Nicola Silva

Homologous recombination-dependent processing of meiotic double-strand breaks is crucial to generate functional gametes. The breast and ovarian cancer susceptibility gene BRCA2 is essential for the loading of RAD51-DMC1 recombinases at DNA breaks to allow accurate repair. Here, we identify the novel protein RIPR-1 as a binding partner of Caenorhabditis elegans BRC-2/BRCA2. RIPR-1 and BRC-2 form an obligate complex both in vitro and in vivo and display interdependent loading in developing oocytes. Loss of ripr-1 results in the complete destabilization of BRC-2, while brc-2 is only partially required to preserve RIPR-1 stability. Strikingly, we found a dramatic accumulation of RAD-51 in late pachytene cells of both ripr-1 and brc-2 nulls, unveiling a BRCA2-independent pathway for recruitment of RAD-51 at resected meiotic DNA breaks. RIPR-1 enhances BRC-2 binding to single-stranded DNA and unlike the individual components, the assembled RIPR-1-BRC-2 complex can also bind double-stranded DNA in vitro. Surprisingly, we observe that RAD-51-dependent crossover designation still occurs in absence of RIPR-1-BRC-2, indicating partial, and stage-dependent constraints on homologous recombination execution. Altogether, our work identifies a novel BRC-2 interactor and sheds new light on HR-mediated regulation of DNA repair during meiosis.

Double-strand breaks (DSBs) are the most deleterious lesions experienced by our genome. Yet, DSBs are intentionally induced during gamete formation to promote the exchange of genetic material between homologous chromosomes. While the conserved topoisomerase-like enzyme Spo11 catalyzes DSBs, additional regulatory proteins—referred to as ‘Spo11 accessory factors’—regulate the number, timing, and placement of DSBs during meiotic prophase, ensuring that SPO-11 does not wreak havoc on the genome. Despite the importance of the accessory factors, they are poorly conserved at the sequence level, suggesting that these factors may adopt unique functions in different species. In this work, we present a detailed analysis of the genetic and physical interactions between the DSB factors in the nematode Caenorhabditis elegans, providing new insights into conserved and novel functions of these proteins. This work shows that HIM-5 is the determinant of X-chromosome-specific crossovers and that its retention in the nucleus is dependent on DSB-1, the sole accessory factor that interacts with SPO-11. We further provide evidence that HIM-5 mediates interactions with the different accessory factors subgroups, providing insights into how components on the DNA loops may interact with the chromosome axis.

Meiotic prophase is characterized by a dynamic program in which germ cells undergo a complex series of associations and dissociations of protein complexes that drive assembly, remodeling, and disassembly of meiosis-specific chromosome structures and dramatic changes in chromosome compaction. Failure to properly coordinate these processes can result in improper chromosome segregation, producing aneuploid gametes and inviable zygotes. Here, we investigate the roles of Caenorhabditis elegans DUO-1, an ortholog of mammalian ubiquitin-specific proteases USP26 and USP29, in mediating these dynamic chromosomal events during meiotic prophase. Cytological analyses of duo-1 null mutants indicate that loss of DUO-1 function leads to impaired assembly of meiotic chromosome axes and synaptonemal complexes (SCs), loss of integrity of meiotic chromosome axes, ineffective homolog pairing, premature separation of sister chromatids, and late-prophase chromosome decompaction. Further, axis/SC instability in duo-1 mutants correlates with depletion of REC-8 cohesin complexes and is accompanied by massive accumulation of early DSB repair intermediates. By using a dual-AID-tagged allele to deplete DUO-1 during meiotic development, we demonstrate that DUO-1 is continually required throughout meiotic prophase progression, to promote proper axis/SC assembly in early prophase, to maintain axis/SC stability during the late pachytene stage, and to promote/maintain chromosome compaction at the end of meiotic prophase. Together, our data emphasize the importance of mechanisms that actively maintain meiotic chromosome structure and meiosis-specific chromosome architecture throughout meiotic prophase and implicate DUO-1 as a key player in these active maintenance processes.

Double-strand breaks (DSBs) are the most deleterious lesions experienced by our genome. Yet, DSBs are intentionally induced during gamete formation to promote the exchange of genetic material between homologous chromosomes. While the conserved topoisomerase-like enzyme Spo11 catalyzes DSBs, additional regulatory proteins— referred to as “Spo11 accessory factors”— regulate the number, timing, and placement of DSBs during meiotic prophase ensuring that SPO11 does not wreak havoc on the genome. Despite the importance of the accessory factors, they are poorly conserved at the sequence level suggesting that these factors may adopt unique functions in different species. In this work, we present a detailed analysis of the genetic and physical interactions between the DSB factors in the nematode Caenorhabditis elegans providing new insights into conserved and novel functions of these proteins. This work shows that HIM-5 is the determinant of X-chromosome-specific crossovers and that its retention in the nucleus is dependent on DSB-1, the sole accessory factor that interacts with SPO-11. We further provide evidence that HIM-5 mediates interactions with the different accessory factors sub-groups, providing insights into how components on the DNA loops may interact with the chromosome axis.

