The tumor suppressor BRCA1-BARD1 complex localizes to the synaptonemal complex and regulates recombination under meiotic dysfunction in Caenorhabditis elegans

Breast cancer susceptibility gene 1 (BRCA1) and binding partner BRCA1-associated RING domain protein 1 (BARD1) form an essential E3 ubiquitin ligase important for DNA damage repair and homologous recombination. The Caenorhabditis elegans orthologs, BRC-1 and BRD-1, also function in DNA damage repair, homologous recombination, as well as in meiosis. Using functional GFP fusions we show that in mitotically-dividing germ cells BRC-1 and BRD-1 are nucleoplasmic with enrichment at foci that partially overlap with the recombinase RAD-51. Co-localization with RAD-51 is enhanced under replication stress. As cells enter meiosis, BRC-1-BRD-1 remains nucleoplasmic and in foci, and beginning in mid-pachytene the complex co-localizes with the synaptonemal complex. Following establishment of the single asymmetrically positioned crossover on each chromosome pair, BRC-1-BRD-1 concentrates to the short arm of the bivalent. Localization dependencies reveal that BRC-1 and BRD-1 are interdependent and the complex fails to properly localize in both meiotic recombination and chromosome synapsis mutants. Consistent with a role for BRC-1-BRD-1 in meiotic recombination in the context of the synaptonemal complex, inactivation of BRC-1 or BRD-1 enhances the embryonic lethality of mutants defective in chromosome synapsis. Our data suggest that under meiotic dysfunction, BRC-1-BRD-1 stabilizes the RAD-51 filament and alters the recombination landscape; these two functions can be genetically separated from BRC-1-BRD-1’s role in the DNA damage response. Together, we propose that BRC-1-BRD-1 serves a checkpoint function at the synaptonemal complex where it monitors and modulates meiotic recombination.


Introduction
BRCA1 was identified twenty-eight years ago as the causative agent of early-onset familial breast cancer [1]. Subsequently, BRCA1 was shown to interact with BARD1 through their RING domains [2], to form an E3 ubiquitin ligase, which adds the small polypeptide ubiquitin to protein substrates [3] (hereafter referred to as BRCA1-BARD1). While BRCA1-BARD1 has been extensively studied with respect to its crucial tumor suppressor activities, we still do not fully understand how this protein complex mediates the diverse functions that have been ascribed to it (e.g., DNA metabolism, checkpoint signaling, chromatin dynamics, centrosome amplification, and transcriptional and translational regulation [4,5]). This is due in part to the diversity of protein-protein interactions involved in generating numerous distinct BRCA1-BARD1 multi-protein complexes [6]. An additional impediment to understanding BRCA1-BARD1 function is that the corresponding mouse knockouts are embryonic lethal [7,8].
The simple metazoan Caenorhabditis elegans offers several advantages to the study of this key complex. First, unlike in mammals, C. elegans BRCA1 and BARD1 orthologs, BRC-1 and BRD-1, are not essential yet play critical roles in DNA replication and the DNA damage response, as well as in homologous recombination, which is critical for repairing programmed double strand breaks (DSBs) during meiosis [9][10][11][12][13][14]. Additionally, attributes of the C. elegans system, including sophisticated genetics, ease of genome editing, and the spatio-temporal organization of the germ line allow us to overcome some challenges inherent in studying this complex in mammalian meiosis.
Meiosis is essential for sexual reproduction and results in the precise halving of the genome for packaging into gametes. During meiosis, homologous chromosomes are connected by crossover recombination to facilitate their alignment and segregation on the meiotic spindle. Recombination is integrated and reinforced with chromosome pairing and synapsis, although the extent of dependencies of these critical meiotic processes are distinct in different organisms (reviewed in [15,16]). While it is well established that BRCA1-BARD1 plays an important role in DNA repair and recombination [5], the specific function of BRCA1-BARD1 in meiotic recombination is not known. In mice, partial deletions of BRCA1 result in early apoptosis of male germ cells due to failures in meiotic sex chromosome inactivation [17,18]. BRCA1 has been shown to co-localize with RAD51 on asynapsed chromosomes in mouse spermatocytes, suggesting it functions in meiotic recombination [19]. In C. elegans, brc-1 and brd-1 mutants have mild meiotic phenotypes consistent with a role in some aspect of meiotic recombination [9,10]. However, the relationship between BRC-1-BRD-1 function in synapsis and recombination has not been explored.
Here, we assessed BRC-1 and BRD-1 dynamics in the C. elegans germ line. Surprisingly, BRC-1-BRD-1 localizes to the synaptonemal complex (SC), becomes concentrated onto chromosome regions upon crossover designation, and at late meiotic prophase is restricted to the short arm of each bivalent as defined by the single crossover site on C. elegans chromosomes. BRC-1 and BRD-1 are interdependent for localization to the SC and proper localization is dependent on meiotic recombination and chromosome synapsis. Further, our data suggest that the BRC-1-BRD-1 complex promotes homologous recombination under meiotic dysfunction by stabilizing the RAD-51 filament and altering the patterning of crossovers. Similar findings are reported by Janisiw et al. in the accompanying paper.

GFP::BRC-1 and BRD-1::GFP are expressed in embryos and the germ line
To examine BRC-1 and BRD-1 expression and localization in C. elegans, we engineered GFP:: BRC-1 and BRD-1::GFP fusions at the endogenous loci using CRISPR-Cas9 [20]. brc-1 and brd-1 mutants have low levels of embryonic lethality, produce slightly elevated levels of male progeny (X0), a readout of X chromosome nondisjunction, and display sensitivity to γ-irradiation (IR) [10]. Worms expressing these fusions as the only source of BRC-1 or BRD-1 produced wild-type levels of viable progeny and males, and were not sensitive to IR (S1A-S1C Fig), suggesting that the fusions are fully functional.
We monitored the localization of GFP::BRC-1 and BRD-1::GFP by live cell imaging. In whole worms, GFP fluorescence was observed in embryos and in the germ line, with very little signal in the soma (note auto-fluorescence of gut granules also observed in wild type; Fig 1A). Immunoblots of whole worm extracts of gfp::brc-1; fog-2, which are true females [21] and therefore do not contain embryos, compared to self-fertilizing gfp::brc-1 hermaphrodites containing embryos, revealed that <10% of the GFP::BRC-1 signal is due to expression in embryos (S1E Fig). Thus, BRC-1 and BRD-1 are expressed predominantly in the germ line.

