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Extensive changes of higher order chromatin arrangements can be observed during prometaphase, terminal cell differentiation and cellular senescence. Experimental systems where major reorganization of nuclear architecture can be induced under defined conditions, may help to better understand the functional implications of such changes. Here, we report on profound chromatin reorganization in fibroblast nuclei by chaetocin, a thiodioxopiperazine metabolite. Chaetocin induces strong condensation of chromosome territories separated by a wide interchromatin space largely void of DNA. Cell viability is maintained irrespective of this peculiar chromatin phenotype. Cell cycle markers, histone signatures, and tests for cellular senescence and for oxidative stress indicate that chaetocin induced chromatin condensation/clustering (CICC) represents a distinct entity among nuclear phenotypes associated with condensed chromatin. The territorial organization of entire chromosomes is maintained in CICC nuclei; however, the conventional nuclear architecture harboring gene-dense chromatin in the nuclear interior and gene-poor chromatin at the nuclear periphery is lost. Instead gene-dense and transcriptionally active chromatin is shifted to the periphery of individual condensed chromosome territories where nascent RNA becomes highly enriched around their outer surface. This chromatin reorganization makes CICC nuclei an attractive model system to study this border zone as a distinct compartment for transcription. Induction of CICC is fully inhibited by thiol-dependent antioxidants, but is not related to the production of reactive oxygen species. Our results suggest that chaetocin functionally impairs the thioredoxin (Trx) system, which is essential for deoxynucleotide synthesis, but in addition involved in a wide range of cellular functions. The mechanisms involved in CICC formation remain to be fully explored

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Aneuploidy is a change in number or structure of one or more chromosomes that are not a multiple of the whole chromosome set. One of the best known pathological aneuploidies is trisomy 21 (Down syndrome), with chromosome 21 present in three instead of two copies. Patients with Down syndrome display severe mental retardation and growth defects. In fact, most abnormal aneuploid karyotypes lead to spontaneous abortions during embryogenesis, indicating that aneuploidy is not well tolerated in humans. Aneuploidy was also shown to be a common hallmark of cancer tissues; however, the debate is ongoing whether aneuploidy is rather a by-product or a trigger of tumorigenesis. Even though aneuploid karyotypes were already identified more than 100 years ago little is understood about cellular physiology of aneuploidy cells, especially in humans. To uncover the consequences of numerical aneuploidy in human cells, I generated aneuploid cell lines derived from the human cell lines HCT116 and RPE-1 hTERT. First, we showed that aneuploid cells proliferate slower compared to their disomic counterparts. A detailed cell cycle analysis revealed that this delay was due to a prolonged G1 and S phase, whereas G2 and M phase remained unperturbed. Furthermore, we conducted an in depth genome wide comparison of DNA, mRNA and protein levels in aneuploid cells. Using CGH, mRNA array and SILAC technology, we quantified the changes in DNA, mRNA and protein abundance. We revealed that extra chromosomes are actively transcribed and translated. However, the abundance of some proteins, particularly subunits of protein complexes and protein kinases, are adjusted towards disomic levels. Additionally, we asked how the cellular physiology is affected by the addition of a specific chromosome. Two scenarios are possible: either the cellular response depends on the additional chromosomes or all aneuploid cells show the same changes of cellular physiology. Indeed, we found that all aneuploid cell lines show similar physiological responses, irrespective of the type of additional chromosome. All aneuploid cell lines down-regulate DNA and RNA metabolism and up-regulate among others energy metabolism, lysosome function and membrane biosynthesis pathways. Lysosomes which are involved in autophagy are besides the ubiquitin-proteasome system important for cellular protein turn over. We found p62-dependent selective autophagy increased in all analyzed cell lines with extra chromosomes suggesting a role of p62-dependent selective autophagy in maintenance of protein homeostasis upon expression of extra protein in these cell lines.

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