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,
Franck Amiot,3
Xavier Gidrol,3
Emmanuel Barillot,2
Alain Nicolas,1 and
Valérie Borde1*
Institut Curie, Recombinaison et Instabilité Génétique, Centre de Recherche, UMR7147 CNRS-Institut Curie-Université P. et M. Curie, 26 rue d'Ulm, 75248 Paris Cedex 05, France,1 Institut Curie, Service de Bioinformatique, 26 rue d'Ulm, 75248 Paris Cedex 05, France,2 and Service de Génomique Fonctionnelle, CEA, 2 rue Gaston Crémieux, CP5722, 91057 Evry Cedex, France3
Received 3 November 2006/ Returned for modification 30 November 2006/ Accepted 12 December 2006
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ABSTRACT |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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INTRODUCTION |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Once recombination is initiated, a decision is made to channel the early intermediates along a pathway ending in crossover formation or in noncrossover events. This step is influenced both by "crossover interference," in which a crossover in one region makes it unlikely that another will occur nearby (18), and by "crossover homeostasis," which regulates the crossover/noncrossover ratio to ensure a minimal amount of crossover per chromosome (34).
Before this stage, the frequency and the localization of the initiation events are the earliest determinants of how meiotic recombination events are distributed along the chromosomes. Extensive studies performed with Saccharomyces cerevisiae have shown the heterogeneous distribution of the initiating DNA double-strand breaks (DSBs) along the chromosomes and the >100-fold variation in cleavage frequency from site to site (39). These findings are consistent with the nonrandom distribution of recombination along the chromosomes observed in all organisms. The factors that determine whether a specific region is prone to DSB formation are not well understood at the molecular level. The primary sequence is not the main determinant of DSB formation since a reporter construct shows various DSB frequencies and recombination rates depending on where it is inserted into the genome (10, 52). Several rules regarding the distribution of DSBs have nevertheless been described. First, DSBs always occur in open chromatin regions, mainly in promoter-containing regions (4, 53). Second, DSBs are formed preferentially in the chromatin loops, as opposed to the loop basis in which cohesins are located (7, 13). Third, DSB frequencies are generally low in a 20-kb region around centromeres (9, 12) and the centromere itself has a strong inhibitory effect on meiotic recombination initiation (30). Fourth, the rate of meiotic recombination is also usually low close to the natural chromosome ends (3, 46) and DSB frequency is very low up to 40 kb from a telomere (9, 12). This effect may be exaggerated by the fact that these DSB measurements were made with rad50S or sae2
mutants, which accumulate unresected DSBs and in which DSB formation is specifically reduced in late-replicated regions (8). Finally, another factor influencing DSB formation at one site is the proximity to another DSB site (11, 19, 52, 54), although this factor may not be a general rule (15).
The catalytic activity for meiotic DSB formation is carried by the widely conserved Spo11 protein (5, 23). Besides Spo11, at least nine additional DSB proteins are absolutely required for DSB formation (reviewed in reference 24). Recent cytological and chromatin immunoprecipitation (ChIP) analyses have begun to dissect the chromatin association of these proteins on meiotic nucleus spreads and their requirement for Spo11 association with DSB hot-spot regions (24). However, how DSB sites are selected and how Spo11 is recruited to the chromatin of the DSB region remain to be understood.
To address these issues, we previously reported that a fusion between the Gal4 DNA binding domain and Spo11 (Gal4BD-Spo11) yields a protein that is able to introduce DSBs and stimulate meiotic recombination in the naturally cold GAL2 promoter, which contains Gal4 consensus binding sequences (CGGN11CCG, where N11 represents 11 various nucleotides) (38). Thus, the normal recruitment of Spo11 to chromatin can be bypassed by the Gal4BD moiety of the fusion protein. However, all the DSB proteins are still required for Gal4BD-Spo11 DSB formation at GAL2, showing that they are all important for Spo11's ability to create a DSB and not only for the recruitment of Spo11 to the DSB site. Importantly, it was shown that Gal4BD-Spo11 also cleaves the three natural Spo11 DSB sites examined.
