RÉSUMÉ
During early mammalian development, transient pools of pluripotent cells emerge that can be immortalised upon stem cell derivation. The pluripotent state, 'naïve' or 'primed', depends on the embryonic stage and derivation conditions used. Here we analyse the temporal gene expression patterns of mouse, cattle and porcine embryos at stages that harbour different types of pluripotent cells. We document conserved and divergent traits in gene expression, and identify predictor genes shared across the species that are associated with pluripotent states in vivo and in vitro Amongst these are the pluripotency-linked genes Klf4 and Lin28b The novel genes discovered include naïve- (Spic, Scpep1 and Gjb5) and primed-associated (Sema6a and Jakmip2) genes as well as naïve to primed transition genes (Dusp6 and Trip6). Both Gjb5 and Dusp6 play a role in pluripotency since their knockdown results in differentiation and downregulation of key pluripotency genes. Our interspecies comparison revealed new insights of pluripotency, pluripotent stem cell identity and a new molecular criterion for distinguishing between pluripotent states in various species, including human.
RÉSUMÉ
The One Touch Pipeline (OTP) is an automation platform managing Next-Generation Sequencing (NGS) data and calling bioinformatic pipelines for processing these data. OTP handles the complete digital process from import of raw sequence data via alignment of sequencing reads to identify genomic events in an automated and scalable way. Three major goals are pursued: firstly, reduction of human resources required for data management by introducing automated processes. Secondly, reduction of time until the sequences can be analyzed by bioinformatic experts, by executing all operations more reliably and quickly. Thirdly, storing all information in one system with secure web access and search capabilities. From software architecture perspective, OTP is both information center and workflow management system. As a workflow management system, OTP call several NGS pipelines that can easily be adapted and extended according to new requirements. As an information center, it comprises a database for metadata information as well as a structured file system. Based on complete and consistent information, data management and bioinformatic pipelines within OTP are executed automatically with all steps book-kept in a database.
Sujet(s)
Systèmes de gestion de bases de données , Bases de données génétiques , Génomique , Séquençage nucléotidique à haut débit , Analyse de séquence d'ADN , Automatisation , Interface utilisateurRÉSUMÉ
Sexual differentiation of malaria parasites into gametocytes in the vertebrate host and subsequent gamete fertilization in mosquitoes is essential for the spreading of the disease. The molecular processes orchestrating these transitions are far from fully understood. Here, we report the first transcriptome analysis of male and female Plasmodium falciparum gametocytes coupled with a comprehensive proteome analysis. In male gametocytes there is an enrichment of proteins involved in the formation of flagellated gametes; proteins involved in DNA replication, chromatin organization and axoneme formation. On the other hand, female gametocytes are enriched in proteins required for zygote formation and functions after fertilization; protein-, lipid- and energy-metabolism. Integration of transcriptome and proteome data revealed 512 highly expressed maternal transcripts without corresponding protein expression indicating large scale translational repression in P. falciparum female gametocytes for the first time. Despite a high degree of conservation between Plasmodium species, 260 of these 'repressed transcripts' have not been previously described. Moreover, for some of these genes, protein expression is only reported in oocysts and sporozoites indicating that repressed transcripts can be partitioned into short- and long-term storage. Finally, these data sets provide an essential resource for identification of vaccine/drug targets and for further mechanistic studies.
