- Email: [email protected]
Links between chromatin structure, DNA repair and chromosome fragility
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Fundamental Mechanisms
ELSEVIER
and
Molecular
of Mutagenesis
Mutation Research 404 (1998) 39-44
Links between chromatin structure, DNA repair and chromosome fragility J. Surrall6s a,*, S. Puerto a M.J. Ramfrez a A. Creus a, R. Marcos a, L.H.F. Mullenders b, A.T. Natarajan b a Group of Mutagenesis, Genetics Unit, Department of Genetics and Microbiology, Edifici Cn, Universitat Autbnoma de Barcelona, 08193 Bellaterra, Cerdanyola del Vallbs, Barcelona, Spain b Department of Radiation Genetics and Chemical Mutagenesis-MGC, Leiden University Medical Centre, Leiden, Netherlands
Received 27 February 1998; accepted 27 March 1998
Abstract This paper is a brief overview of the studies we have recently conducted to unravel how chromatin structure and DNA repair modulate the fragility of diverse chromosomes and chromosomal regions. We have employed a combination of molecular cytogenetic techniques, including interphase and metaphase multicolour FISH, reverse FISH with CpG-rich probes or repaired DNA fractions, and several combinations of FISH and immunocytogenetics with antibodies against acetylated histories. The targets of our investigation were human constitutive and facultative heterochromatin, chromosomes with high and low gene density and human and hamster fragile sites. The role of DNA repair was investigated by using DNA repair deficient mutants and DNA repair inhibitors. We found that intragenomic heterogeneity in DNA repair and chromatin structure may explain a substantial part of the differential fragility of diverse chromosomes and chromosomal regions. 9 1998 Elsevier Science B.V. All rights reserved. Keywords: DNA repair; Chromatin structure; Heterochromatin; Histone; Chromosome aberration; Gene density; Inactive X chromosome
1. Introduction It is generally accepted that chromosome aberrations are causal events in the development of many human neoplasias. It is therefore important to unravel the mechanisms o f chromosome aberrations formation and to determine which are the genetic factors that modulate chromosomal fragility. A m o n g these factors, we focused our investigations on the role of chromatin structure and D N A repair, as it is
* Corresponding author. Tel.: + 34-3-581-25-97; Fax: + 34-3581-23-86; E-mail: [email protected]
well known that D N A repair, particularly nucleotide excision repair (NER), is highly influenced by chromatin structure and transcriptional activity. Our working hypothesis is that intragenomic heterogeneity in D N A repair and chromatin conformation might explain the differential chromosomal fragility, i.e., the formation of D N A damage and chromosomal aberrations, observed at the cytogenetic level. In order to find out to what extent differences in chromatin structure and transcriptional activity influences the formation o f chromosomal aberrations, we have used advanced molecular cytogenetic and immunocytogenetic techniques to assess the induction of
0027-5107/98/$19.00 9 1998 Elsevier Science B.V. All rights reserved. PII: S0027-5107(98)00093-1
40
J. Surrall~s et al. / Mutation Research 404 (1998) 39-44
chromosomal alterations in different chromosomes and chromosomal regions (namely facultative and constitutive heterochromatin), focusing on human and hamster fragile sites and chromosomes with high and low gene density. DNA repair was investigated at the cytogenetic level by using DNA repair inhibitors and DNA repair deficient mutants. With these tools, we intended to shed some light on the following questions: why is constitutive heterochromatin so fragile? Is the inactive X chromosome more radiosensitive than the active X chromosome? Why are some chromosomes more fragile than others? How is DNA excision repair distributed along the chromosomes? Why are fragile sites fragile? Is there any link between histone acetylation and chromosome fragility? This paper is an overview of some of the experiments we have conducted in this research area in the last two years.
2. Formation of chromosomal alterations human heterochromatin
in
2.1. Chromosome breakage and repair in human constitutive heterochromatin Molecular cytogenetics provides us with a tool to study chromosomal damage formation in bands commonly involved in chromosomal aberrations found in cancers, such as constitutive heterochromatin of human chromosome 1 (band lql2). The molecular mechanisms underlying the extreme fragility of l ql 2 are not well understood. It is, however, well known that human lq12 is formed by highly repetitive DNA, which is located in the periphery of the nucleus, and highly condensed in metaphase and interphase. We hypothesized that slow DNA repair in this region could account for the observed fragility as biochemical experiments have shown that the highly repetitive heterochromatic tz-DNA is poorly repaired for some types of DNA damage [1]. To test our hypothesis, we induced DNA lesions with ethyl methanesulfonate (EMS) which were then converted to chromosome breaks using the DNA repair inhibitor, cytosine arabinoside (Ara-C). It is known that the majority of DNA lesions induced by EMS are repaired by base excision repair, although 0 6
ethylguanine is processed by nucleotide excision repair [2,3]. Repair sites in lq12 converted to chromosome breaks were measured in interphase cells by using multicolour fluorescence in situ hybridization (FISH) with tandem probes targeting the chromosome region of interest in an adjacent fashion. With this approach, the loss of adjacency was interpreted as chromosome breaks in lq12 [4,5]. At the overall genome level, repair sites converted to breaks were also monitored by measuring the frequency of micronuclei (MN) in cycling cells after repair synthesis inhibition [6-8]. The suitability of the FISH methodology to detect lq12 breakage was controlled by including two known clastogenic agents: mitomycinC (MMC) and X-rays. After MMC, X-rays or EMS + Ara-C treatments, the overall clastogenicity, as measured by MN counts, was extremely high. However, this overall clastogenicity was reflected in lq12 only after MMC and X-ray treatments but not after EMS + Ara-C treatment. This relative insensitivity of lq12 to cotreatments with EMS and Ara-C was interpreted as the result of a low level of DNA excision repair in constitutive heterochromatin. Thus, low level of DNA repair may account for the extreme fragility of constitutive heterochromatin in humans [9].
