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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Summary and Discussion
Summary and Discussion
Conservation of genomic integrity is essential for correct expression of the genome and for the faithful transmission of genetic information to the next generations.
However, all living organisms are continuously exposed to a variety of endogenous and environmental DNA-damaging agents, which threat the genomic integrity. To counteract the harmful effects of DNA damage, cells trigger an intricate network of signaling pathways resulting in the activation or modulation of protective mechanisms including cell-cycle checkpoints, gene expression, chromatin remodeling, DNA repair and apoptosis. This protection system is termed the DNA damage response (DDR).
Defects in DDR can lead to mutations, chromosomal aberrations, genome instability and human diseases such as cancer.
Chapter 1 of this thesis gives a brief overview of some aspects of the DDR.
DNA damaging agents used in this thesis are described in chapter 1.2. Also the heat shock response is reviewed focusing on the impact of heat shock on transcription and on its possible genotoxic effects. Chapter 1.3 contains a description of mammalian DNA repair pathways involved in the repair of double strand breaks (NHEJ and HR), bulky DNA lesions (NER) and interstrand crosslinks (ICL repair) and the human syndromes associated with defects in these pathways.
Chromatin organization in the interphase nucleus is introduced in chapter 1.4.
Principally, chromatin can be observed in the interphase nuclei as euchromatin and heterochromatin based on the degree of condensation. Recent studies revealed a nonrandom arrangement of chromosome territories in interphase cells. This section describes the clustering and associations of specific chromosomes or chromosomal regions and the possible role of these associations in the regulation of gene expression and induction of chromosomal exchanges. Numerous studies have reported that pairing of homologous chromosomal regions (mainly centromeric heterochromatic regions) occurs in mammalian somatic cells depending on cell type, cell cycle and disease such as cancer. We review these studies with emphasis on the structure of heterochromatin which consists of different classes of satellite DNA repeats and discuss the possible role of the recently reported transcription of chromosome 9 satellite DNA in human cells. In addition, the impact of DNA damage on chromatin organization is also introduced. There is a large descriptive data on homologue
Summary and Discussion
pairing in somatic cells, however the underlying mechanism of this pairing is still unclear. The aim of our studies was to unravel mechanistic aspects of somatic pairing in human cells. The focus was on the effects of DNA damage on the positioning of euchromatic and heterochromatic regions in interphase and the mechanisms underlying the formation of interchanges between homologous chromosomes containing heterochromatic regions. The results obtained in this study are summerized below and also presented in Table 1.
In chapter 2 we examined the mechanisms of mitomycin C (MMC) induced pairing of homologous chromosomes in interphase and metaphase cells. FISH studies of metaphases of human lymphocytes using whole chromosome and band specific DNA probes revealed that chromosome 9 homologues were frequently involved in MMC induced chromatid exchanges and all of these exchanges were formed between the paracentromeric heterochromatic regions (9q12-13). In contrast, chromosome 8 that has a similar size as chromosome 9 and contains a paracentromeric euchromatic region (8p11.2) was not involved in MMC-induced exchanges. To determine whether close proximity of the heterochromatic regions of chromosome 9 underlies the formation of exchanges after MMC exposure, we employed primary human fibroblasts, FISH and 2-D microscopy. Measurement of inter-homologue distances demonstrated that the 9q12-13 heterochromatic regions as well as the euchromatic 8p11.2 regions are randomly distributed in untreated confluent fibroblasts. Treatment with MMC immediately induced repositioning and pairing of the 9q12-13 regions in a sub-population of cells. In contrast, the euchromatic regions of chromosome 8 did not reveal significant pairing. The frequency of chromosome 9 pairing in interphase correlated with exchanges observed in metaphase cells suggesting that chromatin movement is induced to bring the homologous regions in close contact. These contacts might reflect sites of repair of MMC induced DNA damage leading to chromatid-types exchanges between the homologues. Indeed premature chromosome condensation (PCC) analysis of MMC-treated G1 cells indicated the presence of pairing and breaks involving chromosome 9 and provided direct evidence for a physical interaction between the two homologues. To gain more insight into the mechanism of heterochromatin pairing, cells from xeroderma pigmentosum patients were used to examine if homologous pairing is dependent on NER. The outcome of this approach revealed that confluent XPF primary fibroblasts lacked MMC-induced pairing of homologous 9q12-13 regions in contrast to XPA cells. These data suggest
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that NER is not the pathway involved in pairing; the pairing process rather requires the action of the structure specific endonuclease XPF/ERCC1. XPF/ERCC1 has been implicated in the repair of MMC-induced ICLs, particularly in the unhooking step.