Sujet(s)
Paludisme à Plasmodium falciparum/génétique , Plasmodium falciparum/génétique , Protéome/génétique , Transcriptome/génétique , Chromatine/génétique , Réplication de l'ADN/génétique , Femelle , Gamétogenèse/génétique , Régulation de l'expression des gènes/génétique , Humains , Paludisme à Plasmodium falciparum/parasitologie , Mâle , Voies et réseaux métaboliques/génétique , Plasmodium falciparum/croissance et développement , Plasmodium falciparum/pathogénicité , Biosynthèse des protéines , Caractères sexuelsRÉSUMÉ
Soil flooding is a common stress factor affecting plants. To sustain root function in the hypoxic environment, flooding-tolerant plants may form new, aerenchymatous adventitious roots (ARs), originating from preformed, dormant primordia on the stem. We investigated the signaling pathway behind AR primordium reactivation in the dicot species Solanum dulcamara Transcriptome analysis indicated that flooding imposes a state of quiescence on the stem tissue, while increasing cellular activity in the AR primordia. Flooding led to ethylene accumulation in the lower stem region and subsequently to a drop in abscisic acid (ABA) level in both stem and AR primordia tissue. Whereas ABA treatment prevented activation of AR primordia by flooding, inhibition of ABA synthesis was sufficient to activate them in absence of flooding. Together, this reveals that there is a highly tissue-specific response to reduced ABA levels. The central role for ABA in the response differentiates the pathway identified here from the AR emergence pathway known from rice (Oryza sativa). Flooding and ethylene treatment also induced expression of the polar auxin transporter PIN2, and silencing of this gene or chemical inhibition of auxin transport inhibited primordium activation, even though ABA levels were reduced. Auxin treatment, however, was not sufficient for AR emergence, indicating that the auxin pathway acts in parallel with the requirement for ABA reduction. In conclusion, adaptation of S. dulcamara to wet habitats involved co-option of a hormonal signaling cascade well known to regulate shoot growth responses, to direct a root developmental program upon soil flooding.
Sujet(s)
Inondations , Facteur de croissance végétal/métabolisme , Racines de plante/métabolisme , Solanum/métabolisme , Acide abscissique/métabolisme , Transport biologique/effets des médicaments et des substances chimiques , Éthylènes/pharmacologie , Régulation de l'expression des gènes végétaux/effets des médicaments et des substances chimiques , Acides indolacétiques/métabolisme , Spécificité d'organe/effets des médicaments et des substances chimiques , Spécificité d'organe/génétique , Protéines végétales/métabolisme , Racines de plante/effets des médicaments et des substances chimiques , Transduction du signal/effets des médicaments et des substances chimiques , Solanum/effets des médicaments et des substances chimiques , Transcriptome/effets des médicaments et des substances chimiques , Transcriptome/génétiqueRÉSUMÉ
In eukaryotes, the chromatin architecture has a pivotal role in regulating all DNA-associated processes and it is central to the control of gene expression. For Plasmodium falciparum, a causative agent of human malaria, the nucleosome positioning profile of regulatory regions deserves particular attention because of their extreme AT-content. With the aid of a highly controlled MNase-seq procedure we reveal how positioning of nucleosomes provides a structural and regulatory framework to the transcriptional unit by demarcating landmark sites (transcription/translation start and end sites). In addition, our analysis provides strong indications for the function of positioned nucleosomes in splice site recognition. Transcription start sites (TSSs) are bordered by a small nucleosome-depleted region, but lack the stereotypic downstream nucleosome arrays, highlighting a key difference in chromatin organization compared to model organisms. Furthermore, we observe transcription-coupled eviction of nucleosomes on strong TSSs during intraerythrocytic development and demonstrate that nucleosome positioning and dynamics can be predictive for the functionality of regulatory DNA elements. Collectively, the strong nucleosome positioning over splice sites and surrounding putative transcription factor binding sites highlights the regulatory capacity of the nucleosome landscape in this deadly human pathogen.
