Major Reorganization of Chromosome Conformation During Muscle Development in Pig.

The 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.

The 3D nuclear conformation of the major histocompatibility complex changes upon cell activation both in porcine and human macrophages.

BACKGROUND: The crucial role of the major histocompatibility complex (MHC) for the immune response to infectious diseases is well-known, but no information is available on the 3D nuclear organization of this gene-dense region in immune cells, whereas nuclear architecture is known to play an essential role on genome function regulation. We analyzed the spatial arrangement of the three MHC regions (class I, III and II) in macrophages using 3D-FISH. Since this complex presents major differences in humans and pigs with, notably, the presence of the centromere between class III and class II regions in pigs, the analysis was implemented in both species to determine the impact of this organization on the 3D conformation of the MHC. The expression level of the three genes selected to represent each MHC region was assessed by quantitative real-time PCR. Resting and lipopolysaccharide (LPS)-activated states were investigated to ascertain whether a response to a pathogen modifies their expression level and their 3D organization. RESULTS: While the three MHC regions occupy an intermediate radial position in porcine macrophages, the class I region was clearly more peripheral in humans. The BAC center-to-center distances allowed us to propose a 3D nuclear organization of the MHC in each species. LPS/IFNγ activation induces a significant decompaction of the chromatin between class I and class III regions in pigs and between class I and class II regions in humans. We detected a strong overexpression of TNFα (class III region) in both species. Moreover, a single nucleus analysis revealed that the two alleles can have either the same or a different compaction pattern. In addition, macrophage activation leads to an increase in alleles that present a decompacted pattern in humans and pigs. CONCLUSIONS: The data presented demonstrate that: (i) the MHC harbors a different 3D organization in humans and pigs; (ii) LPS/IFNγ activation induces chromatin decompaction, but it is not the same area affected in the two species. These findings were supported by the application of an original computation method based on the geometrical distribution of the three target genes. Finally, the position of the centromere inside the swine MHC could influence chromatin reorganization during the activation process.

Corrigendum: Non integrative strategy decreases chromosome instability and improves endogenous pluripotency genes reactivation in porcine induced pluripotent-like stem cells.

This corrects the article DOI: 10.1038/srep27059.

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

BACKGROUND: 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. RESULTS: 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. CONCLUSION: 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.

Non integrative strategy decreases chromosome instability and improves endogenous pluripotency genes reactivation in porcine induced pluripotent-like stem cells.

The pig is an emerging animal model, complementary to rodents for basic research and for biomedical and agronomical purposes. However despite the progress made on mouse and rat models to produce genuine pluripotent cells, it remains impossible to produce porcine pluripotent cell lines with germline transmission. Reprogramming of pig somatic cells using conventional integrative strategies remains also unsatisfactory. In the present study, we compared the outcome of both integrative and non-integrative reprogramming strategies on pluripotency and chromosome stability during pig somatic cell reprogramming. The porcine cell lines produced with integrative strategies express several pluripotency genes but they do not silence the integrated exogenes and present a high genomic instability upon passaging. In contrast, pig induced pluripotent-like stem cells produced with non-integrative reprogramming system (NI-iPSLCs) exhibit a normal karyotype after more than 12 months in culture and reactivate endogenous pluripotency markers. Despite the persistent expression of exogenous OCT4 and MYC, these cells can differentiate into derivatives expressing markers of the three embryonic germ layers and we propose that these NI-iPSLCs can be used as a model to bring new insights into the molecular factors controlling and maintaining pluripotency in the pig and other non-rodent mammalians.

Sperm DNA methylation analysis in swine reveals conserved and species-specific methylation patterns and highlights an altered methylation at the GNAS locus in infertile boars.

