DNA replication and chromosome positioning throughout the interphase in three-dimensional space of plant nuclei.

Despite much recent progress, our understanding of the principles of plant genome organization and its dynamics in three-dimensional space of interphase nuclei remains surprisingly limited. Notably, it is not clear how these processes could be affected by the size of a plant's nuclear genome. In this study, DNA replication timing and interphase chromosome positioning were analyzed in seven Poaceae species that differ in their genome size. To provide a comprehensive picture, a suite of advanced, complementary methods was used: labeling of newly replicated DNA by ethynyl-2'-deoxyuridine, isolation of nuclei at particular cell cycle phases by flow cytometric sorting, three-dimensional immunofluorescence in situ hybridization, and confocal microscopy. Our results revealed conserved dynamics of DNA replication in all species, and a similar replication timing order for telomeres and centromeres, as well as for euchromatin and heterochromatin regions, irrespective of genome size. Moreover, stable chromosome positioning was observed while transitioning through different stages of interphase. These findings expand upon earlier studies in suggesting that a more complex interplay exists between genome size, organization of repetitive DNA sequences along chromosomes, and higher order chromatin structure and its maintenance in interphase, albeit controlled by currently unknown factors.

Altered three-dimensional organization of sperm genome in DPY19L2-deficient globozoospermic patients.

PURPOSE: To explore the three-dimensional (3D) organization of sperm genome in DPY19L2-deficient globozoospermic patients speculating a link between DPY19L2 and genome organization of sperm nucleus. METHODS: This is a study of chromatin organization in DPY19L2-deficient globozoospermic patients and healthy donors using three-dimensional fluorescence in situ hybridization (3D-FISH) combined with confocal laser scanning microscopy followed by 3D image analysis. The 3D structures of sperm nuclei, chromocenter, telomeric regions and chromosome territories (CTs), were reconstructed using IMARIS software, and the relative radial position for each individual signal was calculated. Statistical analysis used a non-parametric Mann-Whitney test was appropriate with significance at p < 0.05. RESULTS: DPY19L2-deficient globozoospermic patients display impaired sperm chromocenter organization resulting in an increased number of chromocenters (5.4 vs 3.5; p < 0.0001). Moreover, radial positions of telomeres are modified with a more central position in globozoospermic nuclei. 3D-FISH analysis of five chromosome territories (CTs) (X, Y, 7, 17, 18) showed that DPY19L2-deficient globozoospermic sperm nuclei display altered spatial organization of CT X, CT 7 and CT 18. CONCLUSIONS: Our findings strengthen the hypothesis that DPY19L2 might be considered as a LINC-like protein having a crucial role in the organization of nuclear chromatin in sperm nucleus through its interaction with nuclear lamina. Our results might also explain defective embryonic development after intracytoplasmic sperm injection (ICSI) performed with DPY19L2-deficient globozoospermic sperm.

Visualization of the spatial arrangement of nuclear organization using three-dimensional fluorescence in situ hybridization in early mouse embryos: A new "EASI-FISH chamber glass" for mammalian embryos.

The fertilized oocyte begins cleavage, leading to zygotic gene activation (ZGA), which re-activates the resting genome to acquire totipotency. In this process, genomic function is regulated by the dynamic structural conversion in the nucleus. Indeed, a considerable number of genes that are essential for embryonic development are located near the pericentromeric regions, wherein the heterochromatin is formed. These genes are repressed transcriptionally in somatic cells. Three-dimensional fluorescence in situ hybridization (3D-FISH) enables the visualization of the intranuclear spatial arrangement, such as gene loci, chromosomal domains, and chromosome territories (CTs). However, the 3D-FISH approach in mammalian embryos has been limited to certain repeated sequences because of its unfavorable properties. In this study, we developed an easy-to-use chamber device (EASI-FISH chamber) for 3D-FISH in early embryos, and visualized, for the first time, the spatial arrangements of pericentromeric regions, the ZGA-activated gene (Zscan4) loci, and CTs (chromosome 7), simultaneously during the early cleavage stage of mouse embryos by 3D-FISH. As a result, it was revealed that morphological changes of the pericentromeric regions and CTs, and relocation of the Zscan4 loci in CTs, occurred in the 1- to 4-cell stage embryos, which was different from those in somatic cells. This convenient and reproducible 3D-FISH technique for mammalian embryos represents a valuable tool that will provide insights into the nuclear dynamics of development.