Moreover, metaphase analysis revealed that MMC did not induce chromatid exchanges in XPF cells, i.e. XPF is required for both pairing of homologous regions and exchange formation. This supports a correlation between pairing in interphase and exchanges in metaphase and implicates pairing of heterochromatin as a part of the cellular responses to DNA damage, notably the XPF/ERCC1-dependent processing of ICLs.
Chapter 3 presents data obtained after exposure of confluent fibroblasts to X- rays to examine the effect of ionizing radiation (IR) on the positioning of chromosomal regions 9q12-13 and 8p11.2. Measurement of the inter-homologue distances also revealed pairing of the homologous 9q12-13 regions after exposure to X-rays in contrast to 8p11.2 regions. Similar frequencies of pairing were obtained directly after exposure to MMC and X-rays, however at later time points differences in pairing frequencies were observed between both agents reflecting different kinetics.
Pairing after X-rays disappeared within 1 h after exposure whereas it was persistent for up to 20 h after MMC treatment (chapter 2). The disappearance of pairing correlates with the rapid repair kinetics of X-rays-induced double strand breaks (DSBs) in euchromatin and heterochromatin.
In chapter 4 we applied UV irradiation and heat shock to assess whether pairing of heterochromatin is a general stress response. Both agents were able to induce pairing of the heterochromatic regions of chromosome 9 but not the euchromatic regions of chromosome 8. Following local UV irradiation, pairing was also induced in neighboring unexposed nuclei indicating an untargeted (bystander) effect. On the basis of these results two conclusions can be drawn: (1) the presence of DNA damage in the heterochromatin is not strictly required to allow pairing; (2) in cells without any DNA damage pairing also occurs. In contrast to normal human cells pairing was not induced in XPF cells exposed to UV radiation (as observed for MMC). However, pairing was induced after exposure of XPF cells to heat shock or X- rays. These results indicate that depending on the type of DNA damage, certain DNA repair genes have impact on the pairing process.
In chapter 5, we examined whether other DNA repair proteins are involved in the pairing of heterochromatin. The FANCD1 (BRCA2) protein required for HR
Summary and Discussion
appeared to be involved in MMC-induced pairing in contrast to FANCA, supporting the hypothesis that pairing is linked to homology dependent processing of MMC- induced DNA damage in confluent cells. This chapter also presents evidence that processing of MMC induced ICLs is initiated in confluent human cells. We determined the formation of SSBs and DSBs in MMC-exposed confluent human fibroblasts using two approaches, i.e. comet assay and phosphorylation of H2AX. An immediate induction of DNA breaks detected by comet assay was observed after treatment independent of DNA repair activity (i.e. NER and FA proteins). This might represent breaks induced directly by MMC or during processing of the induced ICLs.
A late response manifested by H2AX phosphorylation corresponded to the induction of heterochromatin pairing, i.e. XPF and BRCA2 deficient cells that lack pairing of heterochromatin were also impaired in H2AX phosphorylation suggesting that some steps in the processing of ICLs occur in non-dividing cells.
Table 1: A summary of the results obtained for pairing of heterochromatic regions of chromosome 9 after exposure of cells to different stressors
*Chapter 2 describes the existence of pairing after MMC treatment in G0, G1 and S-phase; all other experiments were done in confluent human fibroblasts.
