Spo11-Accessory Proteins Link Double-Strand Break Sites to The

Spo11-Accessory Proteins Link Double- Strand Break Sites to the Chromosome Axis in Early Meiotic Recombination

Silvia Panizza,1 Marco A. Mendoza,1,4 Marc Berlinger,1,5 Lingzhi Huang,1 Alain Nicolas,2 Katsuhiko Shirahige,3 and Franz Klein1,* 1Department of Chromosome Biology, Max F. Perutz Laboratories, University of Vienna, Dr. Bohr-Gasse 1, A-1030 Vienna, Austria 2Recombinaison et Instabilite´ Ge´ ne´ tique, Institut Curie Centre de Recherche, Centre National de la Recherche Scientiﬁque UMR3244, Universite´ Pierre et Marie Curie, 26 rue d’Ulm, 75248 Paris Cedex 05, France 3Laboratory of Genome Structure and Function, Research Center for Epigenetic Disease, Institute of Molecular and Cellular Biosciences, University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-0032, Japan 4Present address: Institut de Ge´ ne´ tique et de Biologie Mole´ culaire et Cellulaire, 1 rue Laurent Fries, 67404 Illkirch Cedex, Strasbourg, France 5Present address: EUCODIS Bioscience, Viehmarktgasse 2A, A-1030 Vienna, Austria *Correspondence: [email protected] DOI 10.1016/j.cell.2011.07.003

SUMMARY transesterase highly conserved through evolution (Bergerat et al., 1997; Keeney et al., 1997; reviewed in Keeney, 2001). Meiotic recombination between homologous chro- DSB formation also depends on a number of Spo11-accessory mosomes initiates via programmed DNA double- proteins, which have largely been identified in budding yeast, strand breaks (DSBs), generated by complexes but whose function is only partially understood (reviewed in comprising Spo11 transesterase plus accessory Keeney, 2001). These accessory proteins interact with each proteins. DSBs arise concomitantly with the develop- other (Maleki et al., 2007) and are thought to form a complex ment of axial chromosome structures, where the during DSB formation, termed the pre-DSB recombinosome. Among these factors, Rec102, Rec104, and Ski8 are required coalescence of axis sites produces linear arrays of for Spo11 dimerization, DNA binding and efficient nuclear chromatin loops. Recombining DNA sequences retention (Arora et al., 2004; Kee et al., 2004; Prieler et al., 2005; map to loops, but are ultimately tethered to the Sasanuma et al., 2007). Three quite separate Spo11 accessory underlying axis. How and when such tethering proteins Rec114, Mer2, and Mei4 (collectively named here occurs is currently unclear. Using ChIPchip in yeast, RMM), have been shown to form a subcomplex, to partially co- we show that Spo11-accessory proteins Rec114, localize on chromatin and to be required for Spo11 binding to Mer2, and Mei4 stably interact with chromosome sites of DNA cleavage (Li et al., 2006; Sasanuma et al., 2007). axis sequences, upon phosphorylation of Mer2 by Activation of Mer2 requires cyclin dependent kinase-mediated S phase Cdk. This axis tethering requires meiotic phosphorylation (Henderson et al., 2006), suggesting that this axis components (Red1/Hop1) and is modulated protein helps link DSB formation to the progression of the meiotic in a domain-specific fashion by cohesin. Loss of program. In addition to Spo11 and the accessory proteins, which are all essential for DSB formation, a number of other factors are Rec114, Mer2, and Mei4 binding correlates with important for wild-type levels and distribution of DSBs (reviewed loss of DSBs. Our results strongly suggest that hot- in Hunter, 2007). spot sequences become tethered to axis sites by DSBs occur preferentially in clusters of sites called ‘‘hot- the DSB machinery prior to DSB formation. spots’’ where, in yeast, the probability of DSB formation is 100–1000 times higher than at other sites (Baudat and Nicolas, INTRODUCTION 1997; Gerton et al., 2000; Blitzblau et al., 2007; Buhler et al., 2007; Pan et al., 2011). Hotspot activity is primarily determined In sexually reproducing organisms, the production of gametes by local features of chromatin structure, notably absence relies on meiosis, in which a single round of DNA replication is of nucleosomes (Pan et al., 2011; for review see Lichten, followed by two rounds of chromosome segregation. A critical 2008). Indeed, certain histone modifications are required to step in meiosis is recombination between homologous chromo- achieve wild-type levels of DSB formation and their patterns somes. Crossovers resulting from recombination are essential correlate to the DSB landscape (Yamashita et al., 2004; Miecz- for the physical links between homologs that mediate their kowski et al., 2007; Borde et al., 2009). However, factors segregation to opposite poles at the first meiotic division. other than chromatin modifications are required to explain Recombination is initiated by the introduction of DNA double- long-range effects, such as the fact that the activation of a hot- strand breaks (DSBs) catalyzed by Spo11, a meiosis-specific spot can interfere with that of a neighboring one over distances

