Building the synaptonemal complex: Molecular interactions between the axis and the central region.

The successful delivery of genetic material to gametes requires tightly regulated interactions between the parental chromosomes. Central to this regulation is a conserved chromosomal interface called the synaptonemal complex (SC), which brings the parental chromosomes in close proximity along their length. While many of its components are known, the interfaces that mediate the assembly of the SC remain a mystery. Here, we survey findings from different model systems while focusing on insight gained in the nematode C. elegans. We synthesize our current understanding of the structure, dynamics, and biophysical properties of the SC and propose mechanisms for SC assembly.

A suppressor screen in C. elegans identifies a multiprotein interaction that stabilizes the synaptonemal complex.

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.

Synaptonemal Complex dimerization regulates chromosome alignment and crossover patterning in meiosis.

During sexual reproduction the parental homologous chromosomes find each other (pair) and align along their lengths by integrating local sequence homology with large-scale contiguity, thereby allowing for precise exchange of genetic information. The Synaptonemal Complex (SC) is a conserved zipper-like structure that assembles between the homologous chromosomes, bringing them together and regulating exchanges between them. However, the molecular mechanisms by which the SC carries out these functions remain poorly understood. Here we isolated and characterized two mutations in the dimerization interface in the middle of the SC zipper in C. elegans. The mutations perturb both chromosome alignment and the regulation of genetic exchanges. Underlying the chromosome-scale phenotypes are distinct alterations to the way SC subunits interact with one another. We propose a model whereby the SC brings homologous chromosomes together through two activities: obligate zipping that prevents assembly on unpaired chromosomes; and a tendency to extend pairing interactions along the entire length of the chromosomes.

Let's get physical - mechanisms of crossover interference.

The formation of crossovers between homologous chromosomes is key to sexual reproduction. In most species, crossovers are spaced further apart than would be expected if they formed independently, a phenomenon termed crossover interference. Despite more than a century of study, the molecular mechanisms implementing crossover interference remain a subject of active debate. Recent findings of how signaling proteins control the formation of crossovers and about the interchromosomal interface in which crossovers form offer new insights into this process. In this Review, we present a cell biological and biophysical perspective on crossover interference, summarizing the evidence that links interference to the spatial, dynamic, mechanical and molecular properties of meiotic chromosomes. We synthesize this physical understanding in the context of prevailing mechanistic models that aim to explain how crossover interference is implemented.

Heterologous synapsis in C. elegans is regulated by meiotic double-strand breaks and crossovers.

Alignment of the parental chromosomes during meiotic prophase is key to the formation of genetic exchanges, or crossovers, and consequently to the successful production of gametes. In almost all studied organisms, alignment involves synapsis: the assembly of a conserved inter-chromosomal interface called the synaptonemal complex (SC). While the SC usually synapses homologous sequences, it can assemble between heterologous sequences. However, little is known about the regulation of heterologous synapsis. Here, we study the dynamics of heterologous synapsis in the nematode C. elegans. We characterize two experimental scenarios: SC assembly onto a folded-back chromosome that cannot pair with its homologous partner; and synapsis of pseudo-homologs, a fusion chromosome partnering with an unfused chromosome half its size. We observed elevated levels of heterologous synapsis when the number of meiotic double-strand breaks or crossovers were reduced, indicating that the promiscuity of synapsis is regulated by break formation or repair. In addition, our data suggests the existence of both chromosome-specific and nucleus-wide regulation on heterologous synapsis.

Taz1 enforces cell-cycle regulation of telomere synthesis.

The dramatic telomerase-dependent overelongation of telomeres in cells lacking Taz1 (ortholog of human TRF1/TRF2) or Rap1 implicates these proteins in restraint of telomerase activity. However, the modes by which these proteins regulate telomerase remain mysterious. Here we show that the mechanisms underlying excessive telomerase activity differ markedly between taz1Δ and rap1Δ strains. Despite allowing elevated telomerase access, rap1Δ telomeres are processed and synthesized in a cell-cycle-constrained manner similar to that of wild-type cells. In contrast, taz1Δ telomeres are processed with little cell-cycle dependency and recruit telomerase over an abnormally wide range of cell-cycle stages. Furthermore, although taz1Δ telomeres experience transient attrition mediated by replication fork stalling, this is balanced not only by temporal expansion of the telomerase activity period, but also by markedly increased recruitment of telomerase and its accessory factor Est1, suggesting that stalled forks generate robust substrates for telomerase.

Sumoylation of RecQ helicase controls the fate of dysfunctional telomeres.

