Uncovering the genetic basis of adaptive change: on the intersection of landscape genomics and theoretical population genetics

A workshop recently held at the Ecole Polytechnique Federale de Lausanne (EPFL, Switzerland) was dedicated to understanding the genetic basis of adaptive change, taking stock of the different approaches developed in theoretical population genetics and landscape genomics and bringing together knowledge accumulated in both research fields. Indeed, an important challenge in theoretical population genetics is to incorporate effects of demographic history and population structure. But important design problems (e.g. focus on populations as units, focus on hard selective sweeps, no hypothesis-based framework in the design of the statistical tests) reduce their capability of detecting adaptive genetic variation. In parallel, landscape genomics offers a solution to several of these problems and provides a number of advantages (e.g. fast computation, landscape heterogeneity integration). But the approach makes several implicit assumptions that should be carefully considered (e.g. selection has had enough time to create a functional relationship between the allele distribution and the environmental variable, or this functional relationship is assumed to be constant). To address the respective strengths and weaknesses mentioned above, the workshop brought together a panel of experts from both disciplines to present their work and discuss the relevance of combining these approaches, possibly resulting in a joint software solution in the future.

Genomics and the origin of species.

Speciation is a fundamental evolutionary process, the knowledge of which is crucial for understanding the origins of biodiversity. Genomic approaches are an increasingly important aspect of this research field. We review current understanding of genome-wide effects of accumulating reproductive isolation and of genomic properties that influence the process of speciation. Building on this work, we identify emergent trends and gaps in our understanding, propose new approaches to more fully integrate genomics into speciation research, translate speciation theory into hypotheses that are testable using genomic tools and provide an integrative definition of the field of speciation genomics.

Genomics and drug profiling of fatal TCF3-HLF-positive acute lymphoblastic leukemia identifies recurrent mutation patterns and therapeutic options.

TCF3-HLF-positive acute lymphoblastic leukemia (ALL) is currently incurable. Using an integrated approach, we uncovered distinct mutation, gene expression and drug response profiles in TCF3-HLF-positive and treatment-responsive TCF3-PBX1-positive ALL. We identified recurrent intragenic deletions of PAX5 or VPREB1 in constellation with the fusion of TCF3 and HLF. Moreover somatic mutations in the non-translocated allele of TCF3 and a reduction of PAX5 gene dosage in TCF3-HLF ALL suggest cooperation within a restricted genetic context. The enrichment for stem cell and myeloid features in the TCF3-HLF signature may reflect reprogramming by TCF3-HLF of a lymphoid-committed cell of origin toward a hybrid, drug-resistant hematopoietic state. Drug response profiling of matched patient-derived xenografts revealed a distinct profile for TCF3-HLF ALL with resistance to conventional chemotherapeutics but sensitivity to glucocorticoids, anthracyclines and agents in clinical development. Striking on-target sensitivity was achieved with the BCL2-specific inhibitor venetoclax (ABT-199). This integrated approach thus provides alternative treatment options for this deadly disease.

Evolutionary genomics and HIV restriction factors.

PURPOSE OF REVIEW: To provide updated insights into innate antiviral immunity and highlight prototypical evolutionary features of well characterized HIV restriction factors. RECENT FINDINGS: Recently, a new HIV restriction factor, Myxovirus resistance 2, has been discovered and the region/residue responsible for its activity identified using an evolutionary approach. Furthermore, IFI16, an innate immunity protein known to sense several viruses, has been shown to contribute to the defense to HIV-1 by causing cell death upon sensing HIV-1 DNA. SUMMARY: Restriction factors against HIV show characteristic signatures of positive selection. Different patterns of accelerated sequence evolution can distinguish antiviral strategies--offense or defence--as well as the level of specificity of the antiviral properties. Sequence analysis of primate orthologs of restriction factors serves to localize functional domains and sites responsible for antiviral action. We use recent discoveries to illustrate how evolutionary genomic analyses help identify new antiviral genes and their mechanisms of action.

Genomik und personalisierte Medizin [Genomics and personalized medicine].