BRC-1-BRD-1 and RAD-51 become concentrated in foci upon replication stress
The C. elegans germ line is arranged in a spatio-temporal gradient, with proliferating germ cells (premeiotic) and all stages of meiosis arrayed from the distal to proximal end [22] (Fig  1B). We first focused on the premeiotic zone, where germ cells are mitotically proliferating. GFP::BRC-1 and BRD-1::GFP were observed diffusely throughout the nucleus, with occasional foci that partially co-localized with the recombinase RAD-51 (Fig 1C and 1D). In mammalian cells RAD51 marks stalled/collapsed replication forks [23], and BRCA1/BRC-1 has been implicated in repair of damaged forks in both mammals and C. elegans [14,24]. To determine whether BRC-1-BRD-1 responds to stalled/collapsed replication forks, we treated worms with the ribonucleotide reductase inhibitor, hydroxyurea (HU). HU slows replication causing fork stalling and collapse, and cell cycle arrest leading to enlarged nuclei [23,25]. GFP::BRC-1 and BRD-1::GFP fluorescence became enriched in many foci following exposure to HU, and these exhibited substantial co-localization with RAD-51 (Fig 1C and 1D). Consistent with a role in resolving collapsed replication forks, both brc-1 and brd-1 mutants were sensitive to HU as measured by embryonic lethality (S1D Fig). These results suggest that BRC-1-BRD-1 responds to replication stress and concentrates in foci where it co-localizes with RAD-51, presumably to resolve stalled/collapsed replication forks.

BRC-1 and BRD-1 localize to the SC and concentrate to the short arm of the bivalent during meiotic prophase
In early meiotic prophase (transition zone/early pachytene), GFP::BRC-1 and BRD-1::GFP direct fluorescence were observed diffusely on chromatin and in foci (Fig 2A). These foci partially overlapped with RAD-51, which marks meiotic DSBs [26]. We noticed that the relative intensity of the foci was weaker in fixed versus live imaging (see Figs 3 and 4), suggesting that these foci were sensitive to fixation conditions. Beginning at mid-pachytene, GFP::BRC-1 and BRD-1::GFP were observed in tracks along the entire chromosome length, and then concentrated to a portion of each chromosome at late pachytene (Fig 2A). In diplotene and diakinesis, GFP::BRC-1 and BRD-1::GFP were further restricted to six short stretches on the six pairs of homologous chromosomes (Fig 2A). As oocytes continued to mature, GFP::BRC-1 and BRD-1::GFP were disassembled from chromosomes in an asynchronous manner, with some chromosomes losing signal before others. Thus, in diakinesis nuclei we did not always observe six stretches of fluorescence, and the fluorescence intensity varied between chromosomes.
The concentration of BRC-1-BRD-1 into tracks at mid-pachytene suggested that the complex localized to the SC. To investigate this, we co-stained with antibodies against GFP and the SC central region component, SYP-1 [27]. Homologous chromosomes begin synapsing early in meiotic prophase (29); however, GFP::BRC-1 was not observed on tracks until after the SC appeared to be fully formed ( Fig 2B). Interestingly, the concentration of GFP::BRC-1 to a portion of each chromosome preceded the relocalization of SYP-1 to the short arm of the bivalent (arrows in late pachytene images of GFP::BRC-1; Fig 2B). As the SC reorganizes as a consequence of crossover maturation [28], we examined worms co-expressing TagRFP-T::BRC-1 (TagRFP-T is a RFP variant with improved photostability [20,29]) and GFP::COSA-1, a cyclin related protein that marks presumptive crossover sites [30]. TagRFP-T::BRC-1 also appeared to be fully functional (S1A-S1C Fig), although the fluorescent signal was weaker than GFP, and could only be detected in mid-late pachytene through diakinesis. GFP::COSA-1 was observed at one end of each TagRFP-T::BRC-1 stretch (Fig 2C). Thus, BRC-1 and BRD-1 localize to the SC and are redistributed concomitant with crossover designation, suggesting that BRC-1-BRD-1 functions in one or more aspects of meiotic recombination within the context of the SC.
To examine localization dependencies between BRC-1 and BRD-1 in C. elegans germ cells, we monitored GFP::BRC-1 and BRD-1::GFP in the corresponding brd-1(ok1623) and brc-1 (xoe4) null mutant backgrounds by live cell imaging. In the absence of BRD-1 we observed diffuse GFP::BRC-1 fluorescence within the nucleoplasm from proliferative zone to mid-pachytene, with no evidence of tracks ( Fig 3B). In late pachytene, weak GFP::BRC-1 foci were observed; however, in diplotene and diakinesis only a diffuse nucleoplasmic signal was detected, with no concentrated regions of GFP::BRC-1. This result suggests that BRD-1 is required for the correct localization of BRC-1 in meiotic cells. In worms harboring a null allele of brc-1, BRD-1::GFP was largely cytosolic, except at diakinesis where it was observed in the nucleoplasm. Analysis of steady state protein levels by immunoblot revealed that BRC-1 and BRD-1 were present, albeit at reduced levels, in the absence of the other partner (in brc-1 (xoe4), BRD-1::GFP = 60% of wild-type levels; in brd-1(ok1623), GFP::BRC-1 = 50% of wildtype levels; Fig 3C). Thus, BRC-1 and BRD-1 are mutually dependent for localization to meiotic chromosomes.

