metabolic networks is central to biology and medicine. However, existing approaches either require specifying a cellular objective or can only be used for small networks. We introduce new coupling types describing the relations between reaction activities, and develop an efficient computational framework, which does not require any cellular objective for systematic studies of large-scale metabolism. We identify the driver reactions facilitating control of 23 metabolic networks from all kingdoms of life. We find that unicellular organisms require a smaller degree of control than multicellular organisms. Driver reactions are under complex cellular regulation in Escherichia coli, indicating their preeminent role in facilitating cellular control. In human cancer cells, driver reactions play pivotal roles in malignancy and represent potential therapeutic targets. The developed framework helps us gain insights into regulatory principles of diseases and facilitates design of engineering strategies at the interface of gene regulation, signaling, and metabolism.

The function of the mammalian heart depends on the interplay between different cardiac cell types. The deployment of these cells, with precise spatiotemporal regulation, is also important during development to establish the heart structure. In this Review, we discuss the diverse origins of cardiac cell types and the lineage relationships between cells of a given type that contribute to different parts of the heart. The emerging lineage tree shows the progression of cell fate diversification, with patterning cues preceding cell type segregation, as well as points of convergence, with overlapping lineages contributing to a given tissue. Several cell lineage markers have been identified. However, caution is required with genetic-tracing experiments in comparison with clonal analyses. Genetic studies on cell populations provided insights into the mechanisms for lineage decisions. In the past 3 years, results of single-cell transcriptomics are beginning to reveal cell heterogeneity and early developmental trajectories. Equating this information with the in vivo location of cells and their lineage history is a current challenge. Characterization of the progenitor cells that form the heart and of the gene regulatory networks that control their deployment is of major importance for understanding the origin of congenital heart malformations and for producing cardiac tissue for use in regenerative medicine. In this Review, Meilhac and Buckingham discuss the origin of cardiac cell populations, their lineage relationships and the genes that regulate their behaviour and differentiation. Characterizing the progenitor cells that form the heart and the gene regulatory networks controlling their deployment is of major importance for understanding the origin of congenital heart malformations and for developing cardiac regeneration therapies.

International audience; A chronic inflammatory state to a large extent explains sickle cell disease (SCD) pathophysi-ology. Nonetheless, the principal dysregulated factors affecting this major pathway and their mechanisms of action still have to be fully identified and elucidated. Integrating gene expression and genome-wide association study (GWAS) data analysis represents a novel approach to refining the identification of key mediators and functions in complex diseases. Here, we performed gene expression meta-analysis of five independent publicly available microarray datasets related to homozygous SS patients with SCD to identify a consensus SCD transcriptomic profile. The meta-analysis conducted using the MetaDE R package based on combining p values (maxP approach) identified 335 differentially expressed genes (DEGs; 224 upregulated and 111 downregulated). Functional gene set enrichment revealed the importance of several metabolic pathways, of innate immune responses, erythrocyte development, and hemostasis pathways. Advanced analyses of GWAS data generated within the framework of this study by means of the atSNP R package and SIFT tool identified 60 regulatory single-nucleotide polymorphisms (rSNPs) occurring in the promoter of 20 DEGs and a deleterious SNP, affecting CAMKK2 protein function. This novel database of candidate genes, transcription factors, and rSNPs associated with SCD provides new markers that may help to identify new therapeutic targets.

The causative agent of Legionnaires' pneumonia, Legionella pneumophila, colonizes amoebae and biofilms in the environment. The opportunistic pathogen employs the Lqs (Legionella quorum sensing) system and the signaling molecule LAI-1 (Legionella autoinducer-1) to regulate virulence, motility, natural competence, and expression of a 133 kb genomic island, including a putative novel regulator. Here, we show that the regulator termed LvbR is an LqsS-regulated, pleotropic transcription factor that binds to the promoter of lpg1056/hnox1 (encoding an inhibitor of the diguanylate cyclase Lpg1057), and thus regulates proteins involved in c-di-GMP metabolism. LvbR determines biofilm architecture, since L. pneumophila lacking lvbR accumulates less sessile biomass and forms homogeneous mat-like structures, while the parental strain develops more compact bacterial aggregates. Comparative transcriptomics of sessile and planktonic ΔlvbR or ΔlqsR mutant strains revealed concerted (virulence, fitness island, metabolism) and reciprocally (motility) regulated genes in biofilm and broth, respectively. Moreover, ΔlvbR is hyper-competent for DNA uptake, defective for phagocyte infection, outcompeted by the parental strain in amoebae co-infections, and impaired for cell migration inhibition. Taken together, our results indicate that L. pneumophila LvbR is a novel pleotropic transcription factor, which links the Lqs and c-di-GMP regulatory networks to control biofilm architecture and pathogen-host-cell interactions. This article is protected by copyright. All rights reserved.

International audience; Despite considerable advances in cardiovascular disease treatment, heart failure remains a public health challenge. In this context, gene therapy appears as an attractive approach, but clinical trials using single therapeutic molecules result in moderate benefit. With the objective of improving ischemic heart failure therapy, we designed a combined treatment, aimed to simultaneously stimulate angiogenesis, prevent cardiac remodeling, and restore contractile function. We have previously validated IRES-based vectors as powerful tools to co-express genes of interest. Mono- and multicistronic lentivectors expressing fibroblast growth factor 2 (angiogenesis), apelin (cardioprotection), and/or SERCA2a (contractile function) were produced and administrated by intramyocardial injection into a mouse model of myocardial infarction. Data reveal that combined treatment simultaneously improves vessel number, heart function parameters, and fibrosis prevention, due to FGF2, SERCA2a, and apelin, respectively. Furthermore, addition of SERCA2a in the combination decreases cardiomyocyte hypertrophy. Large-scale transcriptome analysis reveals that the triple treatment is the most efficient in restoring angiogenic balance as well as expression of genes involved in cardiac function and remodeling. Our study validates the concept of combined treatment of ischemic heart disease with apelin, FGF2, and SERCA2a and shows that such therapeutic benefit is mediated by a more effective recovery of gene network regulation.