372 Cell 146, 372–383, August 5, 2011 ª2011 Elsevier Inc. of many kb (Wu and Lichten, 1995; Xu and Kleckner, 1995; (Blat et al., 2002) suggests again a role for axial components in Keeney, 2001). this domain-dependent organization, but how chromosome Chromosomal organization plays an important role for meiotic structure and the initiation of recombination are mechanistically recombination. DSBs arise concomitantly with the development coupled is unclear. of axial structures (Padmore et al., 1991) by coalescence of ‘‘axis Here, using chromatin immunoprecipitation on microarray association sites,’’ locally AT-rich regions that are preferential (ChIPchip) analysis in budding yeast, we show that the Spo11- binding sites for meiotic axis components including cohesin, accessory proteins, Rec114, Mer2, and Mei4 (RMM), whose producing a linear array of loops (Blat and Kleckner, 1999; Blat function was previously unclear, bind stably to DNA at chromo- et al., 2002; Glynn et al., 2004). Given that cohesion is estab- some axis association sites, rather than to loop sequences lished coreplicationally, loops of sister chromatids are expected containing DSB hotspots. We show that this association to be in register (Nasmyth, 2005). Co-oriented linear loop arrays depends on axis components Hop1 and Red1 and that cohesin are joined at their base by a single axial structure (reviewed modulates both RMM and axis component binding in a domain- in Zickler and Kleckner, 1999; Kleckner, 2006). Structural axes wise fashion. Importantly, the chromosomal regions that lose include, among other things, meiosis-specific components RMM and axial component binding in the absence of cohesin such as cohesin Rec8 and, in yeast, the proteins Red1 and also exhibit a localized reduction in DSBs. Thus, the activation Hop1 (Smith and Roeder, 1997; Klein et al., 1999; reviewed in of DSB hotspots requires localized tethering of RMM to the chro- Zickler and Kleckner, 1999). By midprophase, structural axes mosome axis via axial element proteins and cohesin, which of homologous chromosomes are intimately linked by trans- implies that loop-axis tethering occurs prior to or at the time of versal filaments forming the synaptonemal complex (SC). The DSB formation. We also show that S-Cdk1 controls recruitment yeast transversal filament component is Zip1. of Spo11 accessory proteins at the interphase between DNA Early electron microscopy (EM) studies identified nodular replication and DSB formation. Our results show that the devel- structures atop or across the SC, whose number and distribution oping chromosome axis plays a critical role in controlling initia- corresponded to crossovers detected genetically or cytologi- tion of meiotic recombination, reveal that a central role for cally. These and subsequent studies showed that recombination RMM proteins is to establish the association between pre-DSB occurs in complexes that are physically associated with the recombinosomes and structural components, implicate this chromosome structural axes/SC (reviewed in Zickler and Kleck- association as a key feature to couple DSB formation to comple- ner, 1999). The DNA sequences at which DSBs occur map to tion of S phase, and suggest that domainal structural organiza- positions in between axis-association sites. By implication, tion along chromosomes is linked to domain-wise control of the DNA sequences that are directly undergoing biochemical DSB formation. Given that Spo11 is highly conserved and changes for recombination are associated to underlying axes mammalian homologs of the RMM complex have recently indirectly, with the involved loop sequences tethered to chromo- been discovered (Kumar et al., 2010), these features are likely some axes by protein-protein interactions between components to be conserved across diverse species. of recombination complexes and components of chromosome axes (Blat et al., 2002; Kleckner, 2006). This tethered loop-axis RESULTS complex (TLAC) model indeed helps to explain several crucial features of meiotic recombination (Kleckner, 2006; Kim et al., Axis Association Sites Are Preferential Binding Sites 2010), but it is currently unknown at which stage of recombina- for Red1, Hop1, Rec8, and Zip1 tion such tethering occurs. Previous studies defined preferred binding sites for meiotic axis- There is indication that chromosome structure influences the component Red1 and cohesin on chromosome 3 (Blat et al., pattern of DSB formation. For example, wild-type levels and 2002). We confirmed and extended these findings using ChIP distribution of DSBs along chromosomes in yeast depend on analysis of Rec8, Red1, Hop1, and Zip1 by high-resolution Affy- the chromosome axis proteins Rec8, Hop1, and Red1 (Schwa- metrix arrays and compared our genome-wide map to that of the cha and Kleckner, 1997; Kugou et al., 2009; Kim et al., 2010). meiotic cohesin subunit Rec8 (Glynn et al., 2004). Unless other- Similarly, in C. elegans, the absence of structure component wise specified, all ChIPchip samples were collected from condensin I induces a large-scale modulation of DSB positions synchronous time courses, 4 hr after transfer to sporulation (Mets and Meyer, 2009). However, there is no direct evidence medium (SPM, t4), when DSBs are most abundant in wild- for pre-DSB establishment of axis association, nor is it known type S. cerevisiae strains under our conditions (data not shown). for any stage of recombination which proteins mediate tethering For all experiments, the progression through nuclear divisions as it evolves through meiotic prophase. was scored by DAPI (data not shown). A third aspect of meiotic recombination, beyond local bio- Results are presented after noise filtering with a 3 kb sliding chemical events and association of recombination complexes window mostly for chromosome 3 (results for all other chromo- with chromosome structure components, is the control at somes can be accessed online). The 3 kb sliding window was domain-wise level. Chromosomes contain recombination-profi- chosen for the purpose of clarity. Analysis at a resolution of cient (hot) and recombination-poor (cold) regions, and when an 500 bp greatly refined the accuracy of the number and position active local hotspot in an active domain is transferred into of peaks, but it did not change the conclusions presented below. a cold domain, it acquires the properties of the cold domain As an example of such 500 bp resolution, two profiles obtained (Wu and Lichten, 1995). The fact that Red1 localizes prefer- from different proteins are provided in Figure S2C (available entially to the DSB-active domains along chromosome III online) for a selection of small and large chromosomes.

Cell 146, 372–383, August 5, 2011 ª2011 Elsevier Inc. 373 Rec8 and Zip1 showed strong centromeric signals on all chromosomes (Figure 1A and Table S2; http://www.univie.ac.at/ SFBChromosomeDynamics/documents.html), consistent with their function at centromeres in early prophase (Bardhan et al., 2010). Furthermore, we found higher Pcorr values between Rec8 and Zip1 (0.68) and between Red1 and Hop1 (0.88), and smaller values for the other combinations. Aside from centromeric regions, Rec8 and Zip1 were distributed more uniformly along the arms than Red1/Hop1. These results further deﬁne the positions of axis-association sites, as well as the distribution of axis-association proteins along these sites, on a genome-wide basis. We note that these results represent population average tendencies. The subset of axis sites bound by axis components in individual cells at any given time may vary.

