Replication stress is a significant contributor to genome instability. Recent studies suggest that the centromere is particularly susceptible to replication stress and prone to rearrangements and genome damage, as well as chromosome loss. This effect is enhanced by loss of heterochromatin. The resulting changes in genetic organization, including chromosome loss, increased mutation and loss of heterozygosity, are important contributors to malignant growth.

Introduction

Certain cancer cells, particularly solid tumours, are known for dramatic CIN (chromosome instability) phenotypes. Aneuploidy and other numerical disruptions (nCIN) probably occur from malfunction of the chromosome segregation apparatus, whereas structural instability (sCIN) is typically manifested as gross chromosome rearrangements, frequently linked to replication stress and DNA breakage (reviewed in [1]). Both forms of CIN can contribute to LOH (loss of heterozygosity), which is also a contributor to malignant transformation (e.g. [2]).

The role of the centromere in maintaining faithful chromosome segregation and preventing nCIN is self-evident: it is a structural domain on the chromosome that is required for proper attachment of the spindle (reviewed in [3]). But it is increasingly clear that the centromere is also a significant contributor to sCIN, particularly under conditions of replication stress. Such stress can be caused by exposure to external DNA-damaging agents, including radiation or chemicals, or chemotherapeutic drugs that block S-phase or stall fork progression. Endogenous stresses can occur in naturally fragile regions of the genome, including repetitive sequences or pause sites that render the genome vulnerable to replication fork stalling or fork collapse. Indeed, a robust system of DNA damage response and repair has been invoked as the primary barrier to malignant transformation (e.g. [4]).

A conserved centromere alternates silencing with synthesis

Fission yeast offers an outstanding genetic model to study how replication stress has an impact on the centromere, because proteins that respond to replication stress and proteins responsible for centromere maintenance are each highly conserved (reviewed in [3,5,6]). As in most eukaryotes, the centromere in fission yeast is a large element, where heterochromatic pericentromere repeats flank a central core domain. This heterochromatin is defined by the presence of histone H3K9me (methylated Lys9 on histone H3), which is bound by two homologues of heterochromatin protein 1, Swi6 and Chp2, via their conserved chromodomains to establish a rigid transcriptionally silenced structure (Figure 1A).

Structure of the fission yeast centromere

Figure 1
Structure of the fission yeast centromere

(A) Heterochromatin forms at the outer repeats, defined by the binding of Swi6 to H3K9me. (B) During early S-phase, H3K9 methylation is reduced. Swi6 binding to DDK promotes early replication initiation. Unopposed binding of Chp1 (in the absence of Swi6) opposes early initiation.

Figure 1
Structure of the fission yeast centromere

(A) Heterochromatin forms at the outer repeats, defined by the binding of Swi6 to H3K9me. (B) During early S-phase, H3K9 methylation is reduced. Swi6 binding to DDK promotes early replication initiation. Unopposed binding of Chp1 (in the absence of Swi6) opposes early initiation.

The methyl mark is established and maintained by the methyltransferase Clr4 (SuVar3-9). Paradoxically, Clr4 is targeted to this domain by transient de-silencing during S-phase, which allows a brief wave of convergent transcription [7,8]. These short RNAs are processed by RNAi mechanisms that are used to target Clr4 back to the site of transcription, re-establishing the methyl mark on newly incorporated histones [9,10]. Clr4 binding to Swi6 and to components of the replisome provide additional mechanisms for recruitment and spreading of the methyl mark [1113]. This is a simplified summary, as additional players that fine-tune the response continue to be identified (reviewed in [3,6]) (Figure 1).

Regulating the timing of centromere DNA replication

The pericentromere is rich in replication origins, which alternate in position with the transcription units that generate the siRNAs [14,15]. Initiation of replication in eukaryotes is a well-conserved process that requires the orderly assembly of a number of proteins that mark potential origins for use (reviewed in [16]). This begins with identification of the origin by ORC (origin recognition complex). The ORC is activated by the Cdc18 (Cdc6) and Cdt1 proteins which load the heterohexameric MCM (minichromosome maintenance) complex, and together these form the preRC (pre-replication complex), which remains poised for activation. The activation signal depends on the combined effects of the CDK (cyclin-dependent kinase) cell cycle kinase, which conveys a global replication signal, and the DDK (Dbf4-dependent kinase) replication kinase, which will activate the preRC directly through phosphorylation of MCM proteins and other substrates. This activation allows the initiation of unwinding, and recruitment of additional proteins to convert the preRC into an initiation complex and finally an intact replisome that processively synthesizes DNA.