2.2. Chromosome breakage and repair in the inactive X chromosome X-chromosome inactivation results in silencing of transcriptional activity in all but one X-chromosome in somatic female cells, hence, allowing dosage compensation of X-linked genes between males and females [10]. In previous studies, comparing euchromatin and heterochromatin in various cells with respect to the formation of chromosomal aberrations, it was not possible to dissect whether heterochromatic state or heterogeneous genetic background accounted for the observed differences between heterochromatin and euchromatin. However, the measurement of repair in the inactive X chromosome (Xi) provides an unique opportunity to overcome this limitation as both X chromosomes (active and inactive) reside in the same cellular environment. We therefore developed a system to detect chromosomal breaks in both the active and Xi. Thus, chromosome aberrations involving the X chromosome were detected by means
J. Surrall[s et a l . / Mutation Research 404 (1998) 39-44
of FISH with an X chromosome specific red painting probe, while the transcriptional status of the X chromosomes involved in the chromosome aberrations was determined by simultaneous immunocytogenetics with antibodies against 5-bromodeoxyuridine (BrdUrd) incorporated at late S-phase [11]. The late replicating Xi was defined by a greater amount of BrdUrd derived fluorescence. This multicolour approach allowed us to study and compare breakage and the extent of repair in the active X and of Xi. We induced chromosome breakage in human lymphocytes with X-rays in the presence or absence of an inhibitor of double strand break repair, adenine 9-/3-arabinofuranoside (Ara-A), and then allow the cells to proceed to mitosis. Our data indicated that both chromosomes are equally radiosensitive. However, the inactive and highly condensed state of the Xi enhanced the inhibitory effect of Ara-A upon the repair of breaks. Thus, the observed fragility is the result of a compromise between the actual number of breaks induced in each chromosome and their differential processing [11 ].
3. Interchromosomal heterogeneity in DNA repair 3.1. CpG islands, gene density and interchromosomal differences in DNA repair
41
tween different chromosomes [18]. The same is true for gene activity, as reflected with antibodies against acetylated histone H4, a cytogenetic maker for gene expression [19,20]. It is expected that nucleotide excision repair is clustered in chromosomes with high gene density and transcriptional activity. We identified these chromosomes by means of distribution of CpG-islands after FISH with a CpG island-rich probe isolated from total human genomic DNA [14]. Thus, three chromosomes with very high gene density (numbers 1, 19 and 20) were identified and compared to two chromosomes with very low gene density (numbers 4 and 18) for clastogenicity and sensitivity to cotreatment with Ara-C and EMS. We again employed the DNA repair inhibitor Ara-C to convert EMS-induced excision repairable lesions to chromosome breaks and, therefore, to check for the existence of heterogeneity of repair at the chromosome level. Multicolour chromosome painting by FISH was used to analyse breakage in chromosomes with diverse gene densities and we found a clear correlation between gene density and sensitivity to Ara-C + EMS. This result indicates that the level of excision repair synthesis is higher in high gene density chromosomes and, therefore, that chromosomes with high gene density are preferentially repaired in human cells [14].
3.2. Towards a 'repair karyotype' A general assumption of the use of painting for quantifying chromosome damage for the whole genome by extrapolation from few painted chromosomes is that the occurrence of breakpoints is randomly distributed within and between chromosomes. However, several studies using banding or FISH methodologies have suggested that random distribution is actually not the case [12-15]. These findings could have important implications in human biomonitoring and biological dosimetry since the actual risk (i.e., chromosomal mutations) cannot be gained from the induction of chromosomal aberrations averaged for the genome overall. It is known that nucleotide excision repair and, to a lesser extent, base excision repair is heterogeneous in human cells since open chromatin, active genes and their transcribed strands are preferentially repaired [16,17]. Human genes are unevenly distributed both within a single chromosome and be-
It is well known that nucleotide excision repair (NER) is preferentially directed to actively transcribed genes and their transcribed strands through transcription coupled mechanisms [16]. Human chromosomes are highly heterogeneous with respect to gene density and transcriptional activity. Thus, chromosomal bands harbouring actively transcribed genes would be expected to be preferentially repaired. In order to visualize the repaired DNA along the arms of the human chromosomes, we used reverse FISH to normal chromosomes with a probe enriched for repaired DNA. Xeroderma pigmentosum group C (XPC) cells are known to be partially deficient in NER since they can only repair the transcribed strands of active genes. Due to the small size of the repair patches and the low resolution of immunocytogenetics, it was not possible to detect the repaired chromosomal regions directly by synchronizing the cells and
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J. Surrall~s et a l . / Mutation Research 404 (1998) 39-44
allowing repair to occur in the presence of BrdUrd only outside of S-phase and then labelling metaphase chromosomes with anti-BrdUrd antibodies. Instead, it was necessary to pool the repaired DNA and then use the repaired DNA as a probe for reverse FISH to normal metaphases. To achieve this, UV-induced repair patches in XPC cells were labelled with BrdUrd and the DNA was centrifuged in CsCI to separate replicating DNA from parental DNA. Subsequently, the parental DNA containing repair sites was isolated selectively with an immunomagnetic system [21]. The extracted repaired fragments were randomly primed with biotin and used as a probe to perform reverse FISH to normal metaphases. This approach allowed us to visualize clusters of transcription coupled repair in light G bands [22].
