Elsevier

DNA Repair

Volume 7, Issue 2, 1 February 2008, Pages 313-320
DNA Repair

Genome-wide demethylation promotes triplet repeat instability independently of homologous recombination

https://doi.org/10.1016/j.dnarep.2007.11.002Get rights and content

Abstract

Trinucleotide repeat instability is intrinsic to a family of human neurodegenerative diseases. The mechanism leading to repeat length variation is unclear. We previously showed that treatment with the demethylating agent 5-aza-2′-deoxycytidine (5-aza-CdR) dramatically increases triplet repeat instability in mammalian cells. Based on previous reports that demethylation increases homologous recombination (HR), and our own observations that HR destabilizes triplet repeats, we hypothesized that demethylation alters repeat stability by stimulating HR. Here, we test that hypothesis at the adenosine phosphoribosyl transferase (Aprt) locus in CHO cells, where CpG demethylation and HR have both been shown to increase CAG repeat instability. We find that the rate of HR at the Aprt locus is not altered by demethylation. The spectrum of recombinants, however, was shifted from the usual 6:1 ratio of conversions to crossovers to more equal proportions in 5-aza-CdR-treated cells. The subtle influences of demethylation on HR at the Aprt locus are not sufficient to account for its dramatic effects on repeat instability. We conclude that 5-aza-CdR promotes triplet repeat instability independently of HR.

Introduction

Expanded tracts of trinucleotide repeats cause at least 18 human diseases [1], [2]. These repeat tracts are unstable and can further expand both in the germline, which causes more severe forms of disease in subsequent generations, and in somatic tissues, which probably contributes to disease progression and pathogenesis in the affected patient [1]. Defining the molecular mechanisms responsible for triplet repeat instability will be critical for development of therapies designed to restrict expansion or promote contraction. Studies in a variety of model organisms have shown that virtually any process that exposes single-stranded DNA – including DNA repair, replication, recombination, and transcription – allows triplet repeats to adopt aberrant secondary structures, which are often misprocessed, changing the length of the repeat [3].

We recently showed that genome-wide demethylation, induced by treatment with 5-aza-2′-deoxycytidine (5-aza-CdR), destabilized CAG triplet repeats in patient-derived cells, causing expansions and contractions [4]. Using a selectable assay for CAG repeat contractions at the adenosine phosphoribosyl transferase (Aprt) locus in CHO cells, we found that treatment with 5-aza-CdR, which leads to passive depletion of DNA methylation [5] and destruction of DNMT1 [6], or with hydralazine, which induces demethylation by a different pathway [7], increased repeat contractions by about 1000- and 50-fold, respectively [4]. Stimulation of repeat contraction by two mechanistically distinct agents, in rough proportion to their ability to decrease genome-wide methylation, argues for a causal link between CpG demethylation and CAG repeat instability [4]. In a separate investigation into the interactions between CAG repeats and homologous recombination (HR), we showed that HR significantly altered repeat stability at the Aprt locus in CHO cells [8]. Among homologous recombinants, 5% had lost large numbers of repeats, whereas no such changes were observed in more than 250 colonies that had not undergone HR [8]. These repeat contractions were evenly distributed between the two types of HR event – crossover and conversion – detected in our assay [8]. These two studies, coupled with the observations summarized below, led us to propose that 5-aza-CdR treatment destabilizes CAG repeats by stimulating HR [8]. Here, we test that hypothesis.

It has been suggested that a major role of DNA methylation – the addition of a methyl group to cytosine within CpG dinucleotides – is to inhibit HR between repeated sequences [9], [10], [11]. Support for this idea is strongest in fungi where crossovers between direct repeats during meiosis are inhibited more than 100-fold by CpG methylation [12]. In mammalian cells, the links between DNA methylation and HR are less direct, and in some cases, contradictory. Treatment with 5-aza-CdR, which induces demethylation, has been shown to stimulate sister-chromatid exchange [5], increase HR within a transgene array [13], and promote double-minute chromosome dimerization [14]. In mouse ES cells, knockout of DNA methyltransferase 1 (DNMT1), the maintenance DNA methyltransferase, reduces CpG methylation and increases loss of heterozygosity (LOH) at the Dnmt1 locus, which was attributed to elevated levels of mitotic HR [15]. In the same study, knockout of Dnmt1 was also shown to increase genomic rearrangements at the hypoxanthine phosphoribosyl transferase (Hprt) locus, suggesting that CpG methylation may inhibit nonhomologous recombination as well [15]. The view that CpG methylation normally inhibits both kinds of recombination can account for several additional observations: patients with mutations in Dnmt3a display increased rearrangements of centromeric repeats [16]; ES cells that lack DNMT1 show an increased efficiency of gene targeting [17]; and DNMT1-deficient human cancer cells have higher frequencies of chromosomal rearrangements [18].

In contrast to these studies, which generally support a role for CpG methylation in genome stability, other reports either failed to detect an effect, or showed that demethylation decreased HR. CpG-methylated and unmethylated DNA constructs, when transfected into COS1 monkey cells, gave identical frequencies of extrachromosomal recombination in an assay designed to detect single-strand annealing (SSA), one kind of HR [19]. Similarly, 5-aza-CdR treatment of HeLa human cells that were modified to carry a chromosomal recombination substrate designed to detect conversion events did not increase the frequency of HR [20]. Finally, DNMT1 deficiency in mouse ES cells, was shown to reduce the rate of loss of an array of selectable transgenes, suggesting that mitotic HR was decreased by demethylation [21].