International audience; Biological processes often manifest themselves as coordinated changes across modules, i.e., sets of interacting genes. Commonly, the high dimensionality of genome-scale data prevents the visual identification of such modules, and straightforward computational search through a set of known pathways is a limited approach. Therefore, tools for the data-driven, computational, identification of modules in gene interaction networks have become popular components of visualization and visual analytics workflows. However, many such tools are known to result in modules that are large, and therefore hard to interpret biologically. Here, we show that the empirically known tendency towards large modules can be attributed to a statistical bias present in many module identification tools, and discuss possible remedies from a mathematical perspective. In the current absence of a straightforward practical solution, we outline our view of best practices for the use of the existing tools.

International audience; Synthesis of the major toxin proteins of the diarrheal pathogen, Clostridium difficile, is dependent on the activity of TcdR, an initiation (sigma) factor of RNA polymerase. The synthesis of TcdR and the activation of toxin gene expression are responsive to multiple components in the bacterium's nutritional environment, such as the presence of certain sugars, amino acids, and fatty acids. This review summarizes current knowledge about the mechanisms responsible for repression of toxin synthesis when glucose or branched-chain amino acids or proline are in excess and the pathways that lead to synthesis of butyrate, an activator of toxin synthesis. The regulatory proteins implicated in these mechanisms also play key roles in modulating bacterial metabolic pathways, suggesting that C. difficile pathogenesis is intimately connected to the bacterium's metabolic state.

International audience; Clostridium difficile, a Gram-positive, anaerobic, spore-forming bacterium, is a major cause of nosocomial infections such as antibiotic-associated diarrhea. Spores are the vector of its transmission and persistence in the environment. Despite the importance of spores in the infectious cycle of C. difficile, little was known until recently about the control of spore development in this entero-pathogen. In this review, we describe recent advances in our understanding of the regulatory network controlling C. difficile sporulation. The comparison with the model organism Bacillus subtilis highlights major differences in the signaling pathways between the forespore and the mother cell and a weaker connection between morphogenesis and gene expression. Indeed, the activation of the SigE regulon in the mother cell is partially independent of SigF although the forespore protein SpoIIR, itself partially independent of SigF, is essential for pro-SigE processing. Furthermore, SigG activity is not strictly dependent on SigE. Finally, SigG is dispensable for SigK activation in agreement with the absence of a pro-SigK sequence. The excision of the C. difficile skin element is also involved in the regulation of SigK activity. The C. difficile sporulation process might be a simpler, more ancestral version of the program characterized for B. subtilis.

To cope with the devastating insults constantly inflicted to their genome by intrinsic and extrinsic DNA damaging sources, cells have evolved a sophisticated network of interconnected DNA caretaking mechanisms that will detect, signal and repair the lesions. Among the underlying molecular mechanisms that regulate these events, PARylation catalyzed by Poly(ADP-ribose) polymerases (PARPs), appears as one of the earliest post-translational modification at the site of the lesion that is known to elicit recruitment and regulation of many DNA damage response proteins. In this review we discuss how the complex PAR molecule operates in stress-induced DNA damage signaling and genome maintenance but also in various physiological settings initiated by developmentally programmed DNA breakage. To illustrate the latter, particular emphasis will be placed on the emerging contribution of PARPs to B cell receptor assembly and diversification.

Identifying genes where a variant allele is preferentially expressed in tumors could lead to a better understanding of cancer biology and optimization of targeted therapy. However, tumor sample heterogeneity complicates standard approaches for detecting preferential allele expression. We therefore developed a novel approach combining genome and transcriptome sequencing data from the same sample that corrects for sample heterogeneity and identifies significant preferentially expressed alleles. We applied this analysis to epithelial ovarian cancer samples consisting of matched primary ovary and peritoneum and lymph node metastasis. We find that preferentially expressed variant alleles include germline and somatic variants, are shared at a relatively high frequency between patients, and are in gene networks known to be involved in cancer processes. Analysis at a patient level identifies patient-specific preferentially expressed alleles in genes that are targets for known drugs. Analysis at a site level identifies patterns of site specific preferential allele expression with similar pathways being impacted in the primary and metastasis sites. We conclude that genes with preferentially expressed variant alleles can act as cancer drivers and that targeting those genes could lead to new therapeutic strategies., Author Summary Identifying genes that contribute to cancer biology is complicated partly because cancers can have dozens of somatic mutations and thousands of germline variants. Somatic mutations are gene variants that arise after conception in an organism while germline variants are gene variants present at conception in an organism. Most methods to identify cancer drivers have focused on determining somatic mutations. In this study we attempt to identify, from a tumor sample, important germline and somatic variants by determining if a variant is expressed (made into RNA) more than expected from the amount of the variant in the genome. The preferred expression of a variant could benefit cancer cells. When applying our analysis to ovarian cancer samples we found that despite the apparent heterogeneity, different patients frequently share the same genes with preferentially expressed variants. These genes in many cases are known to affect cancer processes such as DNA repair, cell adhesion and cell signaling and are targetable with known drugs. We therefore conclude that our analysis can identify germline and somatic gene variants that contribute to cancer biology and can potentially guide individualized therapies.