Here, to determine to what extent the genome-wide Gal4BD-Spo11 DSB profile was modified and to investigate the mechanisms that regulate meiotic DSB distribution, we have further exploited the in vivo properties of the Gal4BD-Spo11 protein to probe the entire genome. We show that DSB distribution is profoundly remodeled upon the introduction of strong new Gal4BD-Spo11 DSBs and that DSB cleavage at a subset of Gal4BD-Spo11-bound sites remains subject to repressing position effects. Globally, the modification of Spo11 cleavage sites leads to a genome-wide redistribution of meiotic double-strand breaks without decreasing the meiotic product viability.
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MATERIALS AND METHODS |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Chromatin immunoprecipitation. For DSB mapping, 50 ml of rad50S mutant cells (about 109 cells) after 6 h in sporulation medium were processed for chromatin immunoprecipitation without cross-linking to select for covalently attached Spo11 (9). For Gal4BD-Spo11 binding, 40 ml of meiotic cells (8 x 108 cells) were treated with 1% freshly prepared formaldehyde for 15 min at room temperature and then 125 mM glycin for 5 min. In both cases, cells were then washed and chromatin immunoprecipitation and DNA purification were performed as described previously (40). Antibodies used were mouse anti-Myc (8 µg; clone 9E10) for Spo11-Myc13 or Gal4BD-Spo11-Myc13 covalent binding and mouse anti-Gal4BD (4 µl; clone 2GV3; Euromedex) for Gal4BD-Spo11 binding. Multiplex PCR was performed on 1/10 of the immunoprecipitate or 1/25,000 of the whole-cell extract, and results were analyzed as described previously (9). Primer sequences are available upon request. For hybridization on microarrays, two-thirds of the immunoprecipitated DNA or 1/4,000 of the DNA from the whole-cell extract was amplified by random primer extension followed by PCR amplification, incorporating amino-allyl dUTP for subsequent dye coupling (9).
Microarray hybridization. The S. cerevisiae open reading frames (ORFs) were reamplified using a pair of universal primers from 6,216 full-length ORFs amplified from genomic DNA by Research Genetics. The intergenic regions were amplified using yeast genomic DNA and 6,220 pairs of specific primers from Research Genetics. After purification by isopropanol precipitation, the sizes and concentrations of the PCR products were measured by agarose gel electrophoresis. DNA was resuspended in a Tris-EDTA-dimethyl sulfoxide (50/50) spotting buffer. DNA printings were performed on Corning Ultra Gaps II slides by using a Microgrid II arraying robot (Biorobotics). Amplified DNA was labeled by coupling with Cy3 or Cy5 and hybridization at 63°C, and washes were done under standard conditions (http://www.derisilab.ucsp.edu).
Data analysis. Array slides were scanned using a 4000B scanner (Axon Instruments) and the images analyzed using the Genepix Pro 5.0 software. Extracted numeric data were transformed as described previously (9). We used the algorithm MANOR (37) to estimate and correct a spatial trend on each chip. Data for ORFs and intergenic regions were normalized separately by fitting the median to 0. Probes corresponding to repeated sequences present at least twice in the genome were removed from the analysis by using the algorithm BLAST (1). A list of these repeated elements is available upon request.
Each experiment was done from three independent cultures (except DSB measurements for the Spo11-Myc pADH1-Gal4BD strain, which were done twice). We computed a weighted average for the replicates, with the standard deviation of the chip as the weight. To determine the enriched probes, we computed the median percentile rank (MPR) among replicates, varying from 0 to 1 for each probe (32), and after examination of the MPR distributions, we chose two criteria such that the estimated numbers of true positives and false positives were optimized. The first defines a stringent category containing most of the true DSB (or binding) sites and very few false positives. We defined primary sites as those sites composed of at least two adjacent chromosomal features with an MPR greater than 0.9 (DSB experiments) or 0.97 (binding experiment). The second category defines secondary sites as those sites composed of all probes with an MPR of >0.8 (DSB experiments) or >0.9 (binding experiment) and not included in a primary site.