Unlike most heterochromatin domains, the fission yeast outer repeats undergo replication early in S-phase [17] and this depends upon Swi6 [18,19]. Swi6 binds to the DDK regulatory subunit Dfp1 [20], and this interaction is required for early replication [18]. Indeed, artificially tethering Dfp1 to the chromatin via the Swi6 chromodomain restores early replication in a swi6Δ cell [18], which indicates that sufficient histone methylation remains in early S-phase, despite the transient de-silencing, to recruit chromodomain proteins. The late replication in swi6Δ is also rescued in a swi6Δclr4Δ double mutant, suggesting that the late effect is linked in some way to unopposed H3K9 methylation [18] (Figure 1B).

In addition to binding to Dfp1, Swi6 also binds to the Cdc18 initiator protein [19] and to ORC (P.-C. Li and S.L. Forsburg, unpublished work). A mutation in Cdc18 that disrupts its association with Swi6 has no effect on centromere silencing or replication in the euchromatin, but leads to even earlier replication in the centromere than in wild-type cells, suggesting that Swi6 interactions with Dfp1 and Cdc18 balance the proper timing of replication. In addition to its connection with components of initiation, there is also a role at the replication fork. Swi6 binds to DNA polymerase α, which is required for silencing [21]. Clr4, the histone methyltransferase, is also associated with the leading strand DNA polymerase ϵ in fission yeast [11], which provides a direct connection for rapid re-methylation of the heterochromatin domain.

Although swi6Δ mutants replicate their centromeres late, we recently showed that clr4Δ mutants replicate early [22]; this is consistent with the observation that clr4Δ rescues the late replication phenotype of swi6Δ [18]. Early replication was also observed in chp1Δ mutants [22]. Chp1 is a chromodomain protein that binds to H3K9me and to siRNA as part of the RITS (RNA-induced transcriptional silencing) complex, which provides one means of recruiting Clr4 [10,23]. Loss of chp1Δ leads to a severe reduction in H3K9 methylation, but does not eliminate it [2426].

Unexpectedly, however, we find that other mutations that significantly reduce H3K9 methylation, such as the RNAi components dcr1Δ, hrr1Δ or rdp1Δ, nevertheless cause late replication similar to swi6Δ [22]. There is residual H3K9me and Chp1 binding in RNAi mutants [27,28], so we speculate that it is not H3K9me itself but Chp1 bound to it that results in late replication [22]. Chp1 binding to H3K9me occurs with higher affinity than that of Swi6, and it needs to be removed to allow proper Swi6 association and assembly of the fully repressed heterochromatin structure [29,30]. This is probably via interactions between adjacent Swi6 chromoshadow domains [31] (Figure 1A). Taken together, these results suggest that Chp1 binding in the absence of Swi6 leads to a structure that is refractory to early replication.

The replication timing results are unexpected, given that ChIP analysis suggests that Swi6 is largely removed from the centromere during mitosis and returns in late S-phase [7,8]. However, it seems likely that a subpopulation of Swi6 is recruited earlier than the bulk of the protein that performs DNA silencing; this can be observed by cytological assays comparing the timing of GFP–Swi6 recruitment to the spindle poles (and thus centromeres) following mitosis. We find that Swi6 is restored to the centromeres very quickly after anaphase, lagging behind the centromere specific histone H3 variant Cnp1 [CENP-A (centromere protein A)] by only approximately 3 min [22]. In contrast, Chp1–GFP recruitment is more prolonged. We suggest that this low level of early Swi6 recruitment allows concentration of Dfp1 (and thus the origin-activating DDK) to facilitate replication directly, and perhaps also works by antagonizing the high-affinity Chp1 protein. By this model, there are two roles for Swi6 in S-phase: one in establishing a permissive replication structure early, and then later, a higher local concentration that locks down the chromatin into typically repressive heterochromatin.