Impairment of either meiotic recombination or synaptonemal complex formation alters GFP::BRC-1 localization
To provide insight into the relationship between BRC-1-BRD-1 and the progression of meiotic recombination, we monitored the localization of GFP::BRC-1 in mutants that impair different steps of meiotic recombination: spo-11 mutants are unable to form meiotic DSBs [32, 33], rad-51 mutants are blocked prior to strand invasion [34-36], and msh-5 mutants fail to form crossovers [37,38]. In live spo-11 mutants, we observed many fewer GFP::BRC-1 foci in transition zone and early pachytene compared to WT (TZ: 1.  (Fig 4). In late pachytene, GFP::BRC-1 fluorescence did not concentrate on a portion of each chromosome pair nor retract to the short arm of the bivalent as in wild type, consistent with these events being dependent on meiotic recombination. However, in 20.23±1.78% of nuclei (n = 4 germ lines) there was enrichment of GFP::BRC-1 on one or sometimes two tracks, in addition to weak staining on other tracks. This is similar to what has been previously reported for synapsis markers, including the phosphorylated form of SYP-4 [39-41], and likely represents spo-11independent lesions capable of recruiting meiotic DNA repair components and altering SC properties. Consistent with this, we observed GFP::BRC-1 enrichment on the phospho-SYP-4-marked chromosome in spo-11 mutants (S2B Fig). However, GFP::BRC-1 did not retract to chromosome subdomains as in wild type in diplotene and diakinesis, suggesting that the relocalization of BRC-1-BRD-1 is dependent on formation of meiotic DSBs. As expected, BRD-1:: GFP was observed in a similar pattern to GFP::BRC-1 in spo-11 mutants throughout meiotic prophase (S2C Fig). fluorescence and RAD-51 signal; green arrows demark foci containing GFP but not RAD-51; red arrows demark foci containing only RAD-51. Scale bar = 5 μm. B) Co-localization between GFP::BRC-1 (green) and SC central component SYP-1 (red) by antibody staining; germ lines at indicated stages were counterstained with DAPI. Blue arrows at late pachytene show chromosomal regions where GFP:: BRC-1 concentrates before SYP-1. Scale bar = 2 μm. C) TagRFP-T::BRC-1 (red) and GFP::COSA-1 (green) at late pachytene showing TagRFP-T::BRC-1 on one side of the GFP::COSA-1 focus, which marks the persumptive crossover. Scale bar = 2 μm. Images are projections through half of the gonad. TZ = transition zone; EP = early pachytene; MP = mid pachytene; LP = late pachytene; DP = diplotene; DK = diakinesis.
https://doi.org/10.1371/journal.pgen.1007701.g002 Following DSB formation and processing, RAD-51 is loaded onto resected single-stranded DNA and facilitates strand exchange [36]. GFP::BRC-1 localization was altered in the rad-51 mutant (Fig 4). Significantly increased levels of GFP::BRC-1 foci were observed throughout the germ line. In the proliferative zone, wild type had 0.55±0.04, while rad-51 had 0.96±0.  BRC-1-BRD-1 regulates meiotic recombination stalled/collapsed replication forks. In transition zone, wild type had 1.29±0.12, while rad-51 had 3.98±0.31 foci/nucleus, and this was further increased in early pachytene (WT: 4.6±0.4 vs. rad-51: 13.3±0.7; S2A Fig). These foci presumably represent resected meiotic DSBs that fail to undergo strand invasion in the absence of RAD-51, as they cannot be solely accounted for by the elevated foci observed in proliferating cells. Track-like structures were not observed until late pachytene in the absence of RAD-51. The punctate nature of GFP::BRC-1 was particularly pronounced in diplotene and diakinesis, with no clear concentration to six regions. This is consistent with the disorganized chromatin masses observed in rad-51 diakinesis nuclei [35], and suggests that RAD-51 is required for the proper organization and retraction of GFP:: BRC-1.
In msh-5 mutants, GFP::BRC-1 appeared similar to wild type from the proliferative zone to mid pachytene, localizing in the nucleoplasm and concentrating in foci before converging on tracks (Fig 4; S2A Fig). Similar to spo-11, 26.27±2.25% of msh-5 late pachytene nuclei (n = 4 germ lines) contained enrichment of GFP::BRC-1 on one or occasionally two chromosomes. In diplotene, GFP::BRC-1 was observed in long tracks, with no evidence of retraction. The presence of more than six stretches of GFP::BRC-1 in diakinesis suggests that BRC-1 remains associated with the univalents in msh-5 mutants. Taken together, our data suggest that GFP:: BRC-1 localizes to the SC and its retraction to the short arm of the bivalent is dependent on processing of meiotic DSBs into crossovers.
We also examined localization of GFP::BRC-1 when synapsis is blocked by mutation of a component of the central region of the SC, syp-1 [27]. GFP::BRC-1 in syp-1 looked similar to wild type in proliferating germ cells (Fig 4). However, as cells entered meiosis GFP::BRC-1 was observed in many foci (in TZ, WT: 1.29±0.12 vs. syp-1: 7.29±0.36 foci/nucleus; S2A Fig). The number of foci increased through early and mid pachytene but GFP::BRC-1 never attained nuclear track staining, supporting a dependency on the SC for track localization. Similarly, the GFP::BRC-1 signal did not localize to sub-regions of condensed (DP and DK) chromosomes, but rather was found in a small number of nuclear foci. Thus, GFP::BRC-1 localization to tracks is dependent on SC formation.
To examine localization under conditions where a subset of chromosomes fail to synapse and recombine, we monitored GFP::BRC-1 localization in the zim-1 mutant, in which chromosomes II and III cannot synapse [42]. In transition zone and early pachytene, GFP::BRC-1 was observed in many foci in the zim-1 mutant, similar to the syp-1 mutant (TZ: WT: 1.29 ±0.12 vs. zim-1: 4.5±0.36 foci/nucleus; Fig 4; S2A Fig). However, as meiosis progressed GFP:: BRC-1 was observed on tracks that condensed to the short arm of the bivalent on multiple chromosomes. Many times we observed more than four stretches of GFP::BRC-1 fluorescence at diplotene/diakinesis (Fig 4), suggesting that there are more than four chiasmata in the zim-1 mutant. We address the role of BRC-1 in chiasmata formation in the zim-1 mutant below.