Genome stability depends upon the RecQ helicases, which are conserved from bacteria to man, but little is known about how their myriad activities are regulated. Fission yeast lacking the telomere protein Taz1 (mammalian TRF1/TRF2 ortholog) lose many hallmarks of telomeres, including accurate replication and local protection from DNA repair reactions. Here we show that the RecQ homolog, Rqh1, is sumoylated. Surprisingly, Rqh1 acts on taz1Delta telomeres in a deleterious way, promoting telomere breakage and entanglement. Mutation of Rqh1 sumoylation sites rescues taz1Delta cells from these hazards without dramatically affecting nontelomeric Rqh1 functions. The prominence of Rqh1 in the etiology of several different telomere defects supports the idea that they originate from a common underlying lesion--aberrant processing of the stalled telomeric replication forks that accumulate in the absence of Taz1. Our work underscores the principle that RecQ helicases are "double-edged swords" whose activity, while necessary for maintaining genome-wide stability, must be vigilantly controlled.

Decoding chromosome organization using CheC-PLS: chromosome conformation by proximity labeling and long-read sequencing.

Genomic approaches have provided detailed insight into chromosome architecture. However, commonly deployed techniques do not preserve connectivity-based information, leaving large-scale genome organization poorly characterized. Here, we developed CheC-PLS: a proximity-labeling technique that indelibly marks, and then decodes, protein-associated sites. CheC-PLS tethers dam methyltransferase to a protein of interest, followed by Nanopore sequencing to identify methylated bases - indicative of in vivo proximity - along reads >100kb. As proof-of-concept we analyzed, in budding yeast, a cohesin-based meiotic backbone that organizes chromatin into an array of loops. Our data recapitulates previously obtained association patterns, and, importantly, exposes variability between cells. Single read data reveals cohesin translocation on DNA and, by anchoring reads onto unique regions, we define the internal organization of the ribosomal DNA locus. Our versatile technique, which we also deployed on isolated nuclei with nanobodies, promises to illuminate diverse chromosomal processes by describing the in vivo conformations of single chromosomes.

Characterization of the Pristionchus pacificus "epigenetic toolkit" reveals the evolutionary loss of the histone methyltransferase complex PRC2.

Comparative approaches have revealed both divergent and convergent paths to achieving shared developmental outcomes. Thus, only through assembling multiple case studies can we understand biological principles. Yet, despite appreciating the conservation-or lack thereof-of developmental networks, the conservation of epigenetic mechanisms regulating these networks is poorly understood. The nematode Pristionchus pacificus has emerged as a model system of plasticity and epigenetic regulation as it exhibits a bacterivorous or omnivorous morph depending on its environment. Here, we determined the "epigenetic toolkit" available to P. pacificus as a resource for future functional work on plasticity, and as a comparison with Caenorhabditis elegans to investigate the conservation of epigenetic mechanisms. Broadly, we observed a similar cast of genes with putative epigenetic function between C. elegans and P. pacificus. However, we also found striking differences. Most notably, the histone methyltransferase complex PRC2 appears to be missing in P. pacificus. We described the deletion/pseudogenization of the PRC2 genes mes-2 and mes-6 and concluded that both were lost in the last common ancestor of P. pacificus and a related species P. arcanus. Interestingly, we observed the enzymatic product of PRC2 (H3K27me3) by mass spectrometry and immunofluorescence, suggesting that a currently unknown methyltransferase has been co-opted for heterochromatin silencing. Altogether, we have provided an inventory of epigenetic genes in P. pacificus to compare with C. elegans. This inventory will enable reverse-genetic experiments related to plasticity and has revealed the first loss of PRC2 in a multicellular organism.

A suppressor screen in C. elegans identifies a multi-protein interaction interface that stabilizes the synaptonemal complex.

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 novel interaction interface in the C. elegans synaptonemal complex, comprised of short segments of three proteins, SYP-1, SYP-3 and SYP-4. We identified the interface through a saturated suppressor screen of a mutant that destabilizes the synaptonemal complex. The specificity and tight distribution of suppressors point to 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 underly meiotic chromosome interactions.

Depicting a cellular space occupied by condensates.

Condensates have emerged as a new way to understand how cells are organized, and have been invoked to play crucial roles in essentially all cellular processes. In this view, the cell is occupied by numerous assemblies, each composed of member proteins and nucleic acids that preferentially interact with each other. However, available visual representations of condensates fail to communicate the growing body of knowledge about how condensates form and function. The resulting focus on only a subset of the potential implications of condensates can skew interpretations of results and hinder the generation of new hypotheses. Here we summarize the discussion from a workshop that brought together cell biologists, visualization and computation specialists, and other experts who specialize in thinking about space and ways to represent it. We place the recent advances in condensate research in a historical perspective that describes evolving views of the cell; highlight different attributes of condensates that are not well-served by current visual conventions; and survey potential approaches to overcome these challenges. An important theme of these discussions is that the new understanding on the roles of condensates exposes broader challenges in visual representations that apply to cell biological research more generally.

Meiotic DNA exchanges in C. elegans are promoted by proximity to the synaptonemal complex.