Two sites were considered common to two experiments if they shared at least one probe. Finally, sites were ranked according to the highest-log-ratio probe within the site.
Microarray data accession number. The microarray data described herein are available at Gene Expression Omnibus (http://www.ncbi.nlm.nih.gov/geo) under the accession number GSE5884.
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RESULTS |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Genome-wide mapping of Gal4BD-Spo11 binding sites. First we mapped the binding sites of the Gal4BD-Spo11 protein. In order to differentiate between the sites that are normally bound and cleaved by Spo11 and the new binding sites driven by Gal4BD, we took advantage of the constitutive Gal4BD-Spo11 expression behind the ADH1 promoter to map binding sites at the beginning of sporulation, about 4 h before DSB formation.
We identified 44 binding sites for Gal4BD-Spo11 (listed in Table S1 in the supplemental material; indicated along chromosomes depicted in Fig. 1A; see Fig. S1 in the supplemental material). The fusion protein is still able to recognize the natural Gal4 binding sites, since only 3 of the 22 strongest targets determined previously for the Gal4-Myc protein in galactose medium (P < 10–5) (41) are not included in our set. Then, using the recently released sequence of our S. cerevisiae background strain, SK1 (sequence data were produced by the Saccharomyces Genome Resequencing Project Sequencing Group at the Sanger Institute and obtained from ftp://ftp.sanger.ac.uk/pub/dmc/yeast), we examined the presence of a Gal4 binding sequence in the Gal4BD-Spo11 binding regions. We found that half of these regions contain at least one occurrence of the Gal4 binding sequence, preferentially (54%) located in intergenic regions at a frequency that is significantly higher than the general distribution of the Gal4 binding sequence (chi-square P value, <0.036). This is most likely due to the better chromatin accessibility of intergenic regions.
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Genome-wide mapping of DSB induced by Gal4BD-Spo11. Next, we used ChIP-chip to map DSBs from meiotic rad50S mutant cells expressing either Spo11-Myc or Gal4BD-Spo11-Myc without prior cross-linking since in this repair-deficient strain background, Spo11 remains covalently attached to the DSB ends (23). We first determined 239 DSB sites for the Spo11 protein (listed in Table S1 in the supplemental material; Fig. 1A; see Fig. S2 in the supplemental material). There was a good overlap with our previously determined Spo11-hemagglutinin DSB sites (9), since among the 100 strongest Spo11-hemagglutinin DSB sites, 71 are detected as DSB sites and 20 are detected as secondary DSB sites in the present study.
We next determined 191 DSB sites for the Gal4BD-Spo11 protein (see Table S1 and Fig. S3 in the supplemental material; Fig. 1A). As expected and as illustrated in Fig. 1B, many of the GalBD-Spo11 and Spo11 DSB sites are common (116 sites) and among the 66 Gal4BD-Spo11-specific DSB sites, we noted that 36 are secondary Spo11 DSB sites. Thus, 152/191 (80%) of Gal4BD-Spo11 DSB sites occur at natural Spo11 DSB sites.
The overlap between the Gal4BD-Spo11 binding sites and the DSB sites is summarized in Fig. 1B. Nine sites are specific to Gal4BD-Spo11, and seven are DSB sites shared by Spo11 and Gal4BD-Spo11. We will hereinafter refer to these 16 Gal4BD binding sites cleaved by Gal4BD-Spo11 as targeted sites. They include the well-known Gal4 targets the GAL1, GAL2, GAL7, GAL10, and GAL80 genes. When coinciding with a Gal4BD binding site, the natural DSB sites have a higher rank in the Gal4BD-Spo11 DSB experiment, suggesting that they are more frequently cleaved (Fig. 2A). The resolution of our data (1 to 2 kb) does not allow us to say if DSBs stimulated by Gal4BD-Spo11 at these sites occur exactly at the same nucleotide positions as those induced by natural Spo11.
