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Additional file 6 Table S3. Nucleus-by-nucleus analysis of pairs of allele conformations and of the effects of LPS/IFNγ activation.

Additional file 1 Fig. S1. BAC probe specificity control by 2D-FISH on porcine metaphases. (a) class I (SLA-1, SBAB-490B10) in green; class II (SLA-DRA, SBAB-591C4) in red; and class III (TNFα, SBAB-493A6) in yellow. (b) porcine chromosome 7 centromere in red (SBAB-437A9). (c) BAC probe specificity control by 2D-FISH on human metaphases: class I (HLA-A, RP11-192H11) in green; class II (HLA-DRA, RP11-379F19) in red; and class III (TNFα, RP11-184F16) in yellow.

Additional file 3 Fig. S2. 3D BAC center-to-center distances in resting and activated swine macrophages. Cumulative frequency graphs of the normalized center-to-center distances between: (i) MHC class III (TNFα) and SSC7 centromere in red; (ii) MHC class II (SLA-DRA) and SSC7 centromere in green. Pairwise comparisons (p-values) of cumulative 3D distance distributions in resting and activated macrophages (Student’s t-test) are indicated in each graph.

Additional file 4 Table S2. Comparison of gene expression level in resting macrophages. mRNA expression level, analyzed by RT-qPCR, were pairwise compared with one-way ANOVA model with repetitions.

Additional file 5 Fig. S3. Schematic representation of the method used to define the conformation of each allele: given that r1 and r2 are the radii of two fluorescent spots, when the 3D distance between two spot centers (d1) is greater than r1 + r2, the allele is assumed to be decompacted (D), whereas when the 3D distance (d2) between two spot centers is less than r1 + r2, the allele is assumed to be compacted (C).

Additional file 2 Table S1. Mean values of 3D normalized center-to-center distances and angles calculated using center-to-center distances.

Additional file 7 Fig. S4. Schematic representation of genes positioned inside survival zones (SZ) as determined by the application of the three-loci algorithm. The probable positioning of three genes (blue, red and green dots) is represented in two cells as an example (in cell 1 shown by dotted lines, and in cell 2 by continuous lines). The different positions of each gene make it possible to define its SZ represented by a circle of the same color. The radius of the circle defined for each locus is denoted by Rb for the blue dot, Rr for the red dot and Rg for the green dot.

International audience; The integration of genetic information in the cellular and nuclear environments is crucial for deciphering how the genome functions in physiological conditions. By combining 3D nuclear mapping, high-flow transcriptomic data analyses, and statistical methods for the development of co-regulated gene networks, it becomes possible to develop an integrated approach to depict the regulation of gene expression. For this purpose, we focused on the mechanisms involved in the transcriptional regulation of genes expressed in muscle during late fetal development in pig (90 and 110 days), a critical period for survival. We published a muscle transcriptomic analysis performed during this perinatal period (Voillet et al. 2014). Data from this previous study obtained from two extreme genetic lines in terms of mortality at birth (Large White and Meishan), were used to construct networks of differentially co-expressed genes. As co-expressed genes are not necessary related to a common biological process, we used information of gene co-localizations (3D DNA FISH) to reinforce observed links in the co-expressed gene network. The innovative network inference method developed, sequentially incorporates biological knowledge on gene spatial co-localization to construct robust networks gathering co-regulated genes. Clustering of nodes (genes) becomes more and more biologically consistent in each iteration. Interestingly, by means of the final network, we particularly uncovered unexpected gene associations in the nuclear space between IGF2 and MYH3 suggesting that they could be subject to similar transcriptional regulation.

Genome sequence alone is not sufficient to explain the overall coordination of nuclear activity in a particular tissue. The nuclear organisation and genomic long-range intra- and inter-chromosomal interactions play an important role in the regulation of gene expression and the activation of tissue- specific gene networks. Here we present an overview of the pig genome architecture in muscle at two late developmental stages. The muscle maturation process occurs between the 90th day and the end of gestation (114 days), a key period for survival at birth. To characterise this period we profiled chromatin interactions genome-wide with in situ Hi-C (High Throughput Chromosome Conformation Capture) in muscle samples collected at 90 and 110 days of gestation, specific moments where a drastic change in gene expression has been reported. About 200 million read pairs per library were generated (3 replicates per condition). This allowed: (a) the design of an experimental Hi-C protocol optimized for frozen fetal tissues, (b) the first Hi-C contact heatmaps in fetal porcine muscle cells, and (c) to profile Topologically Associated Domains (TADs) defined as genomic domains with high levels of chromatin interactions. Using the new assembly version Sus scrofa v11, we could map 82% of the Hi-C reads on the reference genome. After filtering, 49% of valid read pairs were used to infer the genomic interactions in both developmental stages. In addition, ChIP-seq experiments were performed to map the binding of the structural protein CTCF, known to regulate genome structure by promoting interactions between genes and distal enhancers. The Hi-C and ChIP-seq data were analysed in combination with the results of a previous transcriptome analysis, focusing on the hun-dreds of genes that were reported as differentially expressed during muscle maturation. We will report the observed general differences between both developmental stages in terms of transcription and structure.