International audience; The yeast systematic deletion collection offered the basis for a number of different strategies that establish functional links between genes by analyzing the phenotype of cells that combine two different deletions or mutations. A distinguishing feature of the collection is the presence of molecular barcodes at each deleted locus, which can be used to quantify the presence and abundance of cells bearing a given allele in a complex mix. As a result, a large number of mutants can be tested in batch cultures, replacing tedious manipulation of thousands of individual strains with a barcode microarray readout. Barcode-based genetic screens like Genetic Interaction Mapping (GIM) thus require little investment in terms of specific equipment, are fast to perform, and allow precise measurements of double mutant growth rates for both aggravating (synthetic sick) and alleviating (epistatic) effects. We describe here protocols for preparing the pools of haploid double mutant S. cerevisiae cells, testing their composition with barcode microarrays, and analyzing the results to extract useful functional information.

// Ikhlak Ahmed 1 , Thasni Karedath 1 , Simeon S. Andrews 1 , Iman K. Al-Azwani 2 , Yasmin Ali Mohamoud 2 , Denis Querleu 3 , Arash Rafii 4 , Joel A. Malek 1, 2 1 Department of Genetic medicine, Weill Cornell Medicine-Qatar, Education City, Ar-Rayyan, Qatar 2 Genomics Core, Weill Cornell Medicine-Qatar, Education City, Ar-Rayyan, Qatar 3 Department of Gynecologic Oncology, Universite Montepllier 1, Montpellier, France 4 Stem Cell and Microenvironment Laboratory, Weill Cornell Medicine-Qatar, Education City, Ar-Rayyan, Qatar Correspondence to: Joel A. Malek, e-mail: jom2042@qatar-med.cornell.edu Keywords: circular RNA, ovarian cancer, peritoneal metastasis, lymph node metastasis, miRNA Received: December 07, 2015 Accepted: April 02, 2016 Published: April 22, 2016 ABSTRACT Recently, a class of endogenous species of RNA called circular RNA (circRNA) has been shown to regulate gene expression in mammals and their role in cellular function is just beginning to be understood. To investigate the role of circRNAs in ovarian cancer, we performed paired-end RNA sequencing of primary sites, peritoneal and lymph node metastases from three patients with stage IIIC ovarian cancer. We developed an in-house computational pipeline to identify and characterize the circRNA expression from paired-end RNA-Seq libraries. This pipeline revealed thousands of circular isoforms in Epithelial Ovarian Carcinoma (EOC). These circRNAs are enriched for potentially effective miRNA seed matches. A significantly larger number of circRNAs are differentially expressed between tumor sites than mRNAs. Circular and linear expression exhibits an inverse trend for many cancer related pathways and signaling pathways like NFkB, PI3k/AKT and TGF-β typically activated for mRNA in metastases are inhibited for circRNA expression. Further, circRNAs show a more robust expression pattern across patients than mRNA forms indicating their suitability as biomarkers in highly heterogeneous cancer transcriptomes. The consistency of circular RNA expression may offer new candidates for cancer treatment and prognosis.

International audience; In the sea urchin, entry of β-catenin into the nuclei of the vegetal cells at 4th and 5th cleavages is necessary for activation of the endomesoderm gene regulatory network. Beyond that, little is known about how the embryo uses maternal information to initiate specification. Here, experiments establish that of the three maternal Wnts in the egg, Wnt6 is necessary for activation of endodermal genes in the endomesoderm GRN. A small region of the vegetal cortex is shown to be necessary for activation of the endomesoderm GRN. If that cortical region of the egg is removed, addition of Wnt6 rescues endoderm. At a molecular level, the vegetal cortex region contains a localized concentration of Dishevelled (Dsh) protein, a transducer of the canonical Wnt pathway; however, Wnt6 mRNA is not similarly localized. Ectopic activation of the Wnt pathway, through the expression of an activated form of β-catenin, of a dominant-negative variant of GSK-3β or of Dsh itself, rescues endomesoderm specification in eggs depleted of the vegetal cortex. Knockdown experiments in whole embryos show that absence of Wnt6 produces embryos that lack endoderm, but those embryos continue to express a number of mesoderm markers. Thus, maternal Wnt6 plus a localized vegetal cortical molecule, possibly Dsh, is necessary for endoderm specification; this has been verified in two species of sea urchin. The data also show that Wnt6 is only one of what are likely to be multiple components that are necessary for activation of the entire endomesoderm gene regulatory network.

L'article original est publié par The American Society of Plant Biologists; International audience; Plants adapt to different environmental conditions by constantly forming new organs in response to morphogenetic signals. Lateral roots branch from the main root in response to local auxin maxima. How a local auxin maximum translates into a robust pattern of gene activation ensuring the proper growth of the newly formed lateral root is largely unknown. Here, we demonstrate that miR390, TAS3-derived trans-acting short-interfering RNAs (tasiRNAs), and AUXIN RESPONSE FACTORS (ARFs) form an auxin-responsive regulatory network controlling lateral root growth. Spatial expression analysis using reporter gene fusions, tasi/miRNA sensors, and mutant analysis showed that miR390 is specifically expressed at the sites of lateral root initiation where it triggers the biogenesis of tasiRNAs. These tasiRNAs inhibit ARF2, ARF3, and ARF4, thus releasing repression of lateral root growth. In addition, ARF2, ARF3, and ARF4 affect auxin-induced miR390 accumulation. Positive and negative feedback regulation of miR390 by ARF2, ARF3, and ARF4 thus ensures the proper definition of the miR390 expression pattern. This regulatory network maintains ARF expression in a concentration range optimal for specifying the timing of lateral root growth, a function similar to its activity during leaf development. These results also show how small regulatory RNAs integrate with auxin signaling to quantitatively regulate organ growth during development.

