Biology subjects

de Massy, B.

Publications and source records attributed to de Massy, B..

9 recordsLinked to original sources

bioRxiv2024

Fundamental principles of chromosome organization for meiotic recombination

In meiotic cells, chromosomes are organized as chromatin loop arrays anchored to a protein axis. This organization is essential to regulate meiotic recombination, from DNA double-strand break (DSB) formation to their repair 1. In mammals, it is unknown how chromatin loops are organized along the genome and how proteins participating in DSB formation are tethered to the chromosome axes. Here, we identified three categories of axis-associated genomic sites: PRDM9 binding sites, where DSBs form 2, binding sites of the insulator protein CTCF, and H3K4me3-enriched sites. We demonstrated that PRDM9 promotes the recruitment of MEI4 and IHO1, two proteins essential for DSB formation 3,4. In turn, IHO1 anchors DSB sites to the axis components HORMAD1 and SYCP3. We discovered that IHO1, HORMAD1 and SYCP3 are associated at the DSB ends during DSB repair. Our results highlight how interactions of proteins with specific genomic elements shape the meiotic chromosome organization for recombination.

molecular biology↗

bioRxiv2024

PRDM9 drives the location and rapid evolution of recombination hotspots in salmonids

In many eukaryotes, meiotic recombination occurs preferentially at discrete sites, called recombination hotspots. In various lineages, recombination hotspots are located in regions with promoter-like features and are evolutionarily stable. Conversely, in some mammals, hotspots are driven by PRDM9 that targets recombination away from promoters. Paradoxically, PRDM9 induces the self-destruction of its targets and this triggers an ultra-fast evolution of mammalian hotspots. PRDM9 is ancestral to all animals, suggesting a critical importance for the meiotic program, but has been lost in many lineages with surprisingly little effect on meiosis success. However, it is unclear whether the function of PRDM9 described in mammals is shared by other species. To investigate this, we analyzed the recombination landscape of several salmonids, the genome of which harbors one full-length PRDM9 and several truncated paralogs. We identified recombination initiation sites in Oncorhynchus mykiss by mapping meiotic DNA double-strand breaks (DSBs). We found that DNA DSBs clustered at hotspots positioned away from promoters, enriched for the H3K4me3 and H3K4me36 marks and the location of which depended on the genotype of full-length Prdm9. We observed a high level of polymorphism in the zinc finger domain of full-length Prdm9, but not of the truncated paralogs. Moreover, population-scaled recombination maps in O. mykiss, Oncorhynchus kisutch and Salmo salar revealed a rapid turnover of recombination hotspots caused by PRDM9 target motif erosion. Our results imply that PRDM9 function is conserved across vertebrates and that the peculiar evolutionary runaway caused by PRDM9 has been active for several hundred million years.

evolutionary biology↗

bioRxiv2023

TOPOVIBL function in meiotic DSB formation: new insights from its biochemical and structural characterization.

The TOPOVIL complex catalyzes the formation of DNA double strand breaks (DSB) that initiate meiotic homologous recombination, an essential step for chromosome segregation and genetic diversity during gamete production. TOPOVIL is composed of two subunits (SPO11 and TOPOVIBL) and is evolutionarily related to the archaeal TopoVI topoisomerase complex. SPO11 is the TopoVIA subunit orthologue and carries the DSB formation catalytic activity. TOPOVIBL shares homology with the TopoVIB ATPase subunit. TOPOVIBL is essential for meiotic DSB formation, but its molecular function remains elusive, partly due to the lack of biochemical studies. Here, we purified TOPOVIBL{Delta}C25 and characterized its structure and mode of action in vitro. Our structural analysis revealed that TOPOVIBL{Delta}C25 adopts a dynamic conformation in solution and our biochemical study showed that the protein remains monomeric upon incubation with ATP, which correlates with the absence of ATP binding. Moreover, TOPOVIBL{Delta}C25 interacted with DNA, with a preference for some geometries, suggesting that TOPOVIBL senses specific DNA architectures. Altogether, our study identified specific TOPOVIBL features that might help to explain how TOPOVIL function evolved toward a DSB formation activity in meiosis.