The BRC-1-BRD-1 complex is important when chromosome synapsis and crossover formation are perturbed
Given the association of GFP::BRC-1 and BRD-1::GFP with the SC (Fig 4), we next examined the functional consequence of removing BRC-1-BRD-1 when synapsis is perturbed. For these studies we focused on the zim-1 mutant, as the appearance of more than four short tracks of GFP::BRC-1 at diplotene/diakinesis (Fig 4) suggested that these BRC-1-BRD-1-associated regions were altered in the absence of zim-1. Additionally, unlike mutants such as syp-1 that result in a complete failure in synapsis and therefore 95% embryonic lethality [27], loss of ZIM-1 results in 73.9% inviable progeny [42], allowing us to determine whether removal of BRC-1-BRD-1 enhances embryonic lethality.
In contrast to the differential impact of the alleles on embryonic lethality, male progeny, and IR sensitivity, loss of zim-1 in any of the brc-1 or brd-1 mutants resulted in enhanced embryonic lethality compared to the single zim-1 mutant (p<0.0001; Fig 5E). These results suggest that the region C-terminal to the BRC-1 RING domain, which is deleted in brc-1 (tm1145), is important for promoting embryonic viability when chromosome pairing and synapsis are perturbed.
To determine the nature of the enhanced embryonic lethality of zim-1 mutants when BRC-1-BRD-1 is impaired, we first monitored germline apoptosis. Apoptosis is an output of checkpoint signaling and is important for culling defective germ cells [45][46][47]. Previous studies had established that both brc-1 [9] and zim-1 [48] have elevated checkpoint-dependent germline apoptosis. We found that all brc-1 and brd-1 alleles, including brc-1(tm1145), had elevated apoptosis ( Fig 5F). Loss of zim-1 resulted in higher levels of apoptosis than brc-1 and brd-1 mutants; however, the levels of apoptosis in the double brc-1; zim-1 and brd-1; zim-1 mutants were not significantly different than zim-1 alone. We also analyzed SUN-1 phosphorylated on Serine12 (Sun-1 S12P), which is dephosphorylated following establishment of the obligate crossover, and serves as a readout of meiotic progression [49]. Loss of ZIM-1 resulted in persistent SUN-1 S12P, which was unaltered in the absence of BRC-1 (S3C Fig). These results suggest that BRC-1-BRD-1 does not function in known signaling pathways responsible for monitoring unrepaired DSBs or crossovers leading to apoptosis or cell cycle delay.
We next monitored RAD-51 assembly/disassembly in the spatiotemporal organization of the germ line. Previous analyses revealed that brc-1 and brd-1 mutant hermaphrodites have elevated RAD-51 foci in late pachytene, suggesting that repair of a subset of meiotic DSBs is delayed in the absence of BRC-1-BRD-1 [9]; this was also observed in the brc-1(tm1145) brd-1 (dw1) and brd-1(ok1623) mutants (S4A Fig). Further, blocking synapsis on some or all chromosomes results in elevated RAD-51 levels genome wide [26,50], as observed in the zim-1 mutant (Fig 6A and 6B). Surprisingly, brc-1; zim-1 and brd-1; zim-1 double mutants resulted in fewer RAD-51 at mid-late pachytene: RAD-51 foci appeared at similar levels compared to the zim-1 single mutant early in meiotic prophase, but in the latter half of pachytene many fewer RAD-51 were detected on chromosomes (Fig 6A and 6B and S4B Fig). High levels of RAD-51 were observed again at the gonad bend, as nuclei exited pachytene and entered diplotene (Fig 6A and 6B and S4B Fig). Similar patterns were observed when BRC-1-BRD-1 was These results suggest that when synapsis and therefore crossover formation is impaired, BRC-1-BRD-1 plays a role in DSB formation, DNA end resection, RAD-51 loading, and/or stabilization of the RAD-51 filament in mid-late pachytene.
To differentiate between these possible meiotic functions of BRC-1-BRD-1, we analyzed the pattern of the single-stranded binding protein RPA-1 (GFP::RPA-1; [51]). RPA-1 binds resected ends prior to RAD-51 loading [52,53] and is also associated with recombination events at a post-strand-exchange step, which can be observed in chromosome spreads [54]. In the brc-1(tm1145); zim-1 germ line we observed an inverse pattern between RAD-51 and RPA-1 at mid-late pachytene: GFP::RPA-1 foci were prevalent in the region where RAD-51 foci were reduced ( Fig 6A). In the zim-1 single mutant, fewer GFP::RPA-1 foci were observed at this stage, while RAD-51 remained prevalent. We also observed very few RPA-1 foci at midlate pachytene in wild type or brc-1(tm1145) brd-1(dw1) double mutant whole mount gonads (S4C Fig). These results suggest that BRC-1-BRD-1 is not required for DSB formation per se in this region of the germ line, as we observed an increase in GFP::RPA-1 foci, not a decrease as would be expected if BRC-1-BRD-1 mediates DSB formation. Additionally, this result argues against a role for BRC-1-BRD-1 in promoting resection as RPA-1 loads on exposed single stranded DNA [52]. Thus, at mid to late pachytene BRC-1-BRD-1 either facilitates the assembly of RAD-51 on new breaks, and/or stabilizes the RAD-51 filament.

