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.

Lascarez Lagunas, L.I. et al. (2019) International Worm Meeting "Specific chromatin marks regulate the recognition of crossover position for accurate chromosome segregation."

Altendorfer, E., Lascarez Lagunas, L.I., Colaiacovo, M.

[International Worm Meeting, 2019]

A critical step of meiosis I is the repair of DNA double-strand breaks (DSBs) via crossover (CO) recombination. CO position along a chromosome is tightly regulated, since COs too close to a centromere or to a telomere can result in aneuploidy. In C. elegans, as in other organisms, COs occur mostly at the terminal thirds of the chromosomes. Moreover, only one CO occurs between each pair of homologs. This single off-centered CO leads to chromosome remodeling resulting in an asymmetric bivalent configuration required for accurate chromosome segregation in many organisms. However, the regulation of this remodeling process remains poorly understood. Here, we investigated how CO position is assessed to allow for accurate remodeling and faithful chromosome segregation. We used a single inducible DSB system in which DSBs are generated by heat shock-induced excision of a Mos1 transposon inserted at a defined genomic location. We observed that a CO at the center of a chromosome impairs chromosome remodeling in late prophase I and results in chromosome missegregation. The mechanism used in sensing the symmetry-breaking event along the bivalent may involve chromatin expansion forces which can drive changes in chromosome geometry generating the asymmetric bivalent. We assessed different chromatin marks by immunostaining in lines undergoing either a centered or an off-centered DSB/CO. We observed a decrease in H3K9me2-3 signal and an increase in H3K79me3 in late pachytene nuclei following heat-shock in centered DSB/CO lines compared to control. These results indicate there are cytologically detectable changes when a CO is induced at the center compared to at an off-centered position. ChIP/qPCR analysis revealed that H3K9me2-3 remain enriched at the terminal thirds of chromosomes even after heat shock in control lines, however their distribution changes and is observed enriched only at the center of the chromosome undergoing a centered DSB/CO. These results suggest that a centered CO may fail to be sensed as a symmetry-breaking event based on changes on the chromatin landscape. This provides a mechanism involving the distribution of specific chromatin marks for "measuring" distances between chromosome ends and the CO that may establish the asymmetric bivalent configuration.

Andrew Bretscher et al. (2007) International Worm Meeting "CO2 Sensing in C. elegans."

Mario De Bono, Andrew Bretscher

[International Worm Meeting, 2007]

Carbon dioxide (CO₂) is a major by-product of aerobic respiration and is a salient feature of many natural environments. Nematodes, insects and vertebrates all exhibit responses to this gas. As they aggregate C. elegans locally deplete ambient O₂ to create a steep O₂ gradient. This gradient appears to facilitate clump formation in wild strains of C. elegans that carry the npr-1 215F natural allele1-3. As low O₂ and high CO₂ are likely to be coincident in the environment, we decided to examine the response of C. elegans to CO₂ and to investigate whether CO₂ plays a role in aggregation behaviour. On encountering a temporal increase in ambient CO₂ from 0 to 5%, N2 animals rapidly induce reversals and omega turns. Such behaviour typifies the response to a repellent. Consistent with this, C. elegans avoids CO₂ both in solution4 and when presented with a spatial gradient on an agar surface - CO₂ levels higher than 0.5 % are avoided. To identify genes involved in CO₂ sensing we have conducted an EMS mutagenesis screen. The screen yielded 35 Cdad (carbon dioxide avoidance defective) mutants of which 10 were analysed in detail. One of the 10 is strongly attracted to CO₂ and exhibits aggregation behaviour, but is otherwise wild type . In a parallel biased approach we have tested CO₂ avoidance in 275 mutants with known nervous system and/or behavioural defects. About 10% of these have Cdad phenotypes and should help reveal the neural circuits involved in avoidance. Our goal is to determine the genetic and neuronal components of the C. elegans CO₂ sensory apparatus and to analyse how it is modulated in different environmental contexts. (1) Cheung et al., 2005 (2) Rogers et al., 2006 (3) Gray et al., 2004 (4) Dusenbery, 1974.