Personalized medicine has a substantial potential to transform the way diseases will be predicted, prevented and treated. The field will greatly benefit from novel DNA sequencing technologies, in particular commoditization of individual whole genome sequencing. This evolution cannot be stopped, and the medical and scientific community, as well as the society at large, have the responsibility to anticipate the expected benefits from this revolution, but also the potential risks associated with it. Massive investments will be needed for the potential of personalized medicine to be realized, and for the field to come to maturity. In particular, a paradigm change in the way clinical research is done is needed. Switzerland and its Western part pro-actively anticipate these changes.

Gene duplication, population genomics, and species-level differentiation within a tropical mountain shrub.

Gene duplication leads to paralogy, which complicates the de novo assembly of genotyping-by-sequencing (GBS) data. The issue of paralogous genes is exacerbated in plants, because they are particularly prone to gene duplication events. Paralogs are normally filtered from GBS data before undertaking population genomics or phylogenetic analyses. However, gene duplication plays an important role in the functional diversification of genes and it can also lead to the formation of postzygotic barriers. Using populations and closely related species of a tropical mountain shrub, we examine 1) the genomic differentiation produced by putative orthologs, and 2) the distribution of recent gene duplication among lineages and geography. We find high differentiation among populations from isolated mountain peaks and species-level differentiation within what is morphologically described as a single species. The inferred distribution of paralogs among populations is congruent with taxonomy and shows that GBS could be used to examine recent gene duplication as a source of genomic differentiation of nonmodel species.

State of genomics and epigenomics research in the perspective of HIV cure.

PURPOSE OF REVIEW: One of the seven key scientific priorities identified in the road map on HIV cure research is to 'determine the host mechanisms that control HIV replication in the absence of therapy'. This review summarizes the recent work in genomics and in epigenetic control of viral replication that is relevant for this mission. RECENT FINDINGS: New technologies allow the joint analysis of host and viral transcripts. They identify the patterns of antisense transcription of the viral genome and its role in gene regulation. High-throughput studies facilitate the assessment of integration at the genome scale. Integration site, orientation and host genomic context modulate the transcription and should also be assessed at the level of single cells. The various models of latency in primary cells can be followed using dynamic study designs to acquire transcriptome and proteome data of the process of entry, maintenance and reactivation of latency. Dynamic studies can be applied to the study of transcription factors and chromatin modifications in latency and upon reactivation. SUMMARY: The convergence of primary cell models of latency, new high-throughput quantitative technologies applied to the study of time series and the identification of compounds that reactivate viral transcription bring unprecedented precision to the study of viral latency.

Evolutionary insights on C4 photosynthetic subtypes in grasses from genomics and phylogenetics

In plants, an oligogene family encodes NADP-malic enzymes (NADP-me), which are responsible for various functions and exhibit different kinetics and expression patterns. In particular, a chloroplast isoform of NADP-me plays a key role in one of the three biochemical subtypes of C4 photosynthesis, an adaptation to warm environments that evolved several times independently during angiosperm diversification. By combining genomic and phylogenetic approaches, this study aimed at identifying the molecular mechanisms linked to the recurrent evolutions of C4-specific NADP-me in grasses (Poaceae). Genes encoding NADP-me (nadpme) were retrieved from genomes of model grasses and isolated from a large sample of C3 and C4 grasses. Genomic and phylogenetic analyses showed that 1) the grass nadpme gene family is composed of four main lineages, one of which is expressed in plastids (nadpme-IV), 2) C4-specific NADP-me evolved at least five times independently from nadpme-IV, and 3) some codons driven by positive selection underwent parallel changes during the multiple C4 origins. The C4 NADP-me being expressed in chloroplasts probably constrained its recurrent evolutions from the only plastid nadpme lineage and this common starting point limited the number of evolutionary paths toward a C4 optimized enzyme, resulting in genetic convergence. In light of the history of nadpme genes, an evolutionary scenario of the C4 phenotype using NADP-me is discussed.

First insights into the transcriptome and development of new genomic tools of a widespread circum-Mediterranean tree species, Pinus halepensis Mill.

