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International audience; Anatomically modern humans started to exit Africa for the first time at least 60,000 years ago (ya). Along their journey across the globe, they encountered and admixed with other hominins that are now extinct, such as the Neanderthals or Denisovans. Given the deep divergence time between ancient hominins and modern humans, such admixture events left molecular traces in non-African populations that are still visible today in their genomes [1]. Over the past few years, there is accumulating evidence to suggest that these segments of “archaic” DNA have the potential to contribute to phenotypic differences between contemporary individuals and populations [2]. Yet, to understand the genuine contribution of archaic alleles to the genetic architecture of complex traits, it is necessary to account for the diverse selective pressures that have acted upon introgressed alleles. Here, we discuss recent findings on how natural selection—either negative or positive—has shaped the landscape of Neanderthal ancestry in the genomes of modern Eurasians, and comment on the contribution of archaic haplotypes to present-day phenotypic variation.

To date, epimutations reported in man have been somatic and erased in germlines. Here, we identify a cause of the autosomal recessive cblC class of inborn errors of vitamin B12 metabolism that we name “epi-cblC”. The subjects are compound heterozygotes for a genetic mutation and for a promoter epimutation, detected in blood, fibroblasts, and sperm, at the MMACHC locus; 5-azacytidine restores the expression of MMACHC in fibroblasts. MMACHC is flanked by CCDC163P and PRDX1, which are in the opposite orientation. The epimutation is present in three generations and results from PRDX1 mutations that force antisense transcription of MMACHC thereby possibly generating a H3K36me3 mark. The silencing of PRDX1 transcription leads to partial hypomethylation of the epiallele and restores the expression of MMACHC. This example of epi-cblC demonstrates the need to search for compound epigenetic-genetic heterozygosity in patients with typical disease manifestation and genetic heterozygosity in disease-causing genes located in other gene trios., Inborn errors of vitamin B12 metabolism of the cblC class are caused by mutations in the MMACHC gene. Here, Guéant et al. report epi-cblC, a class of cblC in which patients are compound heterozygous for a genetic mutation and a secondary epimutation at the MMACHC locus.

International audience; Genome editing technologies are being widely adopted in plant breeding1. However, a looming challenge of engineering desirable genetic variation in diverse genotypes is poor predictability of phenotypic outcomes due to unforeseen interactions with pre-existing cryptic mutations2-4. In tomato, breeding with a classical MADS-box gene mutation that improves harvesting by eliminating fruit stem abscission frequently results in excessive inflorescence branching, flowering and reduced fertility due to interaction with a cryptic variant that causes partial mis-splicing in a homologous gene5-8. Here, we show that a recently evolved tandem duplication carrying the second-site variant achieves a threshold of functional transcripts to suppress branching, enabling breeders to neutralize negative epistasis on yield. By dissecting the dosage mechanisms by which this structural variant restored normal flowering and fertility, we devised strategies that use CRISPR-Cas9 genome editing to predictably improve harvesting. Our findings highlight the under-appreciated impact of epistasis in targeted trait breeding and underscore the need for a deeper characterization of cryptic variation to enable the full potential of genome editing in agriculture.

International audience; Background: Epistasis and gene-environment interactions are known to contribute significantly to variation of complex phenotypes in model organisms. However, their identification in human association studies remains challenging for myriad reasons. In the case of epistatic interactions, the large number of potential interacting sets of genes presents computational, multiple hypothesis correction, and other statistical power issues. In the case of gene-environment interactions, the lack of consistently measured environmental covariates in most disease studies precludes searching for interactions and creates difficulties for replicating studies.Results: In this work, we develop a new statistical approach to address these issues that leverages genetic ancestry, defined as the proportion of ancestry derived from each ancestral population (e.g., the fraction of European/African ancestry in African Americans), in admixed populations. We applied our method to gene expression and methylation data from African American and Latino admixed individuals, respectively, identifying nine interactions that were significant at P

International audience; PTP1B is an important negative regulator of insulin and other signaling pathways in mammals. However, the role of PTP1B in the regulation of RAS-MAPK signaling remains open to deliberation, due to conflicting evidence from different experimental systems. The Drosophila orthologue of mammalian PTP1B, PTP61F, has until recently remained largely uncharacterized. To establish the potential role of PTP61F in the regulation of signaling pathways in Drosophila and particularly to help resolve its fundamental function in RAS-MAPK signaling, we generated a new allele of Ptp61F as well as employed both RNA interference and overexpression alleles. Our results validate recent data showing that the activity of insulin and Abl kinase signaling is increased in Ptp61F mutants and RNA interference lines. Importantly, we establish negative regulation of the RAS/MAPK pathway by Ptp61F activity in whole animals. Of particular interest, our results document the modulation of hyperactive MAP kinase activity by Ptp61F alleles, showing that the phosphatase intervenes to directly or indirectly regulate MAP kinase itself.

