We used chicken retinospheroids (RS) to study the nuclear architecture of vertebrate cells in a three-dimensional (3D) cell culture system. The results showed that the different neuronal cell types of RS displayed an extreme form of radial nuclear organization. Chromatin was arranged into distinct radial zones which became already visible after DAPI staining. The distinct zones were enriched in different chromatin modifications and in different types of chromosomes. Active isoforms of RNA polymerase II were depleted in the outermost zone. Also chromocenters and nucleoli were radially aligned in the nuclear interior. The splicing factor SC35 was enriched at the central zone and did not show the typical speckled pattern of distribution. Evaluation of neuronal and non-neuronal chicken tissues showed that the highly ordered form of radial nuclear organization was also present in neuronal chicken tissues. Furthermore, the data revealed that the neuron-specific nuclear organization was remodeled when cells spread on a flat substrate. Monolayer cultures of a chicken cell line did not show this extreme form of radial organization. Rather, such monolayer cultures displayed features of nuclear organization which have been described before for many different types of monolayer cells. The finding that an extreme form radial nuclear organization, which has not been described before, is present in RS and tissues, but not in cells spread on a flat substrate, suggests that it would be important to complement studies on nuclear architecture performed with monolayer cells by studies on 3D cell culture systems and tissues.

The human genes CFTR, ASZ1/GASZ, and CTTNBP2/CORTBP2 map to adjacent loci on chromosome 7q31 and display characteristic patterns of nuclear positioning, which strictly correlate with the state of activity. To address the evolutionary conservation of gene positioning, we investigated transcriptional activity and nuclear positioning of the highly conserved murine orthologs and of additional murine genes mapping to the region of conserved synteny on mouse chromosome 6. The results showed that all murine loci investigated constitutively localized in the nuclear interior irrespective of their functional state. Silenced loci did not display preferential association with the nuclear periphery or with chromocenters, respectively, and no differential positioning with respect to the chromosome 6 territory could be observed. This positional behavior of the murine loci was in striking contrast to the positioning of the human orthologs, and the results show that the transcription-dependent positioning of CFTR and adjacent loci has not been conserved. The findings reveal that the nuclear organization of conserved chromosomal regions can change rapidly during evolution and is not always as highly conserved as other features of chromosome organization. Furthermore, the results suggest that the way how nuclear positioning contributes to the regulation of conserved loci can be different in different vertebrate species.

DNA binding transcriptional activators play a central role in gene-selective regulation. In part, this is mediated by targeting local covalent modifications of histone tails. Transcriptional regulation has also been associated with the positioning of genes within the nucleus. We have now examined the role of a transcriptional activator in regulating the positioning of target genes. This was carried out with primary beta-cells and hepatocytes freshly isolated from mice lacking Hnf1 alpha, an activator encoded by the most frequently mutated gene in human monogenic diabetes (MODY3). We show that in Hnf1a(-/-) cells inactive endogenous Hnf1 alpha-target genes exhibit increased trimethylated histone H3-Lys27 and reduced methylated H3-Lys4. Inactive Hnf1 alpha-targets in Hnf1a(-/-) cells are also preferentially located in peripheral subnuclear domains enriched in trimethylated H3-Lys27, whereas active targets in wild-type cells are positioned in more central domains enriched in methylated H3-Lys4 and RNA polymerase II. We demonstrate that this differential positioning involves the decondensation of target chromatin, and show that it is spatially restricted rather than a reflection of non-specific changes in the nuclear organization of Hnf1a-deficient cells. This study, therefore, provides genetic evidence that a single transcriptional activator can influence the subnuclear location of its endogenous genomic targets in primary cells, and links activator-dependent changes in local chromatin structure to the spatial organization of the genome. We have also revealed a defect in subnuclear gene positioning in a model of a human transcription factor disease.