Figure 1. Axis Proteins Bind Identical Sites in vivo The Spo11-Accessory Proteins Rec114, Mer2, and Mei4 ChIPchip profiles of Zip1 (black, FK1), Rec8-HA3 (green, FK1091), V5-Red1 Stably Associate with Axial Sites Rather Than DSB Sites (red, FK3870), and Hop1 (blue, FK3307) for chromosome 3. Black bars, DSB Meiotic DSBs occur between axis association sites, in regions sites (in arbitrary scale) (Baudat and Nicolas, 1997). For all profiles in this that, in organized chromosomes, would comprise the ‘‘loop’’ work ChIP/whole-cell extract (WCE), signal intensity was plotted against regions (Baudat and Nicolas, 1997; Gerton et al., 2000; Blitzblau the chromosomal position after denoising and after decile normalization (the et al., 2007; Buhler et al., 2007; Pan et al., 2011) (see also Fig- 10% lowest values set below 1, see the Experimental Procedures). Numbers ure 1A). It might thus have been expected, analogously, that represent the result of genome-wide comparison between pairs of profiles, presented in all figures in identical format: names of the profiles; number of components required for DSB formation, e.g., Spo11 and its matching peaks/number of peaks of weaker profile and percent peak overlap; accessory proteins, might also bind these regions. To be able overlap between valleys (local minima) of profile 1 and peaks of profile 2; p, the to detect RMM proteins by ChIP analysis, we generated probability to accept a random, hypergeometric model, and Pcorr, Pearson C-terminally tagged versions of Mer2 (tagged with myc9), correlation between the two profiles. Cells were collected 4 hr after transfer to Rec114 (tagged with myc13) and Mei4 (tagged with HA6) (see SPM (t4). A filled oval marks the centromere 3 (CEN3), and a red arrow marks the Experimental Procedures); unless specified otherwise, all a transposon. See also Table S2. experiments described in this work were done with these constructs. Unexpectedly, genome-wide ChIPchip profiles of Mer2, Rec114, and Mei4, closely resembled that of Hop1 and Importantly, the close match between the two profiles shows the Red1 (Figures 2A and 2B). The association profiles of the three low level of experimental noise. Two types of statistical analyses components at t4 were nearly identical (Figure 2A). Almost all were employed to assess the degree of similarities among the 900 peaks of the three profiles matched (>92%), with a genome-wide data sets for different proteins. First, the percent- Pcorr > 0.93 for all three pair-wise comparisons (Figure S1). age of overlapping peaks was calculated (two peaks are Conversely, these binding signals exhibit local minima of DNA considered overlapping if their maxima are less than 1 kb apart binding in the intervening regions, where DSBs are prominent. [d < 1 kb], see the Experimental Procedures). An even more Confirming this, genome-wide peak matching revealed an stringent method is the Pearson correlation (Pcorr), which inverse correlation between DSBs and Rec114 binding: 65% measures the linear correlation between two data sets. Pcorr of DSB peaks (Buhler et al., 2007) matched to local minima of values assume a value of 1 for perfect correlation and À1 for Rec114 (with a relaxed matching distance d < 2 kb), while only a mirror image. A high Pcorr requires that peaks are at corre- 13% of DSB peaks matched a Rec114 peak by the same crite- sponding positions and that their heights are in linear proportion. rion (chromosome 2 is shown as an example, Figure 2E). We

Pcorr was assessed after noise filtering with a 1 kb sliding also obtained a negative Pearson coefficient [Pcorr(10kbsw) = window. À0.37] for the two datasets (see the Experimental Procedures We found that the DNA binding profiles for Red1, Hop1, Zip1, for details). Thus, DNA association peaks of RMM alternate and Rec8 were largely identical (Figure 1A; see Table S2 for with DSBs. numerical data for all chromosomes), revealing that the trans- Given that all ChIPchip profiles analyzed so far correlated versal filament component Zip1 contacts chromosomes at cohe- positively with cohesin and negatively with DSBs, we included sin-binding sites. This result was confirmed by our genome-wide in our analysis a ChIPchip profile of Spo11 (tagged with statistical analysis. For instance, 80% of Zip1 peaks, 81% of myc18) pulled down in the absence of FA-crosslinking in Red1 peaks, and 72% of Hop1 peaks matched Rec8 peaks rad50S cells, as a control. In this mutant background, Spo11 (d < 1 kb, p < 10À10). stays covalently attached to DSB ends, so that binding sites Previous analyses showed that Red1 associates preferentially are expected to positively correlate with DSB positions, which with DSB-rich domains along chromosome 3 (Blat et al., 2002). was indeed the case (Figures S2A and S2B). In the present study, Hop1 and Red1 exhibit the same prefer- Our results show that RMM proteins strongly interact with ence for DSB-rich domains throughout the whole genome. axis-association sites, rather than the intervening regions, which

374 Cell 146, 372–383, August 5, 2011 ª2011 Elsevier Inc. correspond to chromatin loops in condensing chromosomes. This finding strongly implies that one important role of RMM proteins is to mediate the linkage between the DSB-promoting machinery and chromosome axes (Discussion). Importantly, the catalytically dead spo11-Y135F mutant, which can associate with chromosomes but cannot induce DSB formation, exhibits the same Rec114 binding profile as wild-type (81% of wild-type peaks matched, Pcorr = 0.9, Fig- ure 3B and Figure S4B). Thus, RMM localization cannot be a consequence of DSB formation. These findings further imply that RMM/axis association should be established early in meiotic G2/prophase. When followed at 1 hr intervals at four different sites on chromosome 3 by ChIP and qPCR, robust binding was found at an axis site (core 1), after 3, 4, and 5 hr in SPM, whereas binding to two strong DSB sites on loops was weak, but occurred with similar timing (Figure 2C). Similar results were obtained for Mer2 (Figure S1B). ChIPchip profiles at t3 and t4 were almost identical for both Rec114 (91% of peaks at t3 match with t4, Pcorr = 0.91) and Mer2 (90% of peaks match between t3 and t4, Pcorr = 0.89) (Figures 2A and 2B and Figure S5C).

The Spo11-Accessory Proteins Rec114, Mer2, and Mei4 Accumulate in DSB-, Hop1-, and Red1-Rich Genomic Regions Meiotic chromosomes consist of broad DSB-rich and DSB-poor regions (Baudat and Nicolas, 1997; Blitzblau et al., 2007; Buhler et al., 2007). While the local negative correlation between RMM and individual DSB positions was a surprising result, it might be expected that these proteins accumulate preferentially in DSB-rich regions. Indeed, we found that larger DSB-rich chromosomal domains showed an overall accumulation of Rec114 (and Mer2, Mei4 and Hop1, Red1) peaks (Figures 1, 2A, 2B, and 2E). These results are consistent with the previous ﬁnding

