Abstract

Uncovering the motifs of a higher order nuclear architecture and its implications on nuclear function has raised increasing interest in the past decade. The nucleus of higher eukaryotes is considered to display a highly dynamic interaction of DNA and protein factors. There is an emerging view that there are hierarchical levels of gene regulation, reaching from epigenetic modifications at the DNA- and histone level to a higher order functional nuclear topology, in the context of which gene-activating and -repressing processes influence the gene expression profile of an individual cell beyond the sequence information of the DNA. The present work focuses on the analysis of the dynamic aspects of higher order nuclear architecture in living cells. As a prerequisite, an in vivo replication labeling strategy was developed, that enabled the simultaneous visualization of early and mid-to-late replicating chromatin as well as single chromosome territories on the basis of a labeling/segregation approach. The presented scratch replication labeling protocol combines a high labeling efficiency with reduced “damaging” effects and can be successfully applied to a number of adherently growing cell lines, including primary human fibroblasts. In addition, a live cell observation system was developed that facilitates time-lapse confocal (4D) microscopy over elongated time periods which made it possible to follow a complete cell cycle or more. To address possible long-range movements of chromosome territories (CTs) during an entire interphase, fluorescence labeling of a small number of CTs was performed in living HeLa cells stably expressing histone H2B-GFP. This was achieved by in vivo scratch replication labeling with fluorescent nucleotides. Labeled cells were cultivated for several cell cycles until labeled chromatids had segregated. Such cells were followed by time-lapse confocal microscopy over time-scales of up to 20 hours covering major parts or the complete cell cycle. Positional changes of the intensity gravity centers of labeled CTs in the order of several µm were observed in early G1, thereafter, the positions remained within a range of ~1 µm till the end of G2. In conclusion, CT arrangements were highly constrained from mid G1 to late G2 / early prophase, whereas major changes of CT neighborhoods occurred from one cell cycle to the next. More extended movements observed in early G1 might play a role when CTs “home in” to establish a non-random radial CT arrangement. To analyze possible changes of chromosome arrangements from one cell cycle to the next, nuclei were photobleached in G2 maintaining a contiguous zone of unbleached chromatin at one nuclear pole. This zone was stably preserved until the onset of prophase whereas unbleached chromosome segments were often observed to become located at distant sites in the metaphase plates. Accordingly, chromatin patterns observed in daughter nuclei differed significantly from the mother cell nucleus, indicating that CT neighborhoods were not preserved during mitosis. The variability of CT neighborhoods during clonal growth was further confirmed by 3D-FISH experiments. A series of experiments of a more preliminary character looked at the influence of the nuclear lamina in constraining and determining a higher order nuclear architecture by selectively interacting with mid-to-late replicating chromatin. Simultaneous immunodetection of lamin B on two-color replication labeled neuroblastoma cell nuclei revealed specific attachment of the mid-to-late replicating chromatin compartment not only along the periphery but also inside the nucleus along invaginations of the lamina. 4D-live cell observation of lamin C-GFP expressing CHO cells with mid-to-late replicating chromatin labeled simultaneously revealed concomitant movements of replication foci attached to lamin invaginations. Moreover, a functional essay was employed which uses injection of a dominant negative lamin A mutant protein (ΔNLA) to cause a reversible disruption of the nuclear lamina. Initial results point to concomitant distortion of the mid-to-late replication pattern and a preferential attachment of the respective chromatin sites to partially disrupted lamin B (as compared to lamin A and nuclear pore complex). Finally, a model is presented on the chromosome positioning in mammalian nuclei depending on cell cycle and nuclear shape.