4. Stability and persistence of radiation-induced chromosome aberrations Besides differences in DNA damage induction and repair, another factor that might modulate interchromosomal differences in the number of breaks detected after past radiation exposure is a differential persistence of translocations involving different chromosomes. Low persistence of translocations might be related to cell lethality as a result of gene truncation or position effects. A potential position effect that we previously disregarded is the spreading of X inactivation to autosomal material after transitcations between the inactive X chromosome and autosomes [23,24]. If gene truncation/inactivation is involved in the persistence of translocations, then one would expect a lower persistence of translocations involving chromosomes with high gene density. To verify this hypothesis, we analyze the persistence of translocations involving chromosomes 1 and 19 (with high gene density) and 4 and 18 (with very low gene density). Translocations were induced with ionizing radiations in a wild-type lymphoblastoid cell line (TK6) and cell samples were collected and harvested 1, 3, 7, 14, 28, 42 and 56 days after irradiation. After scoring 4000 metaphases per chromosome and time point, about 2 / 3 of the chromosome translocations drastically declined during the first week and then remained stable until day 56. All dicentrics and acentric fragments disappeared in the
first week after treatment. Regarding interchromosomal differences, chromosome aberrations involving chromosome 1 declined slightly faster than those involving chromosome 4, suggesting that aberrations involving chromosome 1 are less stable. Further analysis will allow us to state whether this tendency is biologically relevant and actually related to gene density. Our previous studies showed that tandem labelling failed to detect the clastogenic effect of radioactive iodine in buccal cells from thyroid cancer and hyperthyroidism patients [25], although positive results were obtained with the MN test in human lymphocytes in a similarly exposed population [26,27]. These contradictory results might suggest that chromosome aberrations involving lq12 would be rather unstable.
5. Possible links between histone acetylation and chromosome fragility Histones are the most common proteins in the cell nucleus. They play important biological roles not only in chromatin packaging but also in transcriptional regulation [28]. Histones are subject to a number of modifications that might influence packaging and, therefore gene expression. Among them, reversible acetylation of N-terminal lysines is one of the most widely spread from lower to higher eukaryotes, including all vertebrates and mammals. The suggested mechanisms for the involvement of histone acetylation in trascriptional regulation is the reduction in positive charge of core histones, hence loosening histone-DNA interactions and facilitating gene expression. The use of antibodies against histones acetylated at different N-terminal lysines has greatly improved our understanding of chromosome structure and function. It is known that histone H4 acetylation is a cytogenetic marker for gene expression. The use of these antibodies allowed us to visualize the active and inactive chromosomal domains both in metaphase [19,23,24,29] and interphase [20]. It has been suggested that histone H4 plays a role in maintaining genome integrity through the cell cycle, possibly by a mechanism involving lysine acetylation. Thus, mutants in which all terminal histone H4 lysines are
J. Surralls et al. / Mutation Research 404 (1998) 39-44
substituted with glutamines accumulate increased levels of DNA damage [30]. It has also been reported that the inactive X chromosome shows residual histone H3 and H4 acetylation in the band Xq22, where the common FRAXC fragile site is located [19,31]. On the other hand, the expression of the major X linked fragile site, Xq27.3 is inhibited in vitro by sodium butyrate, a strong inhibitor of histone deacetylases [32]. In addition to the above findings, chromatin decondensed by acetylation showed an elevated radiosensitivity in hamsters [33]. We have investigated the presence of acetylated histones in the commonest Chinese hamster fragile site, Xq21. This band is in the middle of the overall heterochromatic long ann of the hamster X chromosome. By combining immunocytogenetics with antibodies against acetylated histone H4 and FISH with a CpG-island-rich probe isolated from hamster genornic DNA, we found that hamster Xq21 was labelled by both FISH and antibodies against acetylated histone H4. This finding suggests that there is a cluster of active (acetylated H4 positive signal) genes (CpG-island positive signal) in hamster Xq21. Surprisingly, and resembling the human Xq22 band, histone H4 in the hamster Xq21 is also acetylated in the inactive X chromosome which was identified by its general lack of histone H4 acetylation. It has not escaped our notice that this cluster of active genes is indicative of a possible pseudoautosomal region. All these observations taken together support a possible connection between histone acetylation and chromosome fragility. This link could be mediated by the omnipresent DNA repair, as it is also known that DNA repair is influenced by histone acetylation [34]. Further specifically designed experiments are required to determine whether these links are merely coincidental.
Acknowledgements This investigation was partially supported by grants from the European Union (Environmental and Radiation Programmes), the Spanish Ministry of Education and Science (SAF95-0813, CICYT), and the Generalitat de Catalunya (SGR95-00512, CIRIT). J.S. has a Research Postdoctoral Contract (Incorporaci6n de Doctores) by the Spanish Ministry of
43
Education and Culture. J.S.'s visit to Leiden University was made possible, thanks to a long-term postdoctoral fellowship awarded by the Commission of the European Union, Environmental Programme, contract EV5V-CT-94-5240. M.J.R. and S.P.'s contribution to this work was made possible, thanks to fellowship awarded by the Spanish Ministry of Education and Culture and the Autonomous University of Barcelona, respectively. M. McCarthy's secretarial assistance and proofreading is also appreciated.
References [1] C.A. Smith, DNA repair in specific sequences in mammalian cells, J. Cell. Sci. 6 (1987) 225-241. [2] A. Sitaram, G. Plitas, W. Wang, A. Scicchitano, Functional nucleotide excision repair is required for the preferential removal of N-ethylpurines from the transcribed strand of the dihydrofolate reductase gene of Chinese hamster ovary cells, Mol. Cell. Biol. 17 (1997) 564-570. [3] J. Engelbergs, J. Thomale, A. Galhoff, M.F. Rajewsky, Fast repair of O6-ethylguanine, but not O6-methylguanine, in transcribed genes prevents mutation of H-ras in rat mammary tumorigenesis induced by ethylnitrosourea in place of methylnitrosourea, PNAS Abstr. 95 (1998) 1635-1640. [4] D.A. Eastmond, D.S. Rupa, L.S. Hasegawa, Detection of hyperdiploidy and chromosome breakage in interphase human lymphocytes following exposure to the benzene metabolite hydroquinone using multicolor fluorescence in situ hybridization with DNA probes, Mutation Res. 322 (1994) 9-20. [5] D.S. Rupa, L. Hasegawa, D.A. Eastmond, Detection of chromosomal breakage in the lcen-lql2 region of interphase human lymphocytes using multicolour fluorescence in situ hybridization with tandem DNA probes, Cancer Res. 55 (1995) 640-645. [6] M. Fenech, S. Neville, Conversion of excision repairable DNA lesions to micronuclei within one cell cycle in human lymphocytes, Environ. Mol. Matagen. 19 (1992) 27-36. [7] M. Fenech, J. Rinaldi, J. Surrall6s, The origin of micronuclei induced by cytosine arabinoside and its synergistic interaction with hydroxyurea in human lymphocytes, Mutagenesis 9 (1994) 273-277. [8] J. Surrall6s, N. Xamena, A. Creus, R. Marcos, The suitability of the micronucleus assay in human lymphocytes as a new biomarker of excision repair, Mutation Res. 342 (1995) 43-59. [9] J. Surrall~s, F. Darroudi, A.T. Natarajan, Evidence for low level of DNA repair in human chromosome 1 heterochro matin, Genes Chromosomes Cancer 20 (1997) 173-184. [10] M.F. Lyon, Gene action in the X-chromosome of the mouse (Mus musculus L.), Nature 190 (1961) 372-373. [11] J. Surrall6s, A.T. Natarajan, Radiosensitivity and repair of the inactive X chromosome. Insights from FISH and immunocytogenetics, Mutation Res. 414 (1998) 117-124.
