The synaptonemal complex (SC) is a conserved protein structure that holds homologous chromosome pairs together throughout much of meiotic prophase I. It is essential for the formation of crossovers, which are required for the proper segregation of chromosomes into gametes. The assembly of the SC is likely to be regulated by post-translational modifications. The CSN/COP9 signalosome has been shown to act in many pathways, mainly via the ubiquitin degradation/proteasome pathway. Here we examine the role of the CSN/COP9 signalosome in SC assembly in the model organism C. elegans. Our work shows that mutants in three subunits of the CSN/COP9 signalosome fail to properly assemble the SC. In these mutants, SC proteins aggregate, leading to a decrease in proper pairing between homologous chromosomes. The reduction in homolog pairing also results in an accumulation of recombination intermediates and defects in repair of meiotic DSBs to form the designated crossovers. The effect of the CSN/COP9 signalosome mutants on synapsis and crossover formation is due to increased neddylation, as reducing neddylation in these mutants can partially suppress their phenotypes. We also find a marked increase in apoptosis in csn mutants that specifically eliminates nuclei with aggregated SC proteins. csn mutants exhibit defects in germline proliferation, and an almost complete pachytene arrest due to an inability to activate the MAPK pathway. The work described here supports a previously unknown role for the CSN/COP9 signalosome in chromosome behavior during meiotic prophase I.

In meiotic prophase I, homologous chromosome pairing is promoted through chromosome movement mediated by nuclear envelope proteins, microtubules, and dynein. After proper homologue pairing has been established, the synaptonemal complex (SC) assembles along the paired homologues, stabilizing their interaction and allowing for crossing over to occur. Previous studies have shown that perturbing chromosome movement leads to pairing defects and SC polycomplex formation. We show that FKB-6 plays a role in SC assembly and is required for timely pairing and proper double-strand break repair kinetics. FKB-6 localizes outside the nucleus, and in its absence, the microtubule network is altered. FKB-6 is required for proper movement of dynein, increasing resting time between movements. Attenuating chromosomal movement in fkb-6 mutants partially restores the defects in synapsis, in agreement with FKB-6 acting by decreasing chromosomal movement. Therefore, we suggest that FKB-6 plays a role in regulating dynein movement by preventing excess chromosome movement, which is essential for proper SC assembly and homologous chromosome pairing.

During meiotic prophase I, sister chromatid cohesion is established in a way that supports the assembly of the synaptonemal complex (SC). The SC connects homologous chromosomes, directing meiotic recombination to create crossovers. In this paper, we identify two proteins that cooperate to import and load meiotic cohesins, thus indirectly promoting SC assembly. AKIR-1 is a protein with a previously identified meiotic role in SC disassembly. akir-1 mutants have no obvious defects in sister chromatid cohesion. We identified ima-2, a gene encoding for an α-importin nuclear transport protein, as a gene interacting with akir-1 Analysis of akir-1;ima-2 double mutants reveals a decrease in the number of germline nuclei and the formation of polycomplexes (PCs) (an SC protein aggregate). These PCs contain proteins that are part of the two main substructures of the SC: the central region and the lateral element. Unlike typical PCs, they also contain sister chromatid cohesion proteins. In akir-1;ima-2 double mutants, PCs are located in both the nucleus and the cytoplasm. This suggests that the defects observed in the double mutants are both in nuclear import and in the assembly of sister chromatid cohesion. PC formation is also associated with recombination defects leading to reduced numbers of crossovers. Similarly to cohesion mutants, the pairing center protein HIM-8 is mislocalized in akir-1;ima-2 double mutants, forming multiple foci. We propose that AKIR-1 and IMA-2 operate in parallel pathways to import and load chromosomally associated cohesin complex proteins in meiotic nuclei, a novel finding for both of these conserved proteins.

Meiosis is a highly regulated process, partly due to the need to break and then repair DNA as part of the meiotic program. Post-translational modifications are widely used during meiotic events to regulate steps such as protein complex formation, checkpoint activation, and protein attenuation. In this paper, we investigate how proteins that are obligatory components of the SUMO (small ubiquitin-like modifier) pathway, one such post-translational modification, affect the Caenorhabditis elegans germline. We show that UBC-9, the E2 conjugation enzyme, and the C. elegans homolog of SUMO, SMO-1, localize to germline nuclei throughout prophase I. Mutant analysis of smo-1 and ubc-9 revealed increased recombination intermediates throughout the germline, originating during the mitotic divisions. SUMOylation mutants also showed late meiotic defects including defects in the restructuring of oocyte bivalents and endomitotic oocytes. Increased rates of noninterfering crossovers were observed in ubc-9 heterozygotes, even though interfering crossovers were unaffected. We have also identified a physical interaction between UBC-9 and DNA repair protein MRE-11 ubc-9 and mre-11 null mutants exhibited similar phenotypes at germline mitotic nuclei and were synthetically sick. These phenotypes and genetic interactions were specific to MRE-11 null mutants as opposed to RAD-50 or resection-defective MRE-11 We propose that the SUMOylation pathway acts redundantly with MRE-11, and in this process MRE-11 likely plays a structural role.

