In eukaryotes, the interphase nucleus is organized in morphologically and/or functionally distinct nuclear “compartments”. Numerous studies highlight functional relationships between the spatial organization of the nucleus and gene regulation. This raises the question of whether nuclear organization principles exist and, if so, whether they are identical in the animal and plant kingdoms. We addressed this issue through the investigation of the three-dimensional distribution of the centromeres and chromocenters. We investigated five very diverse populations of interphase nuclei at different differentiation stages in their physiological environment, belonging to rabbit embryos at the 8-cell and blastocyst stages, differentiated rabbit mammary epithelial cells during lactation, and differentiated cells of Arabidopsis thaliana plantlets. We developed new tools based on the processing of confocal images and a new statistical approach based on G- and F- distance functions used in spatial statistics. Our original computational scheme takes into account both size and shape variability by comparing, for each nucleus, the observed distribution against a reference distribution estimated by Monte-Carlo sampling over the same nucleus. This implicit normalization allowed similar data processing and extraction of rules in the five differentiated nuclei populations of the three studied biological systems, despite differences in chromosome number, genome organization and heterochromatin content. We showed that centromeres/chromocenters form significantly more regularly spaced patterns than expected under a completely random situation, suggesting that repulsive constraints or spatial inhomogeneities underlay the spatial organization of heterochromatic compartments. The proposed technique should be useful for identifying further spatial features in a wide range of cell types.

The 8.1 haplotype of the HLA complex has been reproducibly associated with several autoimmune diseases and traits, notably with thymus hyperplasia in patients with acquired generalized myasthenia gravis, an autoantibody-mediated disease directed at the muscle acetylcholine receptor. However, the strong linkage disequilibrium across this haplotype has prevented the identification of the causative locus, termed MYAS1. Here, we localized MYAS1 to a 1.2-Mb genome segment by reconstructing haplotypes and assessing their transmission in 73 simplex families. This segment encompasses the class III and proximal class I regions, between the BAT3 and C3-2-11 markers, therefore unambiguously excluding the class II loci. In addition, a case-control study revealed a very strong association with a core haplotype in this same region following an additive model (P ‫؍‬ 7 ؋ 10 ؊11 , odds ratio 6.5 for one copy and 42 for two copies of the core haplotype). Finally, we showed that this region is associated with a marked increase in serum titers of anti-acetylcholine receptor autoantibodies (P ‫؍‬ 8 ؋ 10 ؊6 ). Remarkably, this effect was suppressed by a second locus in cis on the 8.1 haplotype and located toward the class II region. Altogether, these data demonstrate the highly significant but complex effects of the 8.1 haplotype on the phenotype of myasthenia gravis patients and might shed light on its role in other autoimmune diseases.

To investigate the in vivo function of Fas ligand (FasL), we produced a mouse strain with a FasL gene flanked by loxP sequences. Mice with homozygous floxed FasL gene showed no obvious abnormalities. However, germline deletion of the FasL gene, obtained after mating with mice expressing ubiquitous Cre recombinase, resulted in an unexpectedly severe phenotype. FasL−/− mice exhibited an extreme splenomegaly and lymphadenopathy associated with lymphocytic infiltration into multiple organs and autoimmune disease. This severe phenotype led to the premature death at 4 mo of age of >50% of the homozygous mice. It stands in sharp contrast with the milder disease observed in gld (generalized lymphoproliferative disease) mice, indicating that the FasL allele of these mice encodes a protein still able to bind, albeit at a very low level, the Fas receptor.

Whey acidic protein (WAP) and casein (CSN) genes are among the most highly expressed milk protein genes in the mammary gland of the lactating mouse. Their tissue-specific regulation depends on the activation and recruitment of transcription factors, and chromatin modifications in response to hormonal stimulation. We have investigated if another mechanism, such as specific positioning of the genes in the nucleus, could be involved in their functional regulation. Fluorescent in situ hybridization was used to study the nuclear localization of WAP and CSN genes in mouse mammary epithelial cells (HC11) cultured in the absence and presence of lactogenic hormones. Automatic 3D image processing and analysis tools were developed to score gene positions. In the absence of lactogenic hormones, both genes are distributed non-uniformly within the nucleus: the CSN locus was located close to the nuclear periphery and the WAP gene tended to be central. Stimulation by lactogenic hormones induced a statistically significant change to their distance from the periphery, which has been described as a repressive compartment. The detection of genes in combination with the corresponding chromosome-specific probe revealed that the CSN locus is relocated outside its chromosome territory following hormonal stimulation, whereas the WAP gene, which is already sited more frequently outside its chromosome territory in the absence of hormones, is not affected. We conclude that milk protein genes are subject to nuclear repositioning when activated, in agreement with a role for nuclear architecture in gene regulation, but that they behave differently as a function of their chromosomal context.