Initiation de la recombinaison méiotique chez la souris – Hotspots

During meiosis, programmed DNA double strand breaks (DSBs) are induced, which repair by interaction with a chromatid from the homologous chromosome leads to crossover. Thus, in mice, about 300 DSBs are induced in the meiotic prophase of both oocytes and spermatocytes. How the formation of these DSBs is regulated and what determines their location in the genome remain to be understood. We have been working over the last years on the mouse Spo11 protein, the activity that catalyzes DSB formation and on the analysis of a mouse crossover hotspot, named Psmb9. Our present proposal takes advantage of recent advances and unexpected findings leading us to plan a large scale project involving all members of the lab. We thus aim to progress in two directions: 1) Identification of new proteins involved in DSB formation in mice 2) Understanding factors that define initiation sites 1) In S. cerevisiae, in addition to Spo11, several other proteins (Mei4, Rec114, Mer2') are required for DSB formation. Some of them directly interact (for instance Mei4 and Rec114), but their activities are unknown and no homologs have been found in higher eucaryotes. Based on short motifs alignments we have recently found putative Mei4 and Rec114 in M. musculus (and other eucaryotes as well). We will thus undertake the analysis of the genes, the corresponding proteins, Mei4KO and Rec114KO mutant mice. 2) The non random distribution of meiotic recombination has been well described in several species. In particular, in human and mice, crossovers are clustered in small regions (about 1Kb wide) called hotspots, thought to correspond to preferred regions of initiation. Our analysis at Psmb9 has recently revealed the presence of a novel regulatory locus that we named Dsbc1 (for Double strand break control 1) and which has very interesting properties: We have identified two Dsbc1 alleles in two different genetic backgrounds. Comparison of these alleles leads us to conclude that Dsbc1 is regulating the initiation activity in multiple regions in the genome. Depending on the allele, the recombination activity in a given region can be high or low. We now aim to identify the corresponding gene and to elucidate its mechanism of action. We will test several candidate genes by transgenesis, using the property that the introduction of a specific Dsbc1 allele can modify recombination patterns. Furthermore, we have also shown that Dsbc1 influences chromatin structure at Psmb9. We will thus explore the generality of this finding and test if there is a correlation between chromatin structure and recombination activity. We will address this issue by generating histone modification (a subset of them) maps in mouse spermatocytes by ChipSeq. We will thus attempt to predict new hotspots locations. In order to fully integrate the chromatin data, we will attempt a more risky approach with the design of new techniques and protocols to map DSB at the genome scale and at high resolution. In conclusion, our complementary approaches aim to identify new proteins involved in the initiation of meiotic recombination in mice and the features that define the substrate for the initiation machinery. This proposal involves most of the research activity of the group with the full investment of three staff scientists, two postdocs, one technician and one or two student. Several experiments are quite time consuming, in particular those involving mouse transgenesis, and thus scheduled over a four year period.