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The expression of the Gal4BD-Spo11 construct under the constitutive ADH1 promoter raised the possibility that the variation in the DSB profile in the Gal4BD-Spo11 strain resulted from the construct's early expression and/or overexpression. We thus performed two additional control experiments. We first mapped genome-wide DSBs in a strain expressing Spo11-Myc13 from the ADH1 promoter. Among the 285 DSB sites detected, 167 corresponded to Spo11 DSB sites and 93 to secondary Spo11 DSB sites, and importantly, out of the 150 strongest pADH1-Spo11 DSB sites, only one was not detected at all in the endogenous Spo11 experiment. Thus, very few of the pADH1-Gal4BD-Spo11 DSB sites were due to the overexpression of Spo11. Second, we mapped DSBs made by Spo11-Myc in a strain expressing pADH1-GAL4BD alone. None of the 16 targeted Gal4BD-Spo11 DSB sites were stronger than Spo11 DSB sites in this experiment, and among the Gal4BD-Spo11 DSB sites corresponding to secondary Gal4BD binding sites, only one (YCR060W) had a much stronger enrichment in the experiment with Gal4BD than in that with Spo11 (Table S1 in the supplemental material). This indicates that Gal4BD overexpression has no indirect effect and that Gal4BD must be fused with Spo11 in order to induce DSBs at its binding sites.
Long-range cis effects of enhanced DSBs. Despite the substantial overlap between Spo11 and Gal4BD-Spo11 DSB sites, there is a relatively high proportion (24%) of the natural DSBs that are no longer cleaved by Gal4BD-Spo11. Indeed, among the 123 DSB sites cleaved only by Spo11 (Fig. 1B), 61 are not even among our 740 secondary Gal4BD-Spo11 DSB sites. Close examination of specific loci strongly suggests that this finding can be explained at least in part by cis inhibitory effects of Gal4BD-Spo11 DSBs on surrounding natural Spo11 DSBs. Indeed, strong Gal4BD-Spo11 DSBs at GAL2 and MLF3 were accompanied by reduced DSB formation over several kilobases in surrounding regions compared with DSB formation by Spo11 (Fig. 1C). To appreciate the generality of this cis effect, we examined the behavior of the regions surrounding the 12 strongest targeted sites (Fig. 2A). In these regions, DSB formation was significantly reduced compared with that in regions not affected by Gal4BD binding (Fig. 2B). The cumulative reduction effect around targeted sites increased up to 60 kb around the sites and then decreased with increasing distance from the targeted sites (Fig. 2B).
To deepen the analysis of this long-range effect on DSB distribution, we examined the chromosome XIII region around GAL80, which was a strong Gal4BD-Spo11 targeted site and a weaker Spo11 DSB site (Fig. 2A and 3A). In the spo11, pADH1-spo11, and spo11 pADH1-Gal4BD strains, DSBs occurred only in the promoter of the gene adjacent to GAL80 at a frequency of 1%, whereas in the pADH1-Gal4BD-spo11 strain, DSBs occurred exclusively in the GAL80 promoter at a frequency of 10%, near the Gal4 binding sequence (Fig. 3B). This difference in position may reflect local competition between these two adjacent regions. On a larger scale, the chromosome XIII ChIP-chip profile indicates that the number of Gal4BD-Spo11 DSBs around GAL80 was lower than that of Spo11 DSBs (Fig. 3A). Pulsed-field gel electrophoresis analyses confirm that the strongly stimulated GAL80 DSB was accompanied by a reduction in the number of surrounding DSBs, from the left end of chromosome XIII to the centromere on the right (Fig. 3C and D). In the Gal4BD-Spo11 strain, DSBs in the two regions of the left arm surrounding GAL80 occurred at a frequency 50 to 80% that of Spo11 DSBs. Consequently, the total DSB frequencies in the entire left arm were not significantly different in the Spo11 and Gal4BD-Spo11 strains (Fig. 3E). To determine if these differences in the distributions of the Gal4BD-Spo11 and Spo11 DSBs may be due to an impaired ability of Gal4BD-Spo11 to cleave some natural sites, we inactivated by site-directed mutagenesis the Gal4 binding sequence in the GAL80 promoter. This inactivation resulted in a decrease of GAL80 promoter DSB frequency, from 10 to 3%, and DSB formation occurred about 130 bp away in the same intergenic region (gel not shown). This lowered GAL80 promoter cleavage by Gal4BD-Spo11 was accompanied by an increase of DSB formation (between 1.1- and 1.9-fold) in the surrounding regions (Fig. 3F). This result shows that reduced DSB cleavage by Gal4BD-Spo11 away from GAL80 is due to this strongly targeted DSB. Finally, we confirmed the long-range inhibitory cis effect on DSBs surrounding the targeted GAL2 promoter DSB site on chromosome XII by pulsed-field gel electrophoresis with rad50S mutant cells (Fig. 3G, left panel). Interestingly, based on visual inspection of the pulsed-field gel, a DSB reduction was also observed around the targeted GAL2 DSB in another DSB repair-defective mutant, dmc1, which accumulates hyperresected DSBs after the removal of Spo11 (6) (Fig. 3G, right panel).
