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

Profiling the landscape of transcription, chromatin accessibility and chromosome conformation of cattle, pig, chicken and goat genomes [FAANG pilot project]

Functional annotation of livestock genomes is a critical and obvious next step to derive maximum benefit for agriculture, animal science, animal welfare and human health. The aim of the Fr-AgENCODE project is to generate multi-species functional genome annotations by applying high-throughput molecular assays on three target tissues/cells relevant to the study of immune and metabolic traits. An extensive collection of stored samples from other tissues is available for further use (FAANG Biosamples ‘FR-AGENCODE’). From each of two males and two females per species (pig, cattle, goat, chicken), strand-oriented RNA-seq and chromatin accessibility ATAC-seq assays were performed on liver tissue and on two T-cell types (CD3+CD4+&CD3+CD8+) sorted from blood (mammals) or spleen (chicken). Chromosome Conformation Capture (in situ Hi-C) was also carried out on liver. Sequencing reads from the 3 assays were processed using standard processing pipelines. While most (50–70%) RNA-seq reads mapped to annotated exons, thousands of novel transcripts and genes were found, including extensions of annotated protein-coding genes and new lncRNAs (see abstract #69857). Consistency of ATAC-seq results was confirmed by the significant proportion of called peaks in promoter regions (36–66%) and by the specific accumulation pattern of peaks around gene starts (TSS) v. gene ends (TTS). Principal Component Analyses for RNA-seq (based on quantified gene expression) and ATAC-seq (based on quantified chromatin accessibility) highlighted clusters characterised by cell type and sex in all species. From Hi-C data, we generated 40kb-resolution interaction maps, profiled a genome-wide Directionality Index and identified from 4,100 (chicken) to 12,100 (pig) topologically-associating do- mains (TADs). Correlations were reported between RNA-seq and ATAC-seq results (see abstract #71581). In summary, we present here an overview of the first multi-species and -tissue annotations of chromatin accessibility and genome architecture related to gene expression for farm animals

3D genome conformation and gene expression in fetal pig muscle at late gestation

In swine breeding industry, sows have been selected for decades on their prolificacy in order to maximize meat production. However, this selection is associated with a higher mortality of newborns. In this context, the skeletal fetal muscle is essential for the piglet’s survival, as it is necessary for motor functions and thermoregulation. Besides, the three-dimensional structure of the genome has been proven to play an important role in gene expression regulation. Thus, in this project, we have focused our interest on the 3D genome conformation and gene expression in porcine muscle nuclei at late gestation. We have initially developed an original approach in which we combined transcriptome data with information of nuclear locations (assessed by 3D DNA FISH) of a subset of genes, in order to build gene co-expression networks. This study has revealed interesting nuclear associations involving IGF2, DLK1 and MYH3 genes, and highlighted a network of muscle-specific interrelated genes involved in the development and maturity of fetal muscle. Then, we assessed the global 3D genome conformation in muscle nuclei at 90 days and 110 days of gestation by using the High-throughput Chromosome Conformation Capture (Hi-C) method. This study has allowed identifying thousands of genomic regions showing significant differences in 3D conformation between the two gestational ages. Interestingly, some of these genomic regions involve the telomeric regions of several chromosomes that seem to be preferentially clustered at 90 days. More important, the observed changes in genome structure are significantly associated with variations in gene expression between the 90th and the 110th days of gestation

Conformation 3D du génome et expression génique dans la cellule musculaire porcine en fin de gestation