Aleppo pine (Pinus halepensis Mill.) is a relevant conifer species for studying adaptive responses to drought and fire regimes in the Mediterranean region. In this study, we performed Illumina next-generation sequencing of two phenotypically divergent Aleppo pine accessions with the aims of (i) characterizing the transcriptome through Illumina RNA-Seq on trees phenotypically divergent for adaptive traits linked to fire adaptation and drought, (ii) performing a functional annotation of the assembled transcriptome, (iii) identifying genes with accelerated evolutionary rates, (iv) studying the expression levels of the annotated genes and (v) developing gene-based markers for population genomic and association genetic studies. The assembled transcriptome consisted of 48,629 contigs and covered about 54.6 Mbp. The comparison of Aleppo pine transcripts to Picea sitchensis protein-coding sequences resulted in the detection of 34,014 SNPs across species, with a Ka /Ks average value of 0.216, suggesting that the majority of the assembled genes are under negative selection. Several genes were differentially expressed across the two pine accessions with contrasted phenotypes, including a glutathione-s-transferase, a cellulose synthase and a cobra-like protein. A large number of new markers (3334 amplifiable SSRs and 28,236 SNPs) have been identified which should facilitate future population genomics and association genetics in this species. A 384-SNP Oligo Pool Assay for genotyping with the Illumina VeraCode technology has been designed which showed an high overall SNP conversion rate (76.6%). Our results showed that Illumina next-generation sequencing is a valuable technology to obtain an extensive overview on whole transcriptomes of nonmodel species with large genomes.

Pseudogenes in the ENCODE regions: consensus annotation, analysis of transcription, and evolution.

Arising from either retrotransposition or genomic duplication of functional genes, pseudogenes are "genomic fossils" valuable for exploring the dynamics and evolution of genes and genomes. Pseudogene identification is an important problem in computational genomics, and is also critical for obtaining an accurate picture of a genome's structure and function. However, no consensus computational scheme for defining and detecting pseudogenes has been developed thus far. As part of the ENCyclopedia Of DNA Elements (ENCODE) project, we have compared several distinct pseudogene annotation strategies and found that different approaches and parameters often resulted in rather distinct sets of pseudogenes. We subsequently developed a consensus approach for annotating pseudogenes (derived from protein coding genes) in the ENCODE regions, resulting in 201 pseudogenes, two-thirds of which originated from retrotransposition. A survey of orthologs for these pseudogenes in 28 vertebrate genomes showed that a significant fraction ( approximately 80%) of the processed pseudogenes are primate-specific sequences, highlighting the increasing retrotransposition activity in primates. Analysis of sequence conservation and variation also demonstrated that most pseudogenes evolve neutrally, and processed pseudogenes appear to have lost their coding potential immediately or soon after their emergence. In order to explore the functional implication of pseudogene prevalence, we have extensively examined the transcriptional activity of the ENCODE pseudogenes. We performed systematic series of pseudogene-specific RACE analyses. These, together with complementary evidence derived from tiling microarrays and high throughput sequencing, demonstrated that at least a fifth of the 201 pseudogenes are transcribed in one or more cell lines or tissues.

Ancient protostome origin of chemosensory ionotropic glutamate receptors and the evolution of insect taste and olfaction.

Ionotropic glutamate receptors (iGluRs) are a highly conserved family of ligand-gated ion channels present in animals, plants, and bacteria, which are best characterized for their roles in synaptic communication in vertebrate nervous systems. A variant subfamily of iGluRs, the Ionotropic Receptors (IRs), was recently identified as a new class of olfactory receptors in the fruit fly, Drosophila melanogaster, hinting at a broader function of this ion channel family in detection of environmental, as well as intercellular, chemical signals. Here, we investigate the origin and evolution of IRs by comprehensive evolutionary genomics and in situ expression analysis. In marked contrast to the insect-specific Odorant Receptor family, we show that IRs are expressed in olfactory organs across Protostomia--a major branch of the animal kingdom that encompasses arthropods, nematodes, and molluscs--indicating that they represent an ancestral protostome chemosensory receptor family. Two subfamilies of IRs are distinguished: conserved "antennal IRs," which likely define the first olfactory receptor family of insects, and species-specific "divergent IRs," which are expressed in peripheral and internal gustatory neurons, implicating this family in taste and food assessment. Comparative analysis of drosophilid IRs reveals the selective forces that have shaped the repertoires in flies with distinct chemosensory preferences. Examination of IR gene structure and genomic distribution suggests both non-allelic homologous recombination and retroposition contributed to the expansion of this multigene family. Together, these findings lay a foundation for functional analysis of these receptors in both neurobiological and evolutionary studies. Furthermore, this work identifies novel targets for manipulating chemosensory-driven behaviours of agricultural pests and disease vectors.

