Overview

Many aspects of meiotic recombination, such as its initiation via Spo11-generated DSBS, are highly conserved throughout evolution. Nonetheless, there are key differences between organisms that make it essential to study this process directly in mammals. Mouse is an ideal organism for this research. We are examining the mechanisms that control the distribution of crossover recombination products along chromosomes, the factors that connect recombination with the development of specialized higher order chromosome structures, and the checkpoints that monitor the progression of recombination.

This project is an ongoing collaboration with other investigators at MSKCC:

A Spo11 Knockout Mouse

Slideshow
Project Figures
Figures for the Meiotic Recombination in the Mouse project.
Surface-spread spermatocyte nuclei were stained with a serum recognizing mouse Dmc1 and Rad51 (green) and with antibodies that recognizes a component of the mouse chromosome axis, Scp3 (red). Single-channel (A) and two-channel overlay (B) images of a wild

We cloned a mouse ortholog of yeast SPO11 (Keeney et al., Genomics, 1999) and generated mice with a targeted mutation of the Spo11 gene (Baudat et al., Mol. Cell, 2000). This work demonstrated that SPO11 function in meiotic recombination is conserved in mammals. This work also showed that initiation of meiotic recombination precedes and is required for normal synapsis of homologous chromosomes. This issue was in doubt because, at the time, recombination-dependent synapsis was only known in budding yeast, whereas flies and nematodes were known to carry out synapsis that was independent of recombination. Our results thus helped to resolve questions about the generality of yeast meiotic chromosome behavior.

Distinct DNA Damage-dependent and Independent Responses Drive the Loss of Oocytes in Recombination-defective Mouse Mutants

Defects in meiotic recombination in many organisms result in arrest due to activation of a meiotic checkpoint(s). The proximal defect that triggers this checkpoint in mammalian germ cells remains poorly understood. From the initial phenotypic analysis of our Spo11 mutant mice, we inferred that there are distinct DNA damage-dependent and independent mammalian checkpoint responses to meiotic recombination defects, and that these checkpoints operate differently in male vs. female meiosis (Baudat et al., 200). We explored this hypothesis by detailed epistasis analysis combining mutations in Spo11 with mutations in genes necessary for downstream steps in processing of meiotic DSBs.

In the female germ line, we found that absence of DSB formation caused a partial defect in follicle formation, whereas defects in DSB repair caused earlier and more severe meiotic arrest, which could be suppressed by eliminating DSB formation (DiGiacomo et al., PNAS, 2005). This analysis confirmed that there are both DNA damage-dependent and DNA damage-independent responses to recombination errors in mammalian oocytes. Using these findings as a paradigm, we also examined oocyte loss in mutants lacking the DNA damage checkpoint kinase ATM. The absence of ATM caused defects in folliculogenesis that were similar to those in Dmc1 mutants and which could be suppressed by Spo11 mutation, implying that oocyte death in Atm-deficient animals is a response to defective DSB repair.

In the male germ line, fundamentally different recombination defects cause apoptosis of mouse spermatocytes at the same stage in development, stage IV of the seminiferous epithelium cycle, equivalent to mid-pachynema in normal males. To understand the cellular response(s) that triggers apoptosis, we examined markers of spermatocyte development in mice with different recombination defects (Barchi et al., Mol. Cell. Biol.. In Spo11 mutants, spermatocytes express markers of early to mid-pachynema, forming a chromatin domain that contains sex body-associated proteins but that rarely encompasses the sex chromosomes. In Dmc1 mutants, spermatocytes appear to arrest at or about late zygonema. Epistasis analysis reveals that this earlier arrest is a response to unrepaired DSBs, and cytological analysis implicates the BRCT-containing checkpoint protein TOPBP1. Similar to the situation in females, ATM-deficient spermatocytes show similarities to Dmc1 mutants, suggesting that ATM promotes meiotic DSB repair. These results indicate that, despite an equivalent stage of spermatocyte elimination, different recombination-defective mutants manifest distinct responses, providing insight into surveillance mechanisms in male meiosis.

Role of ATM in meiosis

During our investigation of the epistatic relationship between Spo11 and Atm mutations, we found that the testis cellularity of ATM-deficient
mice was markedly increased by Spo11 heterozygosity, accompanied by significantly improved chromosome synapsis. A similar finding with a different Spo11 mutation was reported by the Camerini-Otero laboratory (Bellani et al., J. Cell Science, 2005). Spo11+/-Atm-/- spermatocytes can progress to meiotic metaphase I, although most cells undergo apoptosis at this stage. The rescue of meiotic progression to this stage allowed us to further explore the role of ATM in meiosis (Barchi, Roig, et al., PLoS Genetics, 2008).