arbitrary scale) mark the positions of strong DSB sites (Baudat and Nicolas, 1997). (B) ChIPchip profiles of Rec8-HA3 (FK1091, t4, green), Rec114-myc13 (FK3307, t4, red, repeat R2), Hop1 (FK3307, t4, blue), and Rec104-myc9 (FK4370, t4, ochre) are shown for chromosome 3. Black bars, DSB sites (Baudat and Nicolas, 1997). (C) qPCR results of ChIP of Rec114-myc (FK3307) at four positions on chromosome 3 over a meiotic time course. Core 1 is a Rec8-binding site at 219.5 kb, ADP1 lies in a DSB-cold region (136.5 kb), and DSB1 and DSB3 are hotspots at positions 212 and 225.2 kb. The mean ± standard variation (s) for the ratios of n biological repeats is indicated. (D) The mean Rec114-myc13 (FK3307, t4), Hop1 (FK3307, t4), V5-Red1 (FK3870, t4), and CO (Mancera et al., 2008) signal per chromosome was plotted against chromosome length. r is the slope of the regression line for the 13 larger chromosomes, and p is the probability to accept that regression model for the three smallest chromosomes. (E) DSBs (Buhler et al., 2007) (blue) and Rec114-myc13 profile (FK3307, 4 hR; red) were smoothed at a bandwidth of 3 kb and plotted for chromosome II. Pcorr(10kbsw), genome-wide Pearson correlation, calculated for 10 kb re- Figure 2. DSB-Promoting Factors Map to Cohesin Sites, Rather gions in 5 kb steps and averaged (see the Experimental Procedures). Than DSB Hotspots in DSB-Rich Regions (F) At a bandwidth of 70 kb, a positive correlation between DSBs and Rec114 is (A) ChIPchip profiles of Rec114-myc13 (FK3307, red, repeat R1), Mer2-myc9 found for the whole genome. The SC component Zip1 (green) does not show (FK3222, blue), and Mei4-HA6 (FK2385, green), all t4. Genome-wide correla- a positive correlation with DSBs. Because of the large sliding window, peak tion coefficients are shown in Figure S1A. Regions where qPCR was per- matching is not appropriate. formed, centromere and transposon positions are marked. Black bars (in See also Figures S1, S2, and S3.

Cell 146, 372–383, August 5, 2011 ª2011 Elsevier Inc. 375 Figure 3. Mer2, S-Cdk, and Mei4 Recruit Rec114 to Axial Sites in DSB-Rich Regions (A and B) Profile of Rec114-myc13 in wild-type and different mutant backgrounds: (A) wild-type (FK3307, blue), mer2D (FK3005, red), and mei4D (FK3006, brown) all at t3 (A) and (wild-type (FK3307, blue, t4), spo11-Y135F (FK2993, green, t4), rec104D (FK2999, orange, t3), clb5D, clb6D (FK4196, black, t4) (B). A filled oval marks centromere 3. Genome-wide Pearson correlation coefficients are indicated. (C) qPCR on ChIP of Rec114-myc13 and Mei4-HA6 in MER2 mer2D (FK3219 and FK3214, respectively) and mer2-S30A mer2D cells (FK3220 and FK3215). The western blot in the right panel shows that mer2-S30A does not affect Rec114-myc levels. The mean ± s for the ratios of n biological repeats is indicated. See also Figure S4.

(Mer2 and Mei4 not shown). Red1 and Hop1 exhibited the same behavior (Figure 2D). This observation strongly suggests that the relative abundance of these proteins may cause the increased DSB formation on the three smallest chromosomes.

RMM Localization Is Modulated during Meiotic Progression To determine whether RMM proteins persist at axial sites at later stages of recombination, we performed coimmunostaining of Rec114- myc13 and the SC component Zip1 on spread meiotic nuclei. When the localization of Rec114 and Zip1 foci was compared in early nuclei, hardly any overlap was found (Figure S3A). At later stages, Rec114 foci dimmed and disap- peared in synapsed chromosome regions, while they remained bright in unsynapsed regions of the same nucleus (Figure S3C). This observation has also been reported by others in a different yeast strain background (Li et al., 2006) and suggests a negative regulation of RMM proteins by repair and synapsis. Consis- that the two DSB-rich regions on chromosomes 3 are Red1- tently, we quantified the amount of Rec114 foci in different stages rich domains (Blat et al., 2002). We quantified our results (according to Zip1 staining), and we found that they peak at using the Pearson correlation method after applying a broader the time of DSB formation at late leptotene, followed by a progres- sliding window (70 kb), a method that creates a longer wave- sive decline (Figure S3B). length signal to reveal domain-wide effects (Blat et al., 2002; Dekker, 2007). We found that Rec114 (Figure 2F), as well as Mer2 Recruits Rec114 and Mei4 to Axial Sites Mer2, Mei4, Red1, and Hop1 (data not shown), exhibited To analyze functional dependencies among DSB accessory a binding pattern that mirrored the large-scale distribution proteins with respect to axis association, we analyzed Rec114 of DSBs. Other proteins, such as Rec8 or the SC protein Zip1 ChIPchip profiles in mutants lacking other accessory proteins. did not show a positive correlation with DSBs using this In the absence of either one of Rec114’s binding partners, method (Figure 2F and data not shown). Interestingly, the three Mer2 or Mei4, the interaction of Rec114 with axial sites was smallest yeast chromosomes, recently shown to receive more strongly reduced, resulting in loss of 90% and 85% of matching DSBs (Pan et al., 2011) and more CO per kb (Chen et al., peaks (mer2D and mei4D; Figure 3A and Figure S4A). In contrast, 2008; Mancera et al., 2008) than longer ones, bound signifi- in rec104D cells, binding of Rec114 to DNA was reduced, but not cantly more RMM per unit length than longer chromosomes, as much as in mer2D or mei4D mutants (Figure 3B). A time calculated as the mean of RMM signals per chromosome course analysis of ChIP followed by qPCR confirmed the

376 Cell 146, 372–383, August 5, 2011 ª2011 Elsevier Inc. Figure 4. Axial Element Components Hop1 and Red1 Recruit Mer2 to Axial Sites in DSB-Rich Regions (A and B) Profile of Mer2-myc9 in wild-type and various mutant backgrounds: wild-type (FK3222, blue), mei4D (FK3265, red), rec114D (FK3264, green) (A) and wild- type (FK3222, blue), mer2-S30A (FK4108, black), clb5D, clb6D (FK4099, orange), red1D (FK3858, lilac) (B). A filled oval marks centromere 3. Genome-wide Pearson correlation coefficients are indicated. (C) qPCR of ChIP of Mer2-myc9 of the same DNA used for ChIPchip. The ratio of the signals core 1:ADP1 is plotted for each experiment and the mean ± s for the ratios of n biological repeats is indicated. See also Figure S5.