Sujet(s)
Assemblage et désassemblage de la chromatine , Étapes du cycle de vie/génétique , Nucléosomes/composition chimique , Plasmodium falciparum/génétique , Protéines de protozoaire/génétique , Site d'initiation de la transcription , Composition en bases nucléiques , ADN des protozoaires/composition chimique , ADN des protozoaires/génétique , Érythrocytes/parasitologie , Exons , Régulation de l'expression des gènes , Humains , Introns , Nucléosomes/métabolisme , Cadres ouverts de lecture , Plasmodium falciparum/croissance et développement , Régions promotrices (génétique) , Liaison aux protéines , Sites d'épissage d'ARN , Facteurs de transcription/génétique , Transcription génétiqueRÉSUMÉ
Genetic interactions reveal insights into cellular function and can be used to identify drug targets. Here we construct a new model to predict negative genetic interactions in protein complexes by exploiting the evolutionary history of genes in parallel converging pathways in metabolism. We evaluate our model with protein complexes of Saccharomyces cerevisiae and show that the predicted protein pairs more frequently have a negative genetic interaction than random proteins from the same complex. Furthermore, we apply our model to human protein complexes to predict novel cancer drug targets, and identify 20 candidate targets with empirical support and 10 novel targets amenable to further experimental validation. Our study illustrates that negative genetic interactions can be predicted by systematically exploring genome evolution, and that this is useful to identify novel anti-cancer drug targets.
Sujet(s)
Épistasie , Évolution moléculaire , Génome fongique , Protéines tumorales/génétique , Tumeurs/génétique , Protéines de Saccharomyces cerevisiae/génétique , Saccharomyces cerevisiae/génétique , Antinéoplasiques/usage thérapeutique , Humains , Voies et réseaux métaboliques , Modèles génétiques , Thérapie moléculaire ciblée , Protéines tumorales/antagonistes et inhibiteurs , Protéines tumorales/métabolisme , Tumeurs/traitement médicamenteux , Tumeurs/métabolisme , Cartographie d'interactions entre protéines , Saccharomyces cerevisiae/métabolisme , Protéines de Saccharomyces cerevisiae/métabolisme , Biologie des systèmesRÉSUMÉ
BACKGROUND: Current epigenetic research makes frequent use of whole-genome ChIP profiling for determining the in vivo binding of proteins, e.g. transcription factors and histones, to DNA. Two important and recurrent questions for these large scale analyses are: 1) What is the genomic distribution of a set of binding sites? and 2) Does this genomic distribution differ significantly from another set of sites? FINDINGS: We exemplify the functionality of the PinkThing by analysing a ChIP profiling dataset of cohesin binding sites. We show the subset of cohesin sites with no CTCF binding have a characteristic genomic distribution different from the set of all cohesin sites. CONCLUSIONS: The PinkThing is a web application for fast and easy analysis of the context of genomic loci, such as peaks from ChIP profiling experiments. The output of the PinkThing analysis includes: categorisation of position relative to genes (intronic, exonic, 5' near, 3' near 5' far, 3' far and distant), distance to the closest annotated 3' and 5' end of genes, direction of transcription of the nearest gene, and the option to include other genomic elements like ESTs and CpG islands. The PinkThing enables easy statistical comparison between experiments, i.e. experimental versus background sets, reporting over- and underrepresentation as well as p-values for all comparisons. Access and use of the PinkThing is free and open (without registration) to all users via the website: http://pinkthing.cmbi.ru.nl
Sujet(s)
Immunoprécipitation de la chromatine , Génomique , Sites de fixation , Protéines du cycle cellulaire/métabolisme , Protéines chromosomiques nonhistones/métabolisme , Liaison aux protéines ,RÉSUMÉ
BACKGROUND: The quaternary structure of eukaryotic NADH:ubiquinone oxidoreductase (complex I), the largest complex of the oxidative phosphorylation, is still mostly unresolved. Furthermore, it is unknown where transiently bound assembly factors interact with complex I. We therefore asked whether the evolution of complex I contains information about its 3D topology and the binding positions of its assembly factors. We approached these questions by correlating the evolutionary rates of eukaryotic complex I subunits using the mirror-tree method and mapping the results into a 3D representation by multidimensional scaling. RESULTS: More than 60% of the evolutionary correlation among the conserved seven subunits of the complex I matrix arm can be explained by the physical distance between the subunits. The three-dimensional evolutionary model of the eukaryotic conserved matrix arm has a striking similarity to the matrix arm quaternary structure in the bacterium Thermus thermophilus (rmsd=19 Å) and supports the previous finding that in eukaryotes the N-module is turned relative to the Q-module when compared to bacteria. By contrast, the evolutionary rates contained little information about the structure of the membrane arm. A large evolutionary model of 45 subunits and assembly factors allows to predict subunit positions and interactions (rmsd=52.6 Å). The model supports an interaction of NDUFAF3, C8orf38 and C2orf56 during the assembly of the proximal matrix arm and the membrane arm. The model further suggests a tight relationship between the assembly factor NUBPL and NDUFA2, which both have been linked to iron-sulfur cluster assembly, as well as between NDUFA12 and its paralog, the assembly factor NDUFAF2. CONCLUSIONS: The physical distance between subunits of complex I is a major correlate of the rate of protein evolution in the complex I matrix arm and is sufficient to infer parts of the complex's structure with high accuracy. The resulting evolutionary model predicts the positions of a number of subunits and assembly factors.