The meiotic chromosome axis organizes chromatin and sets the stage for homolog pairing and recombination. Meiotic HORMA domain proteins (mHORMADs) are conserved axis components that conformationally transform during target binding. In C. elegans, four functionally distinct mHORMADs directly interact, but how binding between them is restricted to axis assembly is unknown. Using a mutation in the mHORMADs that delays axis assembly, we isolated a suppressor mutation in a TRiC (Tailless complex peptide 1 Ring Complex) chaperonin subunit that restored mHORMAD localization. CCT-4 associates with meiotic chromatin and forms in vivo complexes with mHORMADs, while germline disruption of TRiC results in axis defects, indicating a nuclear function for TRiC alongside meiotic chromosomes. We propose that chromosome-associated TRiC locally folds mHORMADs into the binding-competent conformation required for axis morphogenesis. More broadly, our results support the model that spatially-restricted folding by TRiC/CCT is a mechanism of controlling the assembly of multimeric complexes that function in tightly co-ordinated events.

A key event in meiosis is the conversion of a small subset of double strand breaks into interhomolog crossovers. In this study, we demonstrate that Caenorhabditis elegans male spermatogenesis has less robust mechanisms than hermaphrodite oogenesis in regulating crossover numbers. This is not a consequence of differences in meiotic prophase timing, sex chromosome genotype, or the presence or absence of germline apoptosis. Using the cyclin-like crossover marker COSA-1, we show that males are less efficient in both converting double strand breaks into crossover designated events and limiting their number, suggesting weakened crossover homeostasis. Surprisingly, we discovered that significant numbers of COSA-1 foci form at the very end of meiotic prophase in the absence of SPO-11 during spermatogenesis. These COSA-1-marked sites are also independent of homologous recombination, and Topoisomerases I and II. We find that the synaptonemal complex, which holds homologs in proximity, differently modulates COSA-1 enrichment to chromosomes in the absence of SPO-11 in males and hermaphrodites. Together, these findings suggest that males have less robust crossover control and that there are previously unrecognized lesions or structures at the end of meiotic prophase in spermatocytes that can accumulate crossover markers.

Meiotic prophase is characterized by a dynamic program in which germ cells undergo a complex series of associations and dissociations of protein complexes that drive assembly, remodeling, and disassembly of meiosis-specific chromosome structures and dramatic changes in chromosome compaction. Failure to properly coordinate these processes can result in improper chromosome segregation, producing aneuploid gametes and inviable zygotes. Here, we investigate the roles of C. elegans DUO-1, an ortholog of mammalian ubiquitin-specific proteases USP26 and USP29, in mediating these dynamic chromosomal events during meiotic prophase. Cytological analyses of duo-1 null mutants indicate that loss of DUO-1 function leads to impaired assembly of synaptonemal complexes (SCs), loss of integrity of meiotic chromosome axes, ineffective homolog pairing, premature separation of sister chromatids, and late-prophase chromosome decompaction. Further, SC instability in duo-1 mutants correlates with depletion of REC-8 cohesin complexes and is accompanied by massive accumulation of early DSB repair intermediates. By using a dual-AID-tagged allele to deplete DUO-1 during meiotic development, we demonstrate that DUO-1 is continually required throughout meiotic prophase progression, to promote proper axis/SC assembly in early prophase, to maintain axis/SC stability during the late pachytene stage, and to promote/maintain chromosome compaction at the end of meiotic prophase. Together, our data reveal that meiotic chromosome structure and meiosis-specific chromosome architecture require active maintenance throughout meiotic prophase, and that this maintenance is necessary for successful meiosis.

