Significance
Gamete production requires tightly regulated interactions between the parental chromosomes, which coalign and exchange information. These events are mediated by the synaptonemal complex—a ladder-like structure that assembles between the parental chromosomes. The molecular interactions that underlie synaptonemal complex assembly remain poorly understood due to rapid sequence divergence and challenges in biochemical reconstitution. Our detailed genetic data suggests a three-component interface in the nematode synaptonemal complex. Destabilization and subsequent restoration of this interface link synaptonemal complex integrity with chromosome alignment and regulation of exchanges. Beyond mechanistic understanding of chromosomal interactions, our work provides a blueprint for genetic probing of large cellular assemblies that are refractory to structural analysis and sheds light on the forces that shape their evolution.
Abstract
Successful chromosome segregation into gametes depends on tightly regulated interactions between the parental chromosomes. During meiosis, chromosomes are aligned end-to-end by an interface called the synaptonemal complex, which also regulates exchanges between them. However, despite the functional and ultrastructural conservation of this essential interface, how protein–protein interactions within the synaptonemal complex regulate chromosomal interactions remains poorly understood. Here, we describe a genetic interaction in the C. elegans synaptonemal complex, comprised of short segments of three proteins, SYP-1, SYP-3, and SYP-4. We identified the interaction through a saturated suppressor screen of a mutant that destabilizes the synaptonemal complex. The specificity and tight distribution of suppressors suggest a charge-based interface that promotes interactions between synaptonemal complex subunits and, in turn, allows intimate interactions between chromosomes. Our work highlights the power of genetic studies to illuminate the mechanisms that underlie meiotic chromosome interactions.
Sexual reproduction requires tightly regulated chromosomal interactions. During meiosis, parental chromosomes (homologs) pair and align along their length. Chromosome alignment sets the stage for reciprocal exchanges called crossovers that physically link the homologs and allow them to correctly segregate during the first meiotic division. Failure to form crossovers, or formation of too many crossovers, can lead to chromosome missegregation, aneuploid progeny, and infertility (1–3).
Crossovers form within, and are regulated by, a dedicated meiotic chromosomal structure called the synaptonemal complex (SC). Upon entry into meiosis, each homolog adopts an elongated morphology organized around a backbone called the axis. The axes of the two homologs are then aligned, end-to-end, concomitant with the assembly of the SC. While the axes are classically considered part of the SC, here we use the term SC to specifically refer to the structure assembled between parallel axes, also known as the central region of the SC. In addition to bringing the homologs, and therefore the DNA sequences that form crossovers, into proximity, the SC directly participates in regulating crossover number and distribution (4, 5).
Our understanding of how the SC brings homologs together and regulates crossovers is limited. While the ultrastructure of the SC is conserved across eukaryotes, appearing as a ~100 nm-wide ladder in electron micrographs (6–11), the protein sequences of its components diverge rapidly (12–14). The poor sequence homology has slowed the identification of SC components, and it is unclear whether the complete set of components has been defined in any organism. Poor sequence homology has also limited the translation of structural findings across model organisms. Finally, we have very limited molecular-genetic information since only a small subset of components has been subjected to systematic mutational analysis or biochemical and structural probing.
The nematode C. elegans has proved a useful model for studying SC structure and function. The SC in worms assembles prior to crossover formation and is essential for all crossovers (8). In addition, the role of the SC in regulating crossover distribution has been directly demonstrated (15). The worm SC includes eight proteins: SYP-1, -2, -3, -4, -5/6, and SKR-1/2 (SYP-5 is partially redundant with SYP-6 and SKR-1 is partially redundant with SKR-2; refs. 8 and 16–21). These components exhibit stereotypical organization within the SC: SYP-1 and -5/6 are organized in a head-to-head fashion to span the distance between the axes, with their N-termini in the middle of the SC and C-termini near the axis (16) and SYP-2, SYP-4, and SKR-1/2 localize to a band in the middle of the SC (21–23). The localization of SYP-3 remains an open discussion, with two studies placing SYP-3 in the middle of the SC (20, 22) and another localizing SYP-3 near the axis (23) (Fig. 1A).