Studies of the repair pathways associated with DNA double strand breaks (DSBs) are numerous, and provide evidence for cell-cycle specific regulation of homologous recombination (HR) by the regulation of its associated proteins. Laser microirradiation is a well-established method to examine in vitro kinetics of repair and allows for live-imaging of DSB repair from the moment of induction. Here we apply this method to whole, live organisms, introducing an effective system to analyze exogenous, microirradiation-induced breaks in the Caenorhabditis elegans germline. Through this method we observed the sequential kinetics of the recruitment of ssDNA binding proteins RPA-1 and RAD-51 in vivo. We analyze these kinetics throughout different regions of the germline, and thus throughout a range of developmental stages of mitotic and meiotic nuclei. Our analysis demonstrates a largely conserved timing of recruitment of ssDNA binding proteins to DSBs throughout the germline, with a delay of RAD-51 recruitment at mid-pachytene nuclei. Microirradiated nuclei are viable and undergo a slow kinetics of resolution. We observe RPA-1 and RAD-51 colocalization for hours post-microirradiation throughout the germline, suggesting that there are mixed RPA-1/RAD-51 filaments. Finally, through live imaging analysis we observed RAD-51 foci movement with low frequency of coalescence.

Akirin, a conserved metazoan protein, functions in muscle development in flies and mice. However, this was only tested in the rodent and fly model systems. Akirin was shown to act with chromatin remodeling complexes in transcription and was established as a downstream target of the NFκB pathway. Here we show a role for Caenorhabditis elegans Akirin/AKIR-1 in the muscle and body length regulation through a different pathway. Akirin localizes to somatic tissues throughout the body of C. elegans, including muscle nuclei. In agreement with its role in other model systems, Akirin loss of function mutants exhibit defects in muscle development in the embryo, as well as defects in movement and maintenance of muscle integrity in the C. elegans adult. We also have determined that Akirin acts downstream of the TGF-β Sma/Mab signaling pathway in controlling body size. Moreover, we found that the loss of Akirin resulted in an increase in autophagy markers, similar to mutants in the TGF-β Sma/Mab signaling pathway. In contrast to what is known in rodent and fly models, C. elegans Akirin does not act with the SWI/SNF chromatin-remodeling complex, and is instead involved with the NuRD chromatin remodeling complex in both movement and regulation of body size. Our studies define a novel developmental role (body size) and a new pathway (TGF-β Sma/Mab) for Akirin function, and confirmed its evolutionarily conserved function in muscle development in a new organism.

Faithful chromosome segregation into gametes depends on Spo11-induced DNA double-strand breaks (DSBs). These yield single-stranded 3' tails upon resection to promote crossovers (COs). While early Mre11-dependent end resection is the predominant pathway in most organisms, Exo1 or Dna2/BLM can also contribute to the efficient processing of meiotic DSBs. Although its enzymatic activity has been thoroughly dissected, the temporal dynamics underlying Spo11 activity have remained mostly elusive. We show that, in Caenorhabditis elegans, SPO-11-mediated DSB induction takes place throughout early meiotic prophase I until mid-late pachynema. We find that late DSBs are essential for CO formation and are preferentially processed by EXO-1 and DNA-2 in a redundant fashion. Further, EXO-1-DNA-2-mediated resection ensures completion of conservative DSB repair and discourages activation of KU-dependent end joining. Taken together, our data unveil important temporal aspects of DSB induction and identify previously unknown functional implications for EXO-1-DNA-2-mediated resection activity in C. elegans.