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[12] C. Cremer, Ch. Miinkel, M. Granzow, A. Jauch, S. Dietzel, R. Eils, X.-Y. Guan, P.S. Meltzer, J.M. Trent, J. Langowsky, T. Cremer, Nuclear architecture and the induction of chromosomal aberrations, Mutation Res. 366 (1996) 97-116. [13] A.T. Natarajan, A.S. Balajee, J.J.W.A. Boei, F. Darroudi, 1. Dominguez, M.P. Hande, C. Meijers, P. Slijepcevic, S. Vermeulen, Y. Xiao, Mechanisms of induction of chromosomal aberrations and their detection by fluorescence in situ hybridization, Mutation Res. 372 (1996) 247-258. [14] J. Surrall~s, S. Sebastian, A.T. Natarajan, Chromosomes with high gene density are preferentially repaired in human cells, Mutagenesis 12 (1997) 437-442. [15] S. Puerto, J. Surrall6s, A. Creus, R. Marcos, lnterchromosomai differences in bleomycin- and Ara-C-induced chromosomal aberrations as detected by multicolour painting FISH, 1998, in preparation. [16] E.C. Friedberg, Relationships between DNA repair and transcription, Annu. Rev. Biochem. 65 (1996) 15-42. [17] L.H.F. Mullenders, H. Vrieling, J. Venema, A.A. van Zeeland, Hierarchies of DNA repair in mammalian cells: biological consequences, Mutation Res. 250 (1991) 223-228. [18] J.M. Craig, W.A. Bickmore, The distribution of CpG islands in mammalian chromosomes, Nat. Genet. 7 (1994) 376-382. [19] P. Jeppesen, B.M. Turner, The inactive X chromosome in female mammals is distinguished by a lack of histone H4 acetylation, a cytogenetic marker for gene expression, Cell 74 (1993) 281-289. [20] J. Surrall6s, P. Jeppesen, H. Morrison, A.T. Natarajan, Analysis of loss of inactive X chromosome in interphase cells, Am. J. Hum. Genet. 59 (1996) 1091-1096. [21] W.H.J. Kalle, A.-M. Hazekamp-van Dokkum, P.H.M. Lohman, A.T. Natarajan, A.A. van Zeeland, LH.F. Mullenders, The use of streptavidin-coated magnetic beads and biotinylated antibodies to investigate induction and repair of DNA damage: analysis of repair patches in specific sequences of uv-irradiated human fibroblasts, Ann. Biochem. 208 (1992) 228-236. [22] J. Surrall6s, P. Karmakar, A.T. Natarajan, L.H.F. Mullenders, Chromosomal distribution of genes subject to transcription coupled DNA repair, Mutation Res. 379 (1997) 36-37.
[23] J. Surrall6s, A.T. Natarajan, Position effects of translocations involving the inactive X chromosome. A study combining FISH and immunocytogenetics, Mutation Res. 379 (1997) 72-73. [24] J. Snrrall6s, A.T. Natarajan, Position effect of translocations involving the inactive X chromosome: physical linkage to XIC/XIST does not lead to long-range de novo inactivation in human somatic cells, 1997, submitted. [25] M.J. Ramlrez, J. Surrall6s, A. Creus, R. Marcos, FISH analysis of lcen-lql2 breakage and micronuclei in buccal cells from radioactive iodine exposed thyroid cancer and hyperthyroidism patients, 1998, in preparation. [26] S. Guti6rrez, E. Carbonell, P. Galofr6, A. Creus, R. Marcos, Micronuclei induction by 131I exposure: study in hyperthyroidism patients, Mutation Res. 373 (1997) 39-45. [27] M.J. Ramlrez, J. Surrall6s, P. Galofr6, A. Creus, R. Marcos, Radioactive iodine induced clastogenic and age-dependent aneugenic effects in exposed patients, Mutagenesis 12 (1997) 449-455. [28] A.P. Wolffe, Transcription: in tune with histones, Cell 77 (1994) 13-16. [29] P. Jeppesen, Histone acetylation: a possible mechanism for inheritance and cell memory at mitosis, Bioessays 19 (1997) 67 -74. [30] P.C. Mcgee, B.A. Morgan, M.M. Smith, Histone H4 and the maintenance of genome integrity, Genes Dev. 9 (1995) 1716-1727. [31] N. Belyaev, A.M. Keohane, B.M. Turner, Differential underacetylation of histones H2A, H3 and H4 on the inactive X chromosome in human female cells, Hum. Genet. 97 (1996) 573-578. [32] M.G. Pomponi, G. Neff, Butyrate and acetyl-camitine inhibit the cytogenetic expression of the fragile X in vitro, Am. J. Med. Genet. 51 (1994) 447-450. [33] Z. Nackerdien, J. Michie, L. Bohm, Chromatin decondensed by acetylation shows an elevated radiation response, Radiat. Res. 117 (1989) 234-244. [34] B. Ramamathan, M.J. Smerdon, Enhanced DNA repair synthesis in hyperacetylated nucleosomes, J. Biol. Chem. 264 (1989) 11026-11034.
