Chromatin folding: from linear chromosomes to the 4D nucleus.

The organization of chromatin within the nucleus influences gene expression during cell differentiation and development. Recent work took advantage of the genome-wide localization of molecular marks on chromosomes to analyze their linear distributions at different length scales. Moreover, chromosome conformation capture techniques detect spatial proximity inside the cell nucleus and allow us to characterize local and long-range chromatin loops as well as interchromosomal contacts. These techniques have improved our understanding of chromatin composition in eukaryotic chromosomes, but the principles governing nuclear organization are still little understood. On the one hand, proteins might localize in stable nuclear structures, such as transcription factories, on which chromatin would have to be targeted to be processed. On the other hand, proteins binding to chromatin might induce the formation of specialized nuclear compartments de novo. Current work is aimed at distinguishing between these possibilities and at elaborating predictive models of chromatin folding.

Dynamics and three-dimensional localization of ribosomal RNA within the nucleolus.

Although rRNA synthesis, maturation, and assembly into preribosomal particles occur within the nucleolus, the route taken by pre-rRNAs from their synthetic sites toward the cytoplasm remains largely unexplored. Here, we employed a nondestructive method for the incorporation of BrUTP into the RNA of living cells. By using pulse-chase experiments, three-dimensional image reconstructions of confocal optical sections, and electron microscopy analysis of ultrathin sections, we were able to describe topological and spatial dynamics of rRNAs within the nucleolus. We identified the precise location and the volumic organization of four typical subdomains, in which rRNAs are successively moving towards the nucleolar periphery during their synthesis and processing steps. The incorporation of BrUTP takes place simultaneously within several tiny spheres, centered on the fibrillar centers. Then, the structures containing the newly synthesized RNAs enlarge and appear as compact ringlets disposed around the fibrillar centers. Later, they form hollow spheres surrounding the latter components and begin to fuse together. Finally, these structures widen and form large rings reaching the limits of the nucleoli. These results clearly show that the transport of pre-rRNAs within the nucleolus does not occur randomly, but appears as a radial flow starting from the fibrillar centers that form concentric rings, which finally fuse together as they progress toward the nucleolar periphery.

The three-dimensional study of chromosomes and upstream binding factor-immunolabeled nucleolar organizer regions demonstrates their nonrandom spatial arrangement during mitosis.

The volumic rearrangement of both chromosomes and immunolabeled upstream binding factor in entire well-preserved mitotic cells was studied by confocal microscopy. By using high-quality three-dimensional visualization and tomography, it was possible to investigate interactively the volumic organization of chromosome sets and to focus on their internal characteristics. More particularly, this study demonstrates the nonrandom positioning of metaphase chromosomes bearing nucleolar organizer regions as revealed by their positive upstream binding factor immunolabeling. During the complex morphogenesis of the progeny nuclei from anaphase to late telophase, the equal partitioning of the nucleolar organizer regions is demonstrated by quantification, and their typical nonrandom central positioning within the chromosome sets is revealed.