Jessica B Bessler et al. (2005) International Worm Meeting "Chromatin Modification and Double-Strand Breaks in C. elegans Meiosis"

Jessica B Bessler, Anne M Villeneuve

[International Worm Meeting, 2005]

Meiosis is the specialized cell division program that allows diploid organisms to generate haploid gametes. Successful meiotic cell divisions require that programmed DNA double-strand breaks (DSBs) are formed and repaired utilizing the homologous chromosome as the repair partner, thereby generating chiasmata and crossover recombination products. While the highly conserved protein SPO-11 is universally required to catalyze meiotic DSB formation, relatively little is known about how chromosomal competence for DSB formation is regulated. Several lines of evidence have suggested a connection between chromosome structure and DSB formation. For example, the C. elegans protein HIM-17 has been identified as a chromatin-associated factor that is required for both the formation of SPO-11-dependent DSBs and for the proper accumulation of histone H3 lysine 9 methylation (H3MeK9) on chromosomes during meiotic prophase progression. The precise nature of the link between HIM-17, DSBs and H3MeK9 is not well understood. Previous analyses have ruled out the possibility that H3MeK9 is a consequence of DSB formation but left open the question of whether acquisition of the H3MeK9 mark might be a prerequisite for either onset or cessation of DSB formation. One way to investigate these links is to determine what proteins interact with HIM-17 in vivo. In experiments currently underway, we are utilizing a HIM-17::GFP fusion protein to immunoprecipitate HIM-17 interacting factors from C. elegans extract. The identification of HIM-17 interacting proteins will allow us to narrow down the potential functions of HIM-17 in DSB formation and chromatin modification. In addition, we are working on localizing C. elegans SPO-11 using both microscopy and extract fractionation coupled with SDS-PAGE and Western analysis in order to determine if the presence or absence of HIM-17 affects SPO-11 localization.

Iuval Clejan et al. (2005) International Worm Meeting "NON-HOMOLOGOUS END-JOINING OF RADIATION-INDUCED DNA DOUBLE STRAND BREAKS IN C. ELEGANS"

Iuval Clejan, Shawn Ahmed

[International Worm Meeting, 2005]

Non-homologous end-joining (NHEJ) of double-strand DNA breaks is an important repair pathway in many organisms, but the particular circumstances under which this pathway is used and the way NHEJ is coordinated with other repair pathways are still unclear. We have observed that C. elegans strains harboring mutations in any of three NHEJ genes, cku-80, cku-70 and lig-4, are defective for repairing DNA double-strand breaks in non-cycling somatic cells. Several phenotypes are observed upon gamma irradiation of these mutants, including Gro, Unc, Pvl, Vul, Rup, and Egl. One possible upstream molecular event for all these phenotypes might be mis-segregation of improperly repaired chromosomes during mitosis following radiation induced DNA breaks. In support of this hypothesis, we observed failure of intestinal nuclei to undergo karyokinesis upon irradiation of NHEJ mutants. Further, the somatic radiation-induced phenotypes of NHEJ mutants synergized with him-10(e1511), a mutation in a known kinetochore component that results in unstable mitotic chromosome segregation. Although NHEJ is important for non-cycling somatic cells, the preferred DNA double-strand break repair pathway in both cycling and non-cycling germline cells and in cycling somatic cells is homologous recombination (HR). Worms depleted for the HR proteins RAD-51, MRE-11 and RAD-54 show sensitivity to gamma radiation in their germlines, and somatic sensitivity to gamma irradiation if irradiated during early embryogenesis. However, we observed no role for HR genes in dsDNA break repair in non cycling somatic cells. This is interesting because it implies MRE-11 does not participate in NHEJ in C. elegans (and possibly higher eukaryotes), as it does in S. cerevisiae. There was no apparent role for NHEJ genes in dsDNA break repair in the germline, even in non-cycling germ cells. In conclusion, we have shown that NHEJ repair of dsDNA breaks occurs preferentially in non-cycling somatic cells, whereas HR repair of dsDNA breaks occurs in cycling somatic cells and in the germline.