Sujet(s)
Complexe I de la chaîne respiratoire/composition chimique , Évolution moléculaire , Modèles moléculaires , Séquence conservée , Humains , Sous-unités de protéines/composition chimique , Thermus thermophilus/métabolismeRÉSUMÉ
BACKGROUND: The relationships between proteins are often asymmetric: one protein (A) depends for its function on another protein (B), but the second protein does not depend on the first. In metabolic networks there are multiple pathways that converge into one central pathway. The enzymes in the converging pathways depend on the enzymes in the central pathway, but the enzymes in the latter do not depend on any specific enzyme in the converging pathways. Asymmetric relations are analogous to the "if->then" logical relation where A implies B, but B does not imply A (A->B). RESULTS: We show that the majority of relationships between enzymes in metabolic flux models of metabolism in Escherichia coli and Saccharomyces cerevisiae are asymmetric. We show furthermore that these asymmetric relationships are reflected in the expression of the genes encoding those enzymes, the effect of gene knockouts and the evolution of genomes. From the asymmetric relative dependency, one would expect that the gene that is relatively independent (B) can occur without the other dependent gene (A), but not the reverse. Indeed, when only one gene of an A->B pair is expressed, is essential, is present in a genome after an evolutionary gain or loss, it tends to be the independent gene (B). This bias is strongest for genes encoding proteins whose asymmetric relationship is evolutionarily conserved. CONCLUSIONS: The asymmetric relations between proteins that arise from the system properties of metabolic networks affect gene expression, the relative effect of gene knockouts and genome evolution in a predictable manner.
Sujet(s)
Évolution biologique , Génome/génétique , Voies et réseaux métaboliques/génétique , Escherichia coli/enzymologie , Régulation de l'expression des gènes codant pour des enzymes , Protéines/génétique , Saccharomyces cerevisiae/enzymologieRÉSUMÉ
The comparison of fully sequenced genomes enables the study of selective constraints that determine genome organisation. We show that, in fungi, adjacent divergently transcribed (<---->) genes are more conserved in orientation than convergent (--><--) or co-oriented (-->-->) gene pairs. Furthermore, the time divergent orientation of two genes is conserved correlates with the degree of their co-expression and with the likelihood of them being functionally related. The functional interactions of the proteins encoded by the conserved divergent gene pairs indicate a potential for protein function prediction in eukaryotes.