International audience; In this study we have identified the target genes of sterol regulatory element binding protein (SREBP)-1a and SREBP-1c in primary cultures of human skeletal muscle cells, using adenoviral vectors expressing the mature nuclear form of human SREBP-1a or SREBP-1c combined with oligonucleotide microarrays. Overexpression of SREBP-1a led to significant changes in the expression of 1,315 genes (655 upregulated and 660 downregulated), whereas overexpression of SREBP-1c modified the mRNA level of 514 genes (310 upregulated and 204 downregulated). Gene ontology analysis indicated that in human muscle cells SREBP-1a and -1c are involved in the regulation of a large number of genes that are at the crossroads of different functional pathways, several of which are not directly connected with cholesterol and lipid metabolism. Six hundred fifty-two of all genes identified to be differentially regulated on SREBP overexpression had a sterol regulatory element (SRE) motif in their promoter sequences. Among these, 429 were specifically regulated by SREBP-1a, 69 by SREBP-1c, and 154 by both 1a and 1c. Because both isoforms recognize the same binding motif, we determined whether some of these functional differences could depend on the environment of the SRE motifs in the promoters. Results from promoter analysis showed that different combinations of transcription factor binding sites around the SRE binding motifs may determine regulatory networks of transcription that could explain the superposition of lipid and cholesterol metabolism with various other pathways involved in adaptive responses to stress like hypoxia and heat shock, or involvement in the immune response.

A small set of core transcription factors (TFs) dominates control of the gene expression program in embryonic stem cells and other well-studied cellular models. These core TFs collectively regulate their own gene expression, thus forming an interconnected auto-regulatory loop that can be considered the core transcriptional regulatory circuitry (CRC) for that cell type. There is limited knowledge of core TFs, and thus models of core regulatory circuitry, for most cell types. We recently discovered that genes encoding known core TFs forming CRCs are driven by super-enhancers, which provides an opportunity to systematically predict CRCs in poorly studied cell types through super-enhancer mapping. Here, we use super-enhancer maps to generate CRC models for 75 human cell and tissue types. These core circuitry models should prove valuable for further investigating cell-type–specific transcriptional regulation in healthy and diseased cells., United States. National Institutes of Health (HG002668)

International audience; Cell differentiation relies on tissue-specific transcription factors (TFs) that cooperate to establish unique transcriptomes and phenotypes. However, the role of ubiquitous TFs in these processes remains poorly defined. Recently, we have shown that the CCCTC-binding factor (CTCF) is required for adipocyte differentiation through epigenomic remodelling of adipose tissue-specific enhancers and transcriptional activation of Peroxisome proliferator-activated receptor gamma (PPARG), the main driver of the adipogenic program (PPARG), and its target genes. Here, we discuss how these findings, together with the recent literature, illuminate a functional role for ubiquitous TFs in lineage-determining transcriptional networks.

International audience; Genomic concepts are based on the assumption that phenotypes arise from the expression of genetic variants. However, the presence of non-Mendelian inheritance patterns provides a direct challenge to this view and suggests an important role for alternative mechanisms of gene regulation and inheritance. Over the past few years, a highly complex and diverse network of noncoding RNAs has been discovered. Research in animal models has shown that RNAs can be inherited and that RNA methyltransferases can be important for the transmission and expression of modified phenotypes in the next generation. We discuss possible mechanisms of RNA-mediated inheritance and the role of these mechanisms for human health and disease.

International audience; Immuno-chemotherapy elicit high response rates in B-cell non-Hodgkin lymphoma but heterogeneity in response duration is observed, with some patients achieving cure and others showing refractory disease or relapse. Using a transcriptome-powered targeted proteomics screen, we discovered a gene regulatory circuit involving the nuclear factor CYCLON which characterizes aggressive disease and resistance to the anti-CD20 monoclonal antibody, Rituximab, in high-risk B-cell lymphoma. CYCLON knockdown was found to inhibit the aggressivity of MYC-overexpressing tumours in mice and to modulate gene expression programs of biological relevance to lymphoma. Furthermore, CYCLON knockdown increased the sensitivity of human lymphoma B cells to Rituximab in vitro and in vivo. Strikingly, this effect could be mimicked by in vitro treatment of lymphoma B cells with a small molecule inhibitor for BET bromodomain proteins (JQ1). In summary, this work has identified CYCLON as a new MYC cooperating factor that autonomously drives aggressive tumour growth and Rituximab resistance in lymphoma. This resistance mechanism is amenable to next-generation epigenetic therapy by BET bromodomain inhibition, thereby providing a new combination therapy rationale for high-risk lymphoma.

In mammals, several genetic pathways have been characterized that govern engagement of multipotent embryonic progenitors into the myogenic program through the control of the key myogenic regulatory gene Myod. Here we demonstrate the involvement of Six homeoproteins. We first targeted into a Pax3 allele a sequence encoding a negative form of Six4 that binds DNA but cannot interact with essential Eya co-factors. The resulting embryos present hypoplasic skeletal muscles and impaired Myod activation in the trunk in the absence of Myf5/Mrf4. At the axial level, we further show that Myod is still expressed in compound Six1/Six4:Pax3 but not in Six1/Six4:Myf5 triple mutant embryos, demonstrating that Six1/4 participates in the Pax3-Myod genetic pathway. Myod expression and head myogenesis is preserved in Six1/Six4:Myf5 triple mutant embryos, illustrating that upstream regulators of Myod in different embryonic territories are distinct. We show that Myod regulatory regions are directly controlled by Six proteins and that, in the absence of Six1 and Six4, Six2 can compensate., Author Summary The onset of skeletal muscle formation is controlled by complex gene regulatory networks. By manipulation of these genetic pathways in the mouse embryo, we have examined the interplay between genes encoding the transcriptional regulator Pax3; the major myogenic determination proteins Myf5, Mrf4, and Myod; as well as genes encoding homeodomain proteins Six1 and Six4. In the absence of Myf5 and Six1/4, Myod expression is compromised. We demonstrate that key regulatory elements of the Myod gene are directly targeted by Six factors, including Six2, which is unexpectedly upregulated in the absence of Six1 and Six4. This work therefore reveals new aspects of the gene regulatory networks that control myogenesis.

