Libuda, Diana E. et al. (2011) International Worm Meeting "Synaptonemal Complex mediates crossover interference during meiosis."

Hayashi, Michiko, Yokoo, Rayka, Villeneuve, Anne M., Libuda, Diana E.

[International Worm Meeting, 2011]

Crossover (CO) recombination events during meiosis are critical for the formation of the chiasmata that link homologous chromosomes together and ensure their proper segregation. Despite an excess of double strand DNA breaks (DSBs) that serve as initiating events for CO formation, most organisms make few COs, and formation of a CO tends to inhibit formation of other COs nearby on the same chromosome, a process known as CO interference. C. elegans exhibits particularly robust CO interference, with only a single CO forming between each pair of homologous chromosomes, making it an ideal model for investigating how COs form and how CO interference is mediated. Here we show that the synaptonemal complex (SC), a proteinaceous structure that forms between homologous chromosomes during meiosis, regulates CO formation both by directing localization of CO-promoting proteins and by mediating CO interference along the lengths of meiotic chromosomes. Our analysis used a GFP-tagged version of COSA-1, a conserved CO-promoting factor that we identified, as a marker of CO sites. Upon onset of the late pachytene stage of meiotic prophase, GFP::COSA-1 localizes to six bright foci per nucleus, corresponding to the single CO site on each homolog pair. These foci represent the sites of eventual concentration of other conserved CO-promoting factors (MSH-5 and ZHP-3) that initially exhibit broader distribution along chromosomes. Analysis of conditions where DSBs are either limiting or in excess demonstrated that COSA-1 foci represent a robust cytological readout of the CO interference process. We exploited this property to investigate involvement of the SC central region proteins (SYPs), in formation and regulation of COs. Our data demonstrate that SYPs both: 1) dictate where CO-promoting proteins will concentrate; and 2) function in inhibiting excess CO formation. In mutants where the SC forms on only a subset of chromosomes, CO-promoting proteins localize only to those chromosomes where SYPs are present. Further, CO-promoting proteins co-localize and become sequestered with SYPs in nuclear aggregates that form when SYPs are prevented from loading onto chromosomes. Most notably, despite the fact that the SYP proteins are required to form COs, we find that partial depletion of the SYP-1 protein actually results in an increase in COSA-1 foci, implying attenuation of CO interference. These and other data implicate the SC as a key player in the robust CO interference mechanism that normally limits the number of CO events to one per homolog pair during C. elegans meiosis.

Libuda, Diana E. et al. (2013) International Worm Meeting "Meiotic chromosome structures constrain and respond to designation of crossover sites."

Villeneuve, Anne M., Meyer, Barbara J., Uzawa, Satoru, Libuda, Diana E.

[International Worm Meeting, 2013]

Proper meiotic chromosome segregation requires processes that promote and constrain the formation of crossovers (COs). CO interference, a process that constrains the number of COs along a chromosome pair, was originally observed a century ago but is still poorly understood. Caenorhabditis elegans exhibits particularly robust CO interference, with only a single CO forming between each pair of homologous chromosomes. Here, we use a cytological marker of CO sites to reveal relationships between COs and the synaptonemal complex (SC), a meiosis-specific structure that assembles between aligned homologous chromosome pairs. We develop a system to assess interference strength quantitatively during wild-type meiosis, demonstrating that interference operates over distances that exceed the length of a normal C. elegans chromosome. Further, we show that partial depletion of the SC central region proteins attenuates CO interference, elevating COs and reducing the effective distance over which interference operates, indicating a role for SC central region proteins in limiting the formation of cytologically-differentiated CO sites. Finally, our analyses reveal a linear relationship between CO number and chromosome axis length, demonstrating that each CO causes a 0.4-0.5 mm increase in axis length. Moreover, we provide additional evidence that this CO-dependent increase in chromosome axis length is a local effect around the CO site. We propose that meiotic chromosome structures establish an environment that promotes CO formation, which in turn alters chromosome structure to inhibit other COs at additional sites.

Libuda, Diana E. et al. (2009) International Worm Meeting "Recombination pathway and partner choice during meiotic double strand break repair in C. elegans."

Libuda, Diana E., Villeneuve, Anne M.

