Charu Jain et al. (2007) International Worm Meeting "Identification of fungal virulence factors using C. elegans as a model host."

Charu Jain, Linsley Kelly, Brendan O'Brien, Reeta Prusty Rao, Samuel M. Politz

[International Worm Meeting, 2007]

Fungal pathogenesis is a significant medical problem. Fungi infect humans, plants and animals; because of their eukaryotic nature, it has been difficult to develop novel antifungal drugs which are not toxic to the host. It would be desirable to establish a model host-fungal pathogen system in order to identify virulence genes in the pathogen and immune response genes in the host. Although several fungal species have been shown to infect C. elegans, none of them is a well-characterized genetic model organism. On that basis we studied interaction between C. elegans as host and the yeast S. cerevisiae as pathogen. On mixed lawns of E. coli and S. cerevisiae, adult wild-type C. elegans showed a tail swelling similar to the deformed anal region (Dar) phenotype described for infection by the bacterium M. nematophilum. This phenotype is not induced by heat-killed yeast, and can be reversed by transferring worms to non-yeast containing plates. As proof of concept we have shown that yeast strains carrying a deletion in gene yap1 fail to induce the Dar phenotype. Yap1 encodes a fungal-specific transcription factor that has been implicated in pathogenesis by indirect evidence. YAP1 has two distinct functions: mediating responses to oxidative stress as well as pleiotropic drug resistance. To investigate which of these is important for producing the Dar phenotype, we tested a yeast strain carrying a deletion in the sod1 superoxide dismutase gene. Like yap1, the sod-1 mutant did not produce the Dar phenotype. In contrast, yeast mutants pdr1 and pdr15, which are involved in pleiotropic drug resistance, and osr5, which is involved in osmotic stress, produced a Dar phenotype similar to wild type, suggesting that the ability to respond to oxidative stress is specifically required for yeast to induce the Dar phenotype. Our working hypothesis is that worms produce reactive oxygen species (ROS) which yeast must counteract in order to be able to induce the Dar phenotype. We are currently testing candidate C. elegans mutants for increased sensitivity or resistance to Dar induction by yeast.

Ming Ming Huang et al. (2001) International C. elegans Meeting "Construction of GFP Tagged Chromosomes"

Don Moerman, Ming Ming Huang, Robert Barstead

[International C. elegans Meeting, 2001]

We have constructed 17 C elegans strains carrying GFP or YFP expressing transgenes integrated randomly in wild type chromosomes. The fluorescent transgenes will be useful as benign dominant markers in crosses to replace chromosomes and as weak balancers for mutations in closely linked essential genes. The integrated transgenes began life as an extrachromosomal array, with GFP fused to promoters for myo-2 and pes-10 or a gut-specific enhancer from F22B7.9. The arrays were integrated using standard methods (1). The resulting strains were outcrossed four times to wild type. Presently, we are mapping the location of each transgene. We will describe their map positions and phenotypes. 1. Mark Edgley, Jun Kelly Liu, Don Riddle, Andy Fire. Worm Breeder's Gazette 15(5): 20 (February 1, 1999).

Alla Grishok et al. (2005) International Worm Meeting "RNAi induced transcriptional silencing in C. elegans"

Alla Grishok, Jina L Sinskey, Phillip A Sharp

[International Worm Meeting, 2005]

RNAi has been implicated in the initiation of chromatin-based silencing of repetitive elements in diverse organisms, ranging from fission yeast to mammals. In C. elegans , repetitive transgenic arrays in the germ-line are maintained at the silenced chromatin state that is transmitted through generations (Kelly et al., 1997; Kelly and Fire, 1998). Involvement of the RNAi pathway in initiation of this silencing has not been extensively studied.Repetitive arrays in the soma of C. elegans allow expression of the transgenes at much higher levels than in the germ-line. However, we show that somatic arrays are prone to transcriptional silencing initiated through an RNAi pathway.We found that transcriptional silencing of the elt-2::gfp/LacZ transgene could be initiated by feeding RNA produced from the commonly used L4440 vector. The transgene and the vector share plasmid backbone sequences. This transgene silencing depends on RNAi pathway genes, including dcr-1, rde-1, rde-4 and rrf-1, and also on alg-1/2 and on the HP1 homolog hpl-2. The latter is a chromatin silencing factor, and expression of the transgene is inhibited at the level of intron-containing precursor mRNA. Chromatin immunoprecipitation experiments showed decreased levels of histone acetylation and less Polymerase II associated with the transgene.This transcriptional silencing in the soma can be distinguished from transgene silencing in the germ-line by its inability to be transmitted across generations and its dependence on the rde-1 gene. Additional chromatin modifying components affecting this process were identified in a candidate RNAi screen. They include histone deacetylases, potential histone methyltransferases, chromodomain-containing genes and genes involved in chromatin remodeling. Interestingly, the sop-2 gene encoding C.elegans Polycomb Group protein with RNA binding properties affected silencing in our system, as well as the gene encoding a protein similar to TAS3, RITS complex component in S. pombe.We observed similarity in the developmental phenotypes of mutants in RNAi-related genes and some mutants in genes encoding chromatin-remodeling components. Specifically, we found similarity in genetic interactions of RNAi pathway genes and chromatin related genes with the lin-35 gene encoding pRb homolog. We are investigating a possible involvement of RNAi pathway in transcriptional regulation of specific genes during C. elegans development.

