Characterization of 3D genomic interactions in fetal pig muscle

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

3D organization of telomeres in porcine neutrophils and analysis of LPS-activation effect

Background: While the essential role of 3D nuclear architecture on nuclear functions has been demonstrated for various cell types, information available for neutrophils, essential components of the immune system, remains limited. In this study, we analysed the spatial arrangements of telomeres which play a central role in cell fate. Our studies were carried out in swine, which is an excellent model organism for both biomedical research and agronomic applications. We isolated bacterial artificial chromosome (BAC)-containing subtelomeric p and q sequences specific to each porcine chromosome. This allowed us to study the behaviour of p and q telomeres of homologous chromosomes for seven pairs chosen for their difference in length and morphology. This was performed using 3D-FISH on structurally preserved neutrophils, and confocal microscopy. Resting and lipopolysaccharide (LPS)-activated states were investigated to ascertain whether a response to a pathogen aggression modifies this organization.[br/] Results: The positions of the p and q telomeres relative to the nuclear outer border were determined in the two states. All p telomeres changed their position significantly during the activation process, although the effect was less pronounced for the q telomeres. The patterns of telomeric associations between homologs and their frequencies were analysed for 7 pairs of chromosomes. This analysis revealed that the distribution of pp, qq and pq associations differs significantly among the 7 chromosomes. This distribution does not fit with the theoretical distribution for each chromosome, suggesting that preferential associations occur between subtelomeres.[br/] Conclusions: The percentage of nuclei harbouring at least one telomeric association between homologs varies significantly among the chromosomes, the smallest metacentric chromosome SSC12, which is also the richest in gene-density, harbouring the highest value. The distribution of types of telomeric associations is highly dependent on the chromosomes and is not affected by the activation process. The frequencies of telomeric associations are also highly dependent on the type of association and the type of chromosome. Overall, the LPS-activation process induces only minor changes in these patterns of associations. When telomeric associations occur, the associations of p and q arms from the same chromosome are the most frequent, suggesting that “chromosome bending” occurs in neutrophils as previously observed in gametes

3D nuclear organization of theCMHcomplex in control and LPS-activated porcine and human macrophages

3D nuclear organization of theCMHcomplex in control and LPS-activated porcine and human macrophages. 22. International Colloquim on Animal Cytogenetics and Genomics (ICACG

Sperm nuclear architecture is locally modified in presence of a Roberstsonian translocation t(13;17)

In mammals, the non-random organization of the sperm nucleus supports an early function during embryonic development. Altering this organization may interfere with the zygote development and reduce fertility or prolificity. Thus, rare studies on sperm cells from infertile patients described an altered nuclear organization that may be a cause or a consequence of their respective pathologies. Thereby, chromosomal rearrangements and aneuploidy can be studied not only for their adverse effects on production of normal/balanced gametes at meiosis but also for their possible impact on sperm nuclear architecture and the epigenetic consequences of altered chromosome positioning. We decided to compare the global architecture of sperm nuclei from boars, either with a normal chromosome composition or with a Robertsonian translocation involving chromosomes 13 and 17. We hypothesized that the fusion between these chromosomes may change their spatial organization and we examined to what extend it could also modify the global sperm nuclear architecture. Analysis of telomeres, centromeres and gonosomes repartition does not support a global nuclear disorganization. But specific analysis of chromosomes 13 and 17 territories highlights an influence of chromosome 17 for the positioning of the fused chromosomes within the nucleus. We also observed a specific clustering of centromeres depending of the chromosome subtypes. Altogether our results showed that chromosome fusion does not significantly alter sperm nucleus architecture but suggest that centromere remodelling after chromosome fusion locally impacts chromosome positioning

Contribution a la carte genique du porc par hybridation moleculaire in situ sur des chromosomes en metaphase et en fin de prophase. Application a l'etude du gene halothane

INIST T 76286 / INIST-CNRS - Institut de l'Information Scientifique et TechniqueSIGLEFRFranc

Localization of beta-galactoside alpha2,6-sialyltransferase gene on pig chromosome 13q4.1

RésuméChantier qualité spécifique "Auteurs Externes" département de Génétique animale : uniquement liaison auteur au référentiel HR-Accessabsen

An innovative method of gene co expression network inference reveals significant biological processes involved in fetal porcine muscle development

International audienceThe 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

Robertsonian translocation t(13;17) affects SSC13 and SSC17 morphologies and localizations.

<p>A–B. Chromosome paints for SSC13 (green) and SSC17 (red) were checked on metaphase spreads and 3D SpermFISH was performed on sperm nuclei. C. SSC13 and SSC17 chromosome territories (CT) were classified in three categories: colocalized when territories partially or completely merged together, adjacent when they are in close contact (but not merged) and distant. Sperm nuclei were classified in these three categories and their distribution tested between control and t(13;17) animals using a χ² test. D. SSC13 and SSC17 CTs were classified regarding their round or elongated morphologies. For each chromosome, sperm nuclei were classified in these two categories and their distribution tested between control and t(13;17) animals using a χ² test.</p

Telomere repartition in sperm nuclei.

<p>A. Morphology and dimensions of the porcine spermatozoa nucleus. B. Chromosome composition of the sperm cell from control animal with 18 autosomes and one sexual chromosome (X or Y), 38 telomeres in total and of the translocated sperm cell t(13;17) with 17 autosomes and one sexual chromosome (X or Y), 36 telomeres in total. C. DNA-FISH with telomere-specific LNA probes on porcine metaphase spreads. D–E. 3D-SpermFISH with a telomere-specific LNA probe and the resulting image segmentation obtained using NEMO. F. Boxplot representing the number of telomeres clusters in control and t(13;17) sperm nuclei. Mean values in each condition are represented above the boxplot with the p-value of the corresponding t-test. G. Observed distribution of telomere associations per nucleus in sperm nucleus of control (gold yellow) and t(13;17) (dark green) animals.</p