During meiosis, programmed double-strand DNA breaks are repaired to form exchanges between the parental chromosomes called crossovers. Chromosomes lacking a crossover fail to segregate accurately into the gametes, leading to aneuploidy. In addition to engaging the homolog, crossover formation requires the promotion of exchanges, rather than non-exchanges, as repair products. However, the mechanism underlying this meiosis-specific preference is not fully understood. Here, we study the regulation of meiotic sister chromatid exchanges in Caenorhabditis elegans by direct visualization. We find that a conserved chromosomal interface that promotes exchanges between the parental chromosomes, the synaptonemal complex, can also promote exchanges between the sister chromatids. In both cases, exchanges depend on the recruitment of the same set of pro-exchange factors to repair sites. Surprisingly, although the synaptonemal complex usually assembles between the two DNA molecules undergoing an exchange, its activity does not rely on a specific chromosome conformation. This suggests that the synaptonemal complex regulates exchanges-both crossovers and sister exchanges-by establishing a nuclear domain conducive to nearby recruitment of exchange-promoting factors.

Telomeres in drag: Dressing as DNA damage to engage telomerase.

The telomere field concentrates both on mechanisms of telomere synthesis and the mechanisms by which telomeres protect chromosome termini from fusion and degradation. Recent studies show that the DNA damage response (DDR) machinery, formerly thought to be the culprit in deleterious telomeric fusion and degradation reactions, plays an active role not only in telomere protection but also in regulating telomere synthesis. Conversely, semi-conservative DNA replication, responsible for the bulk of telomere synthesis, now appears to be a pivotal event on the road to telomere de-protection. These advances prompt the notion that the two guises of telomere function are intricately entangled. Indeed, telomeres appear to expose themselves to the DDR upon passage of the replication fork, in turn attracting telomerase.

Semi-conservative DNA replication through telomeres requires Taz1.

Telomere replication is achieved through the combined action of the conventional DNA replication machinery and the reverse transcriptase, telomerase. Telomere-binding proteins have crucial roles in controlling telomerase activity; however, little is known about their role in controlling semi-conservative replication, which synthesizes the bulk of telomeric DNA. Telomere repeats in the fission yeast Schizosaccharomyces pombe are bound by Taz1, a regulator of diverse telomere functions. It is generally assumed that telomere-binding proteins impede replication fork progression. Here we show that, on the contrary, Taz1 is crucial for efficient replication fork progression through the telomere. Using two-dimensional gel electrophoresis, we find that loss of Taz1 leads to stalled replication forks at telomeres and internally placed telomere sequences, regardless of whether the telomeric G-rich strand is replicated by leading- or lagging-strand synthesis. In contrast, the Taz1-interacting protein Rap1 is dispensable for efficient telomeric fork progression. Upon loss of telomerase, taz1Delta telomeres are lost precipitously, suggesting that maintenance of taz1Delta telomere repeats cannot be sustained through semi-conservative replication. As the human telomere proteins TRF1 and TRF2 are Taz1 orthologues, we predict that one or both of the human TRFs may orchestrate fork passage through human telomeres. Stalled forks at dysfunctional human telomeres are likely to accelerate the genomic instability that drives tumorigenesis.

Single-sister labeling in the C. elegans germline using the nucleotide analog EdU.

Reciprocal exchanges between genetically identical sister chromatids (sister chromatid exchanges or SCEs) have been challenging to study. Here, we describe a protocol that utilizes a pulse/chase of the thymidine analog 5-ethyl-3'-deoxyuridine (EdU) in combination with click chemistry and antibody labeling to selectively label sister chromatids in the C. elegans germline. Labeling has no discernable effects on meiosis, allowing for cytological quantification of SCEs. This protocol can be combined with a variety of imaging approaches, including STED, confocal and super-resolution. For complete details on the use and execution of this protocol, please refer to Almanzar et al. (2021).

Rational engineering of a synthetic insect-bacterial mutualism.

Many insects maintain mutualistic associations with bacterial endosymbionts, but little is known about how they originate in nature. In this study, we describe the establishment and manipulation of a synthetic insect-bacterial symbiosis in a weevil host. Following egg injection, the nascent symbiont colonized many tissues, including prototypical somatic and germinal bacteriomes, yielding maternal transmission over many generations. We then engineered the nascent symbiont to overproduce the aromatic amino acids tyrosine and phenylalanine, which facilitate weevil cuticle strengthening and accelerated larval development, replicating the function of mutualistic symbionts that are widely distributed among weevils and other beetles in nature. Our work provides empirical support for the notion that mutualistic symbioses can be initiated in insects by the acquisition of environmental bacteria. It also shows that certain bacterial genera, including the Sodalis spp. used in our study, are predisposed to develop these associations due to their ability to maintain benign infections and undergo vertical transmission in diverse insect hosts, facilitating the partner-fidelity feedback that is critical for the evolution of obligate mutualism. These experimental advances provide a new platform for laboratory studies focusing on the molecular mechanisms and evolutionary processes underlying insect-bacterial symbiosis.