Male infertility is an increasing health issue in today's society for both human and livestock populations. In livestock, male infertility slows the improvement of animal selection programs and agricultural productivity. There is increasing evidence that epigenetic marks play an important role in the production of good-quality sperm. We therefore screened for specific or common epigenetic signatures of livestock infertility. To do so, we compared DNA methylation level in sperm DNA from fertile and infertile boars. We evaluated first the global level of sperm DNA methylation and found no difference between the two groups of boars. We then selected 42 loci of interest, most of them known to be imprinted in human or mice, and assessed the imprinting status of five of them not previously described in swine tissues: WT1, CNTN3, IMPACT, QPCT, and GRB10. DNA methylation level was then quantified in fertile and infertile boars at these 42 loci. Results from fertile boars indicated that the methylation level of the selected loci is highly conserved between pig, human, and mice, with a few exceptions, including the POU5F1 (OCT4) promoter and RTL1. Comparison between fertile and infertile boars revealed that one imprinted region, the GNAS locus, shows an increase in sperm DNA methylation in three out of eight infertile boars with low semen quality. This increase in DNA methylation is associated with an altered expression of the genes belonging to the GNAS locus, suggesting a new role for GNAS in the proper formation of functional gametes.

Sperm nuclear architecture is locally modified in presence of a Robertsonian 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.

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. 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. 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.

Transcriptomic and nuclear architecture of immune cells after LPS activation.

Changes in the nuclear positioning of specific genes, depending on their expression status, have been observed in a large diversity of physiological processes. However, gene position is poorly documented for immune cells which have been subjected to activation following bacterial infection. Using a pig model, we focused our study on monocyte-derived macrophages and neutrophils, as they are the first lines of defence against pathogens. We examined whether changes in gene expression due to LPS activation imply that genes have repositioned in the nuclear space. We first performed a transcriptomic analysis to identify the differentially expressed genes and then analysed the networks involved during lypopolysaccharide/interferon gamma activation in monocyte-derived macrophages. This allowed us to select four up-regulated (IL1ß, IL8, CXCL10 and TNFα) and four down-regulated (VIM, LGALS3, TUBA3 and IGF2) genes. Their expression statuses were verified by quantitative real-time RT-PCR before studying their behaviour in the nuclear space during macrophage activation by means of 3D fluorescence in situ hybridization. No global correlation was found between gene activity and radial positioning. Only TNFα belonging to the highly transcribed MHC region on chromosome 7 became more peripherally localized in relation to the less decondensed state of its chromosome territory (CT) in activated macrophages. The analysis of gene positioning towards their CT revealed that IL8 increases significantly its tendency to be outside its CT during the activation process. In addition, the gene to CT edge distances increase for the three up-regulated genes (IL8, CXCL10 and TNFα) among the four analysed. Contrarily, the four down-regulated genes did not change their position. The analysis of gene behaviour towards their CT was extended to include neutrophils for three (TNFα, IL8 and IL1ß) up- and two (IGF2 and TUBA3) down-regulated genes, and similar results were obtained. The analysis was completed by studying the four up-regulated genes in fibroblasts, not involved in immune response. Our data suggest that relocation in the nuclear space of genes that are differentially expressed in activated immune cells is gene and cell type specific but also closely linked to the entire up-regulation status of their chromosomal regions.

Nuclear architecture of resting and LPS-stimulated porcine neutrophils by 3D FISH.

Neutrophils are essential components of the innate immune system due to their ability to kill and phagocytose invading microbes. They possess a lobulated nucleus and are capable of extensive and complex changes in response to bacterial stimulation. The aim of our study was to investigate whether the 3D nuclear organization of porcine neutrophils was modified upon stimulation. The organization of centromeres, telomeres, and chromosome territories (chromosomes 2, 3, 7, 8, 12, 13, and 17) was studied on structurally preserved nuclei using 3D fluorescence in situ hybridization, confocal microscopy, and image analysis. By differential labeling of centromeres of acrocentric and metacentric/submetacentric chromosomes, we showed that centromeres associated to form chromocenters but did so preferentially between chromosomes with the same morphology. Upon activation, some of these chromocenters dispersed. Telomeres were also found to form clusters, but their number remained unchanged in lipopolysaccharide-stimulated neutrophils. The analysis of the position of chromocenters and telomere clusters showed a more internal location of the latter compared to the former. The analysis of chromosome territories revealed that homologs were distributed randomly among lobes whatever the cell's status. The volume of these territories was not proportional to chromosome length, and some chromosomes (chr 3, 12, 13, and 17) were more prone to decondensation when neutrophils were stimulated. Thus, our study demonstrated that activation of neutrophils resulted in several modifications of their nuclear architecture: a decrease in the number of non-acrocentric chromocenters and the decondensation of several chromosomes.