Snyder, Matthew P. et al. (2013) International Worm Meeting "Turnover of the H3K9me2 Mark During Late Spermatogenesis."

Snyder, Matthew P., Xu, Xia, Maine, Eleanor

[International Worm Meeting, 2013]

Meiotic silencing is a conserved phenomenon targeting unpaired chromosomes and chromosomal regions during prophase of meiosis I. Meiotic silencing in animals typically occurs at the chromatin level and involves accumulation of histone modifications thought to promote a closed chromatin configuration. This chromatin structure may contribute to transcriptional repression and meiotic chromosomal events such as chromosome disjunction (Bean et al 2004, Jaramillo-Lambert and Engebrecht 2010). During meiosis in C. elegans, non-synapsed chromosomes are enriched for H3K9me2 relative to synapsed chromosomes (Kelly et al. 2002; Bean et al. 2004). Such non-synapsed chromosomes include the male X, homologous chromosomes that fail to synapse due to mutation, and chromosomal translocations/duplications. The pattern of H3K9me2 accumulation during meiosis depends on activity of the small RNA machinery, which may have a role in targeting the mark to unpaired chromosomes and ensuring that the mark does not persist abnormally (Maine et al. 2005; She et al. 2009). Taking a combined biochemical/genetic approach, we are identifying additional factors important for regulating H3K9me2 distribution during meiosis. Through this approach, we are identifying genes that appear to be important for turnover of the H3K9me2 mark during late spermatogenesis.

Katherine M Walstrom et al. (2005) International Worm Meeting "Transcription regulation of endogenous genes by C. elegans RNA Helicase A"

Deborah Schmidt, Christopher R Bauer, Katherine M Walstrom

[International Worm Meeting, 2005]

Transcriptional silencing is an important process required for early germline development in some organisms such as C. elegans and Drosophila (Leatherman and Jongens (2003) BioEssays 25, 326-335). RNA helicase A (RHA) is a conserved protein with roles in transcription regulation and histone modification in numerous organisms such as flies and humans. We are studying the role of C. elegans RHA (RHA-1) in transcription regulation and germline development. A null mutant, rha-1(tm329), has a temperature-dependent defect in germline transcriptional silencing of transgene arrays (Walstrom, Schmidt, Bean, and Kelly (2005) Mech. Dev. In press.). In addition, histone modifications are mis-localized on the transgene arrays, on autosomes, and on the single male X chromosome. These defects in gene regulation lead to a sterile phenotype in hermaphrodite and male worms. We are using real-time RT-PCR to determine if endogenous genes have altered expression in the mutants, and we are focusing on the X chromosome in the mutant males. The rha-1 mutant also has a temperature-dependent sterile phenotype, and we hope to identify genes with altered expression that could lead to the sterile phenotype. Recent results will be presented at the meeting.

Kow, R.L. et al. (2019) International Worm Meeting "Polyadenylation nucleases modulate tau-induced toxicity in tau transgenic Caenorhabditis elegans."

Kow, R.L., Strovas, T.J., Chickering, K., Kraemer, B.C., Saxton, A.