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.

Background Although genome-wide association studies have successfully identified thousands of variants associated to complex traits, these variants only explain a small amount of the entire heritability of the trait. Gene-gene interactions have been proposed as a source to explain a significant percentage of the missing heritability. However, detecting gene-gene interactions has proven to be very difficult due to computational and statistical challenges. The vast number of possible interactions that can be tested induces very stringent multiple hypotheses corrections that limit the power of detection. These issues have been mostly highlighted for the identification of pairwise effects and are even more challenging when addressing higher order interaction effects. In this work we explore the use of local ancestry in recently admixed individuals to find signals of gene-gene interaction on human traits and diseases. Results We introduce statistical methods that leverage the correlation between local ancestry and the hidden unknown causal variants to find distant gene-gene interactions. We show that the power of this test increases with the number of causal variants per locus and the degree of differentiation of these variants between the ancestral populations. Overall, our simulations confirm that local ancestry can be used to detect gene-gene interactions, solving the computational bottleneck. When compared to a single nucleotide polymorphism (SNP)-based interaction screening of the same sample size, the power of our test was lower on all settings we considered. However, accounting for the dramatic increase in sample size that can be achieve when genotyping only a set of ancestry informative markers instead of the whole genome, we observe substantial gain in power in several scenarios. Conclusion Local ancestry-based interaction tests offer a new path to the detection of gene-gene interaction effects. It would be particularly useful in scenarios where multiple differentiated variants at the interacting loci act in a synergistic manner. Electronic supplementary material The online version of this article (doi:10.1186/s12863-015-0283-z) contains supplementary material, which is available to authorized users.

Background Deciphering the genetic architecture of complex traits is still a major challenge for human genetics. In most cases, genome-wide association studies have only partially explained the heritability of traits and diseases. Epistasis, one potentially important cause of this missing heritability, is difficult to explore at the genome-wide level. Here, we develop and assess a tool based on interactive odds ratios (IOR), Fast Odds Ratio-based sCan for Epistasis (FORCE), as a novel approach for exhaustive genome-wide epistasis search. IOR is the ratio between the multiplicative term of the odds ratio (OR) of having each variant over the OR of having both of them. By definition, an IOR that significantly deviates from 1 suggests the occurrence of an interaction (epistasis). As the IOR is fast to calculate, we used the IOR to rank and select pairs of interacting polymorphisms for P value estimation, which is more time consuming. Results FORCE displayed power and accuracy similar to existing parametric and non-parametric methods, and is fast enough to complete a filter-free genome-wide epistasis search in a few days on a standard computer. Analysis of psoriasis data uncovered novel epistatic interactions in the HLA region, corroborating the known major and complex role of the HLA region in psoriasis susceptibility. Conclusions Our systematic study revealed the ability of FORCE to uncover novel interactions, highlighted the importance of exhaustiveness, as well as its specificity for certain types of interactions that were not detected by existing approaches. We therefore believe that FORCE is a valuable new tool for decoding the genetic basis of complex diseases. Electronic supplementary material The online version of this article (doi:10.1186/s12863-015-0174-3) contains supplementary material, which is available to authorized users.

Background: The transcription factor DOF AFFECTING GERMINATION1 (DAG1) is a repressor of seed germination acting downstream of the master repressor PHYTOCROME INTERACTING FACTOR3-LIKE 5 (PIL5). Among others, PIL5 induces the expression of the genes encoding the two DELLA proteins GA INSENSITIVE 1 (GAI) and REPRESSOR OF ga1-3 (RGA). Results: Based on the properties of gai-t6 and rga28 mutant seeds, we show here that the absence of RGA severely increases dormancy, while lack of GAI only partially compensates RGA inactivation. In addition, the germination properties of the dag1rga28 double mutant are different from those of the dag1 and rga28 single mutants, suggesting that RGA and DAG1 act in independent branches of the PIL5-controlled germination pathway. Surprisingly, the dag1gai-t6 double mutant proved embryo-lethal, suggesting an unexpected involvement of (a possible complex between) DAG1 and GAI in embryo development. Conclusions: Rather than overlapping functions as previously suggested, we show that RGA and GAI play distinct roles in seed germination, and that GAI interacts with DAG1 in embryo development.