[International Worm Meeting, 2009]

Recombination is critical for generating genetic variation, for maintaining genome integrity through repair of DNA breaks, and for ensuring chromosome segregation during meiosis, the specialized cell division program by which diploid organisms generate haploid gametes. Meiotic recombination is initiated by double strand DNA breaks (DSBs), which are repaired using meiosis-specific mechanisms that favor utilization of the homologous chromosome (instead of the sister chromatid) as the recombination partner and that promote a crossover (rather than noncrossover) outcome of the DSB repair (DSBR) process. Crossover recombination between homologous chromosomes is required to create temporary physical connections that promote proper meiotic chromosome segregation. Our lab has built on a recent analysis of DSBR following heat-shock induced excision of the Mos1 transposon (Robert et al. 2008) to develop assays to monitor meiotic recombination events between homologous chromosomes initiated at a defined DSB site (see abstract by Rosu and Villeneuve). Since the C. elegans germline is organized in a spatial-temporal gradient, DSBs can be induced simultaneously at all meiotic stages, and the repair outcomes for these DSBs induced at different stages can be subsequently assessed. Currently, we are using a novel Mos1-based assay system to investigate the nature of gene conversion tracts associated with both crossover and noncrossover homologous recombination events generated by DSBs induced at various stages of meiotic prophase. In addition, we are constructing a second-generation version of the assay system of Rosu and Villeneuve; this modified assay system incorporates features that also allow for the detection of recombination events between sister chromatids. With this new meiotic DSBR assay, we will directly test the hypothesis that germ cells undergo a switch in DSBR mode during prophase progression to enable use of the sister chromatids as repair partners during late prophase. Moreover, we will investigate the roles of meiotic recombination machinery components and meiotic chromosome structures in promoting specific outcomes of DSBR by assessing repair events in mutants defective in the genes encoding these proteins. Furthermore, we are developing genetic and RNAi-based screens incorporating this second-generation assay to identify components that play roles in inhibiting the formation of either crossover or intersister recombination events. Overall, these studies will elucidate how meiotic chromosomes are able to utilize specific recombination pathway and partner preferences to yield desired recombination outcomes, thereby enabling proper chromosome segregation and maintaining genome integrity.

Naftaly, Alice et al. (2021) International Worm Meeting "Deciphering the mechanisms of temperature-induced DNA damage in C. elegans spermatocytes"

Libuda, Diana, Naftaly, Alice

[International Worm Meeting, 2021]

Spermatogenesis and oogenesis create genetically unique haploid gametes in most sexually reproducing organisms. In comparison to oogenesis, spermatogenesis must occur in a narrow temperature range, typically 2-7°C below body temperature. In mammalian spermatocytes subjected to acute thermal stress, an increase in DNA damage occurs as well as a reduction in fertility. Despite these well documented effects on genome integrity and fertility, the exact mechanisms that cause temperature-induced heat stress in spermatogenesis remain unclear. Recent work in Caenorhabditis elegans revealed a large increase in DNA double-strand breaks (DSBs) marked by the recombinase protein RAD-51 when spermatocytes are exposed to a two-hour heat shock at 34°C. The temperature-induced DSBs are SPO-11 independent, suggesting other mechanisms are responsible for DSB formation. While a small portion of the temperature-induced DSBs were attributed to the transposition of the DNA transposable element Tc1, the origin of the remaining DSBs are unclear. To determine the other mechanisms that generate the temperature-induced DSBs, we will use RAD-51 chromatin immunoprecipitation followed by sequencing (ChIP-seq) on C. elegans males before and after heat-shock. From these experiments, we will define the wild type DSB landscape in C. elegans males. Also, we will determine where temperature-induced DSBs are found across the genome, therefore indicating how these DSBs are formed, including whether other transposable elements are activated upon heat shock. In addition, we will investigate the repair pathways required for resolving temperature-induced DSBs. In hermaphrodites, interhomolog recombination requires the protein RAD-50 to load RAD-51 onto DSB sites. To determine if interhomolog recombination is required to repair temperature-induced DSBs in males, we are utilizing a rad-50 null mutation. Immunofluorescence of RAD-51 in rad-50 mutant males before and after heat-shock will determine whether males require RAD-50 dependent RAD-51 loading during meiotic prophase I and if the RAD-50 pathway is required for repair of temperature-induced DNA damage in spermatocytes. Together, this work will establish the DSB landscape in C. elegans males and provide insight into how temperature-induced DSBs are both formed and repaired.

