Changes in chromatin organization of human cells in response to genotoxic stress
Abdel-Halim Mahfouz, H.I.
Citation
Abdel-Halim Mahfouz, H. I. (2009, February 24). Changes in chromatin organization of human cells in response to genotoxic stress. Retrieved from https://hdl.handle.net/1887/13517
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License: Licence agreement concerning inclusion of doctoral thesis in the Institutional Repository of the University of Leiden
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Aim and outline of the thesis
Aim and outline of the thesis
Aim and Outline of the Thesis
Cells are constantly under threat of cytotoxic and/or mutagenic events due to exposure to various types of endogenous and exogenous stressors that cause damage to DNA and other macromolecules. To counteract the deleterious effects of DNA damage due to interference with DNA-metabolizing processes, i.e. transcription and replication, cells are equipped with efficient defense mechanisms that constitute the so called DNA damage response (DDR). DNA damage provokes signaling pathways that activate cell cycle checkpoints, DNA repair, gene expression, chromatin remodeling and apoptosis. Proper DDR is essential to maintain genomic integrity.
Recently, the impact of DNA damage on chromatin organization has gained increasing interest. Numerous studies have shown that chromosome domains are non- randomly arranged within the three-dimensional space of the interphase nucleus and that chromatin positioning plays an important role in regulation of gene expression and maintenance of genome stability by preventing undesired chromosome exchanges. There is growing evidence that infliction of DNA damage can lead to spatial repositioning of chromatin (Dolling et al., 1997; Figgit and Savage, 1999; Aten et al., 2004). Mitomycin C (MMC) induced interchanges between homologous chromosomes as shown by conventional cytogenetic studies have been taken as cytological evidence for somatic pairing of human chromosomes (Shaw and Cohen, 1965, Morad et al., 1973). In these studies chromosomes 1, 9, and 16 were frequently involved in MMC-induced homologous chromosomal exchanges involving the heterochromatic blocks of these chromosomes. Pairing of specific chromosomal regions might differ dependent on biological states or activities, i.e. cell type, cell cycle phase, differentiation stage and diseases. However, the biological relevance of pairing is unclear. The aim of this thesis was to dissect the mechanisms that underlie the interaction between homologous chromosomes observed in metaphase and the interphase of the cell cycle. To reach this goal, we assessed the effect of genotoxic agents on the positioning of homologous chromosomal regions in human cells. The relationship between interphase pairing and processing of DNA damage that leads to exchange formation was examined. Specific DNA probes for euchromatic and heterochromatic regions of the human genome were used in concert with 2D
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Aim and outline of the thesis
fluorescence in situ hybridization (FISH) to assess the impact of genotoxic exposure on spatial chromatin organization in normal and DNA repair deficient human cells.
The first chapter of this thesis describes different cellular genotoxic stressors and summarizes the DNA damage response particularly DNA repair pathways.
Furthermore, chromatin organization of the interphase nucleus is reviewed with special emphasis on homologous pairing of specific chromosomal regions in somatic cells and the impact of various genotoxic agents on chromatin organization.
In chapters 2 and 3 we examined the mechanisms underlying DNA damage induced pairing of homologous chromosomes. Chromosome 9 homologues are the most frequently involved in MMC induced chromatid exchanges in human lymphocytes treated in G1 phase and these exchanges are formed between the paracentromeric heterochromatic regions (9q12-13) of chromosome 9. In contrast, the similarly sized chromosome 8 with a paracentromeric euchromatic region (8p11.2) did not reveal exchanges. One of the main questions addressed is whether the heterochromatic regions of chromosome 9 are in close proximity to allow the formation of MMC-induced exchanges. Measurement of inter-homologue distances demonstrated that the 9q12-13 heterochromatic regions as well as the euchromatic 8p11.2 regions are largely randomly distributed in untreated confluent normal fibroblasts. MMC treatment of confluent cells induced repositioning and pairing of the 9q12-13 regions in a sub-population of cells suggesting chromatin movement to bring the homologous regions in close contact leading to exchange formation. To gain insight into the mechanism of heterochromatin pairing, a genetic approach was taken, i.e. cells from DNA repair deficient patients were used to assess whether MMC induced homologous pairing and exchanges are dependent on DNA repair. The outcome of these experiments revealed that DNA repair plays a key role in MMC- induced pairing and exchanges. This implicates pairing of heterochromatin as a part of cellular responses to DNA damage, particularly in the recombinational processing of MMC-induced interstrand cross-links (ICLs). Also ionizing radiation (IR) induced pairing of the homologous regions 9q12-13 in contrast to 8p11.2 regions. The kinetics of pairing after X-rays suggests that homology dependent processing of IR-induced DSBs might occur in non-dividing cells.
Chapter 4 addresses the question whether pairing is a general cellular stress response targeted by DNA damage in the heterochromatic regions that undergo pairing. This was done by assessment of the effect of UV irradiation and heat shock
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Aim and outline of the thesis
treatment on the localization of homologous chromosomes. Both agents were able to induce pairing of the heterochromatic regions of chromosome 9 but not the euchromatic regions of chromosome 8. Local UV irradiation demonstrated that pairing was not targeted by DNA damage in the heterochromatic regions of chromosome 9. Human cells with deficiencies in various repair pathways were employed to elucidate which type of DNA repair is important for pairing. We conclude that pairing of heterochromatin is linked to homologous recombination during processing of MMC-induced ICLs.
In chapter 5, a mechanistic study was carried out to find a relationship between the repair of DNA damage and pairing of heterochromatin. Processing of MMC-induced ICLs, i.e. the formation of DNA breaks was detected in confluent G1 human cells by various approaches. Immediate formation of DNA breaks after MMC treatment either represents DNA breaks induced by MMC or reflects processing of MMC-induced ICLs. These initial breaks were monitored independent of any DNA repair deficiency. However, later formation of phosphorylation of H2AX corresponded with the ability to perform heterochromatin pairing. Cells impaired in MMC-induced H2AX phosphorylation were also lacking the pairing of heterochromatin suggesting that the pairing process is linked to the DNA damage response.
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