Replication as a source of stress

Several aspects of this process expose vulnerabilities that are specific to S-phase. First, the pericentromere is made up of repeated sequences, and such sequences are known to be prone to recombination or replication fork pausing (e.g [32,33]). The presence of heterochromatin is generally refractory to recombination [34]. However, from M- to S-phase, heterochromatin is partly disrupted [7,8], suggesting a window of vulnerability. Transcription during S-phase to generate the siRNAs used to target Clr4 leads to the possibility of collisions between DNA and RNA polymerase; recent work has shown that the RNAi proteins contribute to RNA polymerase eviction to reduce this possibility [15].

That this domain is subject to intrinsic stress even in normal cells with intact heterochromatin is suggested by the constitutive presence of a low level of histone H2AX phosphorylation; this modification is typically associated with regions of DNA damage or double-strand breaks [35]. The Smc5/6 protein complex, which is associated with recombination-mediated replication fork restart, is enriched at the centromere and other repetitive domains {particularly the rDNA (ribosomal DNA) [36,37]}. Brc1, a BRCT (breast cancer early-onset 1 C-terminal)-motif-containing protein that binds γH2AX (phosphorylated histone H2AX) and is also associated with replication fork restart, is also enriched at the centromere, where its presence depends on Clr4 [38]. The pericentromere may be refractive to repair (e.g. [39,40]), making it particularly vulnerable to damage from replication stress.

Interestingly, the repeats of the centromere provide a substrate for BIR (break-induced replication) even in wild-type cells; this has been demonstrated using an elegant genetic system, in which a non-essential minichromosome derivative of chromosome 3 is examined for rearrangements, including isochromosome formation and translocations from the intact chromsome 3 under a variety of conditions [41,42] (Figure 2). These results show that recombination through the centromere is a significant contributor to LOH. Intriguingly, in rad51Δ mutants, there is an increased recombination through the imr repeats to generate iso-chromosomes [42]. This suggests that Rad51 functions to limit the scale of rearrangement, possibly promoting a maintenance recombination-based mechanism to preserve the repetitive centromere structure. There are synthetic growth defects between swi6Δ and rad51Δ [43], which suggests that this mechanism becomes particularly important when heterochromatin formation is impaired. Intriguingly, it has been suggested that recombination in the centromere is an intrinsic part of centromere maintenance, particularly of the inner imr repeat sequences that flank the central core, and are unique to each centromere [44]. Thus the cell must balance between limited recombination and the potential chromosome rearrangements.

Minichromosome model for chromosome rearrangement [41,42]

Figure 2
Minichromosome model for chromosome rearrangement [41,42]

(A) Recombination through the centromere leads to isochromosome formation or acrocentric derivatives through BIR. (B) Internal model for centromere rearrangement uses an interrupted ura4+ gene to monitor recombination. (C) Double mutants that disrupt Swi6 or Chp1 and components of replication fork stability have enhanced defects, using the system in (B). Median recombination rate is reported. Data taken from [47].

Figure 2
Minichromosome model for chromosome rearrangement [41,42]

(A) Recombination through the centromere leads to isochromosome formation or acrocentric derivatives through BIR. (B) Internal model for centromere rearrangement uses an interrupted ura4+ gene to monitor recombination. (C) Double mutants that disrupt Swi6 or Chp1 and components of replication fork stability have enhanced defects, using the system in (B). Median recombination rate is reported. Data taken from [47].

Heterochromatin and replication fork stability proteins make independent contributions to centromere stability

We hypothesized that the outer repeats of the centromere are sources of replication stress in the absence of heterochromatin, and reasoned this should be exacerbated by mutations that destabilize the replication fork. Mrc1 is a non-essential component of the replisome that acts as a processivity factor (part of the fork protection complex), and has a genetically separable function in activating the replication checkpoint kinase Cds1 [45,46]. We found that double mutants between heterochromatin components and mrc1Δ have reduced growth rates and dramatically increased sensitivity to HU (hydroxyurea), which is not seen in double mutants with the checkpoint-specific mrc1-SA allele [45]. This suggests that replication fork processivity associated with Mrc1 is particularly important when repeated domains are destabilized. We also observed that chp1Δmrc1Δ shows significantly lower plating efficiency than chp1Δ or swi6Δmrc1Δ, suggesting that the effect is exacerbated in that strain.