International audience; The typical pattern of morphological evolution associated with the radiation of a group of related species is the emergence of a novel trait and its subsequent diversification. Yet the genetic mechanisms associated with these two evolutionary steps are poorly characterized. Here, we show that a spot of dark pigment on fly wings emerged from the assembly of a novel gene regulatory module in which a set of pigmentation genes evolved to respond to a common transcriptional regulator determining their spatial distribution. The primitive wing spot pattern subsequently diversified through changes in the expression pattern of this regulator. These results suggest that the genetic changes underlying the emergence and diversification of wing pigmentation patterns are partitioned within genetic networks.

In the last 20 years, we have witnessed an exponential number of evidences linking the human mixed lineage leukemia-1 (MLL1) gene to several acute and myelogenous leukemias. MLL1 is one of the founding members of the SET1 family of lysine methyltransferases and is key for the proper control of developmentally regulated gene expression. MLL1 is a structurally complex protein composed of several functional domains. These domains play pivotal roles for the recruitment of regulatory proteins. These MLL1 regulatory proteins (MRPs) dynamically interact with MLL1 and consequently control gene expression. In this review, we summarize recent structural and functional studies of MRPs and discuss emergent structural paradigms for the control of MLL1 activity. © 2012 Wiley Periodicals, Inc. Biopolymers 99: 136–145, 2013.

Sfl1p and Sfl2p are two homologous heat shock factor-type transcriptional regulators that antagonistically control morphogenesis in Candida albicans, while being required for full pathogenesis and virulence. To understand how Sfl1p and Sfl2p exert their function, we combined genome-wide location and expression analyses to reveal their transcriptional targets in vivo together with the associated changes of the C. albicans transcriptome. We show that Sfl1p and Sfl2p bind to the promoter of at least 113 common targets through divergent binding motifs and modulate directly the expression of key transcriptional regulators of C. albicans morphogenesis and/or virulence. Surprisingly, we found that Sfl2p additionally binds to the promoter of 75 specific targets, including a high proportion of hyphal-specific genes (HSGs; HWP1, HYR1, ECE1, others), revealing a direct link between Sfl2p and hyphal development. Data mining pointed to a regulatory network in which Sfl1p and Sfl2p act as both transcriptional activators and repressors. Sfl1p directly represses the expression of positive regulators of hyphal growth (BRG1, UME6, TEC1, SFL2), while upregulating both yeast form-associated genes (RME1, RHD1, YWP1) and repressors of morphogenesis (SSN6, NRG1). On the other hand, Sfl2p directly upregulates HSGs and activators of hyphal growth (UME6, TEC1), while downregulating yeast form-associated genes and repressors of morphogenesis (NRG1, RFG1, SFL1). Using genetic interaction analyses, we provide further evidences that Sfl1p and Sfl2p antagonistically control C. albicans morphogenesis through direct modulation of the expression of important regulators of hyphal growth. Bioinformatic analyses suggest that binding of Sfl1p and Sfl2p to their targets occurs with the co-binding of Efg1p and/or Ndt80p. We show, indeed, that Sfl1p and Sfl2p targets are bound by Efg1p and that both Sfl1p and Sfl2p associate in vivo with Efg1p. Taken together, our data suggest that Sfl1p and Sfl2p act as central “switch on/off” proteins to coordinate the regulation of C. albicans morphogenesis., Author Summary Candida albicans can switch from a harmless colonizer of body organs to a life-threatening invasive pathogen. This switch is linked to the ability of C. albicans to undergo a yeast-to-filament shift induced by various cues, including temperature. Sfl1p and Sfl2p are two transcription factors required for C. albicans virulence, but antagonistically regulate morphogenesis: Sfl1p represses it, whereas Sfl2p activates it in response to temperature. We show here that Sfl1p and Sfl2p bind in vivo, via divergent motifs, to the regulatory region of a common set of targets encoding key determinants of morphogenesis and virulence and exert both activating and repressing effects on gene expression. Additionally, Sfl2p binds to specific targets, including genes essential for hyphal development. Bioinformatic analyses suggest that Sfl1p and Sfl2p control C. albicans morphogenesis by cooperating with two important regulators of filamentous growth, Efg1p and Ndt80p, a premise that was confirmed by the observation of concomitant binding of Sfl1p, Sfl2p and Efg1p to the promoter of target genes and the demonstration of direct or indirect physical association of Sfl1p and Sfl2p with Efg1p, in vivo. Our data suggest that Sfl1p and Sfl2p act as central “switch on/off” proteins to coordinate the regulation of C. albicans morphogenesis.

International audience; Immuno-chemotherapy elicit high response rates in B-cell non-Hodgkin lymphoma but heterogeneity in response duration is observed, with some patients achieving cure and others showing refractory disease or relapse. Using a transcriptome-powered targeted proteomics screen, we discovered a gene regulatory circuit involving the nuclear factor CYCLON which characterizes aggressive disease and resistance to the anti-CD20 monoclonal antibody, Rituximab, in high-risk B-cell lymphoma. CYCLON knockdown was found to inhibit the aggressivity of MYC-overexpressing tumours in mice and to modulate gene expression programs of biological relevance to lymphoma. Furthermore, CYCLON knockdown increased the sensitivity of human lymphoma B cells to Rituximab in vitro and in vivo. Strikingly, this effect could be mimicked by in vitro treatment of lymphoma B cells with a small molecule inhibitor for BET bromodomain proteins (JQ1). In summary, this work has identified CYCLON as a new MYC cooperating factor that autonomously drives aggressive tumour growth and Rituximab resistance in lymphoma. This resistance mechanism is amenable to next-generation epigenetic therapy by BET bromodomain inhibition, thereby providing a new combination therapy rationale for high-risk lymphoma.