Dans le secteur de l’élevage porcin, les truies ont été sélectionnées pendant des décennies pour leur prolificité afin de maximiser la production de viande. Cependant, cette sélection a été associée à une mortalité plus élevée des nouveau-nés. Dans ce contexte, le muscle foetal squelettique est essentiel à la survie du porcelet, car il est nécessaire pour les fonctions motrices et la thermorégulation. Par ailleurs, la structure tridimensionnelle du génome s'est avérée jouer un rôle important dans la régulation de l'expression génique. Ainsi, dans ce projet, nous nous sommes intéressés à la conformation 3D du génome et l'expression des gènes dans les noyaux des cellules musculaires porcines à la fin de la gestation. Nous avons initialement développé une approche originale dans laquelle nous avons combiné des données transcriptomiques avec des informations de localisations nucléaires (évaluées par 3D DNA FISH) d'un sous-ensemble de gènes, afin de construire des réseaux de gènes co-exprimés. Cette étude a révélé des associations nucléaires intéressantes impliquant les gènes IGF2, DLK1 et MYH3, et a mis en évidence un réseau de gènes interdépendants spécifiques du muscle impliqués dans le développement et la maturité du muscle foetal. Nous avons ensuite évalué la conformation globale du génome dans les noyaux musculaires à 90 jours et à 110 jours de gestation en utilisant la méthode de capture de conformation de chromatine à haut débit (Hi-C) couplée au séquençage. Cette étude a permis d'identifier des milliers de régions génomiques présentant des différences significatives dans la conformation 3D entre les deux âges gestationnels. Fait intéressant, certaines de ces régions génomiques impliquent les régions télomériques de plusieurs chromosomes qui semblent former des clusters préférentiellement à 90 jours. Plus important, les changements observés dans la structure du génome sont associés de manière significative à des variations d'expression géniques entre le 90ème et le 110ème jour de gestation.In swine breeding industry, sows have been selected for decades on their prolificacy in order to maximize meat production. However, this selection is associated with a higher mortality of newborns. In this context, the skeletal fetal muscle is essential for the piglet’s survival, as it is necessary for motor functions and thermoregulation. Besides, the three-dimensional structure of the genome has been proven to play an important role in gene expression regulation. Thus, in this project, we have focused our interest on the 3D genome conformation and gene expression in porcine muscle nuclei at late gestation. We have initially developed an original approach in which we combined transcriptome data with information of nuclear locations (assessed by 3D DNA FISH) of a subset of genes, in order to build gene co expression networks. This study has revealed interesting nuclear associations involving IGF2, DLK1 and MYH3 genes, and highlighted a network of muscle specific interrelated genes involved in the development and maturity of fetal muscle. Then, we assessed the global 3D genome conformation in muscle nuclei at 90 days and 110 days of gestation by using the High-throughput Chromosome Conformation Capture (Hi¬ C) method. This study has allowed identifying thousands of genomic regions showing significant differences in 3D conformation between the two gestational ages. Interestingly, some of these genomic regions involve the telomeric regions of several chromosomes that seem to be preferentially clustered at 90 days. More important, the observed changes in genome structure are significantly associated with variations in gene expression between the 90th and the 110th days of gestation

Three-dimensional genome organization via triplex-forming RNAs

An increasing number of long noncoding RNAs (lncRNAs) have been proposed to act as nuclear organization factors during interphase. Direct RNA-DNA interactions can be achieved by the formation of triplex helix structures where a single-stranded RNA molecule hybridizes by complementarity into the major groove of double-stranded DNA. However, whether and how these direct RNA-DNA associations influence genome structure in interphase chromosomes remain poorly understood. Here we theorize that RNA organizes the genome in space via a triplex-forming mechanism. To test this theory, we apply a computational modeling approach of chromosomes that combines restraint-based modeling with polymer physics. Our models suggest that colocalization of triplex hotspots targeted by lncRNAs could contribute to large-scale chromosome compartmentalization cooperating, rather than competing, with architectural transcription factors such as CTCF.This work was supported by the European Research Council under the 7th Framework Program FP7/2007-2013 (ERC grant agreement no. 609989 to M.A.M.-R.) and the Spanish Ministerio de Ciencia, Innovación y Universidades through nos. IJCI-2015-23352 to I.F. and BFU2017-85926-P and PID2020-115696RB-I00 to M.A.M.-R. CRG acknowledges support from ‘Centro de Excelencia Severo Ochoa 2013-2017’, SEV-2012-0208 and the CERCA Program/Generalitat de Catalunya, as well as support from the Spanish Ministry of Science and Innovation through the Instituto de Salud Carlos III and the EMBL partnership, the Generalitat de Catalunya through Departament de Salut and Departament d’Empresa i Coneixement, and cofinancing with funds from the European Regional Development Fund by the Spanish Ministry of Science and Innovation corresponding to the Programa Opertaivo FEDER Plurirregional de España 2014–2020 and by the Secretaria d’Universitats i Recerca, Departament d’Empresa i Coneixement of the Generalitat de Catalunya corresponding to the program Operatiu FEDER Catalunya 2014–2020 and the NIH (to C.T. Wu no. R01HD091797 for supporting I.F.

A new approach of gene co-expression network inference reveals significant biological processes involved in porcine muscle development in late gestation

The integration of genetic information in the cellular and nuclear environments is crucial for deciphering the way in which the genome functions under different physiological conditions. Experimental techniques of 3D nuclear mapping, a high-flow approach such as transcriptomic data analyses, and statistical methods for the development of co-expressed gene networks, can be combined to develop an integrated approach for depicting the regulation of gene expression. Our work focused more specifically on the mechanisms involved in the transcriptional regulation of genes expressed in muscle during late foetal development in pig. The data generated by a transcriptomic analysis carried out on muscle of foetuses from two extreme genetic lines for birth mortality are used to construct networks of differentially expressed and co-regulated genes. We developed an innovative co-expression networking approach coupling, by means of an iterative process, a new statistical method for graph inference with data of gene spatial co-localization (3D DNA FISH) to construct a robust network grouping co-expressed genes. This enabled us to highlight relevant biological processes related to foetal muscle maturity and to discover unexpected gene associations between IGF2, MYH3 and DLK1/MEG3 in the nuclear space, genes that are up-regulated at this stage of muscle development

