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.

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.

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.

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.

The GENETPIG database: a tool for comparative mapping in pig (Sus scrofa).

The GENETPIG database has been established for storing and disseminating the results of the European project: 'GENETPIG: identification of genes controlling economic traits in pig'. The partners of this project have mapped about 630 porcine and human ESTs onto the pig genome. The database collects the mapping results and links them to other sources of mapping data; this includes pig maps as well as available comparative mapping information. Functional annotation of the mapped ESTs is also given when a significant similarity to cognate genes was established. The database is accessible for consultation via the Internet at http://www.infobiogen.fr/services/Genetpig/.

Evolutionary breakpoints through a high-resolution comparative map between porcine chromosomes 2 and 16 and human chromosomes.

This study reports a high-resolution comparative map between human chromosomes and porcine chromosomes 2 (SSC2) and 16 (SSC16), pointing out new homologies and evolutionary breakpoints. SSC2 is of particular interest because of the presence of several important QTLs. Among 226 porcine ESTs selected according to their expected localization, 151 were RH mapped and ordered on SSC2. This study confirmed the extensive conservation between SSC2 and HSA11 and HSA19 and refined the homology with HSA5 (three blocks defined). Furthermore the SSC2q pericentromeric region was shown to be homologous to another human chromosome (HSA1). A complex organization of these syntenies was demonstrated on SSC2q. Our strategy led us to improve also the SSC16 RH map by adding 45 markers. Two-color fluorescence in situ hybridization of markers representative of each synteny confirmed block order. Finally, 29 breakpoints were identified in both species, and porcine BACs containing two breakpoints were isolated.

A high-resolution comparative map between pig chromosome 17 and human chromosomes 4, 8, and 20: identification of synteny breakpoints.

We report on the construction of a high-resolution comparative map of porcine chromosome 17 (SSC17) focusing on evolutionary breakpoints with human chromosomes. The comparative map shows high homology with human chromosome 20 but suggests more limited homologies with other human chromosomes. SSC17 is of particular interest in studies of chromosomal organization due to the presence of QTLs that affect meat quality and carcass composition. A total of 158 pig ESTs available in databases or developed by the Sino-Danish Pig Genome Sequencing Consortium were mapped using the INRA-University of Minnesota porcine radiation hybrid panel. The high-resolution map was further anchored by fluorescence in situ hybridization. This study confirmed the extensive conservation between SSC17 and HSA20 and enabled the gene order to be determined. The homology of the SSC17 pericentromeric region was extended to other human chromosomes (HSA4, HSA8) and the chromosomal breakpoint boundaries were accurately defined. In total 15 breakpoints were identified.

Contribution to high-resolution mapping in pigs with 101 type I markers and progress in comparative map between humans and pigs.

In the frame of the European program GenetPig, we localized on the Pig map 105 coding sequences (type I markers) from different origins, using INRA-University of Minnesota porcine Radiation Hybrid Panel (IMpRH, 101 markers) and somatic cell hybrid panel (SCHP, 93 markers, of which only four were not also mapped using IMpRH). Thus, we contributed to the improvement of the porcine high-resolution map, and we complemented the integration between the RH and cytogenetic maps. IMpRH tools allowed us to map 101 new markers relatively to reference markers of the first generation radiation hybrid map. Ninety out of 101 markers are linked to an already mapped marker with a LOD score greater than 4.8. Seventy-eight markers were informative for comparative mapping. Comparison of marker positions on the RH map with those obtained on the cytogenetic map or those expected by Human-Pig comparative map data suggested to us to be cautious with markers linked with a LOD lower than 6. These results allowed us to specify chromosomal fragments well conserved between humans and pigs and also to suggest new correspondences (Sscr1-Hsap3, Sscr9-Hsap9, Sscr13-Hsap11, Sscr15-Hsap6) confirmed by FISH on pig chromosomes. We examined in more detail the comparative map between Hsap12 and Sscr5 considering gene order, which suggests that rearrangements have occurred within the conserved synteny.