Stressor Chapter Cell line Pairing* Remarks
MMC 2
WT + Persistent pairing and formation of chromatid-type exchanges
XPF - Absence of chromatid-type exchanges
XPA + Persistent pairing
5 FA-A + Persistent pairing
FA-D1 - X-rays
3 WT + Quick recovery of pairing with time 4 XPF + Delayed response, quick recovery of
pairing with time
UV
4
WT + Persistent pairing
XPF -
XPA + Delayed response, persistent pairing 5 FAA +
FA-D1 +
Heat 4 WT + Quick recovery of pairing with time XPF +
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In meiosis, the term pairing refers to the interaction of homologous chromosomes mediating crossing over, which is essential for proper segregation. The fact that MMC produces a preponderance of homologous chromatid-type exchanges involving the paracentromeric heterochromatic regions assumes pairing of these regions in somatic cells. There is increasing evidence that centromeric regions change position during the cell cycle. The proximity of homologous heterochromatic regions during DNA replication was suggested to allow the formation of exchanges observed in these regions after MMC treatment (Morad et al., 1973) (contact first hypothesis). The observations in the present study revealed that (i) heterochromatic regions of chromosome 9 are distributed randomly inside untreated nuclei, (ii) pairing of these regions in G1 cells occurs directly after MMC treatment and (iii) frequencies of pairing in interphase correlate with homologous exchanges in metaphase. Together, these findings argue against the contact first hypothesis. In line with these findings is the observation that the centromeric regions of chromosome 9 are distal to each other in untreated interphase human cells (Gagne and Laberge, 1972). Moreover, recent studies revealed variability in the positioning of homologous chromosomes in interphase (Cremer et al., 2001; Bolzer et al., 2005). The repositioning of the homologous heterochromatic regions as inferred from measurement of the inter- homologue distances and the observed pairing induced by MMC, may ultimately lead to the formation of homologous exchanges.
Pairing of the heterochromatic regions 9q12-13 was induced in response to different genotoxic agents implying a cellular stress response. This response was studied using different mutant cell lines and a combination of cytogenetic and cellular approaches. The rapid induction of pairing, its recovery after certain types of DNA damage and dependency on temperature and DNA repair proteins such as XPF and BRCA2 indicate a genetically regulated enzymatic process. Moreover, data on MMC- induced pairing suggest that pairing of heterochromatin is likely required for homology-dependent processing of MMC-induced ICLs leading to homologous exchange formation. Stalled transcription at the site of UV-induced DNA damage was shown to provoke recombination in yeast (Aboussekhra and al-Sharif, 2005). If the latter applies to human cells, it indicates that stalled transcription sites are targets for stable (recombination-mediated) interactions between homologues manifested by pairing of homologous chromosomes. It is conceivable that the known poor repair of
Summary and Discussion
UV-induced photolesions in heterochromatin in human cells might lead to persistent blockage of transcription and provoke interactions between homologous regions.
Strikingly, in the present study pairing was observed only in a subset of cells suggesting that chromatin movement which brings the homologues together, is constrained by nuclear architecture, i.e. there might be a maximal distance between the homologous regions to carry out pairing. This hypothesis is supported by the observation that the distribution of inter-homologue distances is altered after exposure to DNA damaging agents (Chapter 2 and 3). MMC- and UV-induced pairing at certain time point (up to 24 h) may represent an equilibrium between paired and non- paired homologues. The pairing may be abolished by dissociation due to failure to keep the paired status or after correct repair. SSA is likely to occur in mammalian genomes containing a high proportion of repetitive sequences (McHugh et al., 2001).
XPF/ERCC1 has been suggested to have a role in the recombinational repair of ICLs (De Silva et al., 2002; for review see McHugh et al., 2001; Dronkert and Kanaar, 2001). There is evidence for a role of this heterodimer endonuclease in homology- driven recombination (Adair et al., 2000; Sargent et al., 1997; Niedernhofer et al., 2001) and in removing the 3’ tails during SSA (Sargent et al., 2000; Al-Minawy et al., 2007). In S. cerevisiae, the homologues of XPF and ERCC1 are required for the SSA subpathway of HR (Ivanov and Haber, 1995). We speculate that a strand-annealing pathway involving XPF might underlie the pairing observed in our studies which in case of MMC lead to chromatid-type exchanges (Chapter 2). HR is known to be active in S-phase where the information on the sister chromatids are used for faithful repair. Recombination is potentially dangerous when the homologous chromosome is used as a template as it may lead to homozygosity for recessive mutations or inappropriate rearrangements and consequently this is suppressed to prevent these events. If the DNA-damage induced pairing of homologous chromosomes is dependent on homology-directed DNA repair in G1 cells, it might be restricted due to spatial organization of homologous sequences.