Marcel Tijsterman et al. (2005) International Worm Meeting "Double-strand break responses in C. elegans"

Joris Pothof, Marcel Tijsterman, Evelien Kruisselbrink, Gijs van Haaften, Ronald Plasterk

[International Worm Meeting, 2005]

The DNA within our cells is constantly being damaged by both environmental and endogenous agents; of the many forms of DNA damage, the DNA double strand break (DSB) is considered to be most dangerous. Many genes that play a role in the processing of DSBs have been identified over the past decades, however, it is clear that many components required in higher eukaryotes are not yet known and the identification of those will be a major challenge for future research.To identify the complete set of genes that are crucial for a proper response to DNA DSBs we performed a genome-wide screen in which we fed animals the Ahringer RNAi library and monitored the brood size after exposure to sub-lethal doses of ionizing radiation (see abstract van Haaften). This screen revealed 45 genes, that when knocked down result in ionizing radiation sensitivity; some of these also have defects in the apoptotic and/or cell cycle response.One of the genes we previously found to be required for a proper response to DNA DSBs is C. elegans lin-61, the worm homolog of human L3MBTL2 and MBTD1. lin-61 is essential for ionizing radiation induced cell cycle arrest and apoptosis, has an impaired intra-S-phase checkpoint and is not able to activate the apoptotic program upon E2F deregulation in a lin-35(n745)/Rb background or after cep-1/p53 upregulation. Furthermore we show that LIN-61 is upregulated after DNA damage in an ATM-1 dependent manner. LIN-61 interacts with a SET domain containing histone methyltransferase, a Sharp like protein and to EFL-2, one of the two worm homologs of the transcription factor E2F, and subsequent analyses revealed that both the chromatin remodeling genes and efl-2 are also essential for the DNA damage checkpoint.In a complementing approach, we recently developed a transgenic system that allows the visualisation of repair of a single localized DSB. This system is based on pie-1 driven (germ line) expression of the rare-cutting yeast endonuclease I-SceI that is combined with transgenes containing LacZ reporters that are interrupted with I-SceI restriction sites; homology driven repair of the I-SceI-induced DSB can restore B-galactosidase expression, which is seen as blue staining of somatic sub-lineages.

Stamper, Ericca et al. (2011) International Worm Meeting "Regulation of Meiotic Double-Strand Break Formation in C. elegans."

Stamper, Ericca, Dernburg, Abby

[International Worm Meeting, 2011]

Meiotic recombination is essential for the successful execution of meiosis, and therefore for sexual reproduction. Recombination is initiated by the formation of programmed DNA double-strand breaks (DSBs), catalyzed by the conserved endonuclease Spo11. If not properly repaired, DSBs pose a threat to genomic integrity. The activity of Spo11 is thought to be tightly regulated to control both the number and location of DSBs. In budding yeast, where the regulation of DSB formation is best understood, at least 9 proteins in addition to Spo11 are required for meiotic DSBs. However, little is understood about how recombination initiation and DSB formation is regulated in other organisms, including C. elegans, which has emerged as an important system for studying meiosis. To gain new insights into the regulation of SPO-11 and DSB formation in C. elegans, I have been characterizing a novel gene that we have named dsb-1. Animals lacking dsb-1 function are viable and fertile, but do not make crossovers during meiosis and therefore produce many inviable progeny and a high incidence of males (Him). This defect in recombination can be rescued by irradiation, indicating that they are specifically defective in DSB formation and not repair. DSB-1 shares homology with another C. elegans gene implicated in DSB formation (Rosu et al., 2009), but lacks obvious homology with proteins outside of Caenorhabditis. I have found that epitope-tagged DSB-1, expressed from single-copy transgenes integrated by MosSCI, localizes as foci on meiotic chromosomes during early meiotic prophase, corresponding to the timing of DSB formation. One hypothesis is that DSB-1 may recruit SPO-11 to meiotic chromosomes. However, analysis by yeast two-hybrid (Y2H) assays failed to detect an interaction between DSB-1 and SPO-11. Y2H analysis did detect a weak interaction between DSB-1 and HTP-3, complementing previous studies that suggested a role for HTP-3 in DSB formation through the recruitment of recombination proteins to meiotic chromosomes (Goodyer et al., 2008). I plan to investigate DSB-1 function through co-immunoprecipitation and ChIP experiments. Future goals of my project aim to address some of the many remaining questions regarding the role of DSB-1 in promoting DSBs and the regulation of meiotic recombination initiation in C. elegans. References: Goodyer W, Kaitna S, Couteau F, Ward JD, Boulton SJ, Zetka M. 2008. HTP-3 links DSB formation with homolog pairing and crossing over during C. elegans meiosis. Dev Cell 14:263-74. Rosu S, Tam A, Villeneuve A. 2009. Identification of novel components of the C. elegans meiotic machinery. International Worm Meeting.

Rosu, Simona et al. (2011) International Worm Meeting "A novel gene promoting meiotic double-strand break formation in C. elegans."

