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Biomedical subjects

Daniele Zink

Publications and source records attributed to Daniele Zink.

11 recordsLinked to original sources

Genome restructuring in mouse embryos during reprogramming and early development.

Although a growing number of studies investigates functional genome organization in somatic cell nuclei, it is largely unknown how mammalian genome organization is established during embryogenesis. To address this question, we investigated chromo center formation and the peculiar arrangements of chromosome domains in early mouse embryos. At the one-cell stage, we observed characteristic arrangements of chromosomes and chromo center components. Subsequently, starting with the burst of zygotic genome transcription major rearrangements led to the establishment of somatic type chromo centers with a defined spatio-temporal organization. These processes appeared to be completed at the blastocyst stage with the onset of cell differentiation. During the same developmental period, a fraction of pericentric heterochromatin that was late replicating in the first cycle underwent switches in replication timing, spatial organization and epigenetic marks. Cloning experiments revealed that the genome organization typical for more advanced stages was quickly reverted into the one-cell stage-specific form after nuclear transfer, supporting the idea that reprogramming associated genome remodeling in normal and cloned embryos is determined by cytoplasmic factors. Together, the results suggest that distinct but characteristic forms of nuclear genome organization are required for genome reprogramming in early embryos and for proper regulation of differential gene expression patterns at later stages.

Animals↗

The temporal program of DNA replication: new insights into old questions.

During the last decades it has been shown that the replication timing program in metazoans is related to chromosome structure, the nuclear positioning and AT/GC content of chromosomal loci, their patterns of histone modifications, and their transcriptional regulation. Here, the current state of knowledge concerning these relationships is reviewed. An integrated view on structure-function relationships in the nucleus is provided and the determination and functional role of the replication timing program is discussed in this context. A corresponding comprehensive model is developed and a key aspect of this model is the suggestion that mammalian chromosomes are organized into stable units equivalent to replicon clusters. It is proposed that the nuclear positions of these units would depend on their histone modifications and determine the replication timing of the whole unit. It is furthermore predicted that replication timing is only indirectly linked to transcriptional regulation and contributes to the maintenance of gene expression patterns. These clear predictions, and the fact that the tools are at hand now to further test them, open an avenue towards solving the long standing problem on how replication timing is determined in metazoan cells.

Animals↗

Architectural reorganization of the nuclei upon transfer into oocytes accompanies genome reprogramming.

The ability of cloned embryos to sustain full-term development depends on the ability of the recipient ooplasm to reprogram the donor cell genome. As the nuclear architecture has recently emerged as a key-factor in the regulation of gene expression, we questioned whether early embryos obtained from transfer of ES metaphasic chromosomes into mouse ooplasm would adopt the somatic or embryonic type of nuclear organization. We have particularly focused on the arrangement of chromosomal territories with respect to the nucleolar compartment, and the pericentric heterochromatin domains called chromocenters. We found that nuclear transfer triggers profound chromatin rearrangements including the dispersion of the donor cell chromocenters components. These rearrangements lead to a typical 1-cell pronuclear organization, namely a radial arrangement of the chromosome territories with centromeres attached to the nucleoli, which adopt the compact fibrillar structure of nucleolar precursor bodies (NPBs). Subsequently, during the second cycle, the cloned embryos undergo further reorganization with the establishment of new chromocenters, clustered in one part of the nucleus, as during normal embryogenesis. We could also establish that the adequate distribution of chromosomal territories at the pronuclear stage seems important for the development until blastocyst.

Animals↗

The replication timing of CFTR and adjacent genes.

Correlations between transcriptional activity and replication timing have been observed for the human cystic fibrosis transmembrane conductance regulator (CFTR) gene, as well as for other tissue-specific genes. In addition, the patterns of histone modifications and the nuclear positioning of chromosomal loci appear to be related to their replication timing. It is not understood why and how these different features are functionally linked. To address this problem, we investigated the replication timing of the human CFTR gene and of adjacent genes. Recently, we could show that CFTR and adjacent genes associate independently from each other with different nuclear regions and chromatin fractions, in accordance with their individual transcriptional regulation. Together, the results show that not the transcriptional activity, but rather the nuclear position of CFTR and adjacent genes appears to be a major determinant of their replication timing. Furthermore, the results imply a specific functional order of nuclear changes related to switches in replication timing.

Cell Cycle↗

Stable chromosomal units determine the spatial and temporal organization of DNA replication.

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.

Animals↗

Transcription-dependent spatial arrangements of CFTR and adjacent genes in human cell nuclei.

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.

Adaptor Proteins, Signal Transducing↗

Nascent RNA synthesis in the context of chromatin architecture.

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.

Chromatin↗

Nuclear structure in cancer cells.

Nuclear architecture - the spatial arrangement of chromosomes and other nuclear components - provides a framework for organizing and regulating the diverse functional processes within the nucleus. There are characteristic differences in the nuclear architectures of cancer cells, compared with normal cells, and some anticancer treatments restore normal nuclear structure and function. Advances in understanding nuclear structure have revealed insights into the process of malignant transformation and provide a basis for the development of new diagnostic tools and therapeutics.

Cell Nucleus↗

Visualizing chromatin and chromosomes in living cells.

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.

Animals↗

Comparative analysis of the functional genome architecture of animal and plant cell nuclei.

Many studies have shown that the functional architecture of eukaryotic genomes displays striking similarities in evolutionarily distant organisms. For example, late-replicating and transcriptionally inactive chromatin is associated with the nuclear periphery in organisms as different as budding yeast and man. These findings suggest that eukaryotic genomes are organized in cell nuclei according to conserved principles. In order to investigate this, we examined nuclei of different animal and plant species by comparing replicational pulse-labelling patterns and their topological relationship to markers for heterochromatin and euchromatin. The data show great similarities in the nuclear genome organization of the investigated animal and plant species, supporting the idea that eukaryotic genomes are organized according to conserved principles. There are, however, differences between animals and plants with regard to histone acetylation patterns and the nuclear distribution of late-replicating chromatin.

Animals↗

Association of p53 and MSH2 with recombinative repair complexes during S phase.

Our previous recombination and biochemical analyses have led to the hypothesis that the tumor suppressor p53 monitors homologous recombination, a function which was previously attributed to the mismatch repair protein MSH2. Here, we show that a certain fraction of p53 is concentrated within discrete nuclear foci of cells synchronized in G1 phase, a pattern which becomes even more pronounced in S phase, especially after gamma-ray treatment. p53 foci show some colocalization with MSH2 within distinct foci during G1 phase, while dots formed by BRCA1 display an independent localization pattern. In S phase nuclei, p53 foci almost completely colocalize with MSH2 foci and associate with the recombination surveillance factor BRCA1 in irradiated S phase cells. These p53 and MSH2 foci also show significant overlaps with foci of the recombination enzymes Rad50 and Rad51, which for the first time unveiled recombination-related functions of p53 in replicating cells. During S phase, p53 and MSH2 are maximally active in binding to early recombination intermediates, and coexist within the same nuclear DNA-protein complexes. Our data suggest that p53 is linked similarly to homologous recombination as MSH2 and provide further evidence for the new concept of a dual role of p53 in the regulation of growth and repair.

Animals↗