We analysed the nuclear organization of the Polycomb/Trithorax group response element (PRE/TRE) Fab-7 and of other PRE/TREs in larval tissues of D. melanogaster. The results show that pairing/clustering of transgenic and endogenous Fab-7 elements and of other endogenous PRE/TREs occurs only to a limited degree in a highly locus-specific and tissue-specific manner. However, transgenic Fab-7 elements as well as the Fab-7-regulated Abd-B gene and other endogenous loci preferentially occupied defined nuclear regions. Preferred association with the nuclear periphery was observed in the inactive state. However, also in the active state, Fab-7 was often found associated with the nuclear periphery as well as with the boundary of heterochromatin in a fly line- and tissue-specific manner. The boundary between heterochromatin and euchromatin revealed a highly complex architecture in the three-dimensional nuclear space with a close juxtaposition of active and repressed domains. The results suggest that such complex architectures create nuclear microenvironments sustaining specific states of activity of defined PRE/TREs. However, the data also show that the positional behaviour of the transgenic Fab-7 element does not apply to PRE/TREs in general. Altogether, this finding and the highly locus-, tissue-, and fly line-specific behaviour with regard to nuclear positioning and pairing/clustering suggest that the relationships between nuclear organization and functional regulation of PRE/TREs are highly complex and that simple models making general predictions might not be appropriate.

BACKGROUND INFORMATION:Recent results from a limited number of eukaryotic model organisms suggest that major principles governing spatial organization of the genome in functionally distinct nuclear compartments are conserved through evolution.RESULTS:We examined the in situ spatial organization of major nuclear components and nuclear patterns of gene loci with strictly defined expression patterns in endocycling cells of the transparent urochordate Oikopleura dioica, a complex metazoan with a very compact genome. Endocycling cells with different functions and similar DNA content displayed distinct topologies of nuclear components. However, the generation of the diverse nuclear architectures did not involve specific local organization of active genes or their preferential amplification. Interestingly, endocycling cells lacked nuclear-envelope-associated heterochromatin and prominent splicing-factor domains, which in mammalian cells associate with transcriptionally silent and active loci respectively. In addition, no correlation was found between transcriptional activity of a locus and its association with chromatin domains rich in specific histone modifications.CONCLUSIONS:Together, these findings and the absence of typical eukaryotic replication patterns reveal a surprisingly limited functional compartmentalization of O. dioica endocycling nuclei. This indicates that robust cell-type-specific gene expression does not necessarily require high levels of spatial genome organization.

Based on the idea that chromatin domains provide physical barriers for large molecules and multi-enzyme complexes, including the components of the transcription machinery, it has been proposed that transcription should be confined to the surfaces of chromatin domains. As a consequence nascent RNA should accumulate in the interchromatin space, which is thought to provide a special nuclear compartment involved in transcription, as well as in the processing and export of RNA (Cremer et al. 1993, Cremer & Cremer 2001). To further address the relationships between chromatin organization and RNA synthesis, we investigated the localization of BrUTP-labelled nascent RNA in HeLa cells stably expressing green fluorescent protein (GFP)-tagged histone H2B, which highlights the chromatin structure. Our results showed that nascent RNA does not preferentially localize within the interchromatin space. The findings do not support the idea that the interchromatin space provides a nuclear compartment playing an essential role in nascent RNA synthesis. However, the results are in agreement with the emerging view that even condensed chromatin domains display a highly dynamic organization and are not a physical barrier for transcription factors.

DNA replication occurs in mammalian cells at so-called replication foci occupying defined nuclear sites at specific times during S phase. It is an unresolved problem how this specific spatiotemporal organization of replication foci is determined. Another unresolved question remains as to what extent DNA is redistributed during S phase. To investigate these problems, we visualized the replicating DNA and the replication machinery simultaneously in living HeLa cells. Time-lapse analyses revealed that DNA was not redistributed to other nuclear sites during S phase. Furthermore, the results showed that DNA is organized into stable aggregates equivalent to replication foci. These aggregates, which we call sub-chromosomal foci, stably maintained their replication timing from S phase to S phase. During S-phase progression, the replication machinery sequentially proceeded through spatially adjacent sets of sub-chromosomal foci. These findings imply that the specific nuclear substructure of chromosomes and the order of their stable subunits determine the spatiotemporal organization of DNA replication.