Sujet(s)
Champignons/génétique , Transcription génétique/physiologie , Animaux , Séquence conservée/génétique , Évolution moléculaireRÉSUMÉ
The gap between the amount of genome information released by genome sequencing projects and our knowledge about the proteins' functions is rapidly increasing. To fill this gap, various 'genomic-context' methods have been proposed that exploit sequenced genomes to predict the functions of the encoded proteins. One class of methods, phylogenetic profiling, predicts protein function by correlating the phylogenetic distribution of genes with that of other genes or phenotypic characteristics. The functions of a number of proteins, including ones of medical relevance, have thus been predicted and subsequently confirmed experimentally. Additionally, various approaches to measure the similarity of phylogenetic profiles and to account for the phylogenetic bias in the data have been proposed. We review the successful applications of phylogenetic profiling and analyse the performance of various profile similarity measures with a set of one microsporidial and 25 fungal genomes. In the fungi, phylogenetic profiling yields high-confidence predictions for the highest and only the highest scoring gene pairs illustrating both the power and the limitations of the approach. Both practical examples and theoretical considerations suggest that in order to get a reliable and specific picture of a protein's function, results from phylogenetic profiling have to be combined with other sources of evidence.
Sujet(s)
Évolution moléculaire , Phylogenèse , Protéines/métabolisme , Animaux , Génome , Génotype , Protéines/génétiqueRÉSUMÉ
Different forms of the 6S non-coding RNA (ncRNA) exist in enterobacteria and in B. subtilis but there is only limited information about this RNA from other groups of bacteria. Prochlorococcus is an oceanic, ecologically important, cyanobacterium. It possesses the most streamlined genome within the cyanobacterial phylum, lacking many regulatory proteins and mechanisms well-known from other bacteria. Here we show the accumulation of two distinct types of 6S RNA in Prochlorococcus MED4. One of these RNAs is transcribed from a specific promoter located 23 nucleotides downstream the terminal codon of the purK gene, whereas the longer transcript is produced by processing from a purK-6S RNA precursor. The expression of both 6S transcripts is under diel control, reaching maxima during the day and minima coinciding with the S- and G2-like phases which are typical for synchronized cultures of this prokaryote. Based on data from four closely related Prochlorococcus strains and 11 environmental sequences from the Sargasso Sea, a previously unknown structural element is predicted within the 6S RNA 5' domain by comparative computational analysis. The divergent expression in synchronized cultures and unusual structural domains that were detected based on metagenomic data sets indicate that 6S RNA is an extremely important global regulator in these marine cyanobacteria.
Sujet(s)
Cyanobactéries/génétique , ARN bactérien , Séquence nucléotidique , Techniques de culture cellulaire , Cycle cellulaire , Modèles génétiques , Données de séquences moléculaires , ARN bactérien/composition chimique , Alignement de séquences , Similitude de séquences d'acides nucléiquesRÉSUMÉ
BACKGROUND: Whole genome sequencing of marine cyanobacteria has revealed an unprecedented degree of genomic variation and streamlining. With a size of 1.66 megabase-pairs, Prochlorococcus sp. MED4 has the most compact of these genomes and it is enigmatic how the few identified regulatory proteins efficiently sustain the lifestyle of an ecologically successful marine microorganism. Small non-coding RNAs (ncRNAs) control a plethora of processes in eukaryotes as well as in bacteria; however, systematic searches for ncRNAs are still lacking for most eubacterial phyla outside the enterobacteria. RESULTS: Based on a computational prediction we show the presence of several ncRNAs (cyanobacterial functional RNA or Yfr) in several different cyanobacteria of the Prochlorococcus-Synechococcus lineage. Some ncRNA genes are present only in two or three of the four strains investigated, whereas the RNAs Yfr2 through Yfr5 are structurally highly related and are encoded by a rapidly evolving gene family as their genes exist in different copy numbers and at different sites in the four investigated genomes. One ncRNA, Yfr7, is present in at least seven other cyanobacteria. In addition, control elements for several ribosomal operons were predicted as well as riboswitches for thiamine pyrophosphate and cobalamin. CONCLUSION: This is the first genome-wide and systematic screen for ncRNAs in cyanobacteria. Several ncRNAs were both computationally predicted and their presence was biochemically verified. These RNAs may have regulatory functions and each shows a distinct phylogenetic distribution. Our approach can be applied to any group of microorganisms for which more than one total genome sequence is available for comparative analysis.