Defects in crossover (CO) formation during meiosis are a leading cause of birth defects, embryonic lethality, and infertility. In a wide range of species, maternal aging increases aneuploidy and decreases oocyte quality. In C. elegans which produce oocytes throughout the first half of adulthood, aging both decreases oocytes quality and increases meiotic errors. Phenotypes of mutations in genes encoding double-strand break (DSB)-associated proteins get more severe with maternal age suggesting that early meiosis reflects a particularly sensitive node during reproductive aging in the worm. We observed that aging has a direct effect on the integrity of C. elegans meiotic CO formation, as observed by an increase of univalent chromosomes and fusions at diakinesis, with a considerable increase starting at 4 days. We also characterize the possible causes for the age-related changes in CO formation by analyzing both steady-state levels and kinetics of the ssDNA binding proteins RPA-1 and RAD-51. Profound reductions in numbers of both RPA-1 and RAD-51 foci suggests that both DSB formation and early meiotic repair are compromised in aging worms. Using laser microirradiation and γ-irradiation to induce exogenous damage, we show specifically that recruitment of these homologous recombination proteins is altered. Repair defects can be seen in two-and-one-half day-old adults making the loss of germline repair capacity among the earliest aging phenotypes in the worm.

Accumulation of DNA-RNA hybrids in the form of R-loops can result in replication-transcription conflict that leads to the formation of DNA double strand breaks (DSBs). Using null mutants for the two Caenorhabditis elegans genes encoding for RNaseH1 and RNaseH2, we identify novel effects of R-loop accumulation in the germline. R-loop accumulation leads, as expected, to replication stress, followed by the formation of DSBs. A subset of these DSBs are irreparable. However, unlike irreparable DSBs generated in other systems, which trigger permanent cell cycle arrest, germline irreparable DSBs are propagated to oocytes. Despite DNA damage checkpoint activation in the stem cell niche, the signaling cannot be sustained and nuclei with irreparable DNA damage progress into meiosis. Moreover, unlike other forms of DNA damage that increase germline apoptosis, R-loop-generated DSBs remain undetected by the apoptotic checkpoint. This coincides with attenuation of ATM/ATR signaling in mid-to-late meiotic prophase I. These data altogether indicate that in the germline, DSBs that are generated by R-loops can lead to irreparable DSBs that evade cellular machineries designed for damage recognition. These studies implicate germline R-loops as an especially dangerous driver of germline mutagenesis.

Meiosis is a tightly regulated process requiring coordination of diverse events. A conserved ERK/MAPK-signaling cascade plays an essential role in the regulation of meiotic progression. The Thousand And One kinase (TAO) kinase is a MAPK kinase kinase, the meiotic role of which is unknown. We have analyzed the meiotic functions of KIN-18, the homolog of mammalian TAO kinases, in Caenorhabditis elegans. We found that KIN-18 is essential for normal meiotic progression; mutants exhibit accelerated meiotic recombination as detected both by analysis of recombination intermediates and by crossover outcome. In addition, ectopic germ-cell differentiation and enhanced levels of apoptosis were observed in kin-18 mutants. These defects correlate with ectopic activation of MPK-1 that includes premature, missing, and reoccurring MPK-1 activation. Late progression defects in kin-18 mutants are suppressed by inhibiting an upstream activator of MPK-1 signaling, KSR-2. However, the acceleration of recombination events observed in kin-18 mutants is largely MPK-1-independent. Our data suggest that KIN-18 coordinates meiotic progression by modulating the timing of MPK-1 activation and the progression of recombination events. The regulation of the timing of MPK-1 activation ensures the proper timing of apoptosis and is required for the formation of functional oocytes. Meiosis is a conserved process; thus, revealing that KIN-18 is a novel regulator of meiotic progression in C. elegans would help to elucidate TAO kinase's role in germline development in higher eukaryotes.

Identifying protein localization is a useful tool in analyzing protein function. Using GFP-fusion tags, researchers can study the function of endogenous proteins in living tissue. However, these tags are considerably large, making them difficult to insert, and they can potentially affect the normal function of these proteins. To improve on these drawbacks, we have adopted the split sfGFP system for studying the localization of proteins in the Caenorhabditis elegans germline. This system divides the "super folder" GFP into 2 fragments, allowing researchers to use CRISPR/Cas9 to tag proteins more easily with the smaller subunit, while constitutively expressing the larger subunit from another locus. These two parts are able to stably interact, producing a functional GFP when both fragments are in the same cellular compartment. Our data demonstrate that the split sfGFP system can be adapted for use in C. elegans to tag endogenous proteins with relative ease. Strains containing the tags are homozygous viable and fertile. These small subunit tags produce fluorescent signals that matched the localization patterns of the wild-type protein in the gonad. Thus, our study shows that this approach could be used for tissue-specific GFP expression from an endogenous locus.