Previously, we carried out mutational analysis of the N-terminus of SYP-1 and demonstrated its role in holding the parallel axes together and in regulating crossovers (24). Here, we use a temperature-sensitive single substitution mutant in syp-1 to carry out a suppressor screen that allowed us to suggest an interaction interface in the middle of the SC. This predicted interface ensures efficient assembly of the SC onto chromosomes, and consequently, the formation of tightly regulated crossovers and successful chromosome segregation. Many of the suppressors we isolated did not affect meiosis by themselves despite altering highly conserved residues, illuminating the unusual selective pressures that shape SC protein evolution.
Results
A Suppressor Screen of a Temperature-Sensitive SC Mutant.
We previously identified a lysine-to-glutamic acid substitution, syp-1K42E, that weakens interactions among SC proteins, causing failures in chromosome synapsis and crossover regulation (24). In syp-1K42E, the stability of the SC progressively weakens as the temperature increases, leading to worsening meiotic phenotypes (24). At 15 °C, the SC in syp-1K42E animals is mostly unaffected, and the worms indeed produce many viable self-progeny, although their number is reduced by 53% compared to control animals (Fig. 1B, mean total progeny 15 °C; syp-1+ = 222, syp-1K42E= 104). At 25 °C, a temperature at which wild-type C. elegans can reproduce without discernable meiotic defects, syp-1K42E animals are nearly sterile (Fig. 1B, mean total progeny 25 °C; syp-1+ = 145, syp-1K42E = 1). The SC at this temperature assembles onto unpaired chromosomes, a morphology that is never observed in wild-type animals (24).
We carried out a whole-genome mutagenic screen to identify suppressors of the syp-1K42E fertility defect. We used the alkylating agent N-ethyl-N-nitrosourea (ENU) to mutagenize ~50,000 P0 gfp::cosa-1; syp-1K42E animals grown at 15 °C and divided them among 100 plates. We allowed their F1 progeny (carrying heterozygous mutations) to grow at 15 °C until they reached the young adult (L4) stage, at which point we shifted them to the nonpermissive temperature of 25 °C. Candidate suppressors of the syp-1K42E fertility defect quickly outcompeted their non-suppressed siblings and starved the plate (Fig. 1C). Through this approach, we identified 23 suppressed lines that showed a wide range in their ability to suppress the infertility of syp-1K42E at 25 °C (Fig. 1D).
All Suppressed Lines of syp-1K42E Contain Mutations in SC Proteins.
To identify the causative suppressor mutations, we employed a combination of candidate Sanger sequencing and whole-genome sequencing followed by variant calling (Fig. 2A). We hypothesized that suppressor mutations could be within SC components, so we sequenced the N-terminus of syp-1 and syp-3. [Two studies place SYP-3 in the middle of the SC (20, 22), and a small truncation in syp-3 exhibits phenotypes similar to those of syp-1K42E (18).] This approach had the added advantage of confirming the presence of the original syp-1K42E mutation. Twenty-two out of the 23 suppressed strains had the original syp-1K42E mutation. The remaining strain had acquired a mutation that resulted in reversion to the wild-type SYP-1 sequence (Fig. 2A, syp-1E42K revertant). We could distinguish the revertant from wild-type contamination because the suppressed strain used AAA to encode the lysine (K) at position 42, rather than the AAG codon in the reference genome (Fig. 2B). We also identified seven additional candidate suppressor mutations in syp-1 and three candidate suppressor mutations in syp-3. In syp-1, there were three charge-altering mutations, syp-1E41K and syp-1E45K (recovered twice independently), three polar to hydrophobic mutations, syp-1S24L (twice) and syp-1T35I, and one hydrophobic to hydrophobic mutation, syp-1V39I. The three mutations in syp-3 were at amino acid position 62 and altered the normally negatively charged aspartic acid (D) to either asparagine (N, polar), valine (V, hydrophobic), or glycine (G, small/hydrophobic).