[International Worm Meeting, 2019]

The microtubule-associated protein tau accumulates into toxic aggregates in multiple human neurodegenerative diseases such as Alzheimer's disease. To identify novel genes that modulate human tau-induced toxicity, we have used a Caenorhabditis elegans model in which human tau protein is overexpressed in all C. elegans neurons. This causes significant motor dysfunction, progressive loss of neurons, shortened lifespan, and the accumulation of hyperphosphorylated and insoluble tau protein. Using chemical mutagenesis, we identified and then verified that loss of sut-2 strongly suppresses tau-induced toxic phenotypes in tau transgenic C. elegans. The mammalian homolog of sut-2, MSUT2/ZC3H14, was shown to regulate polyadenylation (poly(A)); loss of MSUT2 leads to longer poly(A) tails (Kelly et al (2014) RNA 20:681-688). We examined whether genes encoding poly(A) nucleases, which regulate poly(A) tail length by removing poly(A), also modulated tau-induced toxicity. We found that loss of function alleles in ccr-4, panl-2, and parn-1 enhanced tau-induced toxicity while loss of parn-2 suppressed tau-induced toxicity. This suggests that although poly(A) nucleases can modulate tau-induced toxicity, they may do so via different mechanisms.

Xingyu She et al. (2008) Development & Evolution Meeting "Localized H3K9me2 Accumulation During Meiosis Appears To Be Regulated By Multiple Mechanisms"

Xingyu She, Eleanor Maine, Xia Xu

[Development & Evolution Meeting, 2008]

Meiotic Silencing of Unpaired Chromatin (MSUC) is thought to be important for maintaining a healthy and stable genome, and for preparing the genome for recombination and subsequent meiotic events (see Kelly & Aramayo, 2007). One hallmark of meiotic silencing in C. elegans is the enrichment of histone H3 lysine 9 dimethylation on unpaired chromatin regions (Kelly et al. 2002). We have previously shown that this process requires the germline specific RNA-directed RNA polymerase, EGO-1 (Maine et al., 2005). In our candidate gene survey for additional genes that affect the MSUC process, we found that the loss of csr-1, drh-3 or ekl-1 function causes ectopic H3K9me2 accumulation on paired chromatin. We observe ectopic accumulation only in meiotic nuclei that contain unpaired chromosomes/chromosomal regions, suggesting that unpaired chromatin triggers the accumulation of ectopic silencing marks. EGO-1 activity is not required for H3K9me2 accumulation in the absence of CSR-1, DRH-3, or EKL-1 function. We find that csr-1, drh-3, and ekl-1 mutants resemble ego-1 mutants in several respects. The mutations cause similar germ line developmental defects and sterility, enhance glp-1(ts), and are enhanced by him-17. Mutations in all four genes disrupt RNAi (Smardon et al., 2000; Kim et al., 2005; Duchaine et al., 2006; Yigit et al., 2006) and several other processes (Robert et al., 2005; Rocheleau et al. 2008; J. Ahringer, p.c.). We hypothesize that these proteins function together in a common pathway. In mouse, the SIN3B deacetylase complex is associated with the XY body and may function in MSUC (Costa et al. 2006). In human cell lines, the inhibition of histone deacetylase activity reduces H3K9 methylation in gene promoters (Wu et al., 2008). SIN-3/PQN-28 is the sole C. elegans SIN-3-like protein. It may recruit histone deacetylase as well as interact with other regulatory proteins (Choy et al. 2007). Our preliminary data suggest that SIN-3 is required for the enrichment of H3K9me2 on chromatin regions that are normally active (e.g., unpaired Xs in him-8 hermaphrodites) but not on chromatin regions that have low acetylation levels (e.g., the male X). Our results suggest that H3K9me2 levels may be regulated by multiple complementary mechanisms to ensure extensive accumulation on unpaired chromatin and limit accumulation on paired chromatin.

Krause, Michael W et al. (2010) C. elegans: Development and Gene Expression, EMBL, Heidelberg, Germany "Studies of transcriptional regulation hit the sweet spot"

Iser, Wendy B, Krause, Michael W, Mondoux, Michelle A, Hanover, John A, Wilson, Mark A, Wang, Peng, Gosh, Salil, Fukushige, Tetsu, Wolkow, Catherine, Love, Donna

[C. elegans: Development and Gene Expression, EMBL, Heidelberg, Germany, 2010]