Our results provided evidence that ATM is essential for proper crossover formation in mouse spermatocytes. Specifically, we found that Spo11+/-Atm-/- spermatocytes are defective in forming the obligate crossover on the sex chromosomes, even though the XY pair was usually incorporated in a sex body and was transcriptionally inactivated as in normal spermatocytes. The XY crossover defect correlated with the appearance of lagging chromosomes at metaphase I, which may trigger the extensive metaphase apoptosis that is observed in these cells. In addition, control of the number and distribution of crossovers on autosomes appeared to be defective in the absence of ATM because there was an increase in the
total number of MLH1 foci, which mark the sites of eventual crossover formation, and because interference between MLH1 foci was perturbed. The axes of autosomes exhibited structural defects that correlate with the positions of ongoing recombination. Together, these findings indicate that ATM plays a role in both crossover control and chromosome axis integrity and further suggests that ATM is important for coordinating these features of meiotic chromosome dynamics.

Crossover hotspots in mouse meiosis

Meiotic recombination occurs preferentially within 1-2 kb regions (hotspots) that are surrounded by recombinationally inert sequences (Kauppi, Jeffreys, & Keeney, Nature Rev. Genetics, 2004). It is likely that mammalian hotspots are the sites where SPO11 preferentially forms DSBs. Understanding mammalian hotspots is important for understanding control of crossover distributions and for understanding the contribution of recombination to genome evolution. Mouse is ideal for these studies, but few hotspots have yet been identified. To address this issue, we developed a genomic approach to identify mouse crossover hotspots, based on targeting haplotype block boundaries (Kauppi et al., PNAS, 2007). Using this method, we successfully predicted the location of two previously uncharacterized crossover hotspots in male mice. As increasing amounts of single-nucleotide polymorphism data emerge, this approach will
be useful for investigating the recombination landscape of the mouse genome. Moreover, we are currently developing additional novel methods for identifying meiotic recombination sites genome-wide in mice, based on amplification and sequencing of SPO11-associated oligonucleotides.

Project Members
Publications

Keeney S, Baudat F, Angeles M, Zhou Z-H, Copeland NG, Jenkins NA, Manova K, Jasin M. 1999 A mouse homolog of the Saccharomyces cerevisiae meiotic recombination DNA transesterase Spo11p. Genomics. 1999;61:170-182.

Baudat F, Manova K, Yuen JP, Jasin M, Keeney S. 2000 Chromosome synapsis defects and sexually dimorphic meiotic progression in mice lacking Spo11. Mol Cell. 2000;6:989-998.

Mahadevaiah SK, Turner JMA, Baudat F, Rogakou EP, de Boer P, Blanco-Rodriguez J, Jasin M, Keeney S, Bonner WM, Burgoyne PS. 2001 Recombinational DNA double strand breaks in mice precede synapsis. Nat Genet. 2001;27:271-276.

Kauppi L, Jeffreys AJ, and Keeney S (2004) Where the crossovers are: Recombination distributions in mammals. Nature Rev. Genet. 5, 413-424.

Di Giacomo M, Barchi M, Baudat F, Edelmann W, Keeney S, and Jasin M (2005) Distinct DNA damage-dependent and independent responses drive the loss of oocytes in recombination-defective mouse mutants. Proc. Natl. Acad. Sci. USA 102, 737-742.

Barchi M, Mahadevaiah S, Di Giacomo M, Baudat F, de Rooij DG, Burgoyne PS, Jasin M, and Keeney S (2005). Surveillance of different recombination defects in mouse spermatocytes yields distinct responses despite elimination at an identical developmental stage. Mol. Cell. Biol. 25, 7203-7215.

Neale MJ, Pan J, and Keeney S (2005) Endonucleolytic processing of covalent protein-linked DNA double-strand breaks. Nature 436, 1053-1057.

Kauppi L, Jasin M, and Keeney S (2007) Meiotic crossover hotspots contained in haplotype block boundaries of the mouse genome. Proc. Natl. Acad. Sci. USA 104, 13396-13401.

Barchi M, Roig I, Cole F, Di Giacomo M, de Rooij DG, Keeney S, and Jasin M (2008) ATM promotes the obligate XY crossover and both crossover control and chromosome axis integrity on autosomes. PLoS Genet. 4, e1000076.