We identified the remaining suppressor mutations through whole-genome sequencing. We identified all homozygous SNPs and indels in the suppressed strains relative to the parental strain and the reference genome, WBcel235 (Fig. 2C). In addition to the 2,036 SNPs present in gfp::cosa-1 relative to the reference genome, each suppressed genome had 199 homozygous mutations on average (Fig. 2D). Using this number, plus the number of mutagenized P0 animals (50,000), the number of F1s per P0 at 15 °C (Fig. 1A, gfp::cosa-1; syp-1K42E average total progeny = 104), and the haploid genome size of C. elegans (1 x 108 base pairs), we estimate that each base pair in the genome was mutagenized 10 times in our screen. Using a Poisson distribution, the probability that a base pair was not screened is 4.5 × 10−5 (Fig. 2E, λ = 10).
Out of the 2,789 homozygous mutations in the 14 independent suppressed lines we sequenced, 1,968 were in intergenic regions and UTRs, 159 were synonymous site mutations and 96 mutations were in gene introns (SI Appendix, Fig. S1A and Dataset S1), in line with the genomic abundance of these elements. We found 537 missense mutations and 29 mutations that were likely to abolish gene function through the loss of a start or a stop codon, a nonsense or frameshift mutation or a mutation of a splice acceptor/donor site (SI Appendix, Fig. S1A). The distribution of SNPs matched the expected mutagenic profile of ENU with an overall bias toward GC > TA transitions, although all transitions and transversions were represented (SI Appendix, Fig. S1B and ref. 25).
To prioritize suppressor mutations, we sorted for genes whose RNAi phenotype, allele phenotype, or gene ontology included the words “meiosis” or “meiotic” (Dataset S2). Each genome had between one and four missense mutations in meiotic genes out of 34 missense mutations per genome on average. Strikingly, every genome had a missense mutation in an SC subunit. These included three mutations in syp-1 [syp-1S24L (identified twice), syp-1T35I, and syp-1V39I] and three mutations in syp-4 [syp-4A81T, syp-4E90G, and syp-4E90K (identified eight times)]. Given the number of ENU-generated missense mutations per genome, the probability of uncovering 21 independent mutations in SC proteins by chance is less than 1 × 10−42.
The location of the suppressors in narrow regions of the same SC proteins and the independent isolation of multiple instances of the same mutation, like syp-4E90K, suggest that the mutations in SYP-1, -3, and -4 are the causative suppressor mutations.
Suppression Strength Correlates with Amino Acid Charge.
We noticed that many of the suppressors change a negatively charged residue to a positively charged residue (e.g., syp-1E41K, syp-1E45K, and syp-4E90K) or neutralize a negatively charged residue (e.g., syp-3D62N, syp-3D62V, syp-3D62G, and syp-4E90G). Accordingly, we divided the suppressors into three categories and carefully analyzed their effects; one, suppressor mutations that neutralized a negative charge (syp-3D62N, syp-3D62V, and syp-4E90K), two, suppressor mutations that altered a polar residue (syp-1T35I and syp-1S24L), and three, suppressor mutations that altered a hydrophobic residue (syp-4A81T and syp-1V39I). Rather than solely rely on homozygous suppressed animals recovered from the screen, we engineered three of the strongest suppressor mutations (syp-3D62N, syp-3D62V, and syp-4E90K) using CRISPR/Cas9 in the gfp::cosa-1; syp-1K42E background. (In the figures, we designated the genotype of the suppressed strains, as opposed to the edited strains, with a gray background.) The suppression we observed in these engineered strains (see below) suggests that non-SC mutations likely have limited effects on the meiotic phenotypes and further confirms the identity of the suppressors in SYP-3 and -4.