To maintain the integrity of the genome, meiotic DNA double strand breaks (DSBs) need to form by the meiosis-specific nuclease Spo11 and be repaired by homologous recombination. One class of products formed by recombination are crossovers, which are required for proper chromosome segregation in the first meiotic division. The synaptonemal complex (SC) is a protein structure that connects homologous chromosomes during meiotic prophase I. The proper assembly of the SC is important for recombination, crossover formation, and the subsequent chromosome segregation. Here we identify the components of Cullin RING E3 ubiquitin ligase 4 (CRL4) that play a role in SC assembly in Caenorhabditis elegans. Mutants of the CRL4 complex (cul-4, ddb-1, and gad-1) show defects in SC assembly manifested in the formation of polycomplexes (PCs), impaired progression of meiotic recombination, and reduction in crossover numbers. PCs that are formed in cul-4 mutants lack the mobile properties of wild type SC, but are likely not a direct target of ubiquitination. In C. elegans, SC assembly does not require recombination and there is no evidence that PC formation is regulated by recombination as well. However, in one cul-4 mutant PC formation is dependent upon early meiotic recombination, indicating that proper assembly of the SC can be diminished by recombination in some scenarios. Lastly, our studies suggest that CUL-4 deregulation leads to transposition of the Tc3 transposable element, and defects in formation of SPO-11-mediated DSBs. Our studies highlight previously unknown functions of CRL4 in C. elegans meiosis and show that CUL-4 likely plays multiple roles in meiosis that are essential for maintaining genome integrity.

Replication Protein A (RPA) is a critical complex that acts in replication and promotes homologous recombination by allowing recombinase recruitment to processed DSB ends. Most organisms possess three RPA subunits (RPA1, RPA2, RPA3) that form a trimeric complex critical for viability. The Caenorhabditis elegans genome encodes RPA-1, RPA-2 and an RPA-2 paralog RPA-4. In our analysis, we determined that RPA-2 is critical for germline replication and normal repair of meiotic DSBs. Interestingly, RPA-1 but not RPA-2 is essential for somatic replication, in contrast to other organisms that require both subunits. Six different hetero- and homodimeric complexes containing permutations of RPA-1, RPA-2 and RPA-4 can be detected in whole animal extracts. Our in vivo studies indicate that RPA-1/4 dimer is less abundant in the nucleus and its formation is inhibited by RPA-2. While RPA-4 does not participate in replication or recombination, we find that RPA-4 inhibits RAD-51 filament formation and promotes apoptosis of a subset of damaged nuclei. Altogether these findings point to sub-functionalization and antagonistic roles of RPA complexes in C. elegans.

DNA double-strand breaks (DSBs) are one of the most dangerous assaults on the genome, and yet their natural and programmed production are inherent to life. When DSBs arise close together they are particularly deleterious, and their repair may require an altered form of the DNA damage response. Our understanding of how clustered DSBs are repaired in the germline is unknown. Using laser microirradiation, we examine early events in the repair of clustered DSBs in germ cells within Caenorhabditis elegans. We use precise temporal resolution to show how the recruitment of MRE-11 to complex damage is regulated, and that clustered DNA damage can recruit proteins from various repair pathways. Abrogation of non-homologous end joining or COM-1 attenuates the recruitment of MRE-11 through distinct mechanisms. The synaptonemal complex plays both positive and negative regulatory roles in these mutant contexts. These findings indicate that MRE-11 is regulated by modifying its accessibility to chromosomes.

DNA double-strand breaks (DSBs) are deleterious lesions, which must be repaired precisely to maintain genomic stability. During meiosis, programmed DSBs are repaired via homologous recombination (HR) while repair using the nonhomologous end joining (NHEJ) pathway is inhibited, thereby ensuring crossover formation and accurate chromosome segregation.1,2 How DSB repair pathway choice is implemented during meiosis is unknown. In C. elegans, meiotic DSB repair takes place in the context of the fully formed, highly dynamic zipper-like structure present between homologous chromosomes called the synaptonemal complex (SC).3,4,5,6,7,8,9 The SC consists of a pair of lateral elements bridged by a central region composed of the SYP proteins in C. elegans. How the structural components of the SC are regulated to maintain the architectural integrity of the assembled SC around DSB repair sites remained unclear. Here, we show that SYP-4, a central region component of the SC, is phosphorylated at Serine 447 in a manner dependent on DSBs and the ATM/ATR DNA damage response kinases. We show that this SYP-4 phosphorylation is critical for preserving the SC structure following exogenous (γ-IR-induced) DSB formation and for promoting normal DSB repair progression and crossover patterning following SPO-11-dependent and exogenous DSBs. We propose a model in which ATM/ATR-dependent phosphorylation of SYP-4 at the S447 site plays important roles both in maintaining the architectural integrity of the SC following DSB formation and in warding off repair via the NHEJ repair pathway, thereby preventing aneuploidy.