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Ten out of the 13 targeted sites with a Gal4 consensus sequence had this sequence located in an intergenic region. DSB targeting thus follows the property of natural DSB sites, which in their vast majority are located in intergenic regions (4).
In agreement with results from previous studies (9, 12), our data show very weak average DSB levels around the centromeres (
20 kb) in the presence of Spo11 as well as in the presence of Gal4BD-Spo11 (see Fig. S4 in the supplemental material). In addition, in the 20-kb regions around centromeres, there were significantly lower numbers of DSB sites induced by Spo11 (two sites; P < 0.0001) and Gal4BD-Spo11 (three sites; P < 0.001) than in the rest of the genome. However, the small number of Gal4BD binding sites within 20 kb from a centromere (YGL007W, CEN4, and GAL3) made it unlikely to see a global change between the two proteins when looking at all centromeric regions. These three sites behaved differently. YGL007W, located 14 kb to the left of CEN7, gave rise to a Gal4BD-Spo11 DSB (Fig. 2A), whereas CEN4 and GAL3, located 14 kb to the right of CEN4, did not (Fig. 1C). GAL3 was the second strongest Gal4BD-Spo11 binding site. The absence of DSB at GAL3 was verified both by ChIP-PCR (Fig. 4A) and by Southern blotting (Fig. 4B). DSB formation at GAL3 was also barely detectable in a dmc1 Gal4BD-Spo11 strain (Fig. 4C). Thus, we conclude that the binding of Gal4BD-Spo11 at GAL3 is not sufficient for DSB formation.
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Finally, the 25 binding sites that did not give rise to a DSB appear to be further away from a natural Spo11 DSB site than those where a Gal4BD-Spo11 DSB was detected (median values, 39 kb versus 11 kb; Wilcoxon signed-rank test; P < 0.014). This suggests that Gal4BD-Spo11 DSBs are better induced if binding occurs close to a natural DSB region. On the contrary, no DSB was formed at the binding site YGR183C, located 9 kb from one of the strongest endogenous Spo11 DSB sites, YGR176W (Fig. 1C). In this case, DSB formation at the Gal4BD binding site may be inhibited by competition with the nearby strong natural site.
In summary, it appears that Gal4BD-Spo11 generally follows the trend of the natural DSB sites with respect to chromosomal context but that cold but potentially permissive regions can be warmed up upon the assisted binding of Spo11.
