Meiosis-specific mechanisms of homologous recombination – MEIOSPEHR
Homologous recombination (HR) is a widely conserved DNA repair mechanism that plays a central role in genome stability, fertility and genetic diversity. In cells with mitotic cycles, HR faithfully repairs damaged DNA by copying information from a template DNA present in the sister chromatid. Defects in HR leads to genome instability and cell death. In meiotic cells, HR is initiated by programmed DNA double strand breaks (DSBs). It favors recombination between parental chromosomes in order to generate crossovers (CO), which are important for accurate segregation of the parental chromosomes. Failures or errors in meiotic HR are associated with infertility and aneuploidy diseases.
Meiotic HR can be defined as a “specialization” of HR, as its main steps resemble the ones of mitotic HR, but it uses ubiquitous and meiosis-specific factors to ensure its specific outcomes including CO formation and genetic diversity. In the last decade, a set of important meiosis-specific factors MEIOB, SPATA22, HSF2BP/MEILB2 and BRME1 were identified. Here, taking advantage of the complementary expertise of the 3 teams, we aim at combining in vivo (mouse model), ex vivo (organ cultures), biochemical, biophysical and structural biology approaches to study the molecular bases of the meiosis specific events that control the early steps of meiotic HR. These events are essential to satisfy meiotic outcomes and preserve genome integrity. We propose to decipher the roles of MEIOB, SPATA22, HSF2BP/MEILB2 and BRME1 by studying their interactions, their sequential recruitment, and their functions in collaboration with canonical HR factors such as RPA, RAD51, and BRCA2 and the meiosis-specific recombinase DMC1. We will also explore how a defect in the function of these proteins affects meiotic HR as well as genome integrity and/or fertility.
Our project is organized in 4 tasks. In task 1, we will study the interactions between SPATA22 and HSF2BP-BRME1. We will identify the interfaces between these proteins in vitro (P3) and will observe the impact of this interaction on the structure of the RPA-MEIOB-SPATA22-ssDNA filament by transmission electron microscopy (TEM) (P2). We will characterize a Spata22 mouse mutant lacking the predicted interaction site for HSF2BP (P1). Finally, we will look for new partners of MEIOB-SPATA22 during meiotic HR by co-immunoprecipitation of SPATA22 in mouse testis (P1). In task 2, we will analyze in vitro the interplay between the ssDNA-binding proteins RPA, MEIOB, SPATA22, the mediator protein BRCA2 and the recombinases RAD51 and DMC1 during the assembly of the presynaptic filament by TEM and a combination of structural biology approaches (P2&3). Next, we will question the role of HSF2BP and BRME1 in the regulation of the assembly of this filament (P2 & P3). In task 3, we will explore the impact of MEIOB-SPATA22 and HSF2BP on HR outcomes. We will use TEM-based experiments to test the impact of MEIOB-SPATA22 and HSF2BP-BRME1 on D-loop formation, architecture and stabilization in vitro. P1&2 will develop a new approach to visualize meiotic intermediates (immunoprecipitation of HR intermediates in testis in culture). In task 4, we will use an immunolocalization approach to study the in vivo distribution of the proteins involved in the regulation of the synaptic filament during meiotic recombination progression and verify whether complexes proposed by our in vitro approaches are consistent with their in vivo distribution (P1).
Our project is timely and innovative as it proposes to use state-of-the-art techniques, as well as to develop new tools, for understanding the role of novel meiosis-specific proteins in the formation of the HR recombinogenic filament, capture of the homologous sequence and stabilization of the D-loop. It will impact public health by contributing to predict fertility defects, and, more generally, identify gene mutations involved in genome instability and inheritable diseases.
Project coordination
Emmanuelle MARTINI (Stabilité Génétique, Cellules Souches et Radiations)
The author of this summary is the project coordinator, who is responsible for the content of this summary. The ANR declines any responsibility as for its contents.
Partner
SGCSR Stabilité Génétique, Cellules Souches et Radiations
IGC Intégrité du génome et cancer
I2BC Institut de Biologie Intégrative de la Cellule
Help of the ANR 601,999 euros
Beginning and duration of the scientific project:
December 2023
- 36 Months