Biology subjects

Rog, O.

Publications and source records attributed to Rog, O..

16 recordsLinked to original sources

bioRxiv2026

T2T genomes of Caenorhabditis nigoni and Caenorhabditis briggsae reveals extensive loss of satellite DNA associated with self-fertilization

The two closely related Caenorhabditis nematode species, C. nigoni and C. briggsae, are commonly used to study the evolution of reproductive modes in animals, with the self-fertile C. briggsae and outcrossing C. nigoni sharing a common ancestor [~]3.5 million years ago. Earlier genomic analyses of these species revealed genome shrinkage associated with selfing and proposed that at least some gene loss can be adaptive. However, the incomplete C. nigoni reference genome limited most comparative analyses to genic regions. Here, we leveraged long-read sequencing to generate a telomere-to-telomere (T2T) assembly for the C. nigoni strain JU1422 and the C. briggsae strain AF16. This new 139Mb C. nigoni genome resolved 57 gaps and 149 unassigned scaffolds from the previous genome assembly. Comparison with the 107Mb T2T C. briggsae genome reveals that the major driver of genome content differences are deletions to satellite DNA arrays, reflecting a loss of 9.6Mb. Interestingly, many of the differences are on the C. nigoni X chromosome, which is >13Mb larger than in the previous assembly. The transition to selfing was thus accompanied by a 37% reduction in the size of the sex chromosome compared to 16-21% shrinkage of the autosomes. We also document a surprising degree of plasticity in the ribosomal DNA, with the X chromosome harboring a second 45S rDNA array that is absent in C. briggsae. Our analysis reveals that obligatory outcrossing may play a major role in the maintenance of satellite DNA arrays.

genomics↗

bioRxiv2025

High-efficiency targeted integration of extrachromosomal arrays in C. elegans using PhiC31 integrase

Extrachromosomal arrays are unique chromosome-like structures created from DNA injected into the C. elegans germline. However, they are unstable unless integrated into a chromosome. Current methods for integration using X-rays or CRISPR can damage DNA and exhibit low efficiency. We demonstrate that the viral integrase PhiC31, which mediates non-mutagenic recombination between short attB and attP sequences, can be used for extremely efficient and targeted integration of arrays. Arrays were integrated by PhIAT (PhiC31-mediated Integration of Arrays of Transgenes) at attB sites on three chromosomes, including at a fluorescent landing pad. Moreover, integrations can be inserted at any arbitrary site in the genome by simultaneously co-injecting Cas9 RNP, an attB repair template, and the DNA components for the array - thereby providing sites on all six chromosomes. Single injections can integrate arrays ranging in size from 1 to 18 megabases. PhIAT makes it practical to study genomes of other organisms in the nematode; one of our strains incorporates 65% of the yeast genome at a single site in the worm genome. PhIAT will accelerate a shift from unstable extrachromosomal arrays to direct integration of arrays in C. elegans.

genetics↗

bioRxiv2025

Early resolution of sister chromatids during C. elegans meiosis

Segregating a complete set of chromosomes into the gametes relies on exchanges of genetic material that occur during meiosis. It is only exchanges that form between the similar parental chromosomes (homologs), rather than between the identical sister chromatids, that enable correct chromosome segregation (Zickler and Kleckner 2023). Despite the crucial role of biasing exchanges toward the homolog and the progress in defining some of its regulators (Niu et al. 2009; Goldfarb and Lichten 2010; Lao and Hunter 2010; Kim et al. 2010; Hong et al. 2013), the mechanism that efficiently identifies the homolog and avoids the sister remains unknown. Understanding homolog bias requires knowledge of how the homologs and sisters are organized relative to each other, and how this positioning is established. Here, we use selective labeling of a single sister in the oogenic germline of the nematode Caenorhabditis elegans to define the organization of the sister chromatids at the time exchanges form. We find that pairs of sisters are already well separated (resolved) early in meiosis, despite being tethered to each other at numerous positions along their length. The sisters are resolved in both aligned and unaligned homologs, and their resolution does not require condensins or a prolonged time in meiotic prophase. However, depleting the cohesin loader NIPBLSCC-2 impairs sister resolution, suggesting that an active process - likely loop extrusion by cohesins - de-mixes and resolves the sisters. Our work shows that inter-homolog meiotic exchanges form when the four sister chromatids occupy distinct volumes, suggesting that homolog bias is unlikely to rely on relative proximity. The conservation of meiotic chromosome organization and of cohesins loop-extruding activity suggests that our findings are broadly applicable.

cell biology↗

bioRxiv2025

Kinetic analysis of strand invasion during C. elegans meiosis reveals similar rates of sister- and homolog-directed repair