Group B Streptococcus (GBS), a common commensal of the female genital tract, is the leading cause of invasive infections in neonates. Expression of major GBS virulence factors, such as the hemolysin operon cyl, is regulated directly at the transcriptional level by the CovSR two-component system. Using a random genetic approach, we identified a multi-spanning transmembrane protein, Abx1, essential for the production of the GBS hemolysin. Despite its similarity to eukaryotic CaaX proteases, the Abx1 function is not involved in a post-translational modification of the GBS hemolysin. Instead, we demonstrate that Abx1 regulates transcription of several virulence genes, including those comprising the hemolysin operon, by a CovSR-dependent mechanism. By combining genetic analyses, transcriptome profiling, and site-directed mutagenesis, we showed that Abx1 is a regulator of the histidine kinase CovS. Overexpression of Abx1 is sufficient to activate virulence gene expression through CovS, overcoming the need for an additional signal. Conversely, the absence of Abx1 has the opposite effect on virulence gene expression consistent with CovS locked in a kinase-competent state. Using a bacterial two-hybrid system, direct interaction between Abx1 and CovS was mapped specifically to CovS domains involved in signal processing. We demonstrate that the CovSR two-component system is the core of a signaling pathway integrating the regulation of CovS by Abx1 in addition to the regulation of CovR by the serine/threonine kinase Stk1. In conclusion, our study reports a regulatory function for Abx1, a member of a large protein family with a characteristic Abi-domain, which forms a signaling complex with the histidine kinase CovS in GBS., Author Summary The gram-positive Streptococcus genus includes three major human pathogens that are members of the normal microflora: Streptococcus pneumoniae (also known as the pneumococcus), Streptococcus pyogenes (Group A Streptococcus), and Streptococcus agalactiae (Group B Streptococcus). Their carriage in the population is highly dynamic and mostly asymptomatic. However, each of these species can cause a wide spectrum of diseases, from local infections to systemic and fatal infections including septicemia and meningitis. Expression of streptococcal virulence-associated genes is tightly regulated at the transcriptional level. However, the signal(s) and the precise molecular events controlling the switch from commensalism to virulence are not yet understood. In this study, we identified and characterized a bacterial protein essential for virulence gene expression in Group B Streptococcus, the main pathogen of neonates. We show that this transmembrane protein, named Abx1, interacts with the histidine kinase CovS to modulate the activity of the major regulator of virulence CovR. We define how a core set of four proteins, Abx1, CovS, CovR, and the serine/threonine kinase Stk1, interact to control the expression of virulence genes in S. agalactiae. We propose that Abx1-like proteins, that are widespread in bacteria, might be part of a conserved mechanism of two-component system regulation.

Rationale: Nkx2.5 is one of the most widely studied cardiac-specific transcription factors, conserved from flies to man, with multiple essential roles in both the developing and adult heart. Specific dominant mutations in NKX2.5 have been identified in adult congenital heart disease patients presenting with conduction system anomalies and recent genome-wide association studies implicate the NKX2.5 locus, as causative for lethal arrhythmias (“sudden cardiac death”) that occur at a frequency in the population of 1 in 1000 per annum worldwide. Haploinsufficiency for Nkx2.5 in the mouse phenocopies human conduction disease pathology yet the phenotypes, described in both mouse and man, are highly pleiotropic, implicit of unknown modifiers and/or factors acting in epistasis with Nkx2.5/NKX2.5. Objective: To identify bone fide upstream genetic modifier(s) of Nkx2.5/NKX2.5 function and to determine epistatic effects relevant to the manifestation of NKX2.5 -dependent adult congenital heart disease. Methods and Results: A study of cardiac function in prospero-related homeobox protein 1 (Prox1) heterozygous mice, using pressure-volume loop and micromannometry, revealed rescue of hemodynamic parameters in Nkx2.5 Cre/+ ; Prox1 loxP/+ animals versus Nkx2.5 Cre/+ controls. Anatomic studies, on a Cx40 EGFP background, revealed Cre-mediated knock-down of Prox1 restored the anatomy of the atrioventricular node and His-Purkinje network both of which were severely hypoplastic in Nkx2.5 Cre/+ littermates. Steady state surface electrocardiography recordings and high-speed multiphoton imaging, to assess Ca 2+ handling, revealed atrioventricular conduction and excitation-contraction were also normalized by Prox1 haploinsufficiency, as was expression of conduction genes thought to act downstream of Nkx2.5. Chromatin immunoprecipitation on adult hearts, in combination with both gain and loss-of-function reporter assays in vitro, revealed that Prox1 recruits the corepressor HDAC3 to directly repress Nkx2.5 via a proximal upstream enhancer as a mechanism for regulating Nkx2.5 function in adult cardiac conduction. Conclusions: Here we identify Prox1 as a direct upstream modifier of Nkx2.5 in the maintenance of the adult conduction system and rescue of Nkx2.5 conduction disease phenotypes. This study is the first example of rescue of Nkx2.5 function and establishes a model for ensuring electrophysiological function within the adult heart alongside insight into a novel Prox1-HDAC3-Nkx2.5 signaling pathway for therapeutic targeting in conduction disease.