International audience; Hepatocellular carcinoma (HCC) represents one of the most frequently diagnosed human cancers; however, there are currently few treatment alternatives to surgical resection. In this study we performed bioinformatic analysis of previously published transcriptomic data in order to characterize liver specific networks, including biological functions, signaling pathways and transcription factors, potentially dysregulated in HCC. By incorporating specific signaling inhibitors into real-time proliferation assays using HepG2 cells, we then validated these in silico results. We found that G protein subunits Gi/G0, protein kinase C, Mek1/2, and Erk1/2 (P42/44), JAK1, PPARA and NFκB p65 subunit were the major signaling molecules required for survival and proliferation of human HCC cell lines. We also found that these pathways regulate the expression of key hepatic transcription factors involved in cell differentiation, such as CEBPA, EGR1, FOXM1 and PPARs. By combining bioinformatic and functional analyses, major signaling pathways related to tumorigenicity in HCC are revealed, thereby elucidating potential targets for drug therapies.

Rapid adaptation to changing environments is key in determining the outcome of infections caused by the opportunistic human pathogen Streptococcus agalactiae. We previously demonstrated that the RofA-like protein (RALP) regulators RogB and Rga activate their downstream divergently transcribed genes, that is, the pilus operon PI-2a and the serine-rich repeat encoding gene srr1, respectively. Characterization of the Rga regulon by microarray revealed that the PI-2a pilus was strongly controlled by Rga, a result confirmed at the protein level. Complementation experiments showed that the expression of Rga, but not RogB, in the double ΔrogB/Δrga mutant, or in the clinical strain 2603V/R displaying frameshift mutations in rogB and rga genes, is sufficient to restore wild-type expression levels of PI-2a pilus and Srr1. Biofilm formation was impaired in the Δrga and Δrga/rogB mutants and restored on complementation with rga. Paradoxically, adherence to intestinal epithelial cells was unchanged in the Δrga mutant. Finally, the existence of several clinical isolates mutated in rga highlights the concept of strain-specific regulatory networks.

International audience; Autism spectrum disorders (ASD) are a group of related neurodevelopmental disorders with significant combined prevalence (∼1%) and high heritability. Dozens of individually rare genes and loci associated with high-risk for ASD have been identified, which overlap extensively with genes for intellectual disability (ID). However, studies indicate that there may be hundreds of genes that remain to be identified. The advent of inexpensive massively parallel nucleotide sequencing can reveal the genetic underpinnings of heritable complex diseases, including ASD and ID. However, whole exome sequencing (WES) and whole genome sequencing (WGS) provides an embarrassment of riches, where many candidate variants emerge. It has been argued that genetic variation for ASD and ID will cluster in genes involved in distinct pathways and protein complexes. For this reason, computational methods that prioritize candidate genes based on additional functional information such as protein-protein interactions or association with specific canonical or empirical pathways, or other attributes, can be useful. In this study we applied several supervised learning approaches to prioritize ASD or ID disease gene candidates based on curated lists of known ASD and ID disease genes. We implemented two network-based classifiers and one attribute-based classifier to show that we can rank and classify known, and predict new, genes for these neurodevelopmental disorders. We also show that ID and ASD share common pathways that perturb an overlapping synaptic regulatory subnetwork. We also show that features relating to neuronal phenotypes in mouse knockouts can help in classifying neurodevelopmental genes. Our methods can be applied broadly to other diseases helping in prioritizing newly identified genetic variation that emerge from disease gene discovery based on WES and WGS.

International audience; BACKGROUND: Genetic abnormalities are common in patients with multiple myeloma, and may deregulate gene products involved in tumor survival, proliferation, metabolism and drug resistance. In particular, translocations may result in a high expression of targeted genes (termed spike expression) in tumor cells. We identified spike genes in multiple myeloma cells of patients with newly-diagnosed myeloma and investigated their prognostic value. DESIGN AND METHODS: Genes with a spike expression in multiple myeloma cells were picked up using box plot probe set signal distribution and two selection filters. RESULTS: In a cohort of 206 newly diagnosed patients with multiple myeloma, 2587 genes/expressed sequence tags with a spike expression were identified. Some spike genes were associated with some transcription factors such as MAF or MMSET and with known recurrent translocations as expected. Spike genes were not associated with increased DNA copy number and for a majority of them, involved unknown mechanisms. Of spiked genes, 36.7% clustered significantly in 149 out of 862 documented chromosome (sub)bands, of which 53 had prognostic value (35 bad, 18 good). Their prognostic value was summarized with a spike band score that delineated 23.8% of patients with a poor median overall survival (27.4 months versus not reached, P

International audience; Although p63 and MYC are important in the control of epidermal homeostasis, the underlying molecular mechanisms governing keratinocyte proliferation or differentiation downstream of these two genes are not completely understood. By analyzing the transcriptional changes and phenotypic consequences of the loss of either p63 or MYC in human developmentally mature keratinocytes, we have characterized the networks acting downstream of these two genes to control epidermal homeostasis. We show that p63 is required to maintain growth and to commit to differentiation by two distinct mechanisms. Knockdown of p63 led to down-regulation of MYC via the Wnt/β-catenin and Notch signaling pathways and in turn reduced keratinocyte proliferation. We demonstrate that a p63-controlled keratinocyte cell fate network is essential to induce the onset of keratinocyte differentiation. This network contains several secreted proteins involved in cell migration/adhesion, including fibronectin 1 (FN1), interleukin-1β (IL1B), cysteine-rich protein 61 (CYR61), and jagged-1 (JAG1), that act downstream of p63 as key effectors to trigger differentiation. Our results characterized for the first time a connection between p63 and MYC and a cell adhesion-related network that controls differentiation. Furthermore, we show that the balance between the MYC-controlled cell cycle progression network and the p63-controlled cell adhesion-related network could dictate skin cell fate.