Numerous post-translational modifications are known to influence the activity of chromatin-associated proteins regulating transcription. One post-translational modification we have been focusing on is O-GlcNAcylation, a dynamic, single sugar alteration of Ser and Thr residues of target proteins. Although linked to fly chromatin in 1989 by Kelly & Hart, the role of GlcNAcylation in transcriptional regulation remained under appreciated until recently. Nutrient-driven GlcNAcylation of key components of the transcription machinery may epigenetically modulate gene expression in metazoans. The global effects of GlcNAcylation on transcription can be addressed directly in C. elegans because knockouts of the GlcNAc cycling enzymes are viable and fertile. Using anti-O-GlcNAc ChIP-on-chip whole-genome tiling arrays on wild-type and mutant strains, we detected over 800 promoters where GlcNAc cycling occurs, including microRNA loci and multigene operons. Intriguingly, GlcNAc-marked promoters are biased toward genes associated with PIP3 signaling, hexosamine biosynthesis, and lipid/carbohydrate metabolism. Whole-genome transcriptional profiling of the OGlcNAc cycling mutants confirmed dramatic deregulation of genes in these key pathways. As predicted, the GlcNAc cycling mutants show altered lifespan and UV stress susceptibility phenotypes. We propose that GlcNAc cycling at promoters participates in a molecular program impacting nutrient-responsive pathways in C. elegans, including stress, pathogen response, and adult lifespan. The observed impact of O-GlcNAc cycling on both signaling and transcription in C. elegans has important implications for human diseases of aging, including diabetes

Katz, D.J. et al. (2019) International Worm Meeting "SPR-5;MET-2 maternal reprogramming antagonizes H3K36me3 in the control of germline versus soma."

Carpenter, B.S., Katz, D.J.

[International Worm Meeting, 2019]

In C. elegans, the H3K4me2 demethylase, SPR-5, and the H3K9 methyltransferase, MET-2, are maternally deposited into the oocyte where they cooperate to reestablish the epigenetic ground state of the newly formed zygote. Here, we show that the progeny of spr-5;met-2 mutants display a severe developmental delay that is associated with the ectopic expression of MES-4 germline genes. Previous work from the Strome and Kelly Labs demonstrates that maternally deposited MES-4 transgenerationally maintains H3K36me2/3 at ~200 germline genes in a transcription-independent manner, and this is required to reactivate germline genes in the subsequent generation. We hypothesized that SPR-5;MET-2 reprogramming may prevent MES-4 from ectopically maintaining H3K36me2/3 at germline genes in somatic tissues. By performing ChIP-seq on L1 progeny from spr-5;met-2 mutants, we find that MES-4 germline genes ectopically accumulate H3K36me3 in somatic tissues. Additionally, knocking down MES-4 suppresses the ectopic expression of MES-4 germline genes and rescues the developmental delay. These data suggest a model where SPR-5;MET-2 maternal reprogramming antagonizes H3K36me2/3 to enable the proper transgenerational control of germline versus somatic cell fates. Without SPR-5;MET-2 reprogramming, somatic cells struggle to specify their proper cell fate amongst the noise of inappropriate germline gene transcription, leading to developmental delay. A similar mechanism may underlie the developmental delay of Soto and Kabuki Syndrome patients, who have mutations in histone modifying enzymes.

Amanda Hughes et al. (2007) International Worm Meeting "C. elegans RNA Helicase A genetically interacts with other proteins involved in RNAi."

Deborah Schmidt, Amanda Hughes, Katherine M. Walstrom

[International Worm Meeting, 2007]

RNA helicase A (RHA) is a conserved protein with roles in transcription regulation and histone modification in numerous organisms such as flies and humans. We are studying the role of C. elegans RHA (RHA-1) in transcription regulation and germline development. A null mutant, rha-1(tm329), has a temperature-dependent defect in repressive histone modifications and in germline transcriptional silencing of transgene arrays (Walstrom, Schmidt, Bean, and Kelly (2005) Mech. Dev. 122, 707). These defects in gene regulation lead to a sterile phenotype in hermaphrodite and male worms. Others have shown that rha-1 is required for RNA interference in the germline (Robert et al. (2005) Genes Dev. 19, 782-787). We found that rha-1 interacts genetically with rde-2 and mut-7. The RDE-2 and MUT-7 proteins are required for RNAi and physically associate with each other (Tops et al. (2005) NAR 33, 347). The precise roles of these two proteins and RHA-1 in RNAi are not well understood. Interestingly, rde-2;rha-1 mutants exhibit mutual repression while mut-7;rha-1 mutant hermaphrodites have an enhanced phenotype. We are further characterizing these double-mutants to determine how the germline histone modifications, germline development, and RNAi phenotypes are affected. We are particularly interested in the histone modifications, to which RNAi has been linked, because transcriptional silencing is an important process required for early germline development in many organisms.