Expression of the DYRK1A gene correlates with its 3D positioning in the interphase nucleus of Down syndrome cells.

Down syndrome is a common birth defect caused by trisomy of chromosome 21. Chromosomes occupy distinct territories in interphase nuclei, and their distribution within the nuclear space is nonrandom. In humans with Down syndrome, two chromosomes 21 frequently localize proximal to one another and distant from the third chromosome. Here, we investigated the nuclear organization of DYRK1A and SOD1, two genes mapping to chromosome 21 that greatly contribute to the pathology. We found that DYRK1A conserves its central positioning between normal and trisomic cells, whereas SOD1 adopts more peripheral distribution in trisomic cells. We also found that the relative position of these genes with respect to each other varies among the different copies of chromosome territories 21 within a cell, and that this distinct distribution is associated with differences in their expression levels. All together, our results may explain, at least in part, the difference in the expression level of these two genes implicated in the pathogenesis of Down syndrome.

Chromosome Aberrations in Lymphocytes of Patients Undergoing Radon Spa Therapy: An Explorative mFISH Study.

In the present exploratory study, we aim to elucidate the action of radon in vivo and to assess the possible health risks. Chromosome aberrations were analyzed in lymphocytes of two patients (P1, P2) undergoing radon spa therapy in Bad Steben (Germany). Both patients, suffering from painful chronic degenerative disorders of the spine and joints, received nine baths (1.2 kBq/L at 34 °C) over a 3-week period. Chromosome aberrations were analyzed before and 6, 12 and 30 weeks after the start of therapy using the high-resolution multiplex fluorescence in situ hybridization (mFISH) technique. For comparison, the lymphocytes from two healthy donors (HD1, HD2) were examined. P1 had a higher baseline aberration frequency than P2 and both healthy donors (5.3 ± 1.3 vs. 2.0 ± 0.8, 1.4 ± 0.3 and 1.1 ± 0.1 aberrations/100 analyzed metaphases, respectively). Complex aberrations, biomarkers of densely ionizing radiation, were found in P1, P2 and HD1. Neither the aberration frequency nor the fraction of complex aberrations increased after radon spa treatment, i.e., based on biological dosimetry, no increased health risk was found. It is worth noting that a detailed breakpoint analysis revealed potentially clonal aberrations in both patients. Altogether, our data show pronounced inter-individual differences with respect to the number and types of aberrations, complicating the risk analysis of low doses such as those received during radon therapy.

Spatial link between nucleoli and expression of the Zac1 gene.

Eukaryotic genomes are highly organized within the cell nucleus. Genome organization not only implies the preferential positioning of genetic elements in the interphase nucleus but also the topographic distribution of biological processes. We have investigated the relationship between spatial organization and genome function in single cells. Myc, c-Met, Igf2r, Asb4, and Zac1 genes have the same radial distribution, but they are not positioned in close proximity with respect to each other. Three-dimensional mapping of their transcription sites uncovered a gene-specific pattern of relative positioning with respect to the nucleolus. We found that the Zac1 gene transcription preferentially occurs juxtaposed to the nucleolus, and that its mRNA accumulates at this site of transcription. Nucleoli isolation followed by qRT-PCR provided evidence for a physical interaction between Zac1 mRNA and the nucleolus. Actinomycin-D treatment induced disassembly of the nucleolus, loss of the RNA-FISH signal, and dramatic increase of the ZAC protein level. However, inhibition of RNA polymerase II had no effect over the Zac1 FISH signal and the protein expression. Induction of cell cycle arrest, which involves participation of the ZAC protein, provoked mRNA release from its retention site and protein synthesis. Our data demonstrate that Zac1 mRNA preferentially accumulates in close proximity to nucleoli within the cell nucleus. In addition, our results suggest a functional link between such spatial distribution and protein expression.

Combined fluorescent-chromogenic in situ hybridization for identification and laser microdissection of interphase chromosomes.

Chromosome territories constitute the most conspicuous feature of nuclear architecture, and they exhibit non-random distribution patterns in the interphase nucleus. We observed that in cell nuclei from humans with Down Syndrome two chromosomes 21 frequently localize proximal to one another and distant from the third chromosome. To systematically investigate whether the proximally positioned chromosomes were always the same in all cells, we developed an approach consisting of sequential FISH and CISH combined with laser-microdissection of chromosomes from the interphase nucleus and followed by subsequent chromosome identification by microsatellite allele genotyping. This approach identified proximally positioned chromosomes from cultured cells, and the analysis showed that the identity of the chromosomes proximally positioned varies. However, the data suggest that there may be a tendency of the same chromosomes to be positioned close to each other in the interphase nucleus of trisomic cells. The protocol described here represents a powerful new method for genome analysis.