Kurhanewicz, Nicole et al. (2019) International Worm Meeting "Temperature increases cause transposon-associated DNA damage specific to spermatocytes and not oocytes."

Chan, Fountane, Libuda, Diana, Kurhanewicz, Nicole

[International Worm Meeting, 2019]

Sexually-reproducing organisms use meiosis to generate haploid gametes, such as sperm and eggs, to transmit their genome to the next generation. All tissues are susceptible to dramatic increases in temperature; however, developing sperm in the testes are unusually sensitive to small temperature fluctuations. In contrast to oogenesis and other developmental processes, spermatogenesis requires a narrow isotherm of 2-7 deg C below core body temperature. Although failure to thermoregulate spermatogenetic tissue and prolonged exposure to elevated temperatures are linked to male infertility in several organisms, the mechanisms of temperature-induced male infertility is largely unknown. Here we show that upon a brief 2 deg C temperature increase, spermatocytes specifically exhibit temperature-induced transposon excision and insertion, and demonstrate a 30-40 fold increase in double strand DNA breaks (DSBs) throughout the genome of spermatocytes, which leads to a decrease in male fertility. Using Caenorhabditis elegans to eliminate sex-specific bias, we demonstrate that these temperature-induced DSBs occur in the spermatocytes of both males and hermaphrodites at all stages of meiotic prophase I. In contrast, oocytes do not display any change in DSB formation or transposon mobility upon temperature increases. We also show that these temperature-induced DSBs are due to spermatocyte-specific temperature sensitivity of the PIWI-interacting RNA (piRNA) pathway which targets transposon sequences for germ line silencing, thereby preventing their mobility. Following a temperature increase, we see a genome-wide decrease in the piRNA-associated chromatin mark histone H3 lysine 9 trimethylation specifically in heat-exposed spermatocytes and not oocytes. In addition, null mutations in the piRNA pathway enhance temperature-induced DSBs in spermatocytes. Notably, when all 12 worm-specific Argonautes (WAGOs) are mutated together, temperature-induced DSBs are no longer spermatocyte-specific and instead occur in both spermatocytes and oocytes. Taken together, our data provide mechanistic insight on how temperature increases specifically compromise the genome integrity of spermatocytes and impairs fertility in males.

Toraason, Erik et al. (2021) International Worm Meeting "DNA repair is altered during C. elegans germline aging"

Libuda, Diana, Toraason, Erik, Adler, Victoria

[International Worm Meeting, 2021]

Reproductive aging leads to a decline in oocyte quality and female fertility. Previous studies in multiple organisms have found altered expression of DNA repair genes during oocyte aging, however it is largely unknown whether DNA repair is defective in aged oocytes. To determine whether DNA break formation and repair changes in oocytes upon aging, we used C. elegans to examine DNA repair in aged germlines of both wild-type hermaphrodites, which continuously produce new oocytes, and fog-2 mutant females, which can hold and age their oocytes in specific stages of meiotic prophase I. Our results found that RAD-51 foci, which mark DNA double-strand breaks (DSBs), were elevated in both aged wild-type and fog-2 germlines, indicating that oocytes accumulate DSBs in old germlines regardless of oocyte age. We further determined that the elevated DSBs in aged germlines were SPO-11-dependent, indicating that age either increases SPO-11 activity or delays DSB repair. To assess efficiency of meiotic DSB repair upon aging, we introduced exogenous DSBs via irradiation to both young and old germlines and examined the kinetics of DSB repair following irradiation. Our results indicate that old germlines maintained a higher number of DSBs in a subset of nuclei following irradiation, supporting a model in which DSB repair is less efficient upon aging. To determine how DSB repair is regulated during oocyte aging, we examined mutants deficient in the E2 ubiquitin ligase variant UEV-2, which is upregulated in mutants with an extended reproductive lifespan and is suggested to function in DSB repair. Our analyses indicate that both young and aged uev-2 mutant germlines exhibit elevated but similar levels of RAD-51 foci, implicating UEV-2 as a key regulator of DSB repair during germline aging. Taken together, our work reveals defects associated with germline aging and identifies novel players ensuring efficient DSB repair in young oocytes.

