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Summary and Discussion
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
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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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Summary and Discussion
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 Xrays 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 Xrays. 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
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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 MMCinduced 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.
Stressor
Chapter
Cell line
WT
Pairing*
+
MMC
2
XPF
XPA
FA-A
FA-D1
WT
XPF
+
+
+
+
WT
XPF
XPA
FAA
FA-D1
WT
XPF
+
+
+
+
+
+
5
X-rays
3
4
4
UV
5
Heat
4
Remarks
Persistent pairing and formation of
chromatid-type exchanges
Absence of chromatid-type exchanges
Persistent pairing
Persistent pairing
Quick recovery of pairing with time
Delayed response, quick recovery of
pairing with time
Persistent pairing
Delayed response, persistent pairing
Quick recovery of pairing with time
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.
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Summary and Discussion
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 interhomologue 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 MMCinduced 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
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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 nonpaired 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 homologydriven 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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Summary and Discussion
gene loci can be restricted by attachment to nuclear compartments such as the
nucleolus; indeed, disruption of nucleoli increases the mobility of nucleolarassociated 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.
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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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Summary and Discussion
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