Expression analysis of the AtPHO1 gene family in Arabidopsis thaliana and the involvement of AtPHO1 in the regulation of stomatal movements

Résumé Le transfert du phosphate des racines vers les feuilles s'effectue par la voie du xylème. Il a été précédemment démontré que la protéine AtPHO1 était indispensable au transfert du phosphate dans les vaisseaux du xylème des racines chez la plante modèle Arabidopsis thaliana. Le séquençage et l'annotation du génome d'Arabidopsis ont permis d'identifier dix séquences présentant un niveau de similarité significatif avec le gène AtPHO1 et constituant une nouvelle famille de gène appelé la famille de AtPHO1. Basée sur une étude moléculaire et génétique, cette thèse apporte des éléments de réponse pour déterminer le rôle des membres de ia famille de AtPHO1 chez Arabidopsis, inconnue à ce jour. Dans un premier temps, une analyse bioinformatique des séquences protéiques des membres de la famille de AtPHO1 a révélé la présence dans leur région N-terminale d'un domaine nommé SPX. Ce dernier est conservé parmi de nombreuses protéines impliquées dans l'homéostasie du phosphate chez la levure, renforçant ainsi l'hypothèse que les membres de la famille de AtPHO1 auraient comme AtPHO1 un rôle dans l'équilibre du phosphate dans la plante. En parallèle, la localisation tissulaire de l'expression des gènes AtPHO dans Arabidopsis a été identifiée par l'analyse de plantes transgéniques exprimant le gène rapporteur uidA sous le contrôle des promoteurs respectifs des gènes AtPHO. Un profil d'expression de chaque gène AtPHO au cours du développement de la plante a été obtenu. Une expression prédominante au niveau des tissus vasculaires des racines, des feuilles, des tiges et des fleurs a été observée, suggérant que les gènes AtPHO pourraient avoir des fonctions redondantes au niveau du transfert de phosphate dans le cylindre vasculaire de ces différents organes. Toutefois, plusieurs régions promotrices des gènes AtPHO contrôlent également un profil d'expression GUS non-vasculaire, indiquant un rôle putatif des gènes AtPHO dans l'acquisition ou le recyclage de phosphate dans la plante. Dans un deuxième temps, l'analyse de l'expression des gènes AtPHO durant une carence en phosphate a établi que seule l'expression des gènes AtPHO1, AtPHO1; H1 et AtPHO1; H10 est régulée par cette carence. Une étude approfondie de leur expression en réponse à des traitements affectant l'homéostasie du phosphate dans la plante a ensuite démontré leur régulation par différentes voies de signalisation. Ensuite, une analyse détaillée de la régulation de l'expression du gène AtPHO1; H1O dans des feuilles d'Arabidopsis blessées ou déshydratées a révélé que ce gène constitue le premìer gène marqueur d'une nouvelle voie de signalisation induite par l'OPDA, pas par le JA et dépendante de la protéine COI1. Ces résultats démontrent pour la première fois que l'OPDA et le JA peuvent activer différents gènes via des voies de signalisation dépendantes de COI1. Enfin, cette thèse révèle l'identification d'un nouveau rôle de la protéine AtPHO1 dans la régulation de l'action de l'ABA au cours des processus de fermeture stomatique et de germination des graines chez Arabidopsis. Bien que les fonctions exactes des protéines AtPHO restent à être déterminées, ce travail de thèse suggère leur implication dans la propagation de différents signaux dans la plante via la modulation du potentiel membranaire et/ou l'affectation de la composition en ions des cellules comme le font de nombreux transporteurs ou régulateur du transport d'ions. Summary Phosphate is transferred from the roots to the shoot via the xylem. The requirement for AtPHO1 protein to transfer phosphate to the xylem vessels of the root has been previously demonstrated in Arabidopsis thaliana. The sequencing and the annotation of the Arabidopsis genome had allowed the identification of ten sequences that show a significant level of similarity with the AtPHO1 gene. These 10 genes, of unknown functions, constitute a new gene family