Adler, Victoria et al. (2019) International Worm Meeting "Characterization of meiotic prophase I in a natural isolate of Caenorhabditis elegans."

Adler, Victoria, Libuda, Diana, Anderson, Erik, Zhang, Gaotian

[International Worm Meeting, 2019]

In most sexually reproducing organisms, meiosis ensures reproductive success and creates genetic variation within a population. Errors in meiosis can lead to birth defects and infertility. Evaluation of meiotic processes in closely related species and natural populations can reveal the ancestral state of critical meiotic processes as well as new variants of meiotic genes. Here, we characterize the key processes of meiotic prophase I in the Caenorhabditis elegans strain ECA701, which has several traits indicative of defects during meiosis (see abstract by G. Zhang et al.). To determine the changes in the specific meiotic processes that cause the defective meiotic phenotypes observed in ECA701, we used immunofluorescence with antibodies generated to N2 meiotic proteins to characterize the germ lines of ECA701 males and hermaphrodites. Preliminary observations suggest that although many meiotic proteins between N2 and ECA701 are well conserved, several aspects of meiotic prophase I differ between the two strains. In ECA701, the synaptonemal complex (SC), a well conserved meiosis-specific complex that holds together homologous chromosomes, assembles along most chromosome pairs, but some developing oocytes have a set of chromosomes that are unable to pair and form the SC. In contrast to ECA701 oocytes and N2 spermatocytes, spermatocytes in ECA701 males have no unpaired chromosomes and instead, consistently pair and form SC on five sets of chromosomes. Notably, pairing-associated chromosome movement is significantly longer during ECA701 oogenesis than is observed in the N2 strain. Moreover, during diakinesis, when sets of homologous chromosomes are held together by crossover recombination events to form six bivalent structures in N2, 4-5 sets of chromosomes are able to form bivalents but 1-2 chromatids form univalent structures during ECA701 oogenesis. Future experiments will determine if these unpaired chromosomes are the sex chromosomes, which may explain the Him phenotype (40-45% male progeny) of ECA701. Also, we characterized loading of the recombinase RAD-51, which localizes to sites of double strand DNA breaks (DSBs). Notably, the number of RAD-51 foci per nucleus in ECA701 is less than what is observed in N2. This reduction in RAD-51 foci indicates that either fewer DSBs are formed in ECA701 or some DSBs are marked by another meiotic recombinase. Finally, we observed a complete loss of sperm in the spermatheca of day-three adult hermaphrodites of ECA701. Overall, our preliminary results and future experiments may reveal the possible ancestral state of Caenorhabditis elegans meiosis, the evolution of an androdioecious species, or natural alleles of key meiotic genes that can underlie recombination variation.

Toraason, Erik et al. (2019) International Worm Meeting "Regulation of sister chromatid crossovers maintains genomic integrity during meiosis."

Toraason, Erik, Glover, Marissa, Libuda, Diana, Clark, Cordell, Horacek, Anna

[International Worm Meeting, 2019]

Maintenance of genomic integrity during meiosis, the specialized form of cell division that generates haploid gametes such as sperm and eggs, is critical for fertility. Meiotic cells preferentially utilize recombination to repair DNA double-strand breaks (DSBs) as crossover or noncrossover outcomes. Meiotic recombination with the homologous chromosome has been extensively studied, but the mechanisms of sister chromatid recombination during meiosis are poorly understood. Using a sister chromatid repair (SCR) assay that we developed to directly detect sister chromatid repair at a defined DSB site during Caenorhabditis elegans meiosis, we reveal mechanisms of sister chromatid recombination during meiotic prophase I progression. We find that the sister chromatid is available as a repair template for both crossover or noncrossover recombination throughout meiotic prophase I, with the sister chromatid as the exclusive recombination partner during late meiotic prophase I. By sequencing the conversion tracts of recombination outcomes from the SCR assay, we have found that: 1) DSBs repaired via sister chromatid recombination are processed similarly throughout prophase I; 2) intersister recombination intermediates do not migrate from site of DSB induction; and, 3) recombination with the sister chromatid appears processive, as we observe no evidence of template switching. Further, we have direct evidence with the SCR assay that the Smc5/6 DNA damage complex is required in late meiotic prophase for efficient recombination with the sister chromatid, and specifically, for formation of intersister crossovers. Conversion tracts arising from recombination events in smc-5 mutants appear similar to wild-type, suggesting that SMC-5/6 acts to promote recombination after DSB processing. Taken together, our research has defined fundamental mechanisms ensuring successful meiosis and preserving genomic fidelity across generations.