Villeneuve, Anne, Rosu, Simona

[International Worm Meeting, 2011]

Meiosis is a fundamental process by which diploid organisms generate haploid gametes. During meiosis, crossovers (COs) between the DNA molecules of homologous chromosomes provide physical links (called chiasma) that hold the homologs together and ensure proper segregation at the meiosis I division. COs are generated by homologous recombination initiated by DNA double strand breaks (DSBs) formed by the meiotic SPO-11 protein. The regulation of meiotic DSB formation is not well understood, however it is important that organisms have mechanisms to guarantee at least one break per chromosome pair to provide the obligate chiasma. We have identified a new gene (defined by the me96 mutation) involved in promoting DSB formation during C. elegans meiosis. The me96 allele was isolated from a cytological screen for meiotic abnormalities visible in oocytes at diakinesis, the last stage of meiotic prophase. Overall, me96 worms produce 60% dead embryos and exhibit a mixture of bivalents (homologous chromosomes joined by a chiasma) and univalents (achiasmate chromosomes) at diakinesis. Notably, the phenotype becomes progressively more severe as the worms get older. The me96 allele contains an early stop mutation in the F26H11.6 gene, defining a novel component of the meiotic machinery. Immunofluorescence experiments show that RAD-51 foci, which mark recombination intermediates, are reduced in the me96 mutant. Providing exogenous breaks by irradiation rescues both RAD-51 foci levels and chiasma formation in me96 worms. This suggests F26H11.6 is involved in promoting DSB formation, a step that is critical for meiotic recombination. An antibody against the F26H11.6 protein localizes to chromatin of nuclei in transition zone and early pachytene, the stages of meiotic prophase corresponding to the presumed timing of DSB formation. This suggests that F26H11.6 may help create an environment competent for SPO-11 dependent DSB formation. Further characterization of this mutant will provide new insights into the regulation of endogenous meiotic DSB formation.

Truong, Lisa et al. (2015) International Worm Meeting "Role of the DNA Damage Sensor, ATR in Meiotic Double Strand Break Repair in C. elegans."

Engebrecht, JoAnne, Truong, Lisa

[International Worm Meeting, 2015]

The ataxia telangiectasia and Rad3 related (ATR) kinase is an essential DNA damage sensor in eukaryotes. While mammalian ATR mutations are lethal, Caenorhabditis elegans ATR (ATL-1) mutants are viable but infertile facilitating study of ATR meiotic function in a multicellular organism. A key event during meiosis is the formation of chiasmata, where genetic information is exchanged between homologous non-sister chromatids, in response to double strand breaks (DSBs); defects in chiasmata lead to chromosome errors, resulting in aneuploid gametes. To test the hypothesis that ATL-1 functions in the repair of DSBs, the number of chromosomes as monitored by DAPI in meiotic nuclei from wild-type and atl-1 mutants was analyzed. A significant increase in the mean number of DAPI-stained bodies in diakinesis nuclei in atl-1 mutants compared to wild-type worms was observed, suggesting that ATL-1 contributes to the repair of DSBs. To determine whether ATL-1 mediates recombinational repair through homologs versus sister chromatids, the function of ATL-1 was reduced in syp-1 mutants, which are unable to repair through the homolog due to a failure in chromosome synapsis. A significant increase in the mean number of DAPI-stained bodies was observed in syp-1;atl-1 compared to syp-1 mutants, suggesting that the absence of ATL-1 results in chromosome fragmentation consistent with a role for ATL-1 in recombinational repair through sister chromatids. To test whether ATL-1 also functions in chiasmata formation, the number of foci in meiotic nuclei from COSA-1:GFP worms on control and atl-1 RNAi was analyzed. COSA-1:GFP localizes to sites of crossovers, resulting in six foci in wild-type worms. A significant decrease in the mean number of foci in nuclei from worms on atl-1 RNAi was observed, suggesting that ATL-1 is important for the formation of the correct number of chiasmata. Thus, ATL-1 contributes to homologous repair through both sister chromatids and homologs for chiasmata formation. The results from these studies provide insight into ATR meiotic function with implication for human reproduction.

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.

Kotwaliwale, C.V. et al. (2011) International Worm Meeting "Genomic and Epigenetic Regulation of Double Strand Breaks During Meiosis in C. elegans."

Kotwaliwale, C.V., Dose, A., Dernburg, A.F.