Mer2 Phosphorylation by Cyclin-Dependent Kinase Is Required for Recruitment of Rec114 and Mei4 to Axis Sites DSB formation normally occurs after S phase (Borde et al., 2000), although DNA replication per se is not essential (Hochwagen et al., 2005). Correct temporal coordination of replication and DSB formation involves phosphorylation of Mer2 at serine 30 by S phase cyclin Clb5-, Clb6-dependent kinase (S-Cdk) (Hender- son et al., 2006). It has been proposed that S-Cdk and Ddk phosphorylate Mer2 along newly replicated DNA as one of several steps toward activation of the DSB machinery (Mura- kami and Keeney, 2008). An important aspect of this model, unad- dressed so far, is whether the localization of Mer2 to DNA requires these kinase activities— that is, whether S-Cdk modifies it in situ. To explore this possibility, we first analyzed ChIP- chip profiles of nonphosphorylatable mer2- S30A and wild-type Mer2 in a mutant lacking S-Cdk (clb5D, clb6D), in which DNA replication is undetectable (Dirick et al., 1998; Stuart and Wittenberg, 1998). The pattern of Mer2 association was very similar to that seen in the wild-type, albeit with a slightly reduced intensity (Figures 4B and 4C). The number of peaks detected in the mer2-S30A and in clb5D, clb6D mutants presence of a reduced but still substantial amount of Rec114 in were 80% and 53% respectively of those of a wild-type profile. rec104D (Figure S4A). Genome-wide analysis showed that However, all major peaks were present in the profiles of both rec104D caused a limited reduction in signal amplitude, as mutants (Figure 4B and Figure S5C). Thus, Mer2 can bind to judged from numbers of peaks still matching (51%) and Pcorr axial sites in the absence of S-Cdk and independently of the (0.6). In contrast, localization of Mer2 did not require either of phosphorylation of its critical residue S30. Further, since DNA its binding partners. By ChIPchip analysis, the Mer2-myc9 profile replication occurs normally in mer2-S30A but is absent in in mei4D and rec114D cells at t4 closely resembled the corre- clb5D, clb6D, Mer2 localization can occur quite faithfully, albeit sponding wild-type profile. Thus, Mer2 binds very similarly to with a slight reduction in quantity, irrespective of replication. axis sites in the presence and absence of Rec114 or Mei4 (Fig- We next asked whether Mer2 S30 phosphorylation and/or ure 4A and Figure S5B). These results imply a hierarchy of inter- S-Cdk are required for axis localization of Rec114 and Mei4. actions in which Mer2 binds to axis sites and then recruits We measured Rec114 and Mei4 association to a major axial Rec114 and Mei4 to those sites. site on chromosome 3 over a meiotic time course, when S30

Cell 146, 372–383, August 5, 2011 ª2011 Elsevier Inc. 377 Figure 5. Cohesin Modifies the Chromosomal Distribution of Hop1 and Rec114 (A) Profile of Rec114-myc13 in wild-type (FK3307, blue) and various mutant backgrounds: rec8D (FK3683, green), hop1D (FK3016, light blue), and red1D (FK3466, lilac), all at t4. (B) Profile of Hop1 in wild-type (FK3307, orange) and rec8D (FK3683, black) at t4. The blue arrow points to a domain in which wild-type Rec114 and Hop1 signals dominate (blue and orange peaks). The green arrow points to a domain where these signals dominate in rec8D (green and black peaks). (C) qPCR of Rec114-myc ChIP at three positions in wild- type (left), and hop1D cells (right), at 3 and 4 hr in SPM. The ratio of the signals core 1:ADP1 is plotted for each experiment and the mean ± s for the ratios of n biological repeats is indicated. (D) qPCR of Rec114-myc ChIP at two positions in wild- type (left), or rec8D cells (right) and of a Hop1 ChIP at two positions in wild-type (left), or rec8D cells (right), at 4 hr in SPM. See also Figure S6.

in red1D and hop1D cells. Indeed, there is no detectable enrichment of Rec114 at a major axial site in hop1D mutant (Figure 5C), and Rec114 binding was nearly abolished in genome-wide in red1D or hop1D (Figure 5A), similarly to what was seen for Mer2 (Fig- ure 4B). Genome-wide profiles of a differently- tagged variant of Mer2 (Mer2-HA3) in wild- type, rec114D, red1D and hop1D confirmed of Mer2 was mutated to alanine (mer2-S30A)(Henderson et al., the results obtained (Figures S5A and S5B). Similarly, ChIP 2006). We found that both proteins were absent from this axial employing an anti-Rec114 antibody (Figure S6C) supported the site in mer2-S30A cells (Figure 3C). Furthermore, in clb5D, conclusions based on the C-terminally tagged construct. clb6D double mutants, Rec114 was almost completely absent Thus, Hop1 and Red1 are the axis site-associated compo- from chromosomes, with only 6% of its peaks matching the nents to which recombinosome proteins bind. More specifically, wild-type (Figure 3B). Mer2 is the primary contact with Red1 and Hop1, and it then Taken together, these results imply that, irrespective of DNA recruits Rec114 and Mei4, which in turn results in the activation replication, Mer2 localizes to chromosomes independent of of Spo11. S-Cdk phosphorylation, but that phosphorylation of Mer2 S30 is essential for recruitment of its binding partners Rec114 and Rec8 Modulates Localization of Hop1 and Rec114 Mei4 in both the presence and absence of DNA replication If cohesin were the primary determinant of axial sites, the local- (Discussion). ization of any protein to these sites should require cohesin. In the absence of Rec8, levels of DSB formation vary with hotspot posi- Localization of Mer2 to Axis Sites Requires Axial tion and temperature. For instance, DSBs occur at wild-type Element Components Red1 and Hop1 levels on chromosome 3 at 23C, but are depleted at other posi- We have shown that RMM bind to the same sites as Red1 and tions (Klein et al., 1999; Kugou et al., 2009; Kim et al., 2010). Hop1, and both factors are known to be required for wild-type When we examined Rec114 association with chromatin in levels of DSBs (reviewed in Hunter, 2007). We therefore asked rec8D cells by genome-wide ChIPchip, we observed an inter- whether Mer2 binding depends on Hop1 and Red1. In red1D esting alteration. Peaks appeared unchanged or even increased mutant cells, 89% of Mer2 peaks were lost, with the remaining in regions where DSBs are not altered relative to wild-type, but 11% hardly passing the threshold (Pcorr = 0.07, Figure 4B). were decreased or abolished in domains where DSBs are Further, no significant enrichment of Mer2 was found by qPCR impaired. For example, previous work described a continuous at a major binding site on chromosome 3 in either red1D or in region between positions 150 and 350 kb on chromosome V, hop1D cells (Figure 4C). Thus, the deposition of Mer2 on axial where DSBs disappear in rec8D mutants (Kugou et al., 2009), sites depends on Hop1 and Red1. and we show that Rec114 peaks disappear in the same region Since Mer2 binding is required for Rec114 localization to axis (Figure 5A, blue arrow), providing an explanation for the lack of sites (above), Rec114 should also be absent from these sites DSBs. Furthermore, the region between 350 and 450 kb has