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Analysis of the interaction between IGF2-DLK1 and IGF2-MEG3 RNAs in muscle cells. Decomposed confocal images and merge showing RNA hybridization signals for: a, I: IGF2 (red), II, DLK1 (green) RNAs and III merge; b, I: MEG3 (red), II: IGF2 (green) RNAs and III merge. White arrows on merge images indicate colocated RNAs. The nucleus is counterstained with DAPI (blue). (PPTX 636 kb)

a, Distribution of DNA spot centers to CT edge distances for both alleles located outside of CT is shown. For both alleles, the maximal distance found between RNA spot center and CT edge reached 2 μm. No statistically significant difference was found between the two alleles; however the distribution shows a tendency of the expressed allele to be more frequently outside the CT; b, Heat map representing the position of pair of alleles in each nucleus. The position of each allele towards its CT is analyzed within the 9 different combinations of position. No statistically significant difference was found (n = 51 nuclei). (PPTX 84 kb)

IGF2 - (DLK1-MEG3) DNA positioning relative to CTs (SSC2 and SSC7) in liver cells. Two examples (a, b) of decomposed confocal images showing 3D DNA FISH of IGF2 and DLK1/MEG3 loci in an undifferentiated color (white) (I) and merge (II) with specific chromosome painting probes (SSC2 in red and SSC7 in green). White arrows on merge images indicate the position of proximal alleles. The nucleus is counterstained with DAPI (blue). (PPTX 540 kb)

Information on the genes studied and mapping results in pig. (DOCX 16 kb)

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The Dinosaurs dominated the terrestrial environment for around 170 million years and are probably the most successful land vertebrate group to have existed. They survived several mass extinction events before finally all non-avian species were wiped out 66 million years ago in the Cretaceous-Paleogene extinction event. The neornithes (modern birds) are their living descendants. Despite the huge phenotypic diversity seen in birds, they, and some non-avian reptiles (e.g. some turtle species) display remarkably similar karyotypes with a characteristic pattern of macro and micro chromosomes, small genome size and few repetitive elements. This suggests that these were features present early in their evolution. \ud \ud The availability whole genome sequences and the recent sequencing of around 50 avian genomes, 6 of which were assembled at sufficient read depth and coverage to permit visualization at the chromosomal level, has facilitated the reconstruction of the overall genome structure (karyotype) of both Saurian (bird-reptile) and Avian ancestors. Subsequent use of bioinformatic tools permitted the retracing of the gross evolutionary changes that have occurred along the Dinosaur (and various avian) lineages. Gene ontology analysis of homologous synteny blocks (HSBs) and evolutionary breakpoint regions (EBRs) of chromosomes has allowed us to search for enrichment for genes involved in chromosome rearrangement (consistent with the formation of the signature fragmented karyotype of birds (and probably dinosaurs)). Preliminary analyses of EBRs suggest that they appear to be enriched for genes involved in body size, consistent with the overall gross reduction in body size as dinosaurs evolved into birds. Our results also suggest a period of inter-and intra-chromosomal rearrangements up until around the divergence of turtles (approximately 210 MYA) with a relatively “fixed” pattern thereafter where intra-chromosomal rearrangement plus a few identifiable fissions predominated. It is reasonable therefore to speculate that this ‘avianstyle’ genome may be one of the key factors in the success of this extraordinarily diverse animal group, allowing rapid speciation through increased propensity for random segregation and genetic recombination.