Using the genetic system developed in [42], we examined the frequency of chromosome loss (nCIN) and rearrangements (sCIN) involving the non-essential mini chromosome in swi6Δ, swi6Δmrc1Δ and Δchp1 (the double mutant chp1Δmrc1Δ could not be maintained with the mini-chromosome) [47]. As expected, the rate of chromosome loss was very high in the heterochromatin mutants relative to wild-type, consistent with the structural role of Swi6 and Chp1 in chromosome transmission. Rearrangements occurred at a low frequency in the heteorchromatin mutants, but in contrast with wild-type, these were largely isochromosomes that involved recombination specifically through the outer repeat. This indicates that the outer repeat is destabilized and prone to rearrangement in a mutant that lacks either swi6Δ or chp1Δ. An mrc1Δ single mutant, conversely, shows a low rate of chromosome loss but a high rate of rearrangement not limited to the centromere repeats. Strikingly, the double mutant swi6Δmrc1Δ has a reduced rate of chromosome loss compared with swi6Δ, accompanied by a dramatic increase in recombination. In at least five out of the 12 independent isolates, this results in loss of normal chromosome 3 and recovery of its two arms as separate chromosomes [47], presumably by a BIR mechanism (e.g. [41,42]).

We extended these observations using a simpler assay in which we scored recombination between two repeats of a partial tandem duplication embedded within the heterochromatin (Figure 2B). Results were consistent with those in the minichromosome system; consistent with the effects of swi6Δmrc1Δ, we found that a double mutant swi6Δswi1Δ which lacks the fork protection complex also significantly enhanced recombination. Mutants that lack Swi1 are sensitive to fork-destabilizing agents such as MMS (methyl methanesulfonate) [48,49]. Similar results were observed for clr4Δ as for chp1Δ or swi6Δ. Double mutants with cds1Δ had a less severe, but still detectable, effect. Unexpectedly, however, the synergy between replication fork-protection proteins and heterochromatin mutants in causing genome rearrangements was not found in RNAi mutants such as dcr1Δ. Further epistatic analysis will be required to determine the source of the differences.

Conclusions

Fission yeast centromeres suffer from endogenous replication stress, and are also susceptible to several different forms of fork stressors including transcriptional collisions and reduced fork processivity. The sensitivity cannot be linked simply to replication timing as both early-replicating mutants (e.g. chp1Δ or clr4Δ) and late-replicating mutants (swi6Δ) show similar synthetic defects combined with mrc1Δ, and some late replicators (e.g. dcr1Δ) do not show the same degree of sensitivity. The centromere suffers intrinsic stress even in heterochromatin-competent cells (e.g. by the recruitment of phosphorylated histone H2A, and the Brc1 and Smc5/6 proteins), which may be related to recombination-mediated mechanisms required for its normal maintenance.

There is evidence that the centromere is structurally unstable in cancer cells, prone to fission or breakage [5052]. It has been proposed that defects in chromosome segregation can contribute to centromere-linked breaks and fusions in cancer (e.g. [1]). However, it is now clear that replication stress contributes to mitotic mis-segregation, especially if the checkpoint is disrupted [5254]. Fission yeast provides important insights into the mechanisms involved in centromere-linked genome damage and rearrangements, and how they contribute to LOH and aneuploidy. Taken together, these data emphasize that centromeres are a weak link in genome maintenance, and the target of multiple converging pathways to manage intrinsic stress and prevent multiple CINs.

The 7th International Fission Yeast Meeting: Pombe 2013: An Independent Meeting/EMBO Conference held at University College London, London, U.K., 24–29 June 2013. Organized and Edited by Jürg Bähler (University College London, U.K.) and Jacqueline Hayles (Cancer Research UK London Research Institute, U.K.).

Abbreviations

     
  • BIR

    break-induced replication

  •  
  • CIN

    chromosome instability

  •  
  • DDK

    Dbf4-dependent kinase

  •  
  • LOH

    loss of heterozygosity

  •  
  • MCM

    minichromosome maintenance

  •  
  • nCIN

    numerical CIN

  •  
  • ORC

    origin recognition complex

  •  
  • preRC

    pre-replication complex

  •  
  • sCIN

    structural CIN

I thank members of my laboratory for helpful comments on the paper. I apologize to those colleagues whose work could not be cited for reasons of space.

Funding

This work is supported by the National Institutes of Health [grant number GM059321] and the National Science Foundation [grant number MCB 0640103].

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