The lack of agreement among these studies could arise for several reasons: differences between the cell lines, or the species from which they were derived; whether the recombination substrate is extrachromosomal, randomly integrated, or targeted to a specific locus; and the particular kind of event the substrate is designed to detect—crossover, conversion, or rearrangement. Because our original observations – 5-aza-CdR treatment and HR destabilize repeats – were made at the Aprt locus in CHO cells, we chose to measure the effects of 5-aza-CdR treatment on HR at the same site, using a well-characterized recombination assay that can simultaneously detect crossovers, conversions, rearrangements, and mutations (Fig. 1A) [22]. In this way we can measure the effects of 5-aza-CdR treatment on overall rates of recombination, as well as determine the differential effects, if any, on individual types of events.

By determining whether 5-aza-CdR treatment stimulates HR, we can distinguish between two alternatives for how genome-wide demethylation stimulates CAG repeat contraction. Because 5-aza-CdR treatment and HR each destabilize repeats, we proposed the hypothesis that they operate as part of a common pathway with 5-aza-CdR stimulating HR, which in turn stimulates repeat contractions. A demonstration that 5-aza-CdR stimulates HR would cleanly distinguish this possibility from the alternative: that 5-aza-CdR and HR have their effects via independent pathways. Here we show that 5-aza-CdR treatment does not significantly alter the rate of HR at the Aprt locus, although it does shift the spectrum of events. We conclude that 5-aza-CdR treatment destabilizes triplet repeats by a mechanism that does not proceed via stimulation of HR.

Section snippets

Cell lines and tissue culture conditions

Chinese hamster ovary (CHO) cells lines AT3-2, which contains a single copy of the wild type Aprt gene, and GSAA5, which was derived from AT3-2 cells and carries a duplication at the Aprt locus, have been described previously [23], [24]. Both cell lines carry a functional Aprt gene and are phenotypically APRT+. These cells were cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum (Gibco), nonessential amino acids, sodium pyruvate, penicillin, and streptomycin.

Experimental design

To assay for the effects of demethylation on HR, we used a well-characterized recombination substrate that contains two direct repeats of the selectable Aprt gene at its endogenous locus in GSAA5 CHO cells (Fig. 1). The upstream copy of the gene is truncated at the 3′ end and has a mutated EcoRV site in exon 2 [23]. The downstream copy is functional and contains the wild type EcoRV site. Selection for APRT cells allows us to detect four types of event – conversion, crossover, rearrangement,

Discussion

In previous studies, we showed that both 5-aza-CdR treatment and HR stimulate contraction of CAG repeat tract at the Aprt locus [4], [8]. Because several observations in the literature suggest that demethylation increases HR, we proposed that 5-aza-CdR treatment promotes repeat instability via stimulation of HR [4]. As a critical test of this hypothesis, we measured the effects of 5-aza-CdR treatment on HR, using a well-characterized recombination substrate at the Aprt locus. We show here that

Acknowledgements

We would like to thank Fung Chan for technical help and members of the Wilson laboratory for helpful comments and discussions. We are also indebted to Fisun Hamaratoğlu and Murat B. Yaylaoğlu for critical reading of the manuscript. This work was supported by a postgraduate scholarship A and D from the Natural Sciences and Engineering Council of Canada to V.D., an NIH T32 training grant EY007102 to B.A.P. and an NIH grant GM38219 to J.H.W.

References (40)

  • B. Segura-Pacheco et al.

    Reactivation of tumor suppressor genes by the cardiovascular drugs hydralazine and procainamide and their potential use in cancer therapy

    Clin. Cancer Res.

    (2003)
  • J.L. Meservy et al.

    Long CTG tracts from the myotonic dystrophy gene induce deletions and rearrangements during recombination at the APRT locus in CHO cells

    Mol. Cell. Biol.

    (2003)
  • V. Colot et al.

    Eukaryotic DNA methylation as an evolutionary device

    Bioessays

    (1999)
  • R. Jaenisch et al.

    Epigenetic regulation of gene expression: how the genome integrates intrinsic and environmental signals

    Nat. Genet.

    (2003)
  • M.C. Kricker et al.

    Duplication-targeted DNA methylation and mutagenesis in the evolution of eukaryotic chromosomes

    Proc. Natl. Acad. Sci. U.S.A.

    (1992)
  • L. Maloisel et al.

    Suppression of crossing-over by DNA methylation in Ascobolus

    Genes. Dev.

    (1998)
  • M.W. McBurney et al.

    Reexpression of a cluster of silenced transgenes is associated with their rearrangement

    Genes Chromosomes Cancer

    (2001)
  • R. Rizwana et al.

    CpG methylation reduces genomic instability

    J. Cell Sci.

    (1999)
  • R.Z. Chen et al.

    DNA hypomethylation leads to elevated mutation rates

    Nature

    (1998)
  • G.L. Xu et al.

    Chromosome instability and immunodeficiency syndrome caused by mutations in a DNA methyltransferase gene

    Nature

    (1999)
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