We are studying the mechanism and regulation of meiotic recombination in mouse. Much of this work is part of a longstanding collaboration with Maria Jasin’s lab in the Developmental Biology Program at Memorial Sloan Kettering Cancer Center, starting with our work together to clone and knock out the mouse homolog of Spo11.
Role of ATM in feedback control of double-strand break formationATM is a Ser/Thr kinase mutated in the cancer-prone disease ataxia telagiectasia (A-T). ATM activated by DSBs triggers cell cycle checkpoints and promotes DNA repair in somatic cells, but ATM is also essential during normal, unperturbed meiosis, for reasons that have been unclear. In earlier work, we showed that Atm–/– mutants resemble mutants lacking DSB repair factors such as DMC1, suggesting that absence of ATM disrupts recombination. We and others also found that meiotic defects are greatly suppressed by reducing Spo11 gene dosage: Spo11+/–Atm–/– spermatocytes pair and recombine autosomes and progress further through prophase, unlike Atm–/–. Using this remarkable phenotype as a tool to dissect ATM function, we found that Spo11+/–Atm–/– spermatocytes are deficient in synapsis and crossing-over of the X and Y, leading to lagging chromosomes and extensive apoptosis at MI. Also, the number and distribution of crossovers on autosomes is abnormal, with more total MLH1 foci and less interference between foci. Autosome axes have gaps and other structural defects at positions of ongoing recombination. We concluded that ATM is needed for both crossover control and chromosome axis integrity, and proposed that ATM coordinates these processes (Barchi, Roig et al., 2008).
Precisely what aspect(s) of meiotic recombination is controlled by ATM remained unclear, however. A breakthrough came from our unexpected discovery that Atm–/– spermatocytes experience greatly elevated numbers of DSBs. Moreover, absence of ATM renders DSB formation extremely sensitive to SPO11 expression level, probably explaining why Spo11 heterozygosity partially rescues Atm deficiency: many defects in Atm-null spermatocytes are likely caused by grossly elevated DSBs, which are lowered by reduced SPO11 expression. We proposed that ATM activation governs a negative feedback loop that restrains SPO11 to limit the number of DSBs (Lange et al., 2011). Independent studies in Drosophila and yeast led to a similar conclusion.
X-Y recombinationSex chromosome segregation poses challenges to the male meiotic cell because the X and Y share only a small region of homology, the pseudoautosomal region (PAR): at least one DSB must form within the PAR; that DSB must locate and engage its homologous partner; and a crossover must form. To understand how cells meet these challenges, we studied the behavior and structure of the PAR in normal meiosis, and explored genetic requirements for ensuring PAR DSB formation (Kauppi et al. 2011). We found that mouse PAR DNA occupies unusually long chromosome axes, potentially as shorter chromatin loops, predicted to promote DSB formation. We also found that most PARs have delayed appearance of the RAD51 foci that mark DSB ends.
Mice and humans express two major mRNA splicing isoforms of Spo11, called alpha and beta. The Spo11-beta transcript includes exon 2, while Spo11-alpha skips it. This splice difference is developmentally regulated, with Spo11-beta expressed earlier as the predominant form present at the time most DSBs are made. Transgenic mice expressing only the Spo11-beta isoform make normal numbers of DSBs on autosomes, but are specifically defective for efficient formation of DSBs on the PAR. These results showed that PAR DSBs are genetically distinct from “global” DSBs. These findings uncover specific mechanisms that surmount the unique challenges of X-Y recombination.