378 Cell 146, 372–383, August 5, 2011 ª2011 Elsevier Inc. also been shown to have reduced DSBs in rec8D mutants like RMMs (Figure S1C, Figure 2B, and Table S2). Rec102 and (Kugou et al., 2009), and indeed we found that Rec114 peaks Rec104 distributions could be explained if these proteins were are below wild-type levels in this region. Similarly, we measured located within pre-DSB recombinosomes in a physically more a local reduction of Rec114 binding at two loci, where DSB peripheral location relative to axes than RMM. formation was recently shown to be reduced in rec8D (CYS3 on chromosome 1 and ARG4 on chromosome 8) (Kim et al., DISCUSSION 2010). Conversely, no change in DSB formation was found distal to 150 kb and 450 kb of chromosome 5 or all along chromosome Tethered Loop Axis Complexes 3(Kugou et al., 2009), and indeed in these regions we observed Cytological studies in many organisms first showed that recom- strong binding of Rec114 in rec8D (Figure 5A, green arrow, and bination complexes are associated with chromosome axes Figures S6A–S6C). already before synapsis (Gillies, 1979; Stack and Anderson, We showed above that Hop1 and Rec114 DNA binding 1986; Carpenter, 1987; Anderson et al., 1997; Tarsounas et al., profiles strongly correlate and that Hop1 is essential for the 1999). More recently, in budding yeast it has been shown that recruitment of Rec114. This raises the possibility that cohesin DSB hotspots (Baudat and Nicolas, 1997; Gerton et al., 2000; might control DSB formation by modifying the pattern of Blitzblau et al., 2007; Buhler et al., 2007; Pan et al., 2011) map Hop1 binding to DNA. We tested this hypothesis and found between axial sites (Blat and Kleckner, 1999; Blat et al., 2002; indeed that Hop1 and Rec114 profiles were almost identical Glynn et al., 2004). Taken together, these results implied that in the rec8D mutant (Pcorr = 0.9, 88% identical peaks), which hotspot sequences from loop regions interact with recombino- means that they are coordinately changed in the absence somes on the axis in indirect tethering (Blat et al., 2002), but at of cohesin. In contrast, Hop1 profiles did not match equally which stage of recombination such tethering arises and how it well in rec8D and REC8 (Pcorr = 0.62), and the same was could be mediated remained unclear. true for Rec114 (Pcorr = 0.61). We conclude that meiotic Our observation that essential components of the pre-DSB re- cohesin is required for the deposition of Hop1 in certain chro- combinosome bind specifically to axis sites, defined by meiotic mosomal domains, but not in others. Interestingly, we found axis components (Red1, Hop1, Rec8) and SC component Zip1, largely unperturbed Hop1/Rec114 binding patterns in rec8D provides strong evidence that such tethered loop-axis com- on the three smallest yeast chromosomes (1, 6, and 3) with plexes (Blat et al., 2002) form before or at the time of DSB forma- only a few locally limited reductions, implying that these chro- tion and reveals the factors involved. mosomes consist of DNA with cohesin-independent propensity to form DSBs. Overall, chromosomes contained at least one Pre-DSB Recombination Complexes Are Linked to domain where Hop1/Rec114 localization was independent of Chromosome Axes via Spo11-Accessory Proteins Mer2, Rec8 (see Table S2). Thus, Rec114 requires cohesin to asso- Rec114, and Mei4, as a Prerequisite to DSB Formation ciate with axial sites specifically in regions of low intrinsic We show that three specific Spo11-accessory proteins, Mer2, DSB potential. Consistently, domains dependent on Rec8 for Rec114, and Mei4 (RMM), all essential for DSB formation, Hop1 deposition correlated with regions known to lose DSB localize to axis sites, rather than DSB sites, in early meiotic formation in rec8D cells. Thus, cohesin likely controls DSB prophase. This binding peaks at the time of DSB formation and formation by recruiting axial element proteins to axial sites, occurs at wild-type levels and with normal kinetics in a spoY135F which in turn recruit RMM. The observation that local modula- mutant that cannot form DSBs, implying that it is not a con- tion of binding to axial sites of an axial element component sequence of DSB formation. The axis-association of the prere- correlates to changes in DSB frequencies in the same combinosome requires the phosphorylation of Mer2 at S30 by regions, likely via recruitment of pre-DSB components, strongly Cdk1, which is also essential for DSB formation (Henderson supports a direct role of these axial associations in DSB et al., 2006). Among RMM components, Mer2 plays a pivotal formation. role, as it can associate to axial sites in the absence of Cdk Other Spo11-accessory proteins, Rec102 and Rec104, also activity and independently of Rec114 and Mei4, but can recruit preferentially associate with axis-association sites rather than its partners to these sites only upon phosphorylation by S-Cdk. DSB sites, but less prominently than RMMs. These findings are in agreement with the model proposing that Rec102 and Rec104 are accessory factors required for modification of chromatin-bound Mer2 during S phase triggers Spo11’s dimerization, DNA binding and efficient nuclear reten- the assembly of the prerecombinosome on DNA, and conse- tion (Kee et al., 2004; Prieler et al., 2005; Sasanuma et al., quently DSB formation (Murakami and Keeney, 2008). Moreover, 2007). Genomic ChIPchip analysis of Rec102 (tagged with the pattern of RMM association to axis sites tightly correlates myc18) and Rec104 (tagged with myc9) reveals that, like RMM with the pattern of DSB formation in wild-type and several proteins, Rec102 and Rec104 exhibit some preference for axis mutant backgrounds. Taken together, our data indicate that association sites: 81% of the 157 significant Rec104 peaks axis localization of Spo11-accessory factors is functional to were also axis-association sites, with local minima at DSB sites DSB formation and that hotspot cleavage on loop sequences (Figure S1C). However, the prominence of these peaks and occurs in the context of an axis-tethered pre-DSB recombino- valleys was lower than that of RMM, pointing to a more even some. We propose that RMM are primary mediators of such distribution along the genome (Figure 2B and Figure S1C). tethering and that they exert their role in DSB formation in the Further, these proteins exhibited neither a preference for centro- context of this association to the axis (Figure 6). However, we meres, like cohesin, nor for DSB-prone chromosomal domains, cannot exclude the possibility that a pool of proteins, which we