Ho, Jamie et al. (2021) International Worm Meeting "Nuclear deformation during P-cell nuclear migration"

Ma, Linda, Gregory, Ellen, Ho, Jamie, Libuda, Diana, Starr, Daniel

[International Worm Meeting, 2021]

Cell migration through constricted spaces is critical for developmental and disease processes, including immune cell intravasation and cancer metastasis, but is limited by nuclear deformation. During C. elegans development, larval P-cell nuclei migrate through a constriction between the muscle and cuticle that is ~5% of the resting diameter of the nucleus. Null mutations in the LINC pathway, consisting of SUN (unc-84), KASH (unc-83), and motors (dynein), disrupt about 50% of P-cell nuclear migrations at restrictive temperatures, but at permissive temperatures most P-cell nuclei migrate normally without LINC complexes. We hypothesized that additional pathways function parallel to the LINC pathway. In support of this hypothesis, a forward genetic screen for enhancers of the nuclear migration defect of unc-83/84 (emu) mutants identified toca-1, cgef-1, and fln-2. Knockdown of the Arp2/3 complex component arx-3 in unc-84 null mutants at the permissive temperature caused a significant nuclear migration defect. Under the same conditions, we observe a similar nuclear migration defect when cdc-42 was knocked down, indicating that both the Arp2/3 complex and CDC-42 are required for nuclear migration in the absence of SUN-KASH. In our model, the F-bar domain protein TOCA-1 binds to the nuclear membrane and recruits CDC-42, a small G-protein which is activated by CGEF-1. CDC-42 in turn activates WAVE/WASP and Arp2/3 to generate branched actin, with the help of FLN-2 acting as either an actin bundler or crosslinker. We hypothesize that these proteins form an actin-based pathway that generates a network of branched actin to compress the nucleus. Additionally, the cgef-1; unc-84; cec-4 triple mutant resulted in complete nuclear migration failure at the permissive temperature. CEC-4 is an inner nuclear membrane protein that tethers H3K9 methylated chromatin to the inner nuclear envelope and is therefore predicted to affect the ability of the nucleus to deform. Altogether, these studies support a model where multiple pathways responsible for modulating nuclear deformation play important roles in nuclear migration through constricted spaces.

Gilchrist EJ et al. (1995) International C. elegans Meeting "A C. ELEGANS MYOTONIC DYSTROPHY GENE HOMOLOGUE."

Gilchrist EJ, Baillie DL

[International C. elegans Meeting, 1995]

Myotonic Dystrophy is an inherited, autosomal dominant disease that results in a progressive wasting of the skele- tal muscles (and sometimes heart and smooth muscles) in humans. The gene has been cloned and sequenced (R. Korneluk, personal communication) and encodes a large protein that has been localised to neuromuscular and myotendinous junctions. The N-terminus domain bears similarity to cAMP-dependent serine/threonine protein kinases. Yuji Kohara, in his cDNA sequencing project, identified a C. elegans gene that is 75% similar (if conserved amino acids are considered) to the kinase domain of the human myotonic dystrophy gene. He mapped the cDNA to LG I (personal communication), and, through Southern analysis, we have placed the cDNA on cosmid K06G1. This cosmid was injected into wild-type N2 hermaphrodites by Diana Collins, and we are currently trying to rescue some of the genes in this region with the extrachromosomal array. One candidate gene was unc-57. Animals homozygous for mutations in unc-57 show no obvious structural muscle defects. This is consistent with unc-57 being a muscle, or neuro- muscular kinase. However, the Unc-57 phenotype is not rescued in animals carrying the K06G1 extrachromo- somal array. We are now focusing on some of the lethal mutations in this region that have been isolated in the laboratory of Ann Rose at UBC.