called the AtPHO1 gene family. Based on a molecular and genetics study, this thesis reveals some information needed to understand the role of the AtPHO1 family members in the plant Arabidopsis. First, a bioinformatics study revealed that the AtPHO sequences contained, in the N-terminal hydrophilic region, a motif called SPX and conserved among multiple proteins involved in phosphate homeostasis in yeast. This finding reinforces the hypothesis that all AtPHO1 family members have, as AtPHO1, a role in phosphate homeostasis. In parallel, we identified the pattern of expression of AtPHO genes in Arabidopsis via analysis of transgenic plants expressing the uidA reporter gene under the control of respective AtPHO promoter regions. The results exhibit a predominant expression of AtPHO genes in vascular tissues of all organs of the plant, implying that these AtPHO genes could have redundant functions in the transfer of phosphate to the vascular cylinder of various organs. The GUS expression pattern for several AtPHO promoter regions was also detected in non-vascular tissue indicating a broad role of AtPHO genes in the acquisition or in the recycling of phosphate in the plant. In a second step, the analysis of the expression of AtPHO genes during phosphate starvation established that only the expression of the AtPHO1, AtPHO1; H1 and AtPHO1; H10 genes were regulated by Pi starvation. Interestingly, different signalling pathways appeared to regulate these three genes during various treatments affecting Pi homeostasis in the plant. The third chapter presents a detailed analysis of the signalling pathways regulating the expression of the AtPHO1; H10 gene in Arabidopsis leaves during wound and dehydrated stresses. Surprisingly, the expression of AtPHO1; H10 was found to be regulated by OPDA (the precursor of JA) but not by JA itself and via the COI1 protein (the central regulator of the JA signalling pathway). These results demonstrated for the first time that OPDA and JA could activate distinct genes via COI1-dependent pathways. Finally, this thesis presents the identification of a novel role of the AtPHO1 protein in the regulation of ABA action in Arabidopsis guard cells and during seed germination. Although the exact role and function of AtPHO1 still need to be determined, these last findings suggest that AtPHO1 and by extension other AtPHO proteins could mediate the propagation of various signals in the plant by modulating the membrane potential and/or by affecting cellular ion composition, as it is the case for many ion transporters or regulators of ion transport.

Stable transmission of targeted gene modification using single-stranded oligonucleotides with flanking LNAs.

Targeted mutagenesis directed by oligonucleotides (ONs) is a promising method for manipulating the genome in higher eukaryotes. In this study, we have compared gene editing by different ONs on two new target sequences, the eBFP and the rd1 mutant photoreceptor betaPDE cDNAs, which were integrated as single copy transgenes at the same genomic site in 293T cells. Interestingly, antisense ONs were superior to sense ONs for one target only, showing that target sequence can by itself impart strand-bias in gene editing. The most efficient ONs were short 25 nt ONs with flanking locked nucleic acids (LNAs), a chemistry that had only been tested for targeted nucleotide mutagenesis in yeast, and 25 nt ONs with phosphorothioate linkages. We showed that LNA-modified ONs mediate dose-dependent target modification and analyzed the importance of LNA position and content. Importantly, when using ONs with flanking LNAs, targeted gene modification was stably transmitted during cell division, which allowed reliable cloning of modified cells, a feature essential for further applications in functional genomics and gene therapy. Finally, we showed that ONs with flanking LNAs aimed at correcting the rd1 stop mutation could promote survival of photoreceptors in retinas of rd1 mutant mice, suggesting that they are also active in vivo.