International audience; The catabolite control protein CcpA is a pleiotropic regulator that mediates the global transcriptional response to rapidly catabolizable carbohydrates, like glucose in Gram-positive bacteria. By whole transcriptome analyses, we characterized glucose-dependent and CcpA-dependent gene regulation in Clostridium difficile. About 18% of all C. difficile genes are regulated by glucose, for which 50% depend on CcpA for regulation. The CcpA regulon comprises genes involved in sugar uptake, fermentation and amino acids metabolism, confirming the role of CcpA as a link between carbon and nitrogen pathways. Using combination of chromatin immunoprecipitation and genome sequence analysis, we detected 55 CcpA binding sites corresponding to ∼140 genes directly controlled by CcpA. We defined the C. difficile CcpA consensus binding site (cre(CD) motif), that is, 'RRGAAAANGTTTTCWW'. Binding of purified CcpA protein to 19 target cre(CD) sites was demonstrated by electrophoretic mobility shift assay. CcpA also directly represses key factors in early steps of sporulation (Spo0A and SigF). Furthermore, the C. difficile toxin genes (tcdA and tcdB) and their regulators (tcdR and tcdC) are direct CcpA targets. Finally, CcpA controls a complex and extended regulatory network through the modulation of a large set of regulators.

International audience; Ten years ago, a population of cardiac progenitor cells was identified in pharyngeal mesoderm that gives rise to a major part of the amniote heart. These multipotent progenitor cells, termed the second heart field (SHF), contribute progressively to the poles of the elongating heart tube during looping morphogenesis, giving rise to myocardium, smooth muscle, and endothelial cells. Research into the mechanisms of SHF development has contributed significantly to our understanding of the properties of cardiac progenitor cells and the origins of congenital heart defects. Here recent data concerning the regulation, clinically relevant subpopulations, evolution and lineage relationships of the SHF are reviewed. Proliferation and differentiation of SHF cells are controlled by multiple intercellular signaling pathways and a transcriptional regulatory network that is beginning to be elucidated. Perturbation of SHF development results in common forms of congenital heart defects and particular progenitor cell subpopulations are highly relevant clinically, including cells giving rise to myocardium at the base of the pulmonary trunk and the interatrial septum. A SHF has recently been identified in amphibian, fish, and agnathan embryos, highlighting the important contribution of these cells to the evolution of the vertebrate heart. Finally, SHF-derived parts of the heart share a lineage relationship with craniofacial skeletal muscles revealing that these progenitor cells belong to a broad cardiocraniofacial field of pharyngeal mesoderm. Investigation of the mechanisms underlying the dynamic process of SHF deployment is likely to yield further insights into cardiac development and pathology.

International audience; The adult mammalian cochlea lacks regenerative ability and the irreversible degeneration of cochlear sensory hair cells leads to permanent hearing loss. Previous data show that early postnatal cochlea harbors stem/progenitor-like cells and shows a limited regenerative/repair capacity. These properties are progressively lost later during the postnatal development. Little is known about the genes and pathways that are potentially involved in this difference of the regenerative/repair potentialities between early postnatal and adult mammalian cochlear sensory epithelia (CSE). The goal of our study is to investigate the transcriptomic profiles of these two stages. We used Mouse Genome 430 2.0 microarray to perform an extensive analysis of the genes expressed in mouse postnatal day-3 (P3) and adult CSE. Statistical analysis of microarray data was performed using SAM (Significance Analysis of Microarrays) software. We identified 5644 statistically significant differentially expressed transcripts with a fold change (FC) >2 and a False Discovery Rate (FDR) ≤0.05. The P3 CSE signature included 3,102 transcripts, among which were known genes in the cochlea, but also new transcripts such as, Hmga2 (high mobility group AT-hook 2) and Nrarp (Notch-regulated ankyrin repeat protein). The adult CSE overexpressed 2,542 transcripts including new transcripts, such as Prl (Prolactin) and Ar (Androgen receptor), that previously were not known to be expressed in the adult cochlea. Our comparative study revealed important genes and pathways differentially expressed between the developing and adult CSE. The identification of new candidate genes would be useful as potential markers of the maintenance or the loss of stem cells and regenerative/repair ability during mammalian cochlear development.

Aspergillus fumigatus is the most common and deadly pulmonary fungal infection worldwide. In the lung, the fungus usually forms a dense colony of filaments embedded in a polymeric extracellular matrix. To identify candidate genes involved in this biofilm (BF) growth, we used RNA-Seq to compare the transcriptomes of BF and liquid plankton (PL) growth. Sequencing and mapping of tens of millions sequence reads against the A. fumigatus transcriptome identified 3,728 differentially regulated genes in the two conditions. Although many of these genes, including the ones coding for transcription factors, stress response, the ribosome, and the translation machinery, likely reflect the different growth demands in the two conditions, our experiment also identified hundreds of candidate genes for the observed differences in morphology and pathobiology between BF and PL. We found an overrepresentation of upregulated genes in transport, secondary metabolism, and cell wall and surface functions. Furthermore, upregulated genes showed significant spatial structure across the A. fumigatus genome; they were more likely to occur in subtelomeric regions and colocalized in 27 genomic neighborhoods, many of which overlapped with known or candidate secondary metabolism gene clusters. We also identified 1,164 genes that were downregulated. This gene set was not spatially structured across the genome and was overrepresented in genes participating in primary metabolic functions, including carbon and amino acid metabolism. These results add valuable insight into the genetics of biofilm formation in A. fumigatus and other filamentous fungi and identify many relevant, in the context of biofilm biology, candidate genes for downstream functional experiments.