We investigated in different human cell types nuclear positioning and transcriptional regulation of the functionally unrelated genes GASZ, CFTR, and CORTBP2, mapping to adjacent loci on human chromosome 7q31. When inactive, GASZ, CFTR, and CORTBP2 preferentially associated with the nuclear periphery and with perinuclear heterochromatin, whereas in their actively transcribed states the gene loci preferentially associated with euchromatin in the nuclear interior. Adjacent genes associated simultaneously with these distinct chromatin fractions localizing at different nuclear regions, in accordance with their individual transcriptional regulation. Although the nuclear localization of CFTR changed after altering its transcription levels, the transcriptional status of CFTR was not changed by driving this gene into a different nuclear environment. This implied that the transcriptional activity affected the nuclear positioning, and not vice versa. Together, the results show that small chromosomal subregions can display highly flexible nuclear organizations that are regulated at the level of individual genes in a transcription-dependent manner.

The dynamic organization of eukaryotic genomes in cell nuclei recently came into the focus of research interest. The kinetics of genome dynamics can be addressed only by approaches involving live cell microscopy. Different methods are available to visualize chromatin, specific chromatin fractions, or individual chromosome territories within nuclei of living mammalian cells. Appropriate labeling procedures as well as cell chamber systems and important controls for live cell microscopy are described.

We investigated the chromatin organization of living cells with a combination of recently developed approaches for histone and DNA labeling. Nucleosomal DNA was labeled with a histone H2B-GFP (green fluorescent protein) fusion protein and the chromatin organization of living HeLa cells was analyzed by high resolution confocal microscopy. Within the perinuclear and perinucleolar regions chromatin was organized into large-scale fibers of 2 to 8 µm in length and 300 to 500 nm in diameter. Within the nuclear interior we observed similar large-scale fibers, but in addition focal as well as diffuse forms of organization. Comparison with standard labeling and detection procedures revealed major differences in the chromatin organization observed. Chromatin organization revealed by the distribution of histone H2B-GFP was directly compared with the functional organization of chromatin by Cy3-dUTP labeling of DNA replicating at a specific time. DNA regions replicating at a specific time display characteristic physical and functional properties. Analysis of Cy3-labeled foci revealed that they are associated with all three forms of chromatin organization (fibrillar, focal and diffuse). In particular, Cy3-labeled foci appeared as discontinuous regions of large-scale fibers. These results demonstrate that large-scale chromatin fibers have discontinuous functional characteristics.

We investigated the nuclear higher order compartmentalization of chromatin according to its replication timing (Ferreira et al., 1997) and the relations of this compartmentalization to chromosome structure and the spatial organization of transcription. Our aim was to provide a comprehensive and integrated view on the relations between chromosome structure and functional nuclear architecture. Using different mammalian cell types, we show that distinct higher order compartments whose DNA displays a specific replication timing are stably maintained during all interphase stages. The organizational principle is clonally inherited. We directly demonstrate the presence of polar chromosome territories that align to build up higher order compartments, as previously suggested (Ferreira et al., 1997). Polar chromosome territories display a specific orientation of early and late replicating subregions that correspond to R- or G/C-bands of mitotic chromosomes. Higher order compartments containing G/C-bands replicating during the second half of the S phase display no transcriptional activity detectable by BrUTP pulse labeling and show no evidence of transcriptional competence. Transcriptionally competent and active chromatin is confined to a coherent compartment within the nuclear interior that comprises early replicating R-band sequences. As a whole, the data provide an integrated view on chromosome structure, nuclear higher order compartmentalization, and their relation to the spatial organization of functional nuclear processes.