Cell 146, 372–383, August 5, 2011 ª2011 Elsevier Inc. 379 Pre-DSB Recombinosomes Are Linked to Axis Sites via Red1/Hop1 Whose Chromosome-wide Distributions Are Modulated in Some, but Not All, Regions by Rec8 We found a tight correlation between RMM and axial element components distribution along meiotic chromosomes and Hop1/Red1 are required for RMM stable association to axis sites. Not only did RMM, Hop1, and Red1 binding sites largely overlap, but they also exhibited a similar domain-wide distribution. DSB-rich domains on chromosome 3 had previously been shown to correspond to Red1-rich domains (Blat et al., 2002). Our analysis shows that DSB-rich regions correlate with domains with high Hop1/Red1/RMM binding at the genome- wide level, favoring a model in which axis components promote recombination in particular chromosomal domains via axis recruitment of the pre-DSB recombinosome. Rec8 is required for DSB formation only in certain domains. Accordingly, we found that the patterns of Hop1/Red1 localization, RMM localization and DSBs varied coordinately along chromosomes in wild-type meiosis, but were all altered coordinately in rec8D. Thus, absence of Rec8 results in an altered distribution of Red1/Hop1, which results in an altered localization of RMMs and ultimately in an altered pattern of DSBs. Inter- estingly, however, in certain regions, Red1/Hop1/RMM localization and DSB formation are independent of Rec8. Overall, Red1/Hop1 patterns determine DSB patterns, while Rec8 is important for DSB patterns in some regions, but not others. In accord with these results, absence of Red1, Hop1, and/or Figure 6. Model of Axis Tethering of the DSB Machinery (A) After premeiotic DNA replication, cohesin (brown rings), axial element Rec8 reduces DSB formation throughout the genome, but differ- components Hop1 and Red1 (gray and green filled circles), and the pre-DSB ently at different sites and in different regions (Kugou et al., 2009; recombinosome subunits Mer2, Rec114, and Mei4 (red, blue, and violet filled Kim et al., 2010). The basis for Rec8/Red1/Hop1 localization, circles) bind to axis sites (blue dotted lines). One particular site may be and the nature of the interplay between these components, occupied by a subset of these components at a given time. Hotspots (blue remains to be determined. Interestingly, physical interaction stars) are located between axis sites. Mer2 recruits Rec114 and Mei4 upon between Hop1 and Rec8 has recently been observed by mass phosphorylation of its S30 by S-Cdk. spectrometric analysis of Rec8 coprecipitating proteins (Katis (B) Ordered, linear arrays of loops (blue) emerge after condensation and sister chromatids are conjoined in the developing axis. Spo11-containing pre-DSB et al., 2010). The overall outcome of this interplay, shown by recombinosome is anchored at the axis and interacts for cleavage with one of this analysis in extension of several other studies (Blat et al., the surrounding hotspots. 2002; Joshi et al., 2009; Kugou et al., 2009; Kim et al., 2010) Two preconditions will lead to competition between neighboring hotspots. is that the yeast genome consists of blocks with different recom- First, based on this work, the pre-DSB recombinosome is not freely diffusible bination capacities, characterized by the abundance of Red1/ and becomes a locally limited resource, so that only a single hotspot can Hop1 binding sites. The three smallest chromosomes, 1, 6, be cleaved at a time. Second, because of the presence of DNA damage response mediators, inactivation of the cleavage activity (dotted line) may and 3, consist mainly of recombination proficient blocks, thus quickly follow the first DSB, likely inhibiting further cleavage of the same and ensuring sufficient levels of DSBs (Pan et al., 2011) and high other hotspots. This effect would exacerbate competition between sur- levels of CO (Chen et al., 2008; Mancera et al., 2008) to ensure rounding hotspots. A strict limitation to a single cleavage by the DSB re- the obligate crossover (Jones and Franklin, 2006). Indeed, we combinosome will also protect the sister-hotspot from being cleaved at the found that these three chromosomes bound more RMM, same time. Hop1, and Red1 per unit length than longer chromosomes. The domain-wise distribution of DSB factors provides an explanation for the observation that the behavior of a hotspot are unable to detect, transiently interacts with DSB hotspots to sequence depends on the chromosomal context. Consistently, initiate DSB formation. we find that the relative abundance of axial element components Two other Spo11-accessory proteins, Rec102 and Rec104, Hop1 and Red1 positively correlates with the previously reported also preferentially bound to axial sites, but showed less promi- strength of DSB formation at particular sites (Wu and Lichten, nent differentiation in spatial localization between axis sites 1995). and loop regions than RMM/Hop1/Red1. In accord with earlier observations (Kee and Keeney, 2002; Kee et al., 2004; Prieler RMM-Mediated Recombinosome/Axis Association et al., 2005; Sasanuma et al., 2007), we conclude that these Could Underlie Interference among Neighboring DSBs two proteins play other roles than being key mediators of axis The location of pre-DSB recombinosome on sequences that association of the pre-DSB recombinosome. form the chromosome axis provides a mechanistic link between