"
-
z , r
g
Fundamental Mechanisms
ELSEVIER
and
Molecular
of Mutagenesis
Mutation Research 404 (1998) 39-44
Links between chromatin structure, DNA repair and chromosome fragility J. Surrall6s a,*, S. Puerto a M.J. Ramfrez a A. Creus a, R. Marcos a, L.H.F. Mullenders b, A.T. Natarajan b a Group of Mutagenesis, Genetics Unit, Department of Genetics and Microbiology, Edifici Cn, Universitat Autbnoma de Barcelona, 08193 Bellaterra, Cerdanyola del Vallbs, Barcelona, Spain b Department of Radiation Genetics and Chemical Mutagenesis-MGC, Leiden University Medical Centre, Leiden, Netherlands
Received 27 February 1998; accepted 27 March 1998
Abstract This paper is a brief overview of the studies we have recently conducted to unravel how chromatin structure and DNA repair modulate the fragility of diverse chromosomes and chromosomal regions. We have employed a combination of molecular cytogenetic techniques, including interphase and metaphase multicolour FISH, reverse FISH with CpG-rich probes or repaired DNA fractions, and several combinations of FISH and immunocytogenetics with antibodies against acetylated histories. The targets of our investigation were human constitutive and facultative heterochromatin, chromosomes with high and low gene density and human and hamster fragile sites. The role of DNA repair was investigated by using DNA repair deficient mutants and DNA repair inhibitors. We found that intragenomic heterogeneity in DNA repair and chromatin structure may explain a substantial part of the differential fragility of diverse chromosomes and chromosomal regions. 9 1998 Elsevier Science B.V. All rights reserved. Keywords: DNA repair; Chromatin structure; Heterochromatin; Histone; Chromosome aberration; Gene density; Inactive X chromosome
1. Introduction It is generally accepted that chromosome aberrations are causal events in the development of many human neoplasias. It is therefore important to unravel the mechanisms o f chromosome aberrations formation and to determine which are the genetic factors that modulate chromosomal fragility. A m o n g these factors, we focused our investigations on the role of chromatin structure and D N A repair, as it is
* Corresponding author. Tel.: + 34-3-581-25-97; Fax: + 34-3581-23-86; E-mail: [email protected]
well known that D N A repair, particularly nucleotide excision repair (NER), is highly influenced by chromatin structure and transcriptional activity. Our working hypothesis is that intragenomic heterogeneity in D N A repair and chromatin conformation might explain the differential chromosomal fragility, i.e., the formation of D N A damage and chromosomal aberrations, observed at the cytogenetic level. In order to find out to what extent differences in chromatin structure and transcriptional activity influences the formation o f chromosomal aberrations, we have used advanced molecular cytogenetic and immunocytogenetic techniques to assess the induction of
0027-5107/98/$19.00 9 1998 Elsevier Science B.V. All rights reserved. PII: S0027-5107(98)00093-1
40
J. Surrall~s et al. / Mutation Research 404 (1998) 39-44
chromosomal alterations in different chromosomes and chromosomal regions (namely facultative and constitutive heterochromatin), focusing on human and hamster fragile sites and chromosomes with high and low gene density. DNA repair was investigated at the cytogenetic level by using DNA repair inhibitors and DNA repair deficient mutants. With these tools, we intended to shed some light on the following questions: why is constitutive heterochromatin so fragile? Is the inactive X chromosome more radiosensitive than the active X chromosome? Why are some chromosomes more fragile than others? How is DNA excision repair distributed along the chromosomes? Why are fragile sites fragile? Is there any link between histone acetylation and chromosome fragility? This paper is an overview of some of the experiments we have conducted in this research area in the last two years.
2. Formation of chromosomal alterations human heterochromatin
in
2.1. Chromosome breakage and repair in human constitutive heterochromatin Molecular cytogenetics provides us with a tool to study chromosomal damage formation in bands commonly involved in chromosomal aberrations found in cancers, such as constitutive heterochromatin of human chromosome 1 (band lql2). The molecular mechanisms underlying the extreme fragility of l ql 2 are not well understood. It is, however, well known that human lq12 is formed by highly repetitive DNA, which is located in the periphery of the nucleus, and highly condensed in metaphase and interphase. We hypothesized that slow DNA repair in this region could account for the observed fragility as biochemical experiments have shown that the highly repetitive heterochromatic tz-DNA is poorly repaired for some types of DNA damage [1]. To test our hypothesis, we induced DNA lesions with ethyl methanesulfonate (EMS) which were then converted to chromosome breaks using the DNA repair inhibitor, cytosine arabinoside (Ara-C). It is known that the majority of DNA lesions induced by EMS are repaired by base excision repair, although 0 6
ethylguanine is processed by nucleotide excision repair [2,3]. Repair sites in lq12 converted to chromosome breaks were measured in interphase cells by using multicolour fluorescence in situ hybridization (FISH) with tandem probes targeting the chromosome region of interest in an adjacent fashion. With this approach, the loss of adjacency was interpreted as chromosome breaks in lq12 [4,5]. At the overall genome level, repair sites converted to breaks were also monitored by measuring the frequency of micronuclei (MN) in cycling cells after repair synthesis inhibition [6-8]. The suitability of the FISH methodology to detect lq12 breakage was controlled by including two known clastogenic agents: mitomycinC (MMC) and X-rays. After MMC, X-rays or EMS + Ara-C treatments, the overall clastogenicity, as measured by MN counts, was extremely high. However, this overall clastogenicity was reflected in lq12 only after MMC and X-ray treatments but not after EMS + Ara-C treatment. This relative insensitivity of lq12 to cotreatments with EMS and Ara-C was interpreted as the result of a low level of DNA excision repair in constitutive heterochromatin. Thus, low level of DNA repair may account for the extreme fragility of constitutive heterochromatin in humans [9].