Fertility issues concern both humans, affecting a growing part of the population, and farm animals including pigs in which they slow down the diffusion of agronomical traits of interest. In this project,we focused on two mechanisms linked to infertility: alterations in gametic DNA methylation and chromosomal rearrangements. We first studied DNA methylation in sperm cells of fertile or infertile boars. We observed that the methylation level of spermatic DNA is conserved between three mammalian species, both at the global and local level as well as between fertile and infertile boars. A specific increase in DNA methylation in the GNAS locus was nevertheless identified, as well as a deregulation of its expression in some boars with low quality semen, linking for the first time hypermethylation of this region and male infertility in mammals. To study the effect of chromosomal rearrangements on fertility, we chose to produce induced pluripotent stem cell lines (iPSCs) derived from fibroblasts of these animals, as a tool for studying their differentiation towards the germ cell lineage. Cell lines derived from an azoospermic boar carrier of a reciprocal translocation t(Y;14) harbor several characteristics of pluripotency including the expression of specific genes, a cell cycle resembling the one of embryonic stem cells, and an ability to evolve into the naïve state in an adapted culture medium. However they revealed a poor differentiation potential and a genomic instability increasing with passaging as control lines derived from fertile and caryotypically normal boars, phenomena that might be associated with the integrative reprogramming strategy. The use of a non-integrative technique allowed us to demonstrate that the cell lines obtained with this method did not harbor this instability. Their preliminary characterization may be predictive of production of more stable cell lines gathering more characters of pluripotency., Les problèmes de fertilité s’appliquent à la fois à l’homme, touchant une part croissante de la population, et aux espèces d’élevage comme le porc, freinant la diffusion de caractères d’intérêt agronomique. Dans ce projet, nous avons étudié deux mécanismes liés à l’infertilité : la dérégulation du profil de méthylation de l’ADN gamétique et la présence de remaniements chromosomiques. Nous avons tout d'abord concentré notre étude sur le profil de méthylation de l’ADN spermatique de verrats fertiles et infertiles. Nous avons ainsi constaté que ce niveau de méthylation est conservé entre trois espèces de mammifères, au niveau global et local, ainsi qu'entre verrats fertiles et infertiles. Une augmentation spécifique de la méthylation dans le locus GNAS a été mise en évidence, ainsi qu’une dérégulation de son expression chez certains animaux infertiles, reliant pour la première fois hyperméthylation de cette région et infertilité chez les mammifères. Pour étudier ensuite l’impact des remaniements chromosomiques sur la fertilité des animaux, nous avons choisi de produire des lignées de cellules souches pluripotentes induites (iPSCs) par reprogrammation cellulaire de fibroblastes issus de ces animaux, afin de pouvoir ensuite étudier leur différentiation dans la voie germinale. Les lignées obtenues pour un verrat azoospermique porteur de la translocation réciproque t(Y;14) possèdent de nombreuses caractéristiques des cellules pluripotentes telles que l’expression de gènes spécifiques, un cycle cellulaire proche de celui des cellules souches embryonnaires, et une capacité à évoluer vers l’état naïf de pluripotence dans un milieu de culture adapté. Toutefois, leur capacité de différenciation est faible et elles présentent une instabilité génomique augmentant avec les passages, comme les lignées issues d'animaux fertiles à caryotype normal, phénomène pouvant être associé au choix d'une stratégie de reprogrammation intégrative. En effet, l’utilisation d'une méthode non intégrative a permis de démontrer que les cellules iPS ainsi obtenues ne présentent pas cette instabilité. Leur caractérisation partielle laisse présager l’obtention de lignées plus stables rassemblant plus de caractéristiques de pluripotence.

Depuis les 15 dernières années, de nombreuses études ont mis en évidence le rôle majeur de l’architecture nucléaire dans la régulation de l’expression des gènes et ceci dans une grande diversité de processus biologiques. Bien que la réponse immunitaire soit un de ces processus, peu de données existent sur l’organisation spatiale du génome dans les cellules immunitaires et son impact sur la régulation des gènes dans le contexte d’une réponse à l’infection bactérienne. En utilisant le porc comme organisme modèle, nous avons concentré notre étude sur les macrophages dérivés de monocytes, premières lignes de défense contre les pathogènes. Les cellules immunitaires étant les cibles des mycotoxines, nous nous sommes également intéressés aux effets de la toxine T-2 sur les macrophages et leur réponse induite par les récepteurs TLR. Un effet cytotoxique de la T-2 à une dose de 10 nM, ainsi que des effets inhibiteurs sur certaines réponses liées aux récepteurs TLR ont été mis en évidence. Nous avons ensuite examiné si les changements dans l'expression des gènes dus à l'activation impliquent un repositionnement dans l'espace nucléaire. Une analyse du transcriptome a permis d’identifier les gènes différentiellement exprimés dans les macrophages activés par le mélange LPS-IFNγ et de mettre en évidence des réseaux de gènes impliqués lors de l’activation. Les 4 gènes les plus sur-exprimés (IL1β, IL8, CXCL10 et TNFα) et les 4 gènes les plus sous-exprimés (VIM, LGALS3, TUBA3 et IGF2) ont été sélectionnés pour analyser leur comportement dans l'espace nucléaire au cours de l’activation des macrophages en utilisant la technique FISH 3D. Parmi les 4 gènes sur-exprimés, 3 présentent des modifications de leur position durant le processus d'activation alors que les 4 gènes sous-exprimés ne montent pas de variation de leur position. L'analyse de la position des gènes par rapport à leurs territoires chromosomiques (TC) a été étendue à un second type de cellules immunitaires : les neutrophiles. Des résultats similaires ont été obtenus. Les analyses ont été ensuite complétées par l'étude des 4 gènes sur-exprimés dans un type cellulaire indépendant de la réponse immunitaire (fibroblastes). Nos données suggèrent que les relocalisations dans l'espace nucléaire des gènes différentiellement exprimés dans les cellules immunitaires activées sont gène spécifique, type cellulaire spécifique et concernent essentiellement ceux qui sont sur-exprimés.