A key event in meiosis is the conversion of a small subset of double strand breaks into interhomolog crossovers. In this study, we demonstrate that Caenorhabditis elegans male spermatogenesis has less robust mechanisms than hermaphrodite oogenesis for ensuring and limiting the conversion of double strand breaks into crossovers. This is not a consequence of differences in meiotic prophase timing, sex chromosome genotype, or the presence or absence of germline apoptosis. Using the cyclin-like crossover marker COSA-1, we show that males have a linear response in converting increasing numbers of double strand breaks into crossovers, suggesting weakened crossover homeostasis. While the topoisomerase SPO-11, responsible for initiating meiotic double strand breaks, has an extended period of activity in males as in hermaphrodites, we discovered that COSA-1 foci form at the very end of meiotic prophase in the absence of SPO-11 during spermatogenesis. These COSA-1-marked sites are also independent of homologous recombination, and Topoisomerases I and II. We find that the synaptonemal complex, which holds homologs in proximity, differently modulates COSA-1 enrichment to chromosomes in the absence of SPO-11 in males and hermaphrodites. Together, these findings suggest that males have less robust crossover control and that there are previously unrecognized lesions or structures at the end of meiotic prophase in spermatocytes that can accumulate CO markers. Author Summary: Formation of healthy gametes depends on the accurate partitioning of genetic material in the daughter cells through meiosis. A hallmark of meiosis is the establishment of crossovers, which arise from physical exchange of DNA between the parental chromosomes during homologous recombination. Recombination is initiated via the induction of physiological DNA damage by the topoisomerase SPO-11 and its auxiliary factors. Abrogating SPO-11 activity prevents crossover formation, resulting in random chromosome segregation and generation of aneuploid gametes. While the underlying mechanisms of crossover formation are conserved between the sexes, several pieces of evidence indicate extensive sexual dimorphism. In our work we describe novel features of C. elegans spermatogenesis that reveal significant differences in the regulation of recombination compared to oogenesis. We find that in spermatogenesis crossover-promoting proteins can be recruited to chromosomes even in the absence of SPO-11 activity, a phenomenon not observed in the oogenic hermaphrodite germ line. Furthermore, removal of some auxiliary factors required for physiological break formation during oogenesis does not prevent crossover designation in spermatocytes. We show that the synaptonemal complex, tasked with keeping homologous chromosomes in proximity, exerts opposing roles in males and hermaphrodites by promoting and limiting the recruitment of SPO-11-independent crossover factors, respectively.

DNA double-strand breaks (DSBs) are formed in meiosis, so their repair in the homologous recombination (HR) pathway will lead to crossover formation, which is essential for successful chromosome segregation. HR contains 2 subpathways: synthesis-dependent strand annealing (SDSA) that creates noncrossover and double Holliday junction (dHJ) that generates crossovers. RAD-51 is a protein essential to the formation of all products of HR, as it assembles on the processed DSB, allowing the invasion of the single-stranded DNA into a region of homology. RAD-51 is removed by RAD-54.L after invasion to allow for repair to occur. Here, we investigate a separation of function allele of rad-51, rad-51::FLAG, as compared to 2 other RAD-51 alleles: rad-51::degron and GFP::rad-51. rad-51::FLAG displays slowed repair kinetics, resulting in an accumulation of RAD-51 foci. rad-51::FLAG worms also activate the DSB checkpoint, but to a less extant than that of rad-51 null mutants. In a proximity ligation assay, RAD-54.L and RAD-51 show enriched colocalization in rad-51::FLAG germlines (but not in rad-51::degron), consistent with stalling at the strand invasion step in HR. The defects in RAD-51 disassembly in rad-51::FLAG mutants lead to formation of chromosomal fragments, similar in their magnitude to ones observed in rad-51 or rad-54.L null mutants. However, rad-51::FLAG mutants (unlike a rad-51 null, GFP::rad-51 or rad-54.L null mutants) displayed no defects in the formation of crossover-designated sites (via GFP::COSA-1 localization). Given that rad-51::FLAG worms show checkpoint activation and chromosomal fragments, these results suggest that crossover repair concludes normally, while the noncrossover pathway is perturbed. This is strikingly different from rad-51::degron and GFP::rad-51 strains, which are proficient or deficient in both pathways, respectively. These results suggest that noncrossovers vs crossovers have distinct recombination intermediates and diverge prior to RAD-51 disassembly.

Faithful meiotic segregation requires pairwise alignment of the homologous chromosomes and their synaptonemal complex (SC) mediated stabilization. Here, we investigate factors that promote and coordinate these events during C. elegans meiosis. We identify BRA-2 (BMP Receptor Associated family member 2) as an interactor of HIM-17, previously shown to promote double-strand break formation. We found that loss of bra-2 impairs synapsis elongation without affecting homolog recognition, chromosome movement or SC maintenance. Epistasis analyses reveal previously unrecognized activities for HIM-17 in regulating homolog pairing and SC assembly in a partially overlapping manner with BRA-2. We show that removing bra-2 or him-17 restores nuclear clustering, recruitment of PLK-2 at the nuclear periphery, and abrogation of ectopic synapsis in htp-1 mutants, suggesting intact CHK-2-mediated signaling and presence of a barrier that prevents SC polymerization in the absence of homology. Our findings shed light on the regulatory mechanisms ensuring faithful pairing and synapsis.