Targeting of Gal4BD-Spo11 DSBs in a reporter cassette and position effect. Our ChIP-chip data show that Gal4BD-Spo11 association with chromatin is not always sufficient for DSB formation. It was previously shown that the chromosomal context has a strong influence on DSB formation, since a reporter cassette containing the URA3 gene and arg4 sequence shows various DSB levels depending on the place of insertion along the chromosome (10, 52); it showed very high levels of DSB formation at his4, located in a hot region of chromosome III, and very low levels when it was inserted at RVS161, in the cold centromere-associated chromosome III region (8). To determine whether the targeting of Gal4BD-Spo11 to binding sites would follow or overcome such position effects, we constructed a similar reporter cassette, URA3-gal2, containing four Gal4 binding sequences (Fig. 5A). This reporter cassette was introduced at the same loci (his4 and RVS161) as the URA3-arg4 cassette in strains expressing either Spo11 or Gal4BD-Spo11. In the Spo11 strains, frequent DSBs located in the plasmid sequence to the right of gal2 were observed upon insertion at his4 but none were observed upon insertion at RVS161 (Fig. 5B and D). In the strain expressing Gal4BD-Spo11, at his4, DSBs also frequently occurred in the junction plasmid sequence but, in addition, targeted cleavage occurred in the vicinity of the Gal4 binding sequences (Fig. 5B and D). Thus, Gal4BD-Spo11-specific DSBs can be targeted within an ectopic insert containing a Gal4 binding sequence. At RVS161, weak cleavage by Gal4BD-Spo11 occurred in the plasmid sequence, but none occurred in the Gal4 binding sequences, indicating a strong repression of target DSBs (Fig. 5B and D). Together, these results indicate that Gal4BD-Spo11 is subjected to DSB repression occurring in this naturally cold region of chromosome III.
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strains and examined meiotic DSB formation. Remarkably, Gal4BD-Spo11-specific DSBs occurring in the vicinity of the Gal4 binding sequences were restored (Fig. 5C and D). Therefore, the absence of DSB formation at the RVS161 locus by tethered Spo11 was due to centromere proximity. This finding further confirms that local factors control the cleavage step. |
DISCUSSION |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Genome-wide modification of DSB distribution. In our previous study of DSB formation by the Gal4BD-Spo11 protein (38), we found that this fusion protein was able to introduce DSBs at a few natural Spo11 sites and in the Gal4 binding site-containing promoters of the GAL2, GAL1, GAL10, and GAL7 genes. We have now established the genome-wide distribution of the cleavage regions of Gal4BD-Spo11 in parallel with those of Spo11. The results confirm the dual and highly proficient properties of the fusion protein to cleave natural and novel sites. Indeed, 80% of the Gal4BD-Spo11 DSBs occur at Spo11 DSB sites, suggesting that although DSB frequency at some endogenous Spo11 sites might be affected, the choice of cleavage sites is extensively conserved between the two proteins. However, this is not a simple additive gain of novel targeted sites, since a large number of Spo11 sites (24%) are no longer cleaved by Gal4BD-Spo11 and the genome-wide distribution of DSBs is profoundly modified. Noteworthily, this redistribution has no consequence either on the sporulation efficiency or the spore viability (96%) of the diploid cells carrying the Gal4BD-Spo11 protein (38). We conclude that the natural profile of the meiotic recombination initiation sites in S. cerevisiae cells is flexible. However, as discussed below, control on potential target sites exists, since DSBs do not occur at all Gal4BD-bound sites.
Long-range cis-effect repression of DSB formation. Despite the overlap between Spo11 and Gal4BD-Spo11 DSB sites, some natural DSBs appeared as no longer cleaved by Gal4BD-Spo11. Part of this nonoverlap can be explained by the use of a stringent threshold criterion since 54% of the Gal4BD-Spo11-specific sites are secondary Spo11 DSB sites, indicating that most of these cases represent bona fide cleavage sites for both proteins but that cleavage occurred at different frequencies. DSB formation is reduced around the strongly targeted Gal4BD-Spo11 sites over long distances. We show that this is not due to an impaired ability of Gal4BD-Spo11 to cleave some natural sites since the mutation of the GAL80 binding site restores DSB formation in the surrounding regions.