2.2. Chromosome breakage and repair in the inactive X chromosome X-chromosome inactivation results in silencing of transcriptional activity in all but one X-chromosome in somatic female cells, hence, allowing dosage compensation of X-linked genes between males and females [10]. In previous studies, comparing euchromatin and heterochromatin in various cells with respect to the formation of chromosomal aberrations, it was not possible to dissect whether heterochromatic state or heterogeneous genetic background accounted for the observed differences between heterochromatin and euchromatin. However, the measurement of repair in the inactive X chromosome (Xi) provides an unique opportunity to overcome this limitation as both X chromosomes (active and inactive) reside in the same cellular environment. We therefore developed a system to detect chromosomal breaks in both the active and Xi. Thus, chromosome aberrations involving the X chromosome were detected by means
J. Surrall[s et a l . / Mutation Research 404 (1998) 39-44
of FISH with an X chromosome specific red painting probe, while the transcriptional status of the X chromosomes involved in the chromosome aberrations was determined by simultaneous immunocytogenetics with antibodies against 5-bromodeoxyuridine (BrdUrd) incorporated at late S-phase [11]. The late replicating Xi was defined by a greater amount of BrdUrd derived fluorescence. This multicolour approach allowed us to study and compare breakage and the extent of repair in the active X and of Xi. We induced chromosome breakage in human lymphocytes with X-rays in the presence or absence of an inhibitor of double strand break repair, adenine 9-/3-arabinofuranoside (Ara-A), and then allow the cells to proceed to mitosis. Our data indicated that both chromosomes are equally radiosensitive. However, the inactive and highly condensed state of the Xi enhanced the inhibitory effect of Ara-A upon the repair of breaks. Thus, the observed fragility is the result of a compromise between the actual number of breaks induced in each chromosome and their differential processing [11 ].
3. Interchromosomal heterogeneity in DNA repair 3.1. CpG islands, gene density and interchromosomal differences in DNA repair
41
tween different chromosomes [18]. The same is true for gene activity, as reflected with antibodies against acetylated histone H4, a cytogenetic maker for gene expression [19,20]. It is expected that nucleotide excision repair is clustered in chromosomes with high gene density and transcriptional activity. We identified these chromosomes by means of distribution of CpG-islands after FISH with a CpG island-rich probe isolated from total human genomic DNA [14]. Thus, three chromosomes with very high gene density (numbers 1, 19 and 20) were identified and compared to two chromosomes with very low gene density (numbers 4 and 18) for clastogenicity and sensitivity to cotreatment with Ara-C and EMS. We again employed the DNA repair inhibitor Ara-C to convert EMS-induced excision repairable lesions to chromosome breaks and, therefore, to check for the existence of heterogeneity of repair at the chromosome level. Multicolour chromosome painting by FISH was used to analyse breakage in chromosomes with diverse gene densities and we found a clear correlation between gene density and sensitivity to Ara-C + EMS. This result indicates that the level of excision repair synthesis is higher in high gene density chromosomes and, therefore, that chromosomes with high gene density are preferentially repaired in human cells [14].
3.2. Towards a 'repair karyotype' A general assumption of the use of painting for quantifying chromosome damage for the whole genome by extrapolation from few painted chromosomes is that the occurrence of breakpoints is randomly distributed within and between chromosomes. However, several studies using banding or FISH methodologies have suggested that random distribution is actually not the case [12-15]. These findings could have important implications in human biomonitoring and biological dosimetry since the actual risk (i.e., chromosomal mutations) cannot be gained from the induction of chromosomal aberrations averaged for the genome overall. It is known that nucleotide excision repair and, to a lesser extent, base excision repair is heterogeneous in human cells since open chromatin, active genes and their transcribed strands are preferentially repaired [16,17]. Human genes are unevenly distributed both within a single chromosome and be-
It is well known that nucleotide excision repair (NER) is preferentially directed to actively transcribed genes and their transcribed strands through transcription coupled mechanisms [16]. Human chromosomes are highly heterogeneous with respect to gene density and transcriptional activity. Thus, chromosomal bands harbouring actively transcribed genes would be expected to be preferentially repaired. In order to visualize the repaired DNA along the arms of the human chromosomes, we used reverse FISH to normal chromosomes with a probe enriched for repaired DNA. Xeroderma pigmentosum group C (XPC) cells are known to be partially deficient in NER since they can only repair the transcribed strands of active genes. Due to the small size of the repair patches and the low resolution of immunocytogenetics, it was not possible to detect the repaired chromosomal regions directly by synchronizing the cells and
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allowing repair to occur in the presence of BrdUrd only outside of S-phase and then labelling metaphase chromosomes with anti-BrdUrd antibodies. Instead, it was necessary to pool the repaired DNA and then use the repaired DNA as a probe for reverse FISH to normal metaphases. To achieve this, UV-induced repair patches in XPC cells were labelled with BrdUrd and the DNA was centrifuged in CsCI to separate replicating DNA from parental DNA. Subsequently, the parental DNA containing repair sites was isolated selectively with an immunomagnetic system [21]. The extracted repaired fragments were randomly primed with biotin and used as a probe to perform reverse FISH to normal metaphases. This approach allowed us to visualize clusters of transcription coupled repair in light G bands [22].
4. Stability and persistence of radiation-induced chromosome aberrations Besides differences in DNA damage induction and repair, another factor that might modulate interchromosomal differences in the number of breaks detected after past radiation exposure is a differential persistence of translocations involving different chromosomes. Low persistence of translocations might be related to cell lethality as a result of gene truncation or position effects. A potential position effect that we previously disregarded is the spreading of X inactivation to autosomal material after transitcations between the inactive X chromosome and autosomes [23,24]. If gene truncation/inactivation is involved in the persistence of translocations, then one would expect a lower persistence of translocations involving chromosomes with high gene density. To verify this hypothesis, we analyze the persistence of translocations involving chromosomes 1 and 19 (with high gene density) and 4 and 18 (with very low gene density). Translocations were induced with ionizing radiations in a wild-type lymphoblastoid cell line (TK6) and cell samples were collected and harvested 1, 3, 7, 14, 28, 42 and 56 days after irradiation. After scoring 4000 metaphases per chromosome and time point, about 2 / 3 of the chromosome translocations drastically declined during the first week and then remained stable until day 56. All dicentrics and acentric fragments disappeared in the
first week after treatment. Regarding interchromosomal differences, chromosome aberrations involving chromosome 1 declined slightly faster than those involving chromosome 4, suggesting that aberrations involving chromosome 1 are less stable. Further analysis will allow us to state whether this tendency is biologically relevant and actually related to gene density. Our previous studies showed that tandem labelling failed to detect the clastogenic effect of radioactive iodine in buccal cells from thyroid cancer and hyperthyroidism patients [25], although positive results were obtained with the MN test in human lymphocytes in a similarly exposed population [26,27]. These contradictory results might suggest that chromosome aberrations involving lq12 would be rather unstable.