BRC-1-BRD-1 alters recombination patterning under meiotic dysfunction
A subset of RAD-51 strand invasions are processed into crossovers, which are marked by CNTD1/COSA-1 [30,58]. Given the reduction in RAD-51 in mid-late pachytene in brc-1; zim-1 and brd-1; zim-1 mutant hermaphrodites, we next analyzed crossover precursor formation in the various mutants. In C. elegans, each of the six chromosome pairs forms a single crossover; consequently, there are six COSA-1 foci in hermaphrodite germ cells at late pachytene [30] (Fig 8A). We also observed six COSA-1 foci in late pachytene nuclei in the brc-1 and brd-1 mutants (Fig 8A), indicating that breaks are efficiently processed into crossovers in the absence of BRC-1-BRD-1 in an otherwise wild-type worm. This is consistent with the presence of six bivalents at diakinesis and the low embryonic lethality of brc-1 and brd-1 [9, 10] ( Fig  5B). In zim-1 mutants we expected to observe four COSA-1 foci per nucleus, one on each of the four paired chromosomes, but not on the unpaired chromosomes II and III. Contrary to our expectations, zim-1 had an average of 6.12±0.12 COSA-1 foci (χ 2 : p<0.005), with a very broad distribution ranging from 2 to 9 foci; such a wide distribution is never observed in wild type [30] (Fig 8A; S5 Fig). Inactivation of BRC-1 and/or BRD-1 in zim-1 reduced the number of GFP::COSA-1 foci to a range of 4.3-4.8 in the various mutants, closer to expectations although still significantly different than expected (χ 2 : p<0.005), and the distribution remained broad (p <0.0001; Fig 8A). These results suggest that when crossovers are unable to form between some homologs, additional COSA-1-marked crossover precursors are generated, and some of these are dependent on BRC-1-BRD-1.
The higher than expected numbers of COSA-1 foci observed in zim-1 mutants could reflect recombination intermediates that do not go on to form chiasmata (i.e., non-crossovers or inter-sister crossovers). Alternatively, COSA-1 could mark bona fide inter-homolog crossovers, such that some chromosomes have more than one chiasma, as has been observed in mutants where the X chromosomes fail to pair and synapse [50]. As these two possibilities are not mutually exclusive, the extra COSA-1 foci could be due to a combination of both recombination outcomes. To provide insight into the nature of the extra COSA-1 foci, we analyzed COSA-1 in syp-1 mutants, where no chiasmata can form as all chromosomes fail to synapse, and found that there were on average 4.85±0.07 COSA-1 foci at late pachytene (Fig 8A; S5  Fig). These results suggest that under conditions of meiotic dysfunction when chromosomes are unable to pair/synapse, COSA-1 is recruited to recombination intermediates that are processed into non-crossovers and/or inter-sister crossovers. Similar numbers of COSA-1 foci, associated with MSH-5, were observed in syp-3 mutants; high resolution cytological analyses indicated that these recombination sites are non-randomly distributed but with some   abnormalities, consistent with the formation of nonproductive intermediates or inter-sister crossovers [59]. As with zim-1 mutants, inactivation of BRC-1-BRD-1 in the syp-1 mutant background led to fewer COSA-1 foci (Fig 8A; S5 Fig), suggesting that BRC-1-BRD-1 promotes COSA-1-associated recombination processing when chiasma formation is impaired.
To determine whether the extra COSA-1 foci on synapsed chromosomes could form chiasmata, we examined zim-1 and brc-1(tm1145); zim-1 diplotene/diakinesis nuclei, where chromosomes are individualized and cross-shaped structures indicative of crossovers between homologs can be observed. Consistent with the formation of extra chiasmata in the zim-1 mutant background, we observed 52% of diplotene/diakinesis nuclei (n = 52) containing at least one ring-shaped structure, and six had two ring-shaped structures. The simplest interpretation is that there was a chiasma on each end of the chromosome pair (arrow; Fig 8B). This was reduced to 21% of diplotene/diakinesis nuclei (n = 43) containing ring-shaped chromosomes in the brc-1(tm1145); zim-1 double mutant (zim-1 vs. brc-1(tm1145); zim-1, p = 0.0028 Mann-Whitney). These results suggest that BRC-1-BRD-1 promotes chiasma formation when some chromosomes are unable to interact with their partner.
To examine genetic crossovers, we monitored linkage between SNP markers on chromosomes V and X in Bristol/Hawaiian hybrid strains to assess both crossover numbers and distribution. While inactivation of brc-1 had no effect on crossover numbers on chromosome V (WT = 48.1cM; brc-1 = 50.8cM), we observed an altered distribution compared to wild type (Fig 8D and 8E; S1 Table). In C. elegans, crossovers are enriched on the arms [28, [60][61][62]; in the brc-1(tm1145) mutant we observed a more even distribution, with more crossovers in the center and fewer on the right arm (Fig 8E; S1 Table). On the other hand, in brc-1(tm1145), neither crossover frequency nor distribution were significantly different on the X chromosome ( Fig 8D  and 8E), which has an altered crossover landscape compared to the autosomes [63,64].
https://doi.org/10.1371/journal.pgen.1007701.g008 BRC-1-BRD-1 regulates meiotic recombination C. elegans exhibits strong interference, which is the phenomenon that a crossover at one position on a chromosome decreases the probability of formation of a crossover nearby, resulting in a single crossover per chromosome [62]. Given the detection of DCOs on chromosome V in the zim-1 and brc-1(tm1145); zim-1 mutants, we calculated the interference ratio. While wild type and brc-1 had absolute intereference of 1, as no double crossovers were observed, the zim-1 mutant displayed reduced interference in the left-center and left-right intervals and negative interference in the center-right interval ( Table 1). Inactivation of BRC-1 in the zim-1 mutant restored positive interference in the center-right interval; however, this fell short of statistical significant (p = 0.064). In addition to the non-randomness in the number and position of crossovers, interference also operates on the level of chromatids such that a crossover between any two non-sister chromatids can affect the probability of those chromatids being involved in other crossovers [65]. Chromatid interference has been shown to occur in fungi, Drosophila, maize and humans [65][66][67][68][69][70]. Since we assayed single products of meiosis, the SCO class includes single crossovers as well as recombinants that are the result of three-or fourstrand double crossovers, while only two strand-events can be detected as DCOs. The elevated numbers of SCOs and reduction in two-strand DCOs on chromosome V in the brc-1(tm1145); zim-1 mutant compared to the zim-1 single mutant (Fig 8C), suggest that there may be more three-and/or four-strand double crossovers when BRC-1 is inactivated. Thus, BRC-1 may counteract chromatid interference under meiotic dysfunction, such that more two-strand double crossovers occur. Taken together, the reduced number of COSA-1 foci and alteration in the genetic map in the brc-1(tm1145); zim-1 mutant suggest that BRC-1-BRD-1 modifies recombination patterning under meiotic dysfunction.

Discussion
Here we show that C. elegans BRC-1 and BRD-1 orthologs localize to the SC and regulate recombination when meiosis is perturbed. Our results suggest that BRC-1-BRD-1 plays an BRC-1-BRD-1 regulates meiotic recombination important role in monitoring and modulating processing of meiotic DSBs into crossovers in the context of the specialized meiotic chromosome structure.