International audience; An association between a common deletion comprising the late cornified envelope LCE3B and LCE3C genes (LCE3C_LCE3B-del) and psoriasis has been reported in Caucasian and Asian populations. To investigate whether this deletion plays a role in the genetic of psoriasis in Tunisian population, we determined the LCE3C_LCE3B-del genotype in 180 Ps patients and 208 healthy controls from different regions of Tunisia. The LCE3B and LCE3C gene variant was determined in the patients through PCR amplification and the SPSS software package. The frequency of the LCE3C_LCE3B-del was similar between patients and healthy controls. Subanalyses by family history revealed that the frequency of LCE3C_LCE3B-del was significantly higher in patients with a positive family history than in control individuals, as well as in individuals with a positive family history versus those without in the case cohort. However, no significant difference was observed between psoriatic patients with no family history and controls. We also evaluated the relationship between LCE3C_LCE3B-del and PSORS1. No significant epistatic effect was observed suggesting that there was no significant epistasis of the two loci in the Tunisian population. Our findings indicate that the LCE3C_LCE3B-del might play a role in familial psoriasis in the Tunisian population.

International audience; BACKGROUND: In budding yeast, the highly conserved Tel2 protein is part of several complexes and its main function is now believed to be in the biogenesis of phosphatidyl inositol 3-kinase related kinases. PRINCIPAL FINDINGS: To uncover potentially novel functions of Tel2, we set out to isolate temperature-sensitive (ts) mutant alleles of TEL2 in order to perform genetic screenings. MED15/GAL11, a subunit of Mediator, a general regulator of transcription, was isolated as a suppressor of these mutants. The isolated tel2 mutants exhibited a short telomere phenotype that was partially rescued by MED15/GAL11 overexpression. The tel2-15 mutant was markedly deficient in the transcription of EST2, coding for the catalytic subunit of telomerase, potentially explaining the short telomere phenotype of this mutant. In parallel, a two-hybrid screen identified an association between Tel2 and Rvb2, a highly conserved member of the AAA+ family of ATPases further found by in vivo co-immunoprecipitation to be tight and constitutive. Transiently overproduced Tel2 and Med15/Gal11 associated together, suggesting a potential role for Tel2 in transcription. Other Mediator subunits, as well as SUA7/TFIIB, also rescued the tel2-ts mutants. SIGNIFICANCE: Altogether, the present data suggest the existence of a novel role for Tel2, namely in transcription, possibly in cooperation with Rvb2 and involving the existence of physical interactions with the Med15/Gal11 Mediator subunit.

International audience; With the finding that brown adipose tissue is present and negatively correlated to obesity in adult man, finding the mechanism(s) of how to activate brown adipose tissue in humans could be important in combating obesity, type 2 diabetes, and their complications. In mice, the main regulator of nonshivering thermogenesis in brown adipose tissue is norepinephrine acting predominantly via β(3)-adrenergic receptors. However, vast majorities of β(3)-adrenergic agonists have so far not been able to stimulate human β(3)-adrenergic receptors or brown adipose tissue activity, and it was postulated that human brown adipose tissue could be regulated instead by β(1)-adrenergic receptors. Therefore, we have investigated the signaling pathways, specifically pathways to nonshivering thermogenesis, in mice lacking β(3)-adrenergic receptors. Wild-type and β(3)-knockout mice were either exposed to acute cold (up to 12 h) or acclimated for 7 wk to cold, and parameters related to metabolism and brown adipose tissue function were investigated. β(3)-knockout mice were able to survive both acute and prolonged cold exposure due to activation of β(1)-adrenergic receptors. Thus, in the absence of β(3)-adrenergic receptors, β(1)-adrenergic receptors are effectively able to signal via cAMP to elicit cAMP-mediated responses and to recruit and activate brown adipose tissue. In addition, we found that in human multipotent adipose-derived stem cells differentiated into functional brown adipocytes, activation of either β(1)-adrenergic receptors or β(3)-adrenergic receptors was able to increase UCP1 mRNA and protein levels. Thus, in humans, β(1)-adrenergic receptors could play an important role in regulating nonshivering thermogenesis.

International audience; The adult thorax of Drosophila melanogaster is covered by a stereotyped pattern of mechanosensory bristles called macrochaetes. Here, we report that the MYST containing protein Chameau (Chm) contributes to the establishment of this pattern in the most dorsal part of the thorax. Chm mutant pupae present extra-dorsocentral (DC) and scutellar (SC) macrochaetes, but a normal number of the other macrochaetes. We provide evidences that chm restricts the singling out of sensory organ precursors from proneural clusters and genetically interacts with transcriptional regulators involved in the regulation of achaete and scute in the DC and SC proneural cluster. This function of chm likely relies on chromatin structure regulation since a protein with a mutation in the conserved catalytic site fails to rescue the formation of supernumerary DC and SC bristles in chm mutant flies. This is further supported by the finding that mutations in genes encoding chromatin modifiers and remodeling factors, including Polycomb group (PcG) and Trithorax group (TrxG) members, dominantly modulate the penetrance of chm extra bristle phenotype. These data support a critical role for chromatin structure modulation in the establishment of the stereotyped sensory bristle pattern in the fly thorax.