5. Possible links between histone acetylation and chromosome fragility Histones are the most common proteins in the cell nucleus. They play important biological roles not only in chromatin packaging but also in transcriptional regulation [28]. Histones are subject to a number of modifications that might influence packaging and, therefore gene expression. Among them, reversible acetylation of N-terminal lysines is one of the most widely spread from lower to higher eukaryotes, including all vertebrates and mammals. The suggested mechanisms for the involvement of histone acetylation in trascriptional regulation is the reduction in positive charge of core histones, hence loosening histone-DNA interactions and facilitating gene expression. The use of antibodies against histones acetylated at different N-terminal lysines has greatly improved our understanding of chromosome structure and function. It is known that histone H4 acetylation is a cytogenetic marker for gene expression. The use of these antibodies allowed us to visualize the active and inactive chromosomal domains both in metaphase [19,23,24,29] and interphase [20]. It has been suggested that histone H4 plays a role in maintaining genome integrity through the cell cycle, possibly by a mechanism involving lysine acetylation. Thus, mutants in which all terminal histone H4 lysines are
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substituted with glutamines accumulate increased levels of DNA damage [30]. It has also been reported that the inactive X chromosome shows residual histone H3 and H4 acetylation in the band Xq22, where the common FRAXC fragile site is located [19,31]. On the other hand, the expression of the major X linked fragile site, Xq27.3 is inhibited in vitro by sodium butyrate, a strong inhibitor of histone deacetylases [32]. In addition to the above findings, chromatin decondensed by acetylation showed an elevated radiosensitivity in hamsters [33]. We have investigated the presence of acetylated histones in the commonest Chinese hamster fragile site, Xq21. This band is in the middle of the overall heterochromatic long ann of the hamster X chromosome. By combining immunocytogenetics with antibodies against acetylated histone H4 and FISH with a CpG-island-rich probe isolated from hamster genornic DNA, we found that hamster Xq21 was labelled by both FISH and antibodies against acetylated histone H4. This finding suggests that there is a cluster of active (acetylated H4 positive signal) genes (CpG-island positive signal) in hamster Xq21. Surprisingly, and resembling the human Xq22 band, histone H4 in the hamster Xq21 is also acetylated in the inactive X chromosome which was identified by its general lack of histone H4 acetylation. It has not escaped our notice that this cluster of active genes is indicative of a possible pseudoautosomal region. All these observations taken together support a possible connection between histone acetylation and chromosome fragility. This link could be mediated by the omnipresent DNA repair, as it is also known that DNA repair is influenced by histone acetylation [34]. Further specifically designed experiments are required to determine whether these links are merely coincidental.
Acknowledgements This investigation was partially supported by grants from the European Union (Environmental and Radiation Programmes), the Spanish Ministry of Education and Science (SAF95-0813, CICYT), and the Generalitat de Catalunya (SGR95-00512, CIRIT). J.S. has a Research Postdoctoral Contract (Incorporaci6n de Doctores) by the Spanish Ministry of
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Education and Culture. J.S.'s visit to Leiden University was made possible, thanks to a long-term postdoctoral fellowship awarded by the Commission of the European Union, Environmental Programme, contract EV5V-CT-94-5240. M.J.R. and S.P.'s contribution to this work was made possible, thanks to fellowship awarded by the Spanish Ministry of Education and Culture and the Autonomous University of Barcelona, respectively. M. McCarthy's secretarial assistance and proofreading is also appreciated.
References [1] C.A. Smith, DNA repair in specific sequences in mammalian cells, J. Cell. Sci. 6 (1987) 225-241. [2] A. Sitaram, G. Plitas, W. Wang, A. Scicchitano, Functional nucleotide excision repair is required for the preferential removal of N-ethylpurines from the transcribed strand of the dihydrofolate reductase gene of Chinese hamster ovary cells, Mol. Cell. Biol. 17 (1997) 564-570. [3] J. Engelbergs, J. Thomale, A. Galhoff, M.F. Rajewsky, Fast repair of O6-ethylguanine, but not O6-methylguanine, in transcribed genes prevents mutation of H-ras in rat mammary tumorigenesis induced by ethylnitrosourea in place of methylnitrosourea, PNAS Abstr. 95 (1998) 1635-1640. [4] D.A. Eastmond, D.S. Rupa, L.S. Hasegawa, Detection of hyperdiploidy and chromosome breakage in interphase human lymphocytes following exposure to the benzene metabolite hydroquinone using multicolor fluorescence in situ hybridization with DNA probes, Mutation Res. 322 (1994) 9-20. [5] D.S. Rupa, L. Hasegawa, D.A. Eastmond, Detection of chromosomal breakage in the lcen-lql2 region of interphase human lymphocytes using multicolour fluorescence in situ hybridization with tandem DNA probes, Cancer Res. 55 (1995) 640-645. [6] M. Fenech, S. Neville, Conversion of excision repairable DNA lesions to micronuclei within one cell cycle in human lymphocytes, Environ. Mol. Matagen. 19 (1992) 27-36. [7] M. Fenech, J. Rinaldi, J. Surrall6s, The origin of micronuclei induced by cytosine arabinoside and its synergistic interaction with hydroxyurea in human lymphocytes, Mutagenesis 9 (1994) 273-277. [8] J. Surrall6s, N. Xamena, A. Creus, R. Marcos, The suitability of the micronucleus assay in human lymphocytes as a new biomarker of excision repair, Mutation Res. 342 (1995) 43-59. [9] J. Surrall~s, F. Darroudi, A.T. Natarajan, Evidence for low level of DNA repair in human chromosome 1 heterochro matin, Genes Chromosomes Cancer 20 (1997) 173-184. [10] M.F. Lyon, Gene action in the X-chromosome of the mouse (Mus musculus L.), Nature 190 (1961) 372-373. [11] J. Surrall6s, A.T. Natarajan, Radiosensitivity and repair of the inactive X chromosome. Insights from FISH and immunocytogenetics, Mutation Res. 414 (1998) 117-124.