BRC-1-BRD-1 undergoes dynamic localization that is coupled to crossover recombination
In mouse spermatocytes BRCA1 is associated with RAD51 and enriched on asynapsed regions of meiotic chromosomes, including the X-Y sex body [18,19]. Here we show that C. elegans BRC-1 and BRD-1 partially co-localize with RAD-51 in early meiotic prophase, but become enriched on synapsed chromosomes as meiosis progresses, co-localizing with SYP-1, a SC central region component (Fig 2B). The enrichment of mammalian BRCA1 on asynapsed chromosomes versus BRC-1 on synapsed chromosomes in C. elegans most likely reflects alteration in the relationship between meiotic recombination and SC formation in these organisms. Meiotic chromosomes can pair and synapse in the absence of meiotic recombination in C. elegans [32], while these events are interdependent in mammals [15,16]. The HORMAD axial components also show differences in chromosome association in mice and worms: in mice, HOR-MAD1 and HORMAD2 are enriched on asynapsed chromosomes [71,72], while C. elegans HORMADS, HIM-3, HTP-1/2, and HTP-3, remain associated with synapsed chromosomes [73][74][75][76]. However, the function of HORMADs in preventing inter-sister recombination and in checkpoint signaling appears to be similar in these different organisms [77][78][79][80][81][82]. Thus, the association of BRC-1-BRD-1 to the SC in C. elegans is likely a consequence of the inter-relationship between SC formation and meiotic recombination in this organism and not due to different functions for this complex in worm versus mammalian meiosis.
Another difference between C. elegans and mammals is the nature of the kinetochore. C. elegans chromosomes are holocentric while in many organisms, including yeast and mice, chromosomes are monocentric. Holocentricity dictates that a single off-centered crossover is formed on each homolog pair to define the long and short arms necessary to ensure regulated sister chromatid cohesion release at meiosis I and II [60][61][62]83]. Interestingly, BRC-1-BRD-1 becomes restricted to the short arm of the bivalent, as defined by the crossover site, and this precedes SC reorganization. While the absence of BRC-1-BRD-1 alone does not affect crossover formation on chromosome V and the X chromosome, it does have a subtle effect on the distribution of crossovers along chromosome V such that more occur in the middle of the chromosome (Fig 8D and 8E). The change in crossover distribution in brc-1 mutants may contribute to the slightly increased nondisjunction observed in the absence of the BRC-1-BRD-1 complex.
We show that the concentration of BRC-1-BRD-1 to a portion of each chromosome track in late pachytene is dependent on meiotic DSB formation and processing into crossovers (spo-11, rad-51 and msh-5; Fig 4). Interestingly, in both spo-11 and msh-5 mutants there are occasional chromosomal tracks in late pachytene, which are highly enriched for BRC-1. While synapsis markers also show occasional enrichment to single tracks in the absence of spo-11, and these partially overlap with BRC-1 (S2B Fig), no enrichment of synapsis markers is observed when crossover factors (i.e., msh-5, cosa-1 or zhp-3) are removed [39][40][41]. While it has been proposed that spo-11-independent lesions can recruit meiotic DNA repair components [39][40][41], the enrichment of BRC-1 in the absence of such crossover factors suggests that BRC-1-BRD-1 can respond to other repair intermediates in addition to those leading to inter-homolog crossovers. One possibility is that when inter-homolog crossover formation is blocked, DSBs are repaired through site-specific nucleases [84][85][86], a subset of which leads to the concentration of BRC-1-BRD-1 on chromosomes in late pachytene. This is also consistent with the observation that BRC-1 is maintained on chromosomes in spo-11, rad-51 and msh-5 mutants in diakinesis nuclei. Perhaps the failure to form interhomolog crossovers in these mutants leads to continued association of BRC-1-BRD-1 on chromosomes.

BRC-1 and BRD-1 associate and are mutually dependent for localization to meiotic chromosomes
BRCA1 forms a potent E3 ubiquitin ligase only in complex with its partner BARD1 [2,3]. Biochemical and structural studies have defined the RING domains and associated helices of these proteins as critical for catalytic activity and BRCA1-BARD1 interaction [43]. However, while the BRCA1-BARD1 heterodimer exhibits substantially greater E3 ligase activity in vitro than BRCA1 alone, only the BRCA1 RING domain interacts with the E2 for ubiquitin transfer, suggesting that BRCA1 is the critical subunit for E3 ubiquitin ligase activity [3,87]. Structurefunction analysis of the BARD1 RING domain suggests that BARD1 may serve to attenuate BRCA1 E3 ligase activity [88]. Interestingly, while the localization of BRC-1 and BRD-1 were interdependent (Fig 3B), BRD-1 appeared to be excluded from the nucleus in the absence of BRC-1, while BRC-1 was nucleoplasmic and formed foci in late pachytene in the absence of BRD-1 (Fig 3B). These differences may reflect the nature of the alleles examined: brc-1(xoe4) produces no protein, while the two brd-1 alleles are predicted to produce truncated proteins, where the RING domain and associated helices remain intact (Fig 5A). In humans, BRCA1 nuclear localization signals in the middle of the protein can directly mediate nuclear import, or import can occur indirectly through interaction with BARD1 [89]. Thus, the truncated BRD-1 protein produced from the brd-1(ok1623) allele could associate with BRC-1 and facilitate nuclear localization of the albeit nonfunctional complex. Alternatively, C. elegans BRC-1 may be uniquely required for nuclear localization or retention, and in its absence BRD-1 cannot enter or be retained in the nucleus. The weak nucleoplasmic BRD-1 signal observed at the end of meiotic prophase in the absence of BRC-1 most likely reflects differences in the nuclear membrane as oogenesis proceeds [90,91].
In addition to the N-terminal RING domains, both BRC-1 and BRD-1 contain long linker and phosphoprotein binding BRCA1 C-terminal (BRCT) domains. BRCT domains are phosphorylation-dependent interacting modules that have been implicated in tumor suppressor activity [92]. Interestingly, only BRD-1 contains Ankyrin (ANK) repeat interaction domains. Recent structural and functional analyses of the ANK domain in TONSL-MMS22L, a complex involved in homologous recombination, revealed that the ANK domain interacts with histone H4 tails [93]. The BARD1 ANK domains have a very similar fold [93], suggesting that BARD1 ANK domains may be important for association with chromatin. The predicted truncated proteins produced in the brd-1 mutants, which behave as nulls (Fig 5 and S4 Fig), lack at least part of the BRCT domains and all of the ANK domains, suggesting that some combination of these domains are critical for BRD-1 function with respect to both DNA damage signaling and meiosis.