International audience; Epistatic interactions between mutations play a prominent role in evolutionary theories. Many studies have found that epistasis is widespread, but they have rarely considered beneficial mutations. We analyzed the effects of epistasis on fitness for the first five mutations to fix in an experimental population of Escherichia coli. Epistasis depended on the effects of the combined mutations--the larger the expected benefit, the more negative the epistatic effect. Epistasis thus tended to produce diminishing returns with genotype fitness, although interactions involving one particular mutation had the opposite effect. These data support models in which negative epistasis contributes to declining rates of adaptation over time. Sign epistasis was rare in this genome-wide study, in contrast to its prevalence in an earlier study of mutations in a single gene.

International audience; Dietary restriction (DR) is the most universal intervention known to extend animal lifespan. DR also prevents tumor development in mammals, and this effect requires the tumor suppressor PTEN. However, the metabolic and cellular processes that underly the beneficial effects of DR are poorly understood. We identified slcf-1 in an RNAi screen for genes that extend Caenorhabditis elegans lifespan in a PTEN/daf-18-dependent manner. We showed that slcf-1 mutation, which increases average lifespan by 40%, mimics DR in worms fed ad libitum. An NMR-based metabolomic characterization of slcf-1 mutants revealed lower lipid levels compared to wild-type animals, as expected for dietary-restricted animals, but also higher pyruvate content. Epistasis experiments and metabolic measurements support a model in which the long lifespan of slcf-1 mutants relies on increased mitochondrial pyruvate metabolism coupled to an adaptive response to oxidative stress. This response requires DAF-18/PTEN and the previously identified DR effectors PHA-4/FOXA, HSF-1/HSF1, SIR-2.1/SIRT-1, and AMPK/AAK-2. Overall, our data show that pyruvate homeostasis plays a central role in lifespan control in C. elegans and that the beneficial effects of DR results from a hormetic mechanism involving the mitochondria. Analysis of the SLCF-1 protein sequence predicts that slcf-1 encodes a plasma membrane transporter belonging to the conserved monocarboxylate transporter family. These findings suggest that inhibition of this transporter homolog in mammals might also promote a DR response.

The neural crest (NC) emerges from combinatorial inductive events occurring within its progenitor domain, the neural border (NB). Several transcription factors act early at the NB, but the initiating molecular events remain elusive. Recent data from basal vertebrates suggest that ap2 might have been critical for NC emergence; however, the role of AP2 factors at the NB remains unclear. We show here that AP2a initiates NB patterning and is sufficient to elicit a NB-like pattern in neuralized ectoderm. In contrast, the other early regulators do not participate in ap2a initiation at the NB, but cooperate to further establish a robust NB pattern. The NC regulatory network uses a multistep cascade of secreted inducers and transcription factors, first at the NB and then within the NC progenitors. Here we report that AP2a acts at two distinct steps of this cascade. As the earliest known NB specifier, AP2a mediates Wnt signals to initiate the NB and activate pax3 ; as a NC specifier, AP2a regulates further NC development independent of and downstream of NB patterning. Our findings reconcile conflicting observations from various vertebrate organisms. AP2a provides a paradigm for the reiterated use of multifunctional molecules, thereby facilitating emergence of the NC in vertebrates.

Hirschsprung disease (HSCR) is a common, multigenic neurocristopathy characterized by incomplete innervation along a variable length of the gut. The pivotal gene in isolated HSCR cases, either sporadic or familial, is RET . HSCR also presents in various syndromes, including Shah–Waardenburg syndrome (WS), Down (DS), and Bardet–Biedl (BBS). Here, we report 3 families with BBS and HSCR with concomitant mutations in BBS genes and regulatory RET elements, whose functionality is tested in physiologically relevant assays. Our data suggest that BBS mutations can potentiate HSCR predisposing RET alleles, which by themselves are insufficient to cause disease. We also demonstrate that these genes interact genetically in vivo to modulate gut innervation, and that this interaction likely occurs through complementary, yet independent, pathways that converge on the same biological process.