molecular biology↗

bioRxiv2023

The FIGNL1-FIRRM complex is required to complete meiotic recombination in the mouse and prevents massive DNA damage-independent RAD51 and DMC1 loading

During meiosis, nucleoprotein filaments of the strand exchange proteins RAD51 and DMC1 are crucial for repairing SPO11-generated DNA double-strand breaks (DSBs) by homologous recombination (HR). A balanced activity of positive and negative RAD51/DMC1 regulators ensures proper recombination. Fidgetin-like 1 (FIGNL1) was previously shown to negatively regulate RAD51 in human cells. However, FIGNL1s role during meiotic recombination in mammals remains unknown. Here, we deciphered the meiotic functions of FIGNL1 and FIGNL1 Interacting Regulator of Recombination and Mitosis (FIRRM) using male germline-specific conditional knock-out (cKO) mouse models. Both FIGNL1 and FIRRM are required for completing meiotic prophase in mouse spermatocytes. Despite efficient recruitment of DMC1 on ssDNA at meiotic DSB hotspots, the formation of late recombination intermediates is defective in Firrm cKO and Fignl1 cKO spermatocytes. Moreover, the FIGNL1-FIRRM complex limits RAD51 and DMC1 accumulation on intact chromatin, independently from the formation of SPO11-catalyzed DSBs. Purified human FIGNL1{Delta}N alters the RAD51/DMC1 nucleoprotein filament structure and inhibits strand invasion in vitro. Thus, this complex might regulate RAD51 and DMC1 association at sites of meiotic DSBs to promote proficient strand invasion and processing of recombination intermediates.

genetics↗

bioRxiv2023

Characterization of the REC114-MEI4-IHO1 complex regulating meiotic DNA double-strand break formation

Meiotic recombination is initiated by the formation of DNA double-strand breaks (DSBs), essential for fertility and genetic diversity. In the mouse, DSBs are formed by the catalytic TOPOVIL complex consisting of SPO11 and TOPOVIBL. To preserve genome integrity, the activity of the TOPOVIL complex is finely controlled by several meiotic factors including REC114, MEI4 and IHO1, but the underlying mechanism is poorly understood. Here, we report that mouse REC114 forms homodimers, that it associates with MEI4 as a 2:1 heterotrimer that further dimerizes, and that IHO1 forms coiled-coil based tetramers. Using AlphaFold2 modelling combined with biochemical characterization we uncovered the molecular details of these assemblies. Finally, we show that IHO1 directly interacts with the PH domain of REC114 by recognizing the same surface as TOPOVIBL and another meiotic factor ANKRD31. These results provide strong evidence for the existence of a ternary IHO1-REC114-MEI4 complex and show that REC114 is a potential regulatory platform mediating mutually exclusive interactions with several partners.

biochemistry↗

bioRxiv2022

Evolution and diversity of the TopoVI and TopoVI-like subunits with extensive divergence of the TOPOVIBL subunit

Type II DNA topoisomerases regulate topology by double-stranded DNA cleavage and ligation. The TopoVI family of DNA topoisomerase, first identified and biochemically characterized in Archaea, represents, with TopoVIII and mini-A, the type IIB family. TopoVI has several intriguing features in terms of function and evolution. TopoVI has been identified in some eucaryotes, and a global view is lacking to understand its evolutionary pattern. In addition, in eucaryotes, the two TopoVI subunits (TopoVIA and TopoVIB) have duplicated and evolved to give rise to Spo11 and TopoVIBL, forming TopoVI-like (TopoVIL), a complex essential for generating DNA breaks that initiation homologous recombination during meiosis. TopoVIL is essential for sexual reproduction. How the TopoVI subunits have evolved to ensure this meiotic function is unclear. Here, we investigated the phylogenetic conservation of TopoVI and TopoVIL. We demonstrate that BIN4 and RHL1, potentially interacting with TopoVIB, have co-evolved with TopoVI. Based on model structures, this observation supports the hypothesis for a role of TopoVI in decatenation of replicated chromatids and predicts that in eucaryotes the TopoVI catalytic complex includes BIN4 and RHL1. For TopoVIL, the phylogenetic analysis of Spo11, which is highly conserved among Eukarya, highlighted a eukaryal-specific N-terminal domain that may be important for its regulation. Conversely, TopoVIBL was poorly conserved and rapidly evolving, giving rise to ATP hydrolysis-mutated or -truncated protein variants, or was undetected in some species. This remarkable plasticity of TopoVIBL provides important information for the activity and function of TopoVIL during meiosis.