BRC-1-BRD-1 function in meiotic recombination can be genetically separated from its established role in the DDR
It has long been appreciated that BRCA1-BARD1 mediates its tumor suppressor activity at least in part through regulating homologous recombination [6]. Given the importance of homologous recombination in repairing DSBs during meiosis, it is not surprising that removing BRC-1-BRD-1 impinges on meiotic recombination. Unexpectedly, we identified a small region C-terminal to the BRC-1 RING and associated helices as being important specifically for meiosis, suggesting that the function of BRC-1-BRD-1 in DNA damage response and meiosis are distinct. While containing no specific fold or homology, this region has several potential phosphorylation sites based on prediction algorithms that may mediate its interaction with key meiotic proteins.
BRCA1-BARD1 associates with the recombinase RAD51 in both mammals and C. elegans [19,31,94]. BRCA1 has also been shown to be required for the assembly of DNA damage induced RAD51 foci on chromatin [95], and this has been interpreted as a requirement for BRCA1 in RAD51 filament assembly. However, recent biochemical analyses using purified proteins found that BRCA1 is not required for RAD51 assembly on RPA coated single stranded DNA and instead promotes DNA strand invasion [94]. Further, a BARD1 mutant that cannot interact with RAD51 does not promote DNA strand invasion, and also does not form foci in vivo. Thus, it is likely that BRCA1-BARD1 is not required for RAD51 filament assembly per se. Our IR time course analysis of C. elegans brc-1 brd-1 mutants is consistent with a function for this complex in stabilizing the RAD-51 filament. It is possible that similar to the mammalian complex, BRC-1-BRD-1 promotes RAD-51 strand invasion; however, in vivo the RAD-51 filament may be subject to disassembly by other proteins in the absence of BRC-1-BRD-1, which would not be recapitulated in vitro. One such protein is the FANCJ/ DOG-1 helicase, which interacts with BRCA1 [96], and can disassemble RAD51 on ssDNA in vitro [97]. It is also likely that BRCA1-BARD1 plays multiple roles during homologous recombination and interacts with, and coordinates the activity, of many proteins, including RAD51, and these interactions are modulated under different conditions, including DNA damage, meiosis, meiotic dysfunction, as well as at different stages of the cell cycle. Consistent with this, Janisiw et al. found that BRC-1 associates with the pro-crossover factor MSH-5.

BRCA1-BARD1 and meiotic checkpoint signaling
brc-1 and brd-1 mutants have very subtle defects in meiosis. These include low levels of X chromosome nondisjunction [10] (Fig 5C), a delay in repair of a subset of DSBs through the intersister pathway [9], and elevated heterologous recombination [12]. However, removing BRC-1-BRD-1 when meiosis is perturbed in mutants that impair chromosome pairing, synapsis and crossover recombination leads to enhanced meiotic dysfunction, including elevated embryonic lethality (Fig 5E), impaired RAD-51 stability (Fig 7), and alteration of COSA-1 numbers and the crossover landscape (Fig 8). These results suggest that BRC-1-BRD-1 plays a critical role in meiotic recombination when meiosis is impaired.
In both C. elegans and Drosophila melanogaster, preventing crossover formation on a subset of chromosomes leads to additional events on other chromosomes, and is referred to as the interchromosomal effect [50,[98][99][100][101]. There is also evidence in humans that Robertsonian translocations elicit the interchromosomal effect [102]. Our analyses of the zim-1 mutant, where chromosomes II and III fail to recombine, revealed elevated COSA-1 foci genome wide and an increase in genetic crossovers on chromosome V (but not the X chromosome, Fig 8), consistent with the interchromosomal effect. Further, our data suggest that when meiosis is impaired as in syp-1, and perhaps zim-1 mutants, COSA-1 can mark events that do not ultimately become interhomolog crossovers (see also [59]). Interestingly, removal of BRC-1-BRD-1 in zim-1 and syp-1 mutants decreased the number of COSA-1 foci. On the other hand, in the brc-1(tm1145); zim-1 mutant we detected elevated levels of single crossovers but reduced levels of two-strand double crossovers on chromosome V compared to the zim-1 single mutant, with no change in the overall map length (Fig 8D). One possibility to explain the observed COSA-1 and crossover pattern is that COSA-1 does not become enriched on a subset of crossovers in brc-1; zim-1 mutants even though these events are dependent on the canonical meiotic crossover pathway, as observed in the rtel-1 and dyp-28 mutants [30, [103][104][105]. Alternatively, the extra crossovers that are not marked by COSA-1 in the absence of BRC-1-BRD-1 may be the result of activation of alternative crossover formation pathways. In either scenario, BRC-1, and presumably BRD-1, appear to dictate the patterning of crossovers among non-sister chromatids. As interference is mediated by meiotic chromosome structure [106], perhaps SCassociated BRC-1-BRD-1 counteracts chromatid interference in the context of meiotic dysfunction.
In conclusion, our results suggest that BRC-BRD-1 serves a critical role in monitoring the progression of meiotic recombination in the context of the SC when meiosis cannot proceed normally, suggesting that BRC-1-BRD-1 serves a checkpoint function. When crossover formation is blocked, BRC-1-BRD-1 stabilizes the RAD-51 filament and promotes processing of recombination intermediates marked by COSA-1. In this context, BRC-1-BRD-1 joins a growing list of proteins that monitor meiotic recombination to promote accurate chromosome segregation, including protein kinases and ubiquitin/SUMO E3 ligases [39, 56,57,[107][108][109][110]. Future work will examine the relationship between BRC-1-BRD-1 and other meiotic checkpoint pathways and identify substrates of BRC-1-BRD-1-ubiquitination to understand how this complex modulates recombination under conditions when meiosis is perturbed.