Background: Trachoma, a chronic keratoconjunctivitis caused by Chlamydia trachomatis, is the world's commonest infectious cause of blindness. Blindness is due to progressive scarring of the conjunctiva (trachomatous scarring) leading to in-turning of eyelashes (trichiasis) and corneal opacification. We evaluated the contribution of genetic variation across the chemokine and cytokine clusters in chromosomes 4q and 5q31 respectively to risk of scarring trachoma and trichiasis in a large case-control association study in a Gambian population. Methods: Linkage disequilibrium (LD) mapping was used to investigate risk effects across the 4q and 5q31 cytokine clusters in relation to the risk of scarring sequelae of ocular Ct infection. Disease association and epistatic effects were assessed in a population based study of 651 case-control pairs by conditional logistic regression (CLR) analyses. Results: LD mapping suggested that genetic effects on risk within these regions mapped to the pro-inflammatory innate immune genes interleukin 8 (IL8) and granulocyte-macrophage colony stimulatory factor (CSF2) loci. The IL8-251 rare allele (IL8-251 TT) was associated with protection from scarring trachoma (OR = 0.29 p = 0.027). The intronic CSF2_27348 A allele in chromosome 5q31 was associated with dose dependent protection from trichiasis, with each copy of the allele reducing risk by 37% (p = 0.005). There was evidence of epistasis, with effects at IL8 and CSF2 loci interacting with those previously reported at the MMP9 locus, a gene acting downstream to IL8 and CSF2 in the inflammatory cascade. Conclusion: innate immune response SNP-haplotypes are linked to ocular Ct sequelae. This work illustrates the first example of epistatic effects of two genes on trachoma.

Complex traits are under the genetic control of multiple genes, often with weak effects and strong epistatic interactions. We developed two new collections of mouse strains to improve genetic dissection of complex traits. They are derived from several backcrosses of the Mus spretus SEG/Pas or STF/Pas strains on the C57BL/6J background. Each of the 55 interspecific recombinant congenic strains (IRCSs) carries up to eight SEG/Pas chromosomal segments with an average size of 11.7 Mb, totalizing 1.37% of the genome. The complete series covers 39.7% of the SEG/Pas genome. As a complementary resource, six partial or complete interspecific consomic strains were developed and increased genome coverage to 45.6%. To evaluate the usefulness of these strains for QTL mapping, 16 IRCSs were compared with C57BL/6J for seven hematological parameters. Strain 66H, which carries three SEG/Pas chromosomal segments, had lower red blood cell volume and higher platelet count than C57BL/6J. Each chromosomal segment was isolated in a congenic strain to evaluate individual effects. Congenic strains were combined to assess epistasis. Our data show that both traits were controlled by several genes with complex epistatic interactions. IRCSs are therefore useful to unravel QTL with small effects and gene-by-gene interactions.

The most consistent result in more than two decades of experimental evolution is that the fitness of populations adapting to a constant environment does not increase indefinitely, but reaches a plateau. Using experimental evolution with bacteriophage, we show here that the converse is also true. In populations small enough such that drift overwhelms selection and causes fitness to decrease, fitness declines down to a plateau. We demonstrate theoretically that both of these phenomena must be due either to changes in the ratio of beneficial to deleterious mutations, the size of mutational effects, or both. We use mutation accumulation experiments and molecular data from experimental evolution to show that the most significant change in mutational effects is a drastic increase in the rate of beneficial mutation as fitness decreases. In contrast, the size of mutational effects changes little even as organismal fitness changes over several orders of magnitude. These findings have significant implications for the dynamics of adaptation., PLoS Biology, 5 (4), ISSN:1544-9173, ISSN:1545-7885

During development, neural cell fate in the vertebrate optic nerve is restricted to the astroglial lineage. However, when isolated from the embryo and explanted in vitro, optic nerve progenitors generate neurons instead of astrocytes, suggesting that neuronal potentialities exist and are repressed in progenitors in vivo. Here we have investigated the mechanisms controlling cell fate in the optic nerve. The optic nerve is characterized by expression of the homeodomain transcription factor Pax2 which is maintained in differentiated astrocytes. We have observed that Pax2 is rapidly down-regulated in explanted optic nerves that generate neurons, and that its overexpression by electroporation in the optic nerve, or ectopically in the neural tube, is sufficient to block neuronal differentiation and allow glial development, showing that Pax2 plays a major role in controlling cell fate in the optic nerve. In vitro and ex vivo experiments further show that a signaling cascade that involves successively Sonic hedgehog and FGF is required to maintain Pax2 expression in optic nerve precursors whereby inhibiting the neuronal fate and promoting astroglial differentiation.