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

Major reorganization of chromosome conformation during muscle development in pig

International audienceThe spatial organization of the genome in the nucleus plays a crucial role in eukaryotic cell functions, yet little is known about chromatin structure variations during late fetal development in mammals. We performed in situ high-throughput chromosome conformation capture (Hi-C) sequencing of DNA from muscle samples of pig fetuses at two late stages of gestation. Comparative analysis of the resulting Hi-C interaction matrices between both groups showed widespread differences of different types. First, we discovered a complex landscape of stable and group-specific Topologically Associating Domains (TADs). Investigating the nuclear partition of the chromatin into transcriptionally active and inactive compartments, we observed a genome-wide fragmentation of these compartments between 90 and 110 days of gestation. Also, we identified and characterized the distribution of differential cis-and trans-pairwise interactions. In particular, trans-interactions at chromosome extremities revealed a mechanism of telomere clustering further confirmed by 3D Fluorescence in situ Hybridization (FISH). Altogether, we report major variations of the three-dimensional genome conformation during muscle development in pig, involving several levels of chromatin remodeling and structural regulation

Whole-genome resequencing of honeybee drones to detect genomic selection in a population managed for royal jelly

Four main evolutionary lineages of A. mellifera have been described including eastern Europe (C) and western and northern Europe (M). Many apiculturists prefer bees from the C lineage due to their docility and high productivity. In France, the routine importation of bees from the C lineage has resulted in the widespread admixture of bees from the M lineage. The haplodiploid nature of the honeybee Apis mellifera, and its small genome size, permits affordable and extensive genomics studies. As a pilot study of a larger project to characterise French honeybee populations, we sequenced 60 drones sampled from two commercial populations managed for the production of honey and royal jelly. Results indicate a C lineage origin, whilst mitochondrial analysis suggests two drones originated from the O lineage. Analysis of heterozygous SNPs identified potential copy number variants near to genes encoding odorant binding proteins and several cytochrome P450 genes. Signatures of selection were detected using the hapFLK haplotype-based method, revealing several regions under putative selection for royal jelly production. The framework developed during this study will be applied to a broader sampling regime, allowing the genetic diversity of French honeybees to be characterised in detail

Expressed alleles of imprinted IGF2, DLK1 and MEG3 colocalize in 3D-preserved nuclei of porcine fetal cells

To explore the relationship between spatial genome organization and gene expression in the interphase nucleus, we used a genomic imprinting model, which offers parental-specific gene expression. Using 3D FISH in porcine fetal liver cells, we compared the nuclear organization of the two parental alleles (expressed or not) of insulin-like growth factor 2 (IGF2), a paternally imprinted gene located on chromosome 2. We investigated whether its nuclear positioning favors specific locus associations. We also tested whether IGF2 is implicated in long-range chromatin trans-associations as previously shown in the mouse model species for its reciprocal imprinted gene H19. We focused on the 3D position of IGF2 alleles, with respect to their individual chromosome 2 territories. The paternally expressed allele was tagged with nascent RNA. There were no significant differences in the position of the two alleles (p = 0.06). To determine long-range chromatin trans-interactions, we chose 12 genes, some of which are known to be imprinted in mammalian model species and belong to a network of imprinted genes (i.e. SLC38A4, DLK1, MEG3, and ZAC1). We screened them and ABCG2, OSBP2, OSBPL1, RPL32, NF1, ZAR1, SEP15, GPC3 for associations with IGF2 in liver cells. All imprinted genes tested showed an association with IGF2. The DLK1/MEG3 locus showed the highest rate of colocalization. This gene association was confirmed by 3D FISH (in 20 % of the nuclei analyzed), revealing also the close proximity of chromosomes 2 and 7 (in 60 % of nuclei). Furthermore, our observations showed that the expressed paternal IGF2 allele is involved in this association. This IGF2-(DLK1/MEG3) association also occurred in a high percentage of fetal muscle cells (36 % of nuclei). Finally, we showed that nascent IGF2, DLK1 and MEG3 RNAs can associate in pairs or in a three-way combination. Our results show that trans-associations occur between three imprinted genes IGF2, DLK1 and MEG3 both in fetal liver and muscle cells. All three expressed alleles associated in muscle cells. Our findings suggest that the 3D nuclear organization is linked to the transcriptional state of these genes