Generation of gfp::brc-1, tag-rfp-T::brc-1, brd-1::gfp and brc-1(xoe4)
Fluorescent protein knock-ins were generated using CRISPR-mediated homology dependent repair with self-excising cassette containing hygromycin resistant as selection [20]. The brc-1 (xoe4) deletion allele was generated using Cas9-snRNPs and an single strand oligonucleotide repair template [111]. Cas9 protein was purchased from Innovative Genomics Institute, UC Berkeley. For a list of sgRNAs and repair templates refer to S2 Table. All CRISPR-generated lines were back crossed a minimum of three times, with the exception of JEL744 brc-1(xoe4) brd-1::gfp, which was only back crossed once.

Genetics
C. elegans var. Bristol (N2), was used as the wild-type strain. Other strains used in this study are listed in S3 Table. Some nematode strains were provided by the Caenorhabditis Genetics Center, which is funded by the National Institutes of Health National Center for Research Resources. Strains were maintained at 20˚C.

Embryonic lethality and production of male progeny
Embryonic lethality in the absence or presence of 5mM hydroxyurea (HU) (16 hrs), or 75 Grays (Gy) of γ-irradiation (IR) from a 137 Cs source, was determined over 3 days by counting eggs and hatched larvae 24 hr after removing the hermaphrodite and calculating percent as eggs/ (eggs + larvae); male progeny was assessed 48 hr after removing the hermaphrodite. A minimum of 10 worms were scored for each condition.

Apoptosis assay
Acridine orange (AO) staining of apoptotic germ cells in WT (N2), brc-1 and brd-1 alleles as well as zim-1 and corresponding double and triple mutants were performed as in [48]. Briefly, 0.5 ml of 50 mg/ml AO (Molecular Probes, Invitrogen; Carlsbad, CA) in M9 was added to 60-mm plates containing 48 hr post L4 worms and incubated at room temperature for 1 hr. Worms were transferred to new 60-mm plates, allowed to recover 15 min, and then mounted under coverslips in M9 on 3% agarose pads containing 1 mM tetramisole (Sigma-Aldrich; St. Louis). Apoptotic bodies were scored by fluorescence microscopy and DIC.

Cytological analysis
Gonads were dissected and fixed with 1% paraformaldehyde in egg buffer plus 0.01% Tween20 for 5 min, freeze-cracked and post-fixed in either ice-cold 100% methanol for indirect immunofluorescence, or ice-cold 100% ethanol for direct fluorescence (GFP::BRC-1, TagRFP-T:: BRC-1, BRD-1::GFP, GFP::RPA-1, GFP::COSA-1) for 1 min [112]. For staining with antibodies against phospho-SYP-4, gonads were dissected, freeze-cracked, incubated in 100% methanol for 1 min and post-fixed in 4% paraformaldehyde in PBS, 80mM HEPES(pH7. Collection of fixed images was performed using an API Delta Vision deconvolution microscope, a Nikon TiE inverted microscope stand equipped with an 60x, NA 1.49 objective lens and Andor Clara interline camera, or were captured on a spinning-disk module of an inverted objective fluorescence microscope [Marianas spinning-disk confocal (SDC) real-time 3D Confocal-TIRF (total internal reflection) microscope; Intelligent Imaging Innovations] equipped with an 63x, NA 1.46 objective lens using a Photometrics QuantiEM electron multiplying charge-coupled device (EMCCD) camera. Z stacks (0.2 μm) were collected from the entire gonad. A minimum of three germ lines was examined for each condition. Images were deconvolved using Applied Precision SoftWoRx or Nikon NIS Elements Offline batch deconvolution software employing either "Automatic3D" or "Richardson-Lucy" deconvolution modes and subsequently processed and analyzed using Fiji (ImageJ) (Wayne Rasband, NIH).
RAD-51 foci were quantified in a minimum of three germ lines of age-matched hermaphrodites (18-24 hr post-L4). As zim-1 mutants have an extended transition zone [42], we divided germ lines into four equal zones from the beginning of the transition zone (leptotene/ zygotene), as counted from the first row with three or more crescent-shaped nuclei, through diplotene ( Fig 6B). The number of foci per nucleus was scored for each region.
To assess formation of RAD-51 foci following IR treatment, 18-24 hrs post-L4 worms were exposed to 10 Gy of IR; gonads were dissected 1, 4, 8, and 12 hr following IR treatment and fixed for immunofluorescence as above.
GFP::COSA-1 foci were quantified from deconvolved 3D data stacks; nuclei were scored individually through z-stacks to ensure that all foci within each individual nucleus were counted. Nuclei with features indicative of apoptosis (compact and DAPI-bright) were excluded. Foci were counted in the last five rows of pachytene nuclei as in [30].
For live cell imaging, 18-24 hr post L4 hermaphrodites were anesthetized in 1mM tetramisole and immobilized between a coverslip and an 2% agarose pad on a glass slide. Z-stacks (0.33 μm) were captured on a spinning-disk module of an inverted objective fluorescence microscope (NIH 1S10RR024543) with a 100×, NA 1.46 objective, and EMCCD camera. Z-projections of stacks were generated, cropped, and adjusted for brightness in Fiji.
Pearson's Correlation Coefficient (PCC) was determined by drawing a Region of Interest (ROI) around a nucleus and using the co-localization function in Fiji.

RNA-mediated interference analysis
RNA-mediated interference (RNAi) was performed at 20˚C, using the feeding method [113]. Cultures were plated onto NGM plates containing 25 μg/ml carbenicillin and 1 mM IPTG and were used within 2 weeks. L4 worms were transferred to RNAi plates, and resulting progeny were exposed to IR as described above. The efficiency of RNAi was tested in parallel by examining embryonic lethality.

Meiotic mapping
Meiotic crossover frequencies and distribution were assayed utilizing single-nucleotide polymorphism (SNP) markers as in [114]. The SNP markers located at the boundaries of the chromosome domains were chosen based on data from WormBase (WS231) and [64], and are indicated in Fig 8D. The SNP markers and primers used are listed in [86]. PCR and restriction digests of single embryo lysates were performed and confirmed with additional SNPs as described in [115,116] (Fig 8D). Statistical analyses were performed using the two-tailed Fisher's Exact test, 95% C.I., as in [117,118]. For statistical analyses of interference we conducted χ 2 tests on 2-by-2 contingency tables of observed and expected DCOs [119].
Supporting information S1