In recent years it has become clear that non-coding RNAs (that is, RNAs that are not translated into proteins) play a vital role in regulating gene expression. A previously unknown manifestation of that control has been found to operate on the human dihydrofolate reductase (DHFR) gene. The interfering RNA forms a complex with the DHFR gene's promoter region, and in so doing it interferes with the binding of transcription factors. The non-coding RNA is produced only in quiescent cells, leading to repression of the DHFR gene in these conditions. The DHFR major promoter is repressed in quiescent cells by a non-coding RNA initiated from an upstream promoter. This RNA molecule forms a complex with the major promoter, interacts with the general transcription factor IIB, and dissociates the pre-initiation complex from the promoter. Alternative promoters within the same gene are a general phenomenon in gene expression1,2. Mechanisms of their selective regulation vary from one gene to another and are still poorly understood. Here we show that in quiescent cells the mechanism of transcriptional repression of the major promoter of the gene encoding dihydrofolate reductase depends on a non-coding transcript initiated from the upstream minor promoter and involves both the direct interaction of the RNA and promoter-specific interference. The specificity and efficiency of repression is ensured by the formation of a stable complex between non-coding RNA and the major promoter, direct interaction of the non-coding RNA with the general transcription factor IIB and dissociation of the preinitiation complex from the major promoter. By using in vivo and in vitro assays such as inducible and reconstituted transcription, RNA bandshifts, RNA interference, chromatin immunoprecipitation and RNA immunoprecipitation, we show that the regulatory transcript produced from the minor promoter has a critical function in an epigenetic mechanism of promoter-specific transcriptional repression.

Characterisation of the interactions between susceptibility loci (epistasis) is central to a full understanding of the genetic aetiology and the molecular pathology of complex diseases. We have examined, in British and French pedigrees, evidence for epistasis between the type 2 diabetes susceptibility loci on chromosomes 1q21-25 and 10q23-26 using two complementary linkage-based approaches. Joint two-locus linkage analysis of 1q and 10q in British pedigrees provided significant evidence for interaction (P < or = 0.003) when comparing a general epistasis model with multiplicative or additive-effects-only models. Conditional linkage analysis (which models epistasis as a deviation from multiplicativity only) confirmed these findings, with significant LOD score increases at the 1q (P = 0.0002) and 10q (P = 0.0023) loci. These analyses provided sizeable reductions in the 1-LOD support intervals for both loci. Analyses of the British and French pedigrees together yielded comparable, but not enhanced, findings, with significant (P < or = 0.003) evidence for epistasis in joint two-locus linkage analysis, and during conditional linkage analysis significant increases in linkage evidence at the 1q (P = 0.0002) and 10q (P = 0.0036) loci. Our findings of epistasis nevertheless substantiate the evidence for genuine genetic effects at both loci, facilitate endeavours to fine-map these loci in population samples, and support further examination of this interaction at the nucleotide level by providing a robust prior hypothesis.

The founding member of the inhibitor of apoptosis protein (IAP) family was originally identified as a cell death inhibitor. However, recent evidence suggests that IAPs are multifunctional signaling devices that influence diverse biological processes. To investigate the in vivo function of Drosophila melanogaster IAP2, we have generated diap2 null alleles. diap2 mutant animals develop normally and are fully viable, suggesting that diap2 is dispensable for proper development. However, these animals were acutely sensitive to infection by gram-negative bacteria. In Drosophila, infection by gram-negative bacteria triggers the innate immune response by activating the immune deficiency (imd) signaling cascade, a NF-kappaB-dependent pathway that shares striking similarities with the pathway of mammalian tumor necrosis factor receptor 1 (TNFR1). diap2 mutant flies failed to activate NF-kappaB-mediated expression of antibacterial peptide genes and, consequently, rapidly succumbed to bacterial infection. Our genetic epistasis analysis places diap2 downstream of or in parallel to imd, Dredd, Tak1, and Relish. Therefore, DIAP2 functions in the host immune response to gram-negative bacteria. In contrast, we find that the Drosophila TNFR-associated factor (Traf) family member Traf2 is dispensable in resistance to gram-negative bacterial infection. Taken together, our genetic data identify DIAP2 as an essential component of the Imd signaling cascade, protecting the organism from infiltrating microbes.

International audience; The cis-acting regulatory sequences of imprinted genes are subject to germline-specific epigenetic modifications, the imprints, so that this class of genes is exclusively expressed from either the paternal or maternal allele in offspring. How genes are differentially marked in the germlines remains largely to be elucidated. Although the exact nature of the mark is not fully known, DNA methylation [at differentially methylated regions (DMRs)] appears to be a major, functional component. Recent data in mice indicate that Dnmt3a, an enzyme with de novo DNA methyltransferase activity, and the related protein Dnmt3L are required for methylation of imprinted loci in germ cells. Maternal methylation imprints, in particular, are strictly dependent on the presence of Dnmt3L. Here, we show that, unexpectedly, methylation imprints can be present in some progeny of Dnmt3L(-/-) females. This incomplete penetrance of the effect of Dnmt3L deficiency in oocytes is neither embryo nor locus specific, but stochastic. We establish that, when it occurs, methylation is present in both embryo and extra-embryonic tissues and results in a functional imprint. This suggests that this maternal methylation is inherited, directly or indirectly, from the gamete. Our results indicate that in the absence of Dnmt3L, factors such as Dnmt3a and possibly others can act alone to mark individual DMRs. However, establishment of appropriate maternal imprints at all loci does require a combination of all factors. This observation can provide a basis to understand mechanisms involved in some sporadic cases of imprinting-related diseases and polymorphic imprinting in human.

