Herbette, Marion et al. (2017) International Worm Meeting "Transgenerational requirement for C. elegans SET-2/SET1 in genome stability and DNA repair."

Burny, Claire, Michal, Fanny, Herbette, Marion, Yvert, Gael, Robert, Valerie, Palladino, Francesca, Cluet, David, Mercier, Marine, Bedet, Cecile

[International Worm Meeting, 2017]

Maintaining the integrity of genetic information across generations is essential for both cell survival and reproduction, and requires the timely repair of DNA damage. Specific post-translational modifications of histone tails play a central role in the DNA repair process through the recruitment of proteins and complexes with specific enzymatic activities, or by altering the chromatin state at the site of DNA lesions. I will present data showing that SET-2, the C. elegans homologue of the conserved Set1 histone methyltransferase (HMT), is required for genome stability in the germline. SET1 family HMTs are responsible for methylation of histone H3 on Lysine 4 (H3K4), a modification universally found at transcriptional start sites and associated with gene transcription. The absence of SET-2 results is a strong reduction of H3K4 di- and tri-methylation in the distal part of the germline and correlates with a mutator phenotype and a sensitivity to DNA damage inducing agents which increases transgenerationally. Following exposure to ionizing radiation, set-2 mutant germlines show a delay in the repair of mitotic DNA double strand breaks (DSBs), and increased fragmentation in oocytes.. These observations stongly suggest that SET-2 and H3K4 methylation play an essential role in genome stability in the germline of C. elegans. Experiments performed to better understand the requirement of SET-2 and H3K4 methylation in genome stability show that the DNA damage response (DDR) pathway is still functional in the absence of SET-2 suggesting that SET-2 acts downstream or in parallel of the DDR to control genome stability in the C. elegans germline. Experiments designed to detect DSB repair defects at CRISPR- induced targets, or de novo mutations by whole-genome resequencing, will also be presented.

Phillips, Carolyn M. et al. (2011) International Worm Meeting "Characterization of Transposon Silencing Pathways."

Ruvkun, Gary B., Phillips, Carolyn M.

[International Worm Meeting, 2011]

Small RNA mediated silencing of transposons is an important mechanism to prevent DNA damage and subsequent mutations in the germline. A subset of the exogenous and endogenous small RNA pathways genes have been demonstrated to be involved in transposon silencing. When these genes are mutated, transposons can be excised, leaving a double-strand break, which must be repaired by the cellular machinery, either through homologous recombination or non-homologous end joining. I have developed a strategy to visualize double-strand breaks in the germline generated by unregulated transposons in the transposon silencing mutants. This assay will allow me to look at transposon hopping in mutants that are sterile and possibly to perform a small-scale cytological screen for new genes in the transposon silencing pathway. Additionally, I am generating fluorescently tagged proteins to perform localization analysis of both new and previously characterized genes in this pathway. Localization may identify particular cells where RNA based immunity is important or sub-cellular structures to which these proteins concentrate. Finally, to identify direct binding partners of these proteins and potentially identify additional components of the transposon silencing machinery, I will biochemically purify the components of this pathway. Interacting proteins will be identified by mass spectrometry. Any components identified through this strategy can be further characterized by mutational and localization analysis. These experiments will examine the relationship between the small RNA pathways and genome integrity, and further elucidate the mechanisms of transposon silencing in the germline. CMP is the Marion Abbe Fellow of the Damon Runyon Cancer Research Foundation.

Genta Ohno et al. (2006) East Asia C. elegans Meeting "Visualization of developmentally regulated alternative splicing events by a GFP/RFP based splicing reporter system"

Masatoshi Hagiwara, Hidehito KUROYANAGI, Genta Ohno

[East Asia C. elegans Meeting, 2006]

Alternative splicing produces huge diversity of mRNAs in multicellular organisms. Recently, we have developed a splicing reporter system by utilizing C. elegans as a model organism, and identified trans-acting factors and a cis-acting element regulating tissue-specific expression of egl-15 exon 5s (1). In order to elucidate molecular mechanisms of developmentally regulated alternative splicing in vivo, we are generating alternative splicing reporter worms by utilizing let-2 gene as a model. let-2 encodes α2 (IV) collagen in C. elegans and its mutually exclusive exons were developmentally regulated. mRNA containing exon 9 is predominant in embryos, while mRNA with exon 10 is predominant in adults (2). In order to monitor expression patterns of exons 9 and 10, we amplified exons 8 through 11 of the let-2 genomic DNA, and fused to GFP and RFP. Termination codons were introduced into exon 9 (E9) or 10 (E10) of these constructs so that usage of E9 leads to expression of GFP-fusion protein (E9-GFP) and the usage of E10 results in the expression of RFP-fusion protein (E10-RFP). When expressed in body wall muscles where endogenous let-2 is primarily expressed, reporter expression patterns gradually shift from E9-GFP to E10-RFP along with the developmental stages, consistent with the splicing patterns of endogenous let-2. When expressed under unc-51 promoter, which drives expression in a variety of tissues, transgenic worms showed differential expression pattern of GFP and RFP. These observations suggest that ectopically expressed let-2 exon 9/10 reporters are differentially spliced in developmentally regulated as well as tissue-specific manners. Through EMS mutagenesis and color-based screening, we isolated several lines of mutants that remain to express E9-GFP in body wall muscles until the adult stage. We are currently mapping the gene and are also identifying cis-elements regulating developmental and tissue-specific property of the let-2 reporter. 1. Kuroyanagi H. et al. Nature Methods, (in press). 2. Marion H. et al. JCB, (1993).