AbstractMeiotic chromosome segregation requires reciprocal exchanges between the parental chromosomes (homologs). Exchanges are formed via tightly-regulated repair of double-strand DNA breaks (DSBs). However, since repair intermediates are mostly quantified in fixed images, our understanding of the mechanisms that control the progression of repair remains limited. Here, we study meiotic repair kinetics in Caenorhabditis elegans by extinguishing new DSBs and following the disappearance of a crucial intermediate - strand invasion mediated by the conserved RecA-family recombinase RAD-51. We find that RAD-51 foci have a half-life of 42-132 minutes for both endogenous and exogenous DSBs. Surprisingly, we find that repair templated by the sister chromatid is not slower than repair templated by the homolog. This suggests that differential kinetics are unlikely to underlie homolog bias: the preferential use of the homolog as a repair template. We also use our kinetic information to revisit the total number of DSBs per nucleus - the substrate for the formation of exchanges - and find an average of 40 DSBs in wild-type meiosis and >50 DSBs when homolog pairing is perturbed. Our work opens the door for analysis of the interplay between meiotic repair kinetics and the fidelity of genome inheritance. Key pointsO_LIBy extinguishing new meiotic DSBs, we define the lifetime of a key repair intermediate. C_LIO_LIWe find similar rates of meiotic DNA repair templated by the sister and the homolog. C_LIO_LIKinetic information allows calculation of the total number of meiotic DSBs in C. elegans. C_LI

genetics↗

bioRxiv2024

The synaptonemal complex assembles between meiotic chromosomes by wetting

AbstractDuring meiosis, the parental chromosomes are drawn together to enable exchange of genetic information. Chromosomes are aligned through the assembly of a conserved interface, the synaptonemal complex, composed of a central region that forms between two parallel chromosomal backbones called axes. Here we identify the axis-central region interface in C. elegans, containing a conserved positive patch on the axis component HIM-3 and the C-terminus of the central region protein SYP-5. Crucially, the canonical ultrastructure of the synaptonemal complex is altered upon weakening this interface. We developed a thermodynamic model that recapitulates our experimental observations, indicating that the liquid-like central region can assemble by wetting the axes without active energy consumption. More broadly, our data show that condensation drives tightly regulated nuclear reorganization during sexual reproduction.

cell biology↗

bioRxiv2024

Skp1 is a conserved structural component of the meiotic synaptonemal complex

SummaryDuring sexual reproduction, the parental chromosomes align along their length and exchange genetic information. These processes depend on a chromosomal interface called the synaptonemal complex. The structure of the synaptonemal complex is conserved across eukaryotes, but, surprisingly, the components that make it up are dramatically different in different organisms. Here we find that a protein well known for its role in regulating protein degradation has been moonlighting as a structural component of the synaptonemal complex in the nematode Pristionchus pacificus, and that it has likely carried out both of these functions for more than 100 million years. The synaptonemal complex (SC) is a meiotic interface that assembles between parental chromosomes and is essential for the formation of gametes. While the dimensions and ultrastructure of the SC are conserved across eukaryotes, its protein components are highly divergent. Recently, an unexpected component of the SC has been described in the nematode C. elegans: the Skp1-related proteins SKR-1/2, which are components of the Skp1, Cullin, F-box (SCF) ubiquitin ligase. Here, we find that the role of SKR-1 in the SC is conserved in nematodes. The P. pacificus Skp1 ortholog, Ppa-SKR-1, colocalizes with other SC proteins throughout meiotic prophase, where it occupies the middle of the SC. Like in C. elegans, the dimerization interface of Ppa-SKR-1 is required for its SC function. A dimerization mutant, Ppa-skr-1F105E, fails to assemble SC and is almost completely sterile. Interestingly, the evolutionary trajectory of SKR-1 contrasts with other SC proteins. Unlike most SC proteins, SKR-1 is highly conserved in nematodes. Our results suggest that the structural role of SKR-1 in the SC has been conserved since the common ancestor of C. elegans and P. pacificus, and that rapidly evolving SC proteins have maintained the ability to interact with SKR-1 for at least 100 million years.

genetics↗

bioRxiv2024

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.

genomics↗

bioRxiv2024

Diffusion within the synaptonemal complex can account for signal transduction along meiotic chromosomes

Meiotic chromosomes efficiently transduce information along their length to regulate the distribution of genetic exchanges within and between chromosomes. However, the mode of signal transduction remains unknown. Recently, a conserved chromosomal interface, the synaptonemal complex, was shown to be a biomolecular condensate, offering an attractive mechanism for signal transduction: diffusion of signaling molecules within the synaptonemal complex to allow transmission of information along each pair of chromosomes. Here, we test the feasibility of this mechanism in live C. elegans gonads. Single-molecule tracking shows that a component of the synaptonemal complex (SYP-3) and a conserved regulator of exchanges (ZHP-3) both diffuse within the synaptonemal complex. However, ZHP-3 diffuses 4- and 9-fold faster than SYP-3 before and after crossovers formation, respectively. We use these measurements to parameterize a physical model for signal transduction. We find that ZHP-3, but not SYP-3, explores the lengths of chromosomes on the time scale of crossover maturation, consistent with a role in the spatial regulation of exchanges. Given the conservation of ZHP-3 paralogs across eukaryotes, we propose that diffusion within the synaptonemal complex may be a conserved mechanism of meiotic regulation. More broadly, our work explores how diffusion contained within condensates regulates crucial cellular functions.