44
J. Surralls et al. / Mutation Research 404 (1998) 39-44
[12] C. Cremer, Ch. Miinkel, M. Granzow, A. Jauch, S. Dietzel, R. Eils, X.-Y. Guan, P.S. Meltzer, J.M. Trent, J. Langowsky, T. Cremer, Nuclear architecture and the induction of chromosomal aberrations, Mutation Res. 366 (1996) 97-116. [13] A.T. Natarajan, A.S. Balajee, J.J.W.A. Boei, F. Darroudi, 1. Dominguez, M.P. Hande, C. Meijers, P. Slijepcevic, S. Vermeulen, Y. Xiao, Mechanisms of induction of chromosomal aberrations and their detection by fluorescence in situ hybridization, Mutation Res. 372 (1996) 247-258. [14] J. Surrall~s, S. Sebastian, A.T. Natarajan, Chromosomes with high gene density are preferentially repaired in human cells, Mutagenesis 12 (1997) 437-442. [15] S. Puerto, J. Surrall6s, A. Creus, R. Marcos, lnterchromosomai differences in bleomycin- and Ara-C-induced chromosomal aberrations as detected by multicolour painting FISH, 1998, in preparation. [16] E.C. Friedberg, Relationships between DNA repair and transcription, Annu. Rev. Biochem. 65 (1996) 15-42. [17] L.H.F. Mullenders, H. Vrieling, J. Venema, A.A. van Zeeland, Hierarchies of DNA repair in mammalian cells: biological consequences, Mutation Res. 250 (1991) 223-228. [18] J.M. Craig, W.A. Bickmore, The distribution of CpG islands in mammalian chromosomes, Nat. Genet. 7 (1994) 376-382. [19] P. Jeppesen, B.M. Turner, The inactive X chromosome in female mammals is distinguished by a lack of histone H4 acetylation, a cytogenetic marker for gene expression, Cell 74 (1993) 281-289. [20] J. Surrall6s, P. Jeppesen, H. Morrison, A.T. Natarajan, Analysis of loss of inactive X chromosome in interphase cells, Am. J. Hum. Genet. 59 (1996) 1091-1096. [21] W.H.J. Kalle, A.-M. Hazekamp-van Dokkum, P.H.M. Lohman, A.T. Natarajan, A.A. van Zeeland, LH.F. Mullenders, The use of streptavidin-coated magnetic beads and biotinylated antibodies to investigate induction and repair of DNA damage: analysis of repair patches in specific sequences of uv-irradiated human fibroblasts, Ann. Biochem. 208 (1992) 228-236. [22] J. Surrall6s, P. Karmakar, A.T. Natarajan, L.H.F. Mullenders, Chromosomal distribution of genes subject to transcription coupled DNA repair, Mutation Res. 379 (1997) 36-37.
[23] J. Surrall6s, A.T. Natarajan, Position effects of translocations involving the inactive X chromosome. A study combining FISH and immunocytogenetics, Mutation Res. 379 (1997) 72-73. [24] J. Snrrall6s, A.T. Natarajan, Position effect of translocations involving the inactive X chromosome: physical linkage to XIC/XIST does not lead to long-range de novo inactivation in human somatic cells, 1997, submitted. [25] M.J. Ramlrez, J. Surrall6s, A. Creus, R. Marcos, FISH analysis of lcen-lql2 breakage and micronuclei in buccal cells from radioactive iodine exposed thyroid cancer and hyperthyroidism patients, 1998, in preparation. [26] S. Guti6rrez, E. Carbonell, P. Galofr6, A. Creus, R. Marcos, Micronuclei induction by 131I exposure: study in hyperthyroidism patients, Mutation Res. 373 (1997) 39-45. [27] M.J. Ramlrez, J. Surrall6s, P. Galofr6, A. Creus, R. Marcos, Radioactive iodine induced clastogenic and age-dependent aneugenic effects in exposed patients, Mutagenesis 12 (1997) 449-455. [28] A.P. Wolffe, Transcription: in tune with histones, Cell 77 (1994) 13-16. [29] P. Jeppesen, Histone acetylation: a possible mechanism for inheritance and cell memory at mitosis, Bioessays 19 (1997) 67 -74. [30] P.C. Mcgee, B.A. Morgan, M.M. Smith, Histone H4 and the maintenance of genome integrity, Genes Dev. 9 (1995) 1716-1727. [31] N. Belyaev, A.M. Keohane, B.M. Turner, Differential underacetylation of histones H2A, H3 and H4 on the inactive X chromosome in human female cells, Hum. Genet. 97 (1996) 573-578. [32] M.G. Pomponi, G. Neff, Butyrate and acetyl-camitine inhibit the cytogenetic expression of the fragile X in vitro, Am. J. Med. Genet. 51 (1994) 447-450. [33] Z. Nackerdien, J. Michie, L. Bohm, Chromatin decondensed by acetylation shows an elevated radiation response, Radiat. Res. 117 (1989) 234-244. [34] B. Ramamathan, M.J. Smerdon, Enhanced DNA repair synthesis in hyperacetylated nucleosomes, J. Biol. Chem. 264 (1989) 11026-11034.