Zawadzki, Karl et al. (2011) International Worm Meeting "Illuminating the Formation and Regulation of Meiotic Crossovers with GFP:COSA-1."

Yokoo, Rayka, Villeneuve, Anne, Zawadzki, Karl

[International Worm Meeting, 2011]

Faithful chromosome segregation during meiosis I requires crossover (CO) recombination events that form the basis of temporary links between homologous chromosomes. Surprisingly, rather than creating many COs between homologs, most organisms create only a small number of widely spaced COs while ensuring that each pair of homologs receives at least one. This regulation of CO number and placement is collectively termed "crossover control" but the underlying mechanisms are poorly understood. In C. elegans CO control is particularly robust, with each pair of homologs receiving one CO. We are investigating the formation and regulation of meiotic COs, building on our recent discovery of COSA-1 (Crossover Site Associated), a novel and conserved CO protein that is required for the formation of COs and localizes to CO sites during the late pachytene and diplotene stages of meiotic prophase. A functional GFP::COSA-1 fusion protein serves as a robust in vivo reporter for the CO control system, as GFP::COSA-1 reliably localizes to six foci per nucleus (i.e. one focus for each pair of homologous chromosomes), even in the context of a large excess of DSBs (which serve as initiating events for meiotic recombination). Further, in worms harboring fusions of two chromosomes GFP::COSA-1 localizes to five foci reflecting the reduced chromosome complement. We are taking two approaches that exploit GFP::COSA-1 to investigate the mechanisms of CO control. First, we are using immunoprecipitation of GFP::COSA-1 to identify proteins and DNA that associate with COSA-1. Second, we are conducting a genetic screen to identify mutations that alter the number of GFP::COSA-1 foci. To date, we have isolated four mutants with altered numbers of GFP::COSA-1 foci: three with reduced numbers of foci, perhaps due to early disruption of the meiotic program, and one with increased numbers of foci. Further characterization of these mutations and others generated by additional screening will help elucidate the factors that control CO number.

Huang, Xiaobing et al. (2019) International Worm Meeting "A transgenic co-expressed Amyloid-beta and alpha-Synuclein dementia model in Caenorhabditis elegans."

Huang, Xiaobing, Leung, Ka Lai, Wang, Changliang, Chen, Liang, Shen, Linjing, Wong, Garry

[International Worm Meeting, 2019]

Purpose: Dementia with Lewy bodies (DLB) is the second most common type of progressive dementia after Alzheimer's disease dementia. Lewy bodies develop in nerve cells in brain regions where thinking, memory and movement are lost. Still, the pathology of DLB remains vague. For exploring the pathogenesis of DLB, we constructed a disease model of DLB which co-expressed Amyloid-? (A?) and ?-synuclein in pan-neurons in C. elegans. Methods: We used the locomotion, thrashing, lifespan, development and egg laying behavior assays to evaluate whether co-expressed A? and ?-synuclein would alter phenotypes in worms. Furthermore, we used confocal microscopy to analyze the neurodegeneration of worms and Thioflavin-S staining to observe the misfolded protein aggregation. Moreover, we used RNA-Seq to explore the effects of co-expressed A? and ?-synuclein in worms on transcription. Results: Our results showed that co-expressed A? and ?-synuclein severely impaired the behaviors and affected neurons in C. elegans. Worms that co-expressed A? and ?-synuclein showed uncoordinated and slower movement, shorter lifespan, less progeny and egg laying defects. Moreover, misfolded protein aggregation and damage of dopaminergic neurons were found in co-expressed A? and ?-synuclein worms, suggesting that co-expression of these 2 transgenes can cause damage to neurons beyond those observed in Alzheimer's disease or Parkinson's disease models. RNA-seq data showed that co-expression of transgenes mainly altered expression levels of genes in the TGF-beta signaling pathway, Wnt signaling pathway, ubiquitin mediated proteolysis, and protein processing pathways in endoplasmic reticulum. Conclusion: Our model might be a useful tool to discover and investigate the etiology of Dementia with Lewy bodies. Moreover, it might be an excellent model to screen drugs that can ameliorate the symptoms of dementia. Acknowledgment: This research was supported in part by the Research grant of Garry Wong MYRG2017-00123-FHS.