Data from the literature suggest that modulation of gene expression may lead to the reorganization of nuclear components (for reviews see Singer et al., 1997;
Soutoglou and Misteli, 2007). Spatial positioning and association of specific genes or chromatin domains with internal nuclear compartments such as the nucleolus, PML bodies, Cajal bodies, or stress granules suggests functional importance for these associations in relation to gene expression (Parada et al., 2004). The movement of
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gene loci can be restricted by attachment to nuclear compartments such as the nucleolus; indeed, disruption of nucleoli increases the mobility of nucleolar- associated loci (Chubb et al., 2002). Mitotic and meiotic pairing have been suggested to be triggered by transcription (Cook, 1997). Satellite DNA transcripts in the form of small interfering RNAs participate in the epigenetic chromatin modulation, heterochromatin formation and control of gene expression (for a review see Ugarkovic, 2005). The region 9q12 that contains mostly satellite III DNA has the features of both euchromatin and heterochromatin (Tagarro et al., 1994; Gilbert et al., 2004). Stress granules containing the heat shock transcription factor 1 (HSF1) were shown to form on the heterochromatic regions of chromosome 9 after different types of cellular stress (Jolly et al., 2002). In response to heat shock, transcription of human satellite III DNA repeats on chromosome 9 heterochromatic regions occurs in the form of long single stranded transcripts (Jolly et al., 2004; Metz et al., 2004).
Moreover, a recent report (Valgardsdottir et al., 2008) showed that non-coding satellite III transcripts are induced in human cells by a wide range of stress treatments including DNA damaging agents: UVC, MMS and etoposide. Changes in the characteristics related to the transcriptional repressed state of heterochromatin such as DNA methylation, histone deacetylation, methylation of histone H3 at lysine 9 (H3K9me) and the presence of HP1 might direct the transcriptional activation of the heterochromatic regions of chromosome 9. The satellite III transcripts are associated with the stress granules, comprise acetylated histones (but not H3K9me or HP1), involved in the recruitment of splicing factors to the granules and were suggested to have an epigenetic regulatory role (Jolly et al., 2004; Rizzi et al., 2004; Biamonti, 2004). We hypothesize that the repositioning (pairing) of chromosome 9 heterochromatic regions after infliction of DNA damage may depend on transcriptional activation of DNA sequences inside the heterochromatin. In other words, functional clustering of specific chromosome regions into transcriptional domains may underlie pairing of the homologues. The repositioning of the heterochromatic regions of chromosome 9 toward a nuclear substructure such as transcription factories or stress granules may also explain the fact that pairing is not observed in all cells: once the chromosomal region become associated with the transcriptional factory or stress granule, chromatin movement is restricted and pairing occurs when the two chromosomal regions move toward the same or adjacent transcriptional factories.
Summary and Discussion
The precise biochemical mechanisms of pairing of heterochromatin and its biological relevance require further investigations. Moreover, the complex nature of the pairing process as demonstrated by differences in the recovery of pairing after certain types of stressors remains to be explained. Also elucidation of the relationship between pairing of heterochromatin and transcriptional activation of the satellite sequences and the precise role of these sequences in pairing is important. The development of molecular assays to study the dynamics of the pairing process using GFP-tagged proteins (that preferentially associate with satellite DNA sequences) in living cells may provide a great deal of new information about somatic pairing. The identification of the mechanisms responsible for the dynamics of chromatin, the pairing process and the formation of chromosomal aberrations will certainly be an exciting area for future research that may be important factors in understanding the complexity of gene regulation and genome stability.
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