The interplay between genome organization and nuclear architecture of primate evolutionary neo-centromeres.

An Evolutionary Neo-Centromere (ENC) is a centromere that emerged in an ectopic region of a chromosome during evolution. It is thought that the old centromere must be inactivated because dicentric chromosomes are not viable. The aim of the present study was to investigate whether 3D arrangement in the interphase nucleus of the novel and old centromeric domains was affected by the repositioning event. The data we present here strongly indicate that the ENC phenomenon does not affect the 3D location of either novel or old centromeres. Very likely, other features, such as gene density, rather than the newly acquired or lost functions, define positioning in the nucleus.

Remodeling of the chromatin structure of the facioscapulohumeral muscular dystrophy (FSHD) locus and upregulation of FSHD-related gene 1 (FRG1) expression during human myogenic differentiation.

BACKGROUND: Facioscapulohumeral muscular dystrophy (FSHD) is an autosomal dominant neuromuscular disorder associated with the partial deletion of integral numbers of 3.3 kb D4Z4 DNA repeats within the subtelomere of chromosome 4q. A number of candidate FSHD genes, adenine nucleotide translocator 1 gene (ANT1), FSHD-related gene 1 (FRG1), FRG2 and DUX4c, upstream of the D4Z4 array (FSHD locus), and double homeobox chromosome 4 (DUX4) within the repeat itself, are upregulated in some patients, thus suggesting an underlying perturbation of the chromatin structure. Furthermore, a mouse model overexpressing FRG1 has been generated, displaying skeletal muscle defects. RESULTS: In the context of myogenic differentiation, we compared the chromatin structure and tridimensional interaction of the D4Z4 array and FRG1 gene promoter, and FRG1 expression, in control and FSHD cells. The FRG1 gene was prematurely expressed during FSHD myoblast differentiation, thus suggesting that the number of D4Z4 repeats in the array may affect the correct timing of FRG1 expression. Using chromosome conformation capture (3C) technology, we revealed that the FRG1 promoter and D4Z4 array physically interacted. Furthermore, this chromatin structure underwent dynamic changes during myogenic differentiation that led to the loosening of the FRG1/4q-D4Z4 array loop in myotubes. The FRG1 promoter in both normal and FSHD myoblasts was characterized by H3K27 trimethylation and Polycomb repressor complex binding, but these repression signs were replaced by H3K4 trimethylation during differentiation. The D4Z4 sequences behaved similarly, with H3K27 trimethylation and Polycomb binding being lost upon myogenic differentiation. CONCLUSION: We propose a model in which the D4Z4 array may play a critical chromatin function as an orchestrator of in cis chromatin loops, thus suggesting that this repeat may play a role in coordinating gene expression.

Replication-timing-correlated spatial chromatin arrangements in cancer and in primate interphase nuclei.

Using published high-resolution data on S-phase replication timing, we determined the three-dimensional (3D) nuclear arrangement of 33 very-early-replicating and 31 very-late-replicating loci. We analyzed diploid human, non-human primate and rearranged tumor cells by 3D fluorescence in situ hybridization with the aim of investigating the impact of chromosomal structural changes on the nuclear organization of these loci. Overall, their topology was found to be largely conserved between cell types, species and in tumor cells. Early-replicating loci were localized in the nuclear interior, whereas late-replicating loci showed a broader distribution with a higher preference for the periphery than for late-BrdU-incorporation foci. However, differences in the spatial arrangement of early and late loci of chromosome 2, as compared with those from chromosome 5, 7 and 17, argue against replication timing as a major driving force for the 3D radial genome organization in human lymphoblastoid cell nuclei. Instead, genomic properties, and local gene density in particular, were identified as the decisive parameters. Further detailed comparisons of chromosome 7 loci in primate and tumor cells suggest that the inversions analyzed influence nuclear topology to a greater extent than the translocations, thus pointing to geometrical constraints in the 3D conformation of a chromosome territory.

Multicolor 3D fluorescence in situ hybridization for imaging interphase chromosomes.

Fluorescence in situ hybridization (FISH) of specific DNA probes has become a widely used technique mostly for chromosome analysis and for studies of the chromosomal location of specific DNA segments in metaphase preparations as well as in interphase nuclei. FISH on 3D-preserved nuclei (3D-FISH) in combination with 3D-microscopy and image reconstruction is an efficient tool to analyze the spatial arrangement of targeted DNA sequences in the nucleus. Recent developments of a "new generation" of confocal microscopes that allow the distinct visualization of at least five different fluorochromes within one experiment opened the way for multicolor 3D-FISH experiments. Thus, numerous differently labeled nuclear targets can be delineated simultaneously and their spatial interrelationships can be analyzed on the level of individual nuclei.In this chapter, we provide protocols for the preparation of complex DNA-probe sets suitable for 3D-FISH with up to six different fluorochromes, for 3D-FISH on cultured mammalian cells (growing in suspension or adherently) as well as on tissue sections, and for 3D immuno-FISH.In comparison with FISH on metaphase chromosomes and conventional interphase cytogenetics, FISH on 3D-preserved nuclei requires special demands with regard to probe quality, fixation, and pretreatment steps of cells in order to achieve the two goals, namely the best possible preservation of the nuclear structure and at the same time an efficient probe accessibility.

FISH on Histological Sections.

IntroductionHere we describe fluorescence in situ hybridization (FISH) of DNA probes to histological sections, which allows the visualization of specific DNA targets (chromosome territories and their subregions) in the context of functional tissue organization. Separate protocols are provided for hybridization using paraffin-embedded tissue sections and for hybridization using vibratome or frozen sections. Pretreatment with heat or protease is necessary to allow unmasking of the target DNA and efficient penetration of reagents in the nuclei. Because the goal of the technique is to obtain data on the native 3D structure of the genome, close attention is paid to the preservation of nuclear morphology.