Lee, Teresa W. et al. (2011) International Worm Meeting "Condensin-mediated chromosome architecture & crossover regulation."

Meyer, Barbara J., Lee, Teresa W.

[International Worm Meeting, 2011]

During meiosis, chromosomes undergo complex morphological changes that ensure their proper segregation. Crossover recombination (CO) supplies a physical connection between homologs critical for successful meiosis. Due to their importance, COs are subject to strict control that guarantees at least one per homolog and ensures wide spacing between multiple COs (interference), which requires communication along an entire chromosome's length. C. elegans exhibits an extreme form of interference: only one CO occurs on each homolog. Meiotic disruption of condensin I or condensin II - complexes that structure chromosomes in preparation for cell division - perturbs CO regulation: CO frequencies are increased, and their distribution along the chromosome is altered. This increase is strongly correlated with an overall extension of the chromosome axis, an increase in DNA double-strand breaks (DSBs), and a shift in DSB distribution to the same genetic intervals as the shifted COs. Condensins have non-redundant roles in CO regulation. Disruption of each causes a different distribution of DSBs and COs, and disruption of both perturbs axis length greater than disruption of either. Both complexes may be deployed differentially while sharing an underlying mechanism for structuring meiotic chromosomes. To evaluate the independent roles of condensin I and II, and possible functional redundancy between subunit paralogs, we are analyzing changes in CO and DSB distributions, and effects on chromosome structure, in different backgrounds that disrupt both complexes. We have also found that depletion of the post-translational modification SUMO perturbs regulation of COs and DSBs, while lengthening chromosomal axes. However, the means by which loss of sumoylation increases COs remains unclear. Sumoylation and condensins can influence global chromosome architecture, permitting chromosome-wide communication that may inform CO regulation.

Simona Rosu et al. (2007) International Worm Meeting "Analyzing meiotic recombination initiated by transposon excision-induced double-strand breaks at defined loci."

Valerie ROBERT, Jean-Louis BESSEREAU, Simona Rosu, Anne Villeneuve

[International Worm Meeting, 2007]

Meiosis is a fundamental process by which diploid organisms generate haploid gametes. Central to successful completion of meiosis is the formation of crossovers (COs) between DNA molecules of homologous chromosomes. CO events result in chiasmata, the physical links that hold chromosomes together and ensure proper segregation at the meiosis I division. Most organisms make very few COs per chromosome pair, indicating the process must be tightly regulated. The mechanism must ensure that each pair will undergo at least one CO, and at the same time, the formation of a CO will inhibit other COs nearby. COs are generated by homologous recombination initiated by DNA double strand breaks (DSBs) formed by the meiotic topoisomerease-like SPO-11 protein. There are more DSBs formed than COs, indicating that a subset of recombination precursors enter a CO pathway while the rest are repaired to give noncrossover (NCO) products. Despite the importance of crossing over for ensuring chromosome inheritance, the mechanisms that convert DSBs into COs and that regulate crossing over remain poorly understood. The goal of my project is to investigate the mechanism and regulation of meiotic recombination by analyzing recombination events at a defined locus. As an assay system, I am using worms heterozygous for two different alleles of the unc-5 gene, one of which contains a Mos1 transposon insertion. Induction of transposase causes transposon excision, thus forming a DSB that can lead to recombination. Intragenic recombination events at the unc-5 locus are identified by restoration of WT movement. Further, the CO vs. NCO status of the recovered recombinants is assessed using closely linked flanking markers. This will allow me to determine at what time point in meiosis DSBs are most likely to be converted into CO products and if a CO at this spot will cause interference. Furthermore, I will perform experiments in a spo11 background, which lacks the ability to initiate endogenous meiotic DSBs. In this context the transposon-excision break should be the only site of COs, which will occur on only one chromosome pair. This will allow the opportunity to test the relationship between the CO site and cytological markers of the emerging chiasma, to assess chromosome-wide changes initiated by the CO, and to evaluate the timing of how DSBs are processed into COs. Finally, I will perform these experiments in strains carrying mutations affecting other meiotic components to test how each component affects the outcome of the transposon-induced DSB.