International audience; Human T-cell Lymphotropic Viruses type 1 (HTLV-1) is the etiological agent of Adult T-cell Leukemia/Lymphoma. Although associated with lymphocytosis, HTLV-2 infection is not associated with any malignant hematological disease. Similarly, no infection-related symptom has been detected in HTLV-3-infected individuals studied so far. Differences in individual Tax transcriptional activity might account for these distinct physiopathological outcomes. Tax-1 and Tax-3 possess a PDZ binding motif in their sequence. Interestingly, this motif, which is critical for Tax-1 transforming activity, is absent from Tax-2. We used the DNA microarray technology to analyze and compare the global gene expression profiles of different T- and non T-cell types expressing Tax-1, Tax-2 or Tax-3 viral transactivators. In a T-cell line, this analysis allowed us to identify 48 genes whose expression is commonly affected by all Tax proteins and are hence characteristic of the HTLV infection, independently of the virus type. Importantly, we also identified a subset of genes (n = 70) which are specifically up-regulated by Tax-1 and Tax-3, while Tax-1 and Tax-2 shared only 1 gene and Tax-2 and Tax-3 shared 8 genes. These results demonstrate that Tax-3 and Tax-1 are closely related in terms of cellular gene deregulation. Analysis of the molecular interactions existing between those Tax-1/Tax-3 deregulated genes then allowed us to highlight biological networks of genes characteristic of HTLV-1 and HTLV-3 infection. The majority of those up-regulated genes are functionally linked in biological processes characteristic of HTLV-1-infected T-cells expressing Tax such as regulation of transcription and apoptosis, activation of the NF-κB cascade, T-cell mediated immunity and induction of cell proliferation and differentiation. In conclusion, our results demonstrate for the first time that, in T- and non T-cells types, Tax-3 is a functional analogue of Tax-1 in terms of transcriptional activation and suggest that HTLV-3 might share pathogenic features with HTLV-1 in vivo.

International audience; Complex phenotypes are often controlled by many interacting genes. One question emerging from such organization is how selection, acting at the phenotypic level, shapes the evolution of genes involved in regulatory networks controlling the phenotypes. We studied this issue through a matrix model of such networks. In a population submitted to selection, we simulated the evolution of a quantitative trait controlled by a set of loci that regulate each other through positive or negative interactions. Investigating several levels of selection intensity on the trait, we studied the evolution of regulation intensity between the genes and the evolution of the genetic diversity of those genes as an indirect measure of the strength of selection acting on them. We show that an increasing intensity of selection on the phenotype leads to an increased level of regulation between the loci. Moreover, we found that the genes responding more strongly to selection within the network were those evolving towards stronger regulatory action on the other genes and/or those that are the less regulated by the other genes. This observation is strongest for an intermediate level of selection. This may explain why several experimental studies have shown evidence of selection on regulatory genes inside gene networks.

Morphological consistency in metazoans is remarkable given the pervasive occurrence of genetic variation, environmental effects, and developmental noise. Developmental stability, the ability to reduce developmental noise, is a fundamental property of multicellular organisms, yet its genetic bases remains elusive. Imperfect bilateral symmetry, or fluctuating asymmetry, is commonly used to estimate developmental stability. We observed that Drosophila melanogaster overexpressing Cyclin G (CycG) exhibit wing asymmetry clearly detectable by sight. Quantification of wing size and shape using geometric morphometrics reveals that this asymmetry is a genuine—but extreme—fluctuating asymmetry. Overexpression of CycG indeed leads to a 40-fold increase of wing fluctuating asymmetry, which is an unprecedented effect, for any organ and in any animal model, either in wild populations or mutants. This asymmetry effect is not restricted to wings, since femur length is affected as well. Inactivating CycG by RNAi also induces fluctuating asymmetry but to a lesser extent. Investigating the cellular bases of the phenotypic effects of CycG deregulation, we found that misregulation of cell size is predominant in asymmetric flies. In particular, the tight negative correlation between cell size and cell number observed in wild-type flies is impaired when CycG is upregulated. Our results highlight the role of CycG in the control of developmental stability in D. melanogaster. Furthermore, they show that wing developmental stability is normally ensured via compensatory processes between cell growth and cell proliferation. We discuss the possible role of CycG as a hub in a genetic network that controls developmental stability., Author Summary Developing organisms face genetic, environmental, and stochastic variations, and yet their ability to reach stereotyped phenotypes is pervasive. Although genetic bases of this robustness are actively investigated, no consensus has been achieved, some authors attributing it to particular genes such as Hsp90, while others to emerging properties of complex genetic networks. A particularly puzzling component of robustness is developmental stability, i.e. the reduction of developmental noise. Genetic bases of developmental stability have remained equally elusive. In bilaterians, developmental stability is commonly measured by fluctuating asymmetry (FA), where the same organ is compared on both sides of the same individual. Here, we show that ubiquitous overexpression of the Cyclin G gene induces a 40-fold increase of wing size FA, which is an unprecedented effect, for any organ and in any animal model. Investigating the cellular bases of this asymmetry, we show that the tight negative correlation between cell size and cell number that occurs in wild-type flies was lost. Since not only wings but also legs were affected, Cyclin G appears a candidate gene for a general mechanism of developmental stability in Drosophila, suggesting that phenotypic robustness can be strongly influenced by individual genes.

International audience; Human embryonic stem cells can be differentiated along different lineages, providing the possibility of a precise analysis of genes profiles associated with specific commitments. Subtractive gene expression profiling between differentiated and undifferentiated cells provides lists of potential actors in this commitment. This combines, however, genes that are specifically associated with development and others that are over expressed because of nonlineage-specific differentiation systems. As a way to establish gene profiles associated with the neural and/or to the mesodermal commitments of human embryonic stem cells more precisely, we have carried out a 2-step analysis. We first performed a subtractive analysis of gene profiles of each of these lineages as compared to the undifferentiated stage. Then, we extended the analysis by comparing the 2 sets of results with each other. This strategy has allowed us to eliminate large numbers of genes that were over expressed in both sets of results and to uniquely associate different gene networks with either the neural or the mesodermal commitments.