The cumulated DSB frequency in a chromosomal region is thus kept constant. This DSB homeostasis occurring at the level of initiation over a chromosomal region likely proceeds through mechanisms very different from the classical crossover interference, in which a crossover in one region makes it less likely that another will be found nearby between the same chromosome pairs (reviewed in reference 24). Here, it is unlikely that the DSB reduction occurs on the same DNA molecule as the one that is cleaved. Indeed, such double cutting events are predicted to be very rare (for example 0.1% for two sites occurring each with a 10% frequency). Thus, the modification induced by the targeted DSB site may occur much more frequently than the cleavage itself. Alternatively, if one considers as one unit both sister chromatids or even all four duplexes of a bivalent, a site that shows up with breakage of 10% of the DNA may be considered as a single break at that locus in 40% of meiotic cells. Given the distances over which this phenomenon occurs, one can envisage that the suppression might occur in trans along all (or some) copies of a chromosome within a cell. Thus, it may be that suppression is linked directly to DSB formation, rather than reflecting competition for limiting factors independent of DNA cleavage.
As suggested before for short distances, cis-acting effects on DSB frequencies may be viewed as a competition phenomenon in which the DSB-forming factors available over a given chromosomal region would be attracted to the new and stronger site, rendering the surrounding sites less likely to be cleaved (52). Candidate factors are the DSB proteins Rec102, Rec104, and Mer2 which at early times of meiosis are chromatin associated but do not appear to preferentially localize at hot spots (17, 22). Perhaps, upon DSB site selection or cleavage, these proteins transiently diffuse to the chosen site and deplete other potential sites. Alternatively, preferential and perhaps earlier selection of the prominent DSB sites may induce a cis-acting inhibitory signal, for example, conformational changes or nucleosome modifications.
Position effects for cleavage of sites that are bound by Gal4BD-Spo11. This study allows us to distinguish three types of chromosomal regions with respect to DSB formation: the naturally proficient regions, the cold but permissive regions which can be warmed up by tethering Spo11 through a heterologous DNA binding domain, and the refractory cold regions that remain cold even in the presence of bound Gal4BD-Spo11 protein.
The most spectacular examples of a repressive position effect on Gal4BD-Spo11 potential cleavage sites occur at the natural Gal4BD binding site GAL3 and in the gal2 promoter-containing reporter cassette inserted at RVS161. Both are located in very cold DSB regions and close to a centromere (14 kb [or 23 kb with the Gal4BD-Spo11 construct] and 17 kb away, respectively). In both cases, ChIP analyses clearly demonstrate that Gal4BD-Spo11 is bound there throughout meiosis but that no detectable DSB formation is observed, whereas bringing Gal4BD-Spo11 to other regions like HIS4 is sufficient to produce DSBs. This position effect could reflect either a local inability of cleavage by Spo11 or a persistence of the "tight binding" intermediate, in which Spo11 is active and makes breaks but reseals them without engaging in the subsequent step, the irreversible cleavage and formation of stable DSBs (40). The molecular mechanisms underlying this position effect on DSBs remain to be identified, but most interestingly, we found that moving the CEN3 sequence 21 kb away was sufficient to restore a targeted Gal4BD-Spo11 DSB site in the URA3-gal2 reporter construct at RVS161.