Joshua M Stuart et al. (2003) International Worm Meeting "Discovering conserved pathways and core molecular machinery on a global scale."

Stuart Kim, Joshua M Stuart, Daphne Koller, Eran Segal

[International Worm Meeting, 2003]

The genome projects have paved the way for new genome-wide functional studies on several organisms. We can now use the rich supply of DNA microarray data to find expression patterns that reflect physiologically meaningful relationships. We have developed a computational technique for identifying conserved co-expression patterns in multiple organisms. Co-expression that represents a functionally-relevant coupling between two genes will confer a selective advantage to the organism and hence be selected over the course of evolution while irrelevant co-expression will be lost. We constructed a multi-species expression network combining microarray data from over 3000 microarray experiment from four evolutionarily distant organisms H. sapiens, S. cerevisiae, C. elegans, and D. melanogaster. We found over 10,000 examples of pairs of genes that are strongly co-expressed in microarray data from at least two organisms. These gene correlations form a network revealing many relationships between genes involved in ancient pathways and complexes, spanning several cellular and multicellular core processes. The network includes over 300 genes that encode novel proteins, and we can assign potential functions to these genes based on their co-expression with genes of known function. We are in the process of making the network available as a resource for the academic community.

Gin Fonte et al. (2001) International C. elegans Meeting "Identification of C. elegans proteins that directly interact with the human beta amyloid peptide."

Chris Link, Vadim Kapulkin, David Friedman, Gin Fonte

[International C. elegans Meeting, 2001]

Although the beta amyloid peptide (Abeta) has been implicated as causal in the etiology of Alzheimer's disease (AD), the mechanism of its cell toxicity has not been established. Constitutive or inducible expression of Abeta in C. elegans muscle leads to paralysis. To gain insight into the molecular mechanisms underlying this toxicity, we have sought to identify proteins that directly interact with Abeta in vivo, using a novel co-immunoprecipitation protocol. These proteins may be involved in transducing or ameliorating Abeta toxicity. For our initial experiments, we have investigated whether HSP16 directly associates with Abeta. HSP16 is specifically induced in transgenic animals with constitutive or induced Abeta expression, and closely co-localizes with intracellular Abeta in muscle or neurons as assayed by immunohistochemistry. (The human homologs of HSP16, HSP27 and alpha-B-crystallin, have also been reported to be upregulated in AD brains.) Immunoblot analysis of preparations from transgenic animals demonstrates that HSP16-2 co-immunoprecipitates with Abeta, and Abeta co-immunoprecipitates with HSP-16-2. Immunoprecipitation of synthetic Abeta added to heat-shocked wild type animal lysate also co-immunoprecipitates HSP16. Fractionation of immunoprecipitates from transgenic animals on one dimensional polyacrylamide gradient gels reveals >10 protein bands not observed in control immunoprecipitations. Several of these proteins have been identified by MALDI-TOF mass spectrometry combined with database searching. We are currently trying to establish a role for these proteins in Abeta toxicity by RNAi experiments. Co-immunoprecipitation followed by mass spectrometry may be an effective general approach for identifying interacting proteins in C. elegans