International audience; Background: Genetic determinism of cranial morphology in the mouse is still largely unknown, despite the localization of putative QTLs and the identification of genes associated with Mendelian skull malformations. To approach the dissection of this multigenic control, we have used a set of interspecific recombinant congenic strains (IRCS) produced between C57BL/6 and mice of the distant species Mus spretus (SEG/Pas). Each strain has inherited 1.3% of its genome from SEG/Pas under the form of few, small-sized, chromosomal segments.Results: The shape of the nasal bone was studied using outline analysis combined with Fourier descriptors, and differential features were identified between IRCS BcG-66H and C57BL/6. An F2 cross between BcG-66H and C57BL/6 revealed that, outof the three SEG/Pas-derived chromosomal regions present in BcG-66H, two were involved. Segments on chromosomes 1~32 Mb) and 18 (~13 Mb) showed additive effect on nasal bone shape. The three chromosomal regions present in BcG-66H were isolated in congenic strains to study their individual effect. Epistatic interactions were assessed in bicongenicstrains.Conclusions: Our results show that, besides a strong individual effect, the QTL on chromosome 1 interacts with genes onchromosomes 13 and 18. This study demonstrates that nasal bone shape is under complex genetic control but can beefficiently dissected in the mouse using appropriate genetic tools and shape descriptors.

International audience; The Bacillus subtilis dinR gene product is homologous to the LexA protein of Escherichia coli and regulates the expression of dinR and dinC. Using transcriptional fusions in the dinR and the recA genes, we have investigated the epistatic relationship between these two genes during the SOS response induced either by DNA damage or by competence. The results show that after DNA damage, induction of the expression of both recA and dinR is dependent on the activity of the DinR and RecA proteins. A RecA-dependent activity on DinR is proposed as the initial event in the induction of the SOS network. In contrast, the competence-related induction of dinR and recA appears to involve two distinct mechanisms. While one mechanism corresponds to the classical regulation of the SOS response, the other appears to involve an activating factor. Moreover, this factor is active in cells in which competence is prevented by a mutation in the regulatory gene comA.

International audience; High-throughput DNA sequencing technologies are fuelling an accelerating trend to assemble de novo or resequence the genomes of numerous species as well as to complete unfinished assemblies. While current DNA sequencing technologies remain limited to reading stretches of a few hundreds or thousands of base pairs, experimental and computational methods are continuously improving with the goal of assembling entire genomes from large numbers of short DNA sequences. However, the algorithms that piece together DNA strands face important limitations due, notably, to the presence of repeated sequences or of multiple haplotypes within one genome, thus leaving many assemblies incomplete. Recently, the realization that the physical contacts experienced by a portion of a DNA molecule could be used as a robust and quantitative assay to determine its genomic position has led to the emerging field of contact genomics, which promises to revolutionize current genome assembly approaches by exploiting the flexible polymer properties of chromosomes. Here we review the current applications of contact genomics to genome scaffolding, haplotyping and metagenomic assembly, then outline the future developments we envision.

Using new computational tools in yeast, multi-protein complexes were identified that share an unusually high number of synthetic genetic interactions., Background Synthetic lethality defines a genetic interaction where the combination of mutations in two or more genes leads to cell death. The implications of synthetic lethal screens have been discussed in the context of drug development as synthetic lethal pairs could be used to selectively kill cancer cells, but leave normal cells relatively unharmed. A challenge is to assess genome-wide experimental data and integrate the results to better understand the underlying biological processes. We propose statistical and computational tools that can be used to find relationships between synthetic lethality and cellular organizational units. Results In Saccharomyces cerevisiae, we identified multi-protein complexes and pairs of multi-protein complexes that share an unusually high number of synthetic genetic interactions. As previously predicted, we found that synthetic lethality can arise from subunits of an essential multi-protein complex or between pairs of multi-protein complexes. Finally, using multi-protein complexes allowed us to take into account the pleiotropic nature of the gene products. Conclusions Modeling synthetic lethality using current estimates of the yeast interactome is an efficient approach to disentangle some of the complex molecular interactions that drive a cell. Our model in conjunction with applied statistical methods and computational methods provides new tools to better characterize synthetic genetic interactions.