Single-Molecule Tracking of Chromatin-Associated Proteins in the C. elegans Gonad.

Biomolecules are distributed within cells by molecular-scale diffusion and binding events that are invisible in standard fluorescence microscopy. These molecular search kinetics are key to understanding nuclear signaling and chromosome organization and can be directly observed by single-molecule tracking microscopy. Here, we report a method to track individual proteins within intact C. elegans gonads and apply it to study the molecular dynamics of the axis, a proteinaceous backbone that organizes meiotic chromosomes. Using either fluorescent proteins or enzymatically ligated dyes, we obtain multisecond trajectories with a localization precision of 15-25 nm in nuclei actively undergoing meiosis. Correlation with a reference channel allows for accurate measurement of protein dynamics, compensating for movements of the nuclei and chromosomes within the gonad. We find that axis proteins exhibit either static binding to chromatin or free diffusion in the nucleoplasm, and we separately quantify the motion parameters of these distinct populations. Freely diffusing axis proteins selectively explore chromatin-rich regions, suggesting they are circumventing the central phase-separated region of the nucleus. This work demonstrates that single-molecule microscopy can infer nanoscale-resolution dynamics within living tissue, expanding the possible applications of this approach.

Unconventional conservation reveals structure-function relationships in the synaptonemal complex.

Functional requirements constrain protein evolution, commonly manifesting in a conserved amino acid sequence. Here, we extend this idea to secondary structural features by tracking their conservation in essential meiotic proteins with highly diverged sequences. The synaptonemal complex (SC) is a ~100-nm-wide ladder-like meiotic structure present in all eukaryotic clades, where it aligns parental chromosomes and regulates exchanges between them. Despite the conserved ultrastructure and functions of the SC, SC proteins are highly divergent within Caenorhabditis. However, SC proteins have highly conserved length and coiled-coil domain structure. We found the same unconventional conservation signature in Drosophila and mammals, and used it to identify a novel SC protein in Pristionchus pacificus, Ppa-SYP-1. Our work suggests that coiled-coils play wide-ranging roles in the structure and function of the SC, and more broadly, that expanding sequence analysis beyond measures of per-site similarity can enhance our understanding of protein evolution and function.

Meiotic sister chromatid exchanges are rare in C. elegans.

Sexual reproduction shuffles the parental genomes to generate new genetic combinations. To achieve that, the genome is subjected to numerous double-strand breaks, the repair of which involves two crucial decisions: repair pathway and repair template.1 Use of crossover pathways with the homologous chromosome as template exchanges genetic information and directs chromosome segregation. Crossover repair, however, can compromise the integrity of the repair template and is therefore tightly regulated. The extent to which crossover pathways are used during sister-directed repair is unclear because the identical sister chromatids are difficult to distinguish. Nonetheless, indirect assays have led to the suggestion that inter-sister crossovers, or sister chromatid exchanges (SCEs), are quite common.2-11 Here we devised a technique to directly score physiological SCEs in the C. elegans germline using selective sister chromatid labeling with the thymidine analog 5-ethynyl-2'-deoxyuridine (EdU). Surprisingly, we find SCEs to be rare in meiosis, accounting for <2% of repair events. SCEs remain rare even when the homologous chromosome is unavailable, indicating that almost all sister-directed repair is channeled into noncrossover pathways. We identify two mechanisms that limit SCEs. First, SCEs are elevated in the absence of the RecQ helicase BLMHIM-6. Second, the synaptonemal complex-a conserved interface that promotes crossover repair12,13-promotes SCEs when localized between the sisters. Our data suggest that crossover pathways in C. elegans are only used to generate the single necessary link between the homologous chromosomes. Noncrossover pathways repair almost all other breaks, regardless of the repair template.

Identification of novel synaptonemal complex components in C. elegans.

The synaptonemal complex (SC) is a tripartite protein scaffold that forms between homologous chromosomes during meiosis. Although the SC is essential for stable homologue pairing and crossover recombination in diverse eukaryotes, it is unknown how individual components assemble into the highly conserved SC structure. Here we report the biochemical identification of two new SC components, SYP-5 and SYP-6, in Caenorhabditis elegans. SYP-5 and SYP-6 are paralogous to each other and play redundant roles in synapsis, providing an explanation for why these genes have evaded previous genetic screens. Superresolution microscopy reveals that they localize between the chromosome axes and span the width of the SC in a head-to-head manner, similar to the orientation of other known transverse filament proteins. Using genetic redundancy and structure-function analyses to truncate C-terminal tails of SYP-5/6, we provide evidence supporting the role of SC in both limiting and promoting crossover formation.