[International Worm Meeting, 2011]

Sexual reproduction depends on meiosis, which enables the formation of haploid gametes from diploid cells. In order to segregate to different daughter cells, homologous chromosomes must first establish physical connections with each other via the process of homologous recombination. Meiotic recombination is not distributed randomly across the genome, but occurs in regions that are thought to be preferred sites for programmed DSB formation by the SPO-11 enzyme. In C. elegans, recombination is strongly biased to occur within distal regions (or "arms") of the chromosome. The central regions, which constitute roughly half of the chromosome length, are relatively "cold" for recombination events. It has been unclear whether this bias is a consequence of DSB distribution, or instead reflects a mechanism that biases the downstream repair outcome of DSBs. To address this, we generated a genome-wide map of meiotic DSBs by mapping the distribution of the sole C. elegans RecA-like recombinase, RAD-51, by ChIP-seq (chromatin immunoprecipitation/Illumina sequencing). RAD-51 is a good proxy for DSBs, since it binds to the resected ends and mediates the search for a homologous repair template. We have found that the RAD-51 binding pattern is strikingly similar, but not identical, to the known recombination distribution pattern in C. elegans. RAD-51 binding is highly enriched on chromosome arms. Previous work has shown that the arms of each chromosome differ in recombination rate. We find that these differences are mirrored by the distribution of DSBs. In addition, we find that DSB sites occur in regions of active histone marks such as H3K4 trimethylation and H3K27 acetylation. This is surprising because in general, silencing marks are more abundant on chromosome arms, while active marks are enriched on chromosome centers. This suggests that local gene expression activity may contribute to DSB formation. Consistent with this, we have found a correlation between recombination rate and gene expression. Previous work has shown that recombination rate strongly correlates with intron size. Strikingly, a large number of RAD-51 binding sites occur within introns. Although the DSB pattern is highly correlated with crossover frequency, there are interesting exceptions. In particular, the sub-telomeric regions are completely devoid of crossovers but are active DSB sites. This raises the possibility that additional mechanisms act downstream of DSBs in C. elegans to bias the repair outcome. Taken together, our data provide interesting insights into the role of recombination on the evolution of the C. elegans genome.

Park, sojin et al. (2011) International Worm Meeting "Detection of DNA Strand Breaks detection in Caenorhabditis elegans using Comet assay."

Hyun, Moonjung, Park, sojin, Kim, eunsun, Park, hyejin, Ahn, Byungchan

[International Worm Meeting, 2011]

DNA damage responses (DDR) are important for organisms to maintain genome stability and to survive. DDR is activated in the presence of DNA damage and leads to cell cycle arrest, apoptosis and DNA repair. In C. elegans, double strand breaks (DSBs) induced by DNA damaging agents have been detected indirectly by antibody against DSB recognizing protein, RAD-51. To detect DNA strand breaks induced by IR and CPT, UV at single cells in C. elegans, we developed COMET assay. We treated C. elegans DDR gene mutants (atm-1, brc-1, hus-1) with IR, CPT, and UV. C. elegans atm-1, brc-1 and hus-1 mutants were more sensitive to CPT than N2. DNA strand breaks in a single cell of these mutants atm-1, brc-1,and hus-1 could be detected using COMET assay and more DNA strand breaks accumulate than N2, which corresponds to more RAD-51 foci formation in a single cell in each mutant. This study is the first report in a direct measuring DNA strand breaks in a single cell of C. elegans and this developed assay can be applied to detect DNA strand breaks in different C. elegans mutants which are sensitive to DNA damaging agents.

Sojin Park et al. (2010) East Asia Worm Meeting "COMET assay: DNA Strand Breaks detection in Caenorhabditis elegans"

Sojin Park, Eunsun Kim, Jina Lee, Byungchan Ahn

[East Asia Worm Meeting, 2010]

DNA damage responses (DDR) are important for organisms to maintain genome stability and to survive. DDR is activated In the presence of DNA damage and leads to cell cycle arrest, apoptosis and DNA repair. In C. elegans, double strand breaks (DSBs) induced by DNA damaging agents have been detected indirectly by antibody against DSB recognizing protein, Rad51. To detect DNA strand breaks induced by IR and CPT at single cells in C. elegans, we developed COMET assay.We treated C. elegans DDR gene mutants (hus-1, atm-1, brc-1, rad-51) with IR and CPT. C. elegans atm-1, brc-1 and hus-1 mutants were more sensitive to CPT than N2. Furthermore, atm-1, brc-1, and rad-51, mutants showed more CPT-induced cell cycle arrest than N2. DNA strand breaks in a single cell of these mutants atm-1, rad-51, hus-1, and brc-1 could be detected using COMET assay and more DNA strand breaks accumulate than N2, which corresponds to more Rad-51 foci formation in a single cell in each mutant. This study is the first report in measuring DNA strand breaks in single cells of C. elegans and this developed assay can be applied to detect DNA strand breaks in different C. elegans mutants sensitive to DNA damaging agents.