The original mutation, syp-1K42E, has very low total progeny and a high incidence of male self-progeny at 25 °C (Fig. 1B and ref. 24), caused by meiotic nondisjunction of the X chromosomes. Animals suppressed with syp-3D62N, syp-3D62V, or syp-4E90K showed complete suppression of defects in total progeny and percent males at 25°C (Fig. 3 A and B). Like wild-type animals, they had large brood sizes (Fig. 3A, blue-filled circles, gfp::cosa-1; syp-1K42E; syp-3D62N = 223, gfp::cosa-1; syp-1K42E; syp-3D62V = 210, gfp::cosa-1; syp-1K42E; syp-4E90K = 189) and rare male self-progeny (Fig. 3B, blue-filled circles, all strains <1% male progeny). Animals suppressed with syp-1T35I and syp-1S24L showed an intermediate phenotype with average total progeny of 178 and 99, respectively, and a slightly elevated percentage of male self-progeny (Fig. 3 A and B, teal-filled circles, gfp::cosa-1; syp-1K42E+T35I = 1.4% and gfp::cosa-1; syp-1K42E+S24L = 4% male progeny). Finally, animals suppressed with syp-4A81T and syp-1V39I provided the weakest suppression and had relatively low total progeny and a high percentage of male offspring, albeit significantly suppressed compared to gfp::cosa-1; syp-1K42E (Fig. 3 A and B, yellow filled circles, gfp::cosa-1; syp-1K42E; syp-4A81T average total progeny = 25, average percent males = 9.5%, gfp::cosa-1; syp-1K42E+V39I average total progeny = 31, average percent males = 14.8%).
Suppressor Mutations Restore SC Assembly, SC Stability, and Crossover Regulation.
We next verified that the suppressors restored the SC defects in female meiosis in syp-1K42E animals. At 20 °C, syp-1K42E nuclei in mid-pachytene fail to synapse homologs end-to-end and typically have at least one asynapsed and one partially synapsed (forked) chromosome pair; at 25 °C, the SC assembles onto unpaired chromosomes (24). We found that strains suppressed by syp-3D62N, syp-3D62V, syp-4E90K, syp-1T35I, and syp-1S24L achieved complete synapsis by mid-pachytene at 25 °C (Fig. 3 C and E). We observed almost no forked chromosomes or unpaired chromosomes lacking SC (Fig. 3 C and E and SI Appendix, Fig. S2). In contrast, strains suppressed by syp-4A81T and syp-1V39I failed to achieve full synapsis and only had a few synapsed chromosomes per nucleus (Fig. 3 C and E and SI Appendix, Fig. S2), likely accounting for their failure to form crossovers between all homolog pairs and, consequently, their smaller brood sizes and high percentage of male self-progeny.
We next assessed the ability of the suppressed strains to carry out accurate crossover regulation. syp-1K42E exhibits weaker crossover interference, with more than one crossover observed on the same chromosome and the same stretch of SC (24). We leveraged a cytological marker of crossovers, GFP::COSA-1, which localizes to each of the designated crossovers, numbering six in wildtype C. elegans (one per chromosome) (26). Mutations that impact the integrity of the SC, including syp-1K42E (24), syp-1 RNAi (15), and syp-4CmutFlag (ie25) (22), reduce crossover interference so that there are more than six crossovers per nucleus. We found that strains suppressed by syp-3D62N, syp-3D62V, syp-4E90K, syp-1T35I, and syp-1S24L all had six crossovers per nucleus (Fig. 3 D and E), indicating restoration of crossover interference. In contrast, the weak suppressors syp-4A81T and syp-1V39I showed a wide distribution of crossover number, with some nuclei having only one or two crossovers and some having more than 10 (Fig. 3 D and E). This is likely not just a reflection of the incomplete synapsis in these mutants, since both harbored instances of chromosomes (i.e., a stretch of SC) with more than one crossover (SI Appendix, Fig. S3).