380 Cell 146, 372–383, August 5, 2011 ª2011 Elsevier Inc. chromosome structure and recombination and may help to viability 90%), Mer2-myc9 (spore viability 95%), and Mei4-HA6 (spore viability explain ‘‘long-range effects’’ in the control of recombination. 88%) were generated by PCR-mediated C-terminal tagging at the endoge- For instance, previous studies have shown that the presence nous chromosomal locus (Zachariae et al., 1996). For all constructs, the effi- ciency of sporulation at 30C was comparable to that of an untagged wild- of a strong hot spot at one position along a chromosome can type strain. reduce the probability of DSBs at nearby positions, over distances of up to 60 kb (Wu and Lichten, 1995; Xu and Kleckner, Cytology and Western Blotting 1995; Fan et al., 1997; Jessop et al., 2005; Robine et al., 2007). Yeast chromosome spreads were performed as described (Nairz and Klein, To explain this effect, it was proposed that hotspots might 1997) and stained with anti-myc 9E10 mouse antibody (1:30), followed by compete for ‘‘rate limiting factors, which are not freely diffusible anti-mouse cy3-conjugated secondary antibody (Jackson ImmunoResearch, in the nucleus’’ (Wu and Lichten, 1995). Our findings provide 1:1000) for Rec114-myc and with affinity purified rabbit anti-Zip1 followed by anti-rabbit Alexa 488-conjugated antibody (Molecular Probes, 1:300). a possible candidate for such an entity: the pre-DSB recombino- For western blotting, protein extracts were prepared after addition of some. This complex is clearly rate limiting for DSB formation, trichloroacetic acid, as previously described (Penkner et al., 2005). Proteins and, as we now show that it is anchored at the chromosome were detected with anti-myc 9E10 (mouse, 1:300) and anti-mouse (Pierce, axis, it is not freely diffusible. Moreover, the pre-DSB recombino- 1:10000) secondary horseradish peroxidase-conjugated antibody. some activity could be regulated in a way to serve just a single hotspot. This would further reduce the chance of a DSB oc- ChIPchip curring on a neighboring loop, even if it fulfilled the criteria of Progression through meiosis was monitored by scoring of nuclear divisions, m accessibility to Spo11 (Figure 6B). upon staining of cells with DAPI (0.2 g/ml), to discard aberrantly slow or asynchronous experiments. ChIP from meiotic cultures was performed as described in detail in Katou et al. (2006) and Mendoza et al. (2009). In brief, DSB Formation and Interhomolog Bias for each sample, 50 ml Dynabeads Pan mouse IgG (Invitrogen), 50 ml Dyna- Axis anchoring of the pre-DSB recombinosome and its conse- beads M-280 Sheep anti-Rabbit IgG (Invitrogen), or 25ml ChIP-Adembeads quence, loop/axis tethering, are functionally coupled to DSB (Ademtech) were incubated with the appropriate antibody for 6–15 hr at 4C. Primary antibodies used for ChIP were 9E11 anti-myc (mouse), anti- formation, but what roles might this coupling serve? All mutants Hop1(rabbit), anti-Zip1 (rabbit), anti-HA antibody 12CA5 (mouse), and anti- with defective RMM-axis anchoring are strongly defective in SV5 mouse (Serotec). The precipitated DNA was used as a template for DSB formation. However, reduced levels of DSBs form in quantitative real-time PCR, using the MESA GREEN qPCR MasterMix red1D and hop1D mutants (reviewed in Hunter, 2007), where Plus for SYBR assay (Eurogentec) (primers sequence is available upon no anchoring of RMM components was observed. The implica- request). For genome-wide analysis (ChIPchip), one-tenth of the IP DNA was tion is that DSB formation in these mutants may occur in the analyzed by real-time PCR (qChIP), while the remaining DNA was concen- trated by precipitation in ethanol, and processed further. Two rounds of ampli- absence of axis tethering of the prerecombinosome, although fication, DNA fragmentation, and end-labeling were performed. GeneChip to a clearly reduced extent. S. cerevisiae Tiling Arrays (reverse) from Affymetrix were used. The hybridiza- There is an important process, however, downstream of DSB tion was carried out at 42C for 16 hr. The following washing and staining steps formation for which Red1 and Hop1 are essential, known as the were done with an Affymetrix 450 station. All reagents and protocols used are interhomolog bias of meiotic repair (Schwacha and Kleckner, described in the Affymetrix ChIP Assay Protocol. 1995, 1997; Wan et al., 2004; Niu et al., 2005; Carballo et al., For a detailed description of the microarray data processing, see the Extended Experimental Procedures. 2008; Kim et al., 2010). red1D and hop1D mutants repair their remaining DSBs preferentially using the sister chromatid as ACCESSION NUMBERS template, strongly exacerbating the recombination defect caused by subnormal levels of DSBs. Our work reveals that Microarray data for the complete genome complement are available from the axis tethering of the pre-DSB recombinosome is abolished in GEO repository, accession number GSE29860. Numerical data for all chromo- red1D and hop1D mutants, where the interhomolog bias is somes after 3 kb smoothing are provided in Table S2 and at http://www.univie. known to be abolished, raising the possibility that their earlier ac.at/SFBChromosomeDynamics/documents.html. defect in tethering is responsible for their later defect in interhomolog bias. SUPPLEMENTAL INFORMATION In summary, we found evidence linking DSB formation with the chromosome axis, providing a critical first clue of how chromo- Supplemental Information includes Extended Experimental Procedures, six figures, and two tables and can be found with this article online at doi:10. some structure and DSB regulation are interrelated. The fact 1016/j.cell.2011.07.003. that at least some RMM components are conserved from yeast to human, and appear to associate preferentially to axis struc- ACKNOWLEDGMENTS tures (Lorenz et al., 2006; Kumar et al., 2010), suggests that this mechanism plays indeed an important role. Further work We would like to thank Y. Katou and S. Mori-Uehara for initial help with will be required to properly study the implications of these ChIPchip, B. Hu¨ ttel for advice, and M. and A. Matzke for sharing the Affymetrix observations. facility. Thanks to I. Tamir and D. Chen for important advice with bioinfor- matics. We thank N. Uematsu for the Rec114-myc13 construct, N. Hollings- worth, S. Keeney, K. Nasmyth, and T. F. Wang for plasmids, strains, and anti- EXPERIMENTAL PROCEDURES bodies, B. Byers for early foundational input and N. Kleckner for valuable, critical comments on the manuscript. We are grateful to B. Wohlrab and Yeast Strains C. Zierhut for making Zip1 and Hop1 antibody and to M. Madalinsky for All strains used in this study are derivatives of SK1 background (Kane and Rec114 peptide synthesis. We are indebted to G. Ammerer and the Verein Roth, 1974). Genotypes are provided in Table S1. Rec114-myc13 (spore zur Fo¨ rderung der Genomforschung for generously supplying most of the

Cell 146, 372–383, August 5, 2011 ª2011 Elsevier Inc. 381 microarrays used in this study. This work was funded by SFB grant F34 and in the yeast Saccharomyces cerevisiae. Proc. Natl. Acad. Sci. USA 97, P18847 of the Austrian Science Foundation to F.K. and by aGrant-in-Aid for 11383–11390. Scientific Research (S) from the MEXT and Strategic International Cooperative Gillies, C.B. (1979). The Relationship between Synaptinemal Complexes, Program from JST to K.S. Recombination Nodules and Crossing over in NEUROSPORA CRASSA Bivalents and Translocation Quadrivalents. Genetics 91, 1–17. Received: May 5, 2010 Glynn, E.F., Megee, P.C., Yu, H.G., Mistrot, C., Unal, E., Koshland, D.E., Revised: April 19, 2011 DeRisi, J.L., and Gerton, J.L. (2004). Genome-wide mapping of the cohesin Accepted: July 5, 2011 complex in the yeast Saccharomyces cerevisiae. PLoS Biol. 2, E259. Published: August 4, 2011 Henderson, K.A., Kee, K., Maleki, S., Santini, P.A., and Keeney, S. (2006). 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