One can envisage several nonexclusive ways for the centromere to exert this inhibitory effect. This inhibition might be through chromatin modifications, although no differences between cold centromere-associated and hot regions have been detected by measuring DNase I hypersensibility (10). No specific histone modifications have so far been identified at the proximity of budding yeast centromeres, except for a single nucleosome containing the centromere-specific H3-like protein, Cse4, on the core centromere sequence (35). Another possible mechanism is protection due to the binding of the cohesin complex over a 30- to 50-kb region centered on centromeres (13, 25, 31). This does not appear to be the case, since we examined genome-wide DSB formation in a rec8 rad50S strain and found that DSB formation was still low close to centromeres (data not shown). A third hypothesis would be that the centromere is located in a chromosome territory inaccessible to the DSB-forming machinery. Early in meiosis I, centromeres are paired in nonhomologous pairs (50). This pairing is then disrupted, and chromosome ends become clustered in a limited region of the nuclear envelope at the bouquet stage (44). This complex process may spatially exclude centromeric regions from DSB formation. Since DSB distribution is not affected in mutants suppressing the bouquet (48, 51) and DSB formation likely naturally occurs before the bouquet stage (45, 49), DSB formation may occur rather at the time of centromere pairing. Although we see a global reduction of DSB formation around centromeres when examining all chromosomes together, we have noticed that DSB repression close to an individual centromere is often asymmetrically focused on one side of the centromere. For instance, the left side of CEN3 is a hot region and the other side is cold. Similarly, some genes located near centromeres, like GAL3, are cold, whereas a DSB can be targeted at others (like YGL007W). Previous experiments have shown that the orientation of the centromere does not affect the directionality of recombination repression (29). One attractive explanation for these observations would be that centromeres may act as a boundary separating a DSB-permissive region from a DSB-refractory region, although they may not be a barrier for recombination intermediates since coconversion has been observed across the CEN3 region (47).
The centromere regions are not the only regions in the genome to be repressed while Gal4BD-Spo11 is bound. None of the Gal4BD-Spo11 binding sites in subtelomeric regions give rise to a DSB. However, we cannot rule out the possibility that for some of these sites, DSB frequency is underestimated due to the rad50S mutation (8).
To better understand these cleavage position effects, it will be interesting to determine which DSB factors bind the sites that are bound but not cleaved by Gal4BD-Spo11 and which proteins cannot be recruited to these sites.
Potential benefits of limiting DSB formation. From the data reported here and others before, we can identify two limitations of DSB formation: repression of DSB formation in certain regions of the chromosome (for example, at GAL3 and CEN3) and a balance among DSB sites such that the total number of DSBs in a chromosomal region is not changed (for example, on chromosome XIII upon DSB formation at GAL80). What could be the benefit of such DSB limitation? It has been suggested that too much recombination may be associated with missegregation (16, 33). Making about 150 DSBs in the genome is potentially dangerous for the cell as a single unrepaired DSB can be lethal. Thus, limiting the total amount of DSB formation might be an additional way to ensure that all the DSBs will be repaired properly. Restriction on the initiation position might facilitate the optimal function of chiasmata in chromosome segregation, for example, by avoiding crossing over too closely to a centromere. Our results obtained when we mutated the GAL80 promoter sequence also suggest that the reciprocal control exists which allows new DSBs to appear when DSB activity is lowered or inhibited in a chromosomal region. Similar effects on DSB homeostasis seem to exist in mammals, where the disappearance of one hot spot is often accompanied by the apparition of a new, nearby hot spot (latent hot spot) such that the global recombination frequency is unchanged (21).
Identifying the factors responsible for maintaining a constant level of recombination initiation over a particular chromosomal region and those repressing DSB formation in the subdomains of the chromosome remains an important challenge, but our ability to modulate the number and locations of sites bound by Spo11 provides new insights into the central issue of the nonrandom distribution of meiotic recombination events and a powerful method to modify meiotic recombination by a simple modification of the Spo11 protein.
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ACKNOWLEDGMENTS |
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strain and communicating results before publication and Richard Durbin from the Sanger Institute and Ed Louis for allowing us to use the unpublished SK1 sequence information. We thank Michèle Vedel for constructing the pAP11 plasmid. We also thank Christine Mézard and Pierre-Antoine Defossez for comments that improved the manuscript and the anonymous reviewers for helpful suggestions. This work was supported by grants from the Association pour la Recherche sur le Cancer (to A.N.). N.R. was supported by a predoctoral fellowship from Institut Curie. N.U. was supported by postdoctoral fellowships from the CNRS and the Institut Curie.
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FOOTNOTES |
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Published ahead of print on 22 December 2006.
Supplemental material for this article may be found at http://mcb.asm.org/.
Present address: Kyoto University, Radiation Biology Center, Late Effect Studies Konoe-cho, Yoshida, Sakyo-ku, Kyoto-shi 606-8501, Japan.
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