The mammalian cell nucleus displays a distinct spatial segregation of active euchromatic from inactive heterochromatic genomic regions 1,2. In conventional nuclei, microscopy shows that euchromatin is localized in the nuclear interior and heterochromatin at the nuclear periphery 1,2. Hi-C shows this segregation as a plaid pattern of enriched contacts between A (euchromatic) and B (heterochromatic) compartments 3. Many mechanisms of compartment formation have been proposed, such as attraction of heterochromatin to the nuclear lamina 2,4 , preferential attraction of similar chromatin to each other 1,4-12 , higher levels of chromatin mobility in the active chromatin 13-15 , and transcription-related clustering of euchromatin 16,17. Still, these hypotheses have remained inconclusive due to the difficulty of disentangling intra-chromatin and chromatin-#

For the first time, DNA double-strand breaks (DSBs) were directly visualized in functionally and structurally different chromatin domains of human cells. The results show that genetically inactive condensed chromatin is much less susceptible to DSB induction by gamma-rays than expressed, decondensed domains. Higher sensitivity of open chromatin for DNA damage was accompanied by more efficient DSB repair. These findings follow from comparing DSB induction and repair in two 11 Mbp-long chromatin regions, one with clusters of highly expressed genes and the other, gene-poor, containing mainly genes having only low transcriptional activity. The same conclusions result from experiments with whole chromosome territories, differing in gene density and consequently in chromatin condensation. It follows from our further results that this lower sensitivity of DNA to the damage by ionizing radiation in heterochromatin is not caused by the simple chromatin condensation but very probably by the presence of a higher amount of proteins compared to genetically active and decondensed chromatin. In addition, our results show that some agents potentially used for cell killing in cancer therapy (TSA, hypotonic and hypertonic) influence cell survival of irradiated cells via changes in chromatin structure and efficiency of DSB repair in different ways.

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We show that double strand breaks (DSBs) induced in chromatin of low as well as high density by exposure of human cells to gamma-rays are repaired in low-density chromatin. Extensive chromatin decondensation manifested in the vicinity of DSBs by decreased intensity of chromatin labelling, increased H4K5 acetylation, and decreased H3K9 dimethylation was observed already 15 min after irradiation. Only slight movement of sporadic DSB loci for short distances was noticed in living cells associated with chromatin decondensation around DSBs. This frequently resulted in their protrusion into the low-density chromatin domains. In these regions, the clustering (contact or fusion) of DSB foci was seen in vivo, and in situ after cell fixation. The majority of these clustered foci were repaired within 240 min, but some of them persisted in the nucleus for several days after irradiation, indicating damage that is not easily repaired. We propose that the repair of DSB in clustered foci might lead to misjoining of ends and, consequently, to exchange aberrations. On the other hand, the foci that persist for several days without being repaired could lead instead to cell death.

Biological effects of high-LET (linear energy transfer) radiation have received increasing attention, particularly in the context of more efficient radiotherapy and space exploration. Efficient cell killing by high-LET radiation depends on the physical ability of accelerated particles to generate complex DNA damage, which is largely mediated by LET. However, the characteristics of DNA damage and repair upon exposure to different particles with similar LET parameters remain unexplored. We employed high-resolution confocal microscopy to examine phosphorylated histone H2AX (γH2AX)/p53-binding protein 1 (53BP1) focus streaks at the microscale level, focusing on the complexity, spatiotemporal behaviour and repair of DNA double-strand breaks generated by boron and neon ions accelerated at similar LET values (∼135 keV μm) and low energies (8 and 47 MeV per n, respectively). Cells were irradiated using sharp-angle geometry and were spatially (3D) fixed to maximize the resolution of these analyses. Both high-LET radiation types generated highly complex γH2AX/53BP1 focus clusters with a larger size, increased irregularity and slower elimination than low-LET γ-rays. Surprisingly, neon ions produced even more complex γH2AX/53BP1 focus clusters than boron ions, consistent with DSB repair kinetics. Although the exposure of cells to γ-rays and boron ions eliminated a vast majority of foci (94% and 74%, respectively) within 24 h, 45% of the foci persisted in cells irradiated with neon. Our calculations suggest that the complexity of DSB damage critically depends on (increases with) the particle track core diameter. Thus, different particles with similar LET and energy may generate different types of DNA damage, which should be considered in future research.

A complex study of the spatial arrangement of different genetic elements (genes, centromeres and chromosomal domains) in the cell nucleus is presented and the principles of this arrangement are discussed. We show that the radial location of genetic elements in the three-dimensional (3D) space between the center of the nucleus and the nuclear membrane is element specific and dependent on the position of the element on the chromosome. In contrast, mutual angular positioning of both homologous and heterologous genetic elements is, in the majority of cases, random. In several cases, tethering of heterologous genetic elements was observed. This close proximity of specific loci may be responsible for their mutual rearrangement and the development of cancer. Comparison of our results with transcriptome maps shows that the nuclear location of chromosomal domains with highly expressed genes is more central when compared with chromosomes with low expression. The higher-order chromatin structure is strikingly similar in various human cell types, which correlates with the fact that the profiles of gene expression are also similar.

Amifostine protects normal cells from DNA damage induction by ionizing radiation or chemotherapeutics, whereas cancer cells typically remain uninfluenced. While confirming this phenomenon, we have revealed by comet assay and currently the most sensitive method of DNA double strand break (DSB) quantification (based on γH2AX/53BP1 high-resolution immunofluorescence microscopy) that amifostine treatment supports DSB repair in γ-irradiated normal NHDF fibroblasts but alters it in MCF7 carcinoma cells. These effects follow from the significantly lower activity of alkaline phosphatase measured in MCF7 cells and their supernatants as compared with NHDF fibroblasts. Liquid chromatography-mass spectrometry confirmed that the amifostine conversion to WR-1065 was significantly more intensive in normal NHDF cells than in tumor MCF cells. In conclusion, due to common differences between normal and cancer cells in their abilities to convert amifostine to its active metabolite WR-1065, amifostine may not only protect in multiple ways normal cells from radiation-induced DNA damage but also make cancer cells suffer from DSB repair alteration.

Cellular transition to senescence is associated with extensive chromatin reorganization and changes in gene expression. Recent studies appear to imply an association of lamin B1 (LB1) reduction with chromatin rearrangement in human fibroblasts promoted to senescence, while the mechanisms and structural features of these relationships have not yet been clarified. In this work, we examined the functions of LB1 and the lamin B receptor (LBR) in human cancer cells. We found that both LB1 and LBR tend to deplete during cancer cell transfer to senescence by γ-irradiation. A functional study employing silencing of LBR by small hairpin ribonucleic acid (shRNA) constructs revealed reduced LB1 levels suggesting that the regulation of both proteins is interrelated. The reduced expression of LBR resulted in the relocation of centromeric heterochromatin (CSH) from the inner nuclear membrane (INM) to the nucleoplasm and is associated with its unfolding. This indicates that LBR tethers heterochromatin to INM in cycling cancer cells and that LB1 is an integral part of this tethering. Down-regulation of LBR and LB1 at the onset of senescence are thus necessary for the release of heterochromatin binding to lamina, resulting in changes in chromatin architecture and gene expression. However, the senescence phenotype was not manifested in cell lines with reduced LBR and LB1 expression suggesting that other factors, such as deoxyribonucleic acid (DNA) damage, are needed to trigger senescence. We conclude that the primary response of cells to various stresses leading to senescence consists of the down-regulation of LBR and LB1 to attain reversal of the chromatin architecture.