evolutionary biology↗

bioRxiv2021

TOPOVIBL-REC114 interaction regulates meiotic DNA double-strand breaks

Meiosis requires the formation of programmed DNA double strand breaks (DSBs), essential for fertility and for generating genetic diversity. DSBs are induced by the catalytic activity of the TOPOVIL complex formed by SPO11 and TOPOVIBL. To ensure genomic integrity, DNA cleavage activity is tightly regulated, and several accessory factors (REC114, MEI4, IHO1, and MEI1) are needed for DSB formation in mice. How and when these proteins act is not understood. Here, we show that REC114 is a direct partner of TOPOVIBL, and identified their conserved interacting domains by structural analysis. We then analysed the role of this interaction by monitoring meiotic DSBs in female and male mice carrying point mutations in TOPOVIBL that decrease or disrupt its binding to REC114. In these mutants, DSB activity was strongly reduced genome-wide in oocytes, and only in sub-telomeric regions in spermatocytes. In addition, in mutant spermatocytes, DSB activity was delayed in autosomes. These results provide evidence that REC114 is a key member of the TOPOVIL catalytic complex, and that the REC114/TOPOVIBL interaction ensures the efficiency and timing of DSB activity.

molecular biology↗

bioRxiv2018

ANKRD31 regulates spatiotemporal patterning of meiotic recombination initiation and ensures recombination between heterologous sex chromosomes in mice

Orderly segregation of chromosomes during meiosis requires that crossovers form between homologous chromosomes by recombination. Programmed DNA double-strand breaks (DSBs) initiate meiotic recombination. We identify ANKRD31 as a critical component of complexes of DSB-promoting proteins which assemble on meiotic chromosome axes. Genome-wide, ANKRD31 deficiency causes delayed recombination initiation. In addition, loss of ANKRD31 alters DSB distribution owing to reduced selectivity for sites that normally attract DSBs. Strikingly, ANKRD31 deficiency also abolishes uniquely high rates of recombination that normally characterize pseudoautosomal regions (PARs) of X and Y chromosomes. Consequently, sex chromosomes do not form crossovers leading to chromosome segregation failure in ANKRD31-deficient spermatocytes. These defects are accompanied by a genome-wide delay in assembling DSB-promoting proteins on axes and a loss of a specialized PAR-axis domain that is highly enriched for DSB-promoting proteins. Thus, we propose a model for spatiotemporal patterning of recombination by ANKRD31-dependent control of axis-associated complexes of DSB-promoting proteins.

cell biology↗

bioRxiv2018

Mouse REC114 is essential for meiotic DNA double-strand break formation and forms a complex with MEI4

Programmed formation of DNA double strand breaks (DSBs) initiates the meiotic homologous recombination pathway. This pathway allows homologous chromosomes to find each other and the formation of crossing overs, the products of reciprocal exchanges, which are required for proper chromosome segregation at the first meiotic division. Meiotic DSBs are catalyzed by Spo11 that forms a complex with a second subunit, TopoVIBL, and mediates a DNA type II topoisomerase-like cleavage. Several other proteins are essential for meiotic DSB formation, including three evolutionarily conserved proteins first identified in Saccharomyces cerevisiae (Mer2, Mei4 and Rec114). These three S. cerevisiae proteins and their mouse orthologs (IHO1, MEI4 and REC114) co-localize on the axes of meiotic chromosomes, and mouse IHO1 and MEI4 are essential for meiotic DSB formation. Here, we show that mouse Rec114 is required for meiotic DSB formation. Moreover, MEI4 forms a complex with REC114 and IHO1 in mouse spermatocytes, consistent with cytological observations. We then demonstrated in vitro the formation of a stable complex between REC114 C-terminal domain and MEI4 N-terminal domain. We further determine the structure of REC114 N-terminal domain that revealed similarity with Pleckstrin Homology domains and its property to dimerize. These analyses provide direct insights into the architecture of these essential components of the meiotic DSB machinery.

molecular biology↗