Previous work has shown that meiotic nuclei sense unpaired chromosomes, and, in response, spend a longer time in the so-called transition zone, where homology search and SC assembly occur (corresponding to the classically defined leptotene and zygotene stages of meiosis; ref. 27). The extended transition zone could allow for more time to complete SC assembly. We found that most suppressed strains failed to suppress the extended transition zone of syp-1K42E animals (Fig. 3 F and G and SI Appendix, Fig. S4). In the case of the weak suppressors, that is likely due to partial synapsis (Fig. 3 C and E). Interestingly, the strong and intermediate suppressors exhibited different transition zone lengths—extended in the strong suppressors and shorter in the intermediate suppressors—even though almost all nuclei in these strains achieved complete synapsis by mid-pachytene (Fig. 3 C and E). The mechanism that senses and responds to asynapsed chromosomes depends on axis proteins, likely involving exposed axes not associated with the SC (28, 29). A possible reason for the difference in transition zone length between the strong and intermediate suppressors might be their different propensities to associate with the axes, either in the context of assembled SC or with aberrant conformations, such as forked chromosomes. Alternatively, the difference between the strong and intermediate suppressors may reflect slower kinetics of synapsis or defects that cannot be discerned at the resolution of confocal microscopy.
Finally, we tested whether the suppressor mutations restore the unstable SC in syp-1K42E animals (24). We measured the proportion of GFP::SYP-3 on chromosomes in mid-pachytene relative to the total nucleoplasmic amount in syp-1K42E and suppressed (syp-1T35I + K42E) live animals. We identified interchromosomal regions using HTP-3::wrmScarlet, a component of the axis that assembles independently of the SC. Consistent with our published findings, we found that less GFP::SYP-3 is recruited to chromosomes in syp-1K42E compared to a wild-type control (Fig. 4 A and B and ref. 24). The suppressor mutation (syp-1T35I) restored the amount of SC recruited to chromosomes to wild-type levels (Fig. 4 A and B). Notably, since we analyzed animals at the semipermissive temperature of 20 °C, where mostly normal synapsis occurs in all tested strains, we likely underestimated the full destabilizing effect of syp-1K42E.
In summary, the suppressors of the fertility defect of syp-1K42E also suppressed its other meiotic phenotypes: incomplete synapsis, unstable SC, and weakened crossover interference. Furthermore, weaker suppressors consistently exhibited incomplete suppression. Our data thus suggest that SYP-1 physically interacts with SYP-3 and SYP-4 via charge–charge interactions between the N-terminus of SYP-1, position 62 in SYP-3, and position 90 in SYP-4. SYP-1K42E weakens this interaction and, as a result, the SC is less stable and fails to fully assemble onto chromosomes and to confer robust crossover interference (24). The suppressors restore this predicted charge interface and consequently rescue the chromosomal and meiotic defects.
Suppressors Alter Conserved Residues in SC Proteins.
We next wished to address the molecular mechanism of suppression. Unfortunately, atomic structures for C. elegans SC proteins have not been solved, in a complex or in isolation, preventing us from localizing the mutated sites relative to one another [beyond their colocalization in the middle of the SC (22, 23)]. It has also been challenging to derive structural insight from other model organisms through modeling, due to limited sequence homology between clades (12–14). Our attempts to model a docking interface between the regions surrounding the mutations using AlphaFold did not yield a high-confidence interface.
Lacking tertiary structural information, we examined the potential impact of the suppressor mutations on coiled-coil domains—a conserved feature of SC proteins throughout eukaryotes (12). The suppressor mutations in SYP-1 occur before the start of the coiled-coil domain (Fig. 5 A and B). In SYP-3 and SYP-4, the suppressor mutations lie within extended coiled-coil domains but do not alter their predicted structure (Fig. 5 C–F and SI Appendix, Fig. S5 A and B). In SYP-3, the site of the suppressor mutation is not contained within a heptad repeat—the distinguishing feature of coiled-coils. So, even though one of the mutations introduces a glycine, which can disrupt alpha helices (30), the suppressors are not likely to impact the overall coiled-coil structure of the protein (Fig. 5 C and D and SI Appendix, Fig. S5A). In SYP-4, the suppressor mutations are found within perfect heptad repeats, where the “a” and “d” (first and fourth) positions are occupied by hydrophobic residues, and the “e” and “g” (fifth and seventh) positions are polar. The suppressor mutations do not change the underlying heptad structure: They either alter a residue in the nonsignificant “c” position (A->T) or swap one polar residue for another in the “e” position (E->K; Fig. 5 E and F and SI Appendix, Fig. S5B)….