cell biology↗

bioRxiv2023

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 C. 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 enable reverse-genetic experiments related to plasticity, and in doing so have described the first loss of PRC2 in a multicellular organism.

genetics↗

bioRxiv2022

Meiotic DNA exchanges 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. 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, also promotes exchanges between the sister chromatids. In both cases, exchanges depend on recruitment of the same set of pro-exchange factors to repair sites. Surprisingly, while the synaptonemal complex usually assembles between the two DNA molecules undergoing an exchange, its activity does not rely on a specific chromosome conformation and it can also promote sister exchanges when assembling next to the sisters. This suggests that the synaptonemal complex regulates exchanges by establishing a nuclear domain conducive to nearby coalescence of exchange-promoting factors.

cell biology↗

bioRxiv2022

BRCA1/BRC-1 and SMC-5/6 regulate DNA repair pathway engagement during C. elegans meiosis

The preservation of genome integrity during sperm and egg development is vital for reproductive success. During meiosis, the tumor suppressor BRCA1/BRC-1 and structural maintenance of chromosomes 5/6 (SMC-5/6) complex genetically interact to promote high fidelity DNA double strand break (DSB) repair, but the specific DSB repair outcomes these proteins regulate remain unknown. Here we show that BRCA1/BRC-1 and the SMC-5/6 complex limit intersister crossover recombination as well as error-prone repair pathways during meiotic prophase I. Using genetic and cytological methods to monitor repair of DSBs with different repair partners in Caenorhabditis elegans, we demonstrate that both BRC-1 and SMC-5/6 repress intersister crossover recombination events, with meiotic cells becoming more dependent upon these proteins to repair DSBs in late meiotic prophase I. Sequencing of conversion tracts from homolog-independent DSB repair events indicates that BRC-1 regulates intersister/intrachromatid noncrossover conversion tract length. Moreover, we find that BRC-1 also specifically inhibits error prone repair of DSBs induced at mid-pachytene. Finally, we reveal that functional BRC-1 enhances DSB repair defects in smc-5 mutants by repressing theta-mediated end joining (TMEJ). Taken together, our study illuminates the coordinate interplay of BRC-1 and SMC-5/6 to regulate DSB repair outcomes in the germline.

genetics↗

bioRxiv2021

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. By manipulating the levels of breaks and crossovers, we infer both chromosome-specific and nucleus-wide regulation on heterologous synapsis. Finally, we identify differences between the two conditions, suggesting that attachment to the nuclear envelope plays a role in regulating heterologous synapsis.

cell biology↗

bioRxiv2021

Unconventional conservation reveals structure-function relationships in the synaptonemal complex

Functional requirements constrain protein evolution, commonly manifesting in conserved primary 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) aligns parental chromosome pairs and regulates exchanges between them. In electron micrographs of meiocytes from all eukaryotic clades, the SC appears as a ~100 nm-wide ladder-like structure with regular striations. Despite the conserved ultrastructure and functions, the proteins that make up the SC are highly divergent in sequence. Here we found that, within the Caenorhabditis genus, SC proteins are significantly more diverged than other proteins. However, SC proteins have highly conserved protein length and coiled-coil domain structure. The same unconventional conservation signature holds true for SC proteins in Drosophila and mammals, suggesting it could be a universal feature of SC proteins. We used this evolutionary signature to identify a novel SC protein in the nematode Pristionchus pacificus, Ppa-SYP-1, which has no significant homology to any protein outside of Pristionchus. Our work suggests that the length and relative arrangement of coiled-coils play a key role in the structure and function of the SC. Furthermore, our analysis implies that expanding sequence analysis beyond measures of per-site identity or similarity can enhance our understanding of protein evolution and function. Short abstractFunctional 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.

evolutionary biology↗

bioRxiv2021

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

1.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 multi-second 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 chromatin 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 technique.

biophysics↗

bioRxiv2020

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. This phase-separated interface brings chromosomes together and regulates 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 that the SC brings homologous chromosomes together through two biophysical activities: obligate dimerization that prevents assembly on unpaired chromosomes; and a tendency to phase-separate that extends pairing interactions along the entire length of the chromosomes.

cell biology↗

bioRxiv2020

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. 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. 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, sister-directed repair intermediates are efficiently inhibited by the RecQ helicase BLMHIM-6. Second, the Synaptonemal Complex-a conserved interface that promotes crossover repair- localizes between the homologous chromosomes and not the sister chromatids. Our data suggest that in C. elegans crossover pathways are only used to generate the single necessary link between the homologous chromosomes. Almost all other breaks, regardless of which repair template they use, are repaired by noncrossover pathways.

cell biology↗