Mark A Wilson et al. (2003) International Worm Meeting "A Screen to Identify Genetic Modifiers of daf-2(lf) Lifespan"

Mark A Wilson, Cathy Wolkow

[International Worm Meeting, 2003]

Lifespan is affected by both environmental and genetic factors, as well as by the interactions between them. C. elegans lifespan is modulated by an insulin-like signaling pathway that includes DAF-2, an insulin-receptor (IR) like protein, and AGE-1, the PI3K p110 catalytic subunit. Mild reduction-of-function mutations in daf-2 or age-1 extend adult lifespan, while more severe mutations result in constitutive developmental arrest. Signaling via the DAF-2/AGE-1 pathway antagonizes the DAF-16/forkhead transcription factor, which is required for increased longevity and dauer arrest in daf-2 and age-1 mutants. A working hypothesis is that, in animals lacking DAF-2/IR signaling, DAF-16/FKHD is active and directs the expression of target genes that confer long adult lifespan and controls the dauer developmental decision. Why do animals lacking DAF-2/IR signaling live longer than wild-type? To address this question, we are screening for mutations that alter lifespan in daf-2 mutants. Because lifespan is a post-reproductive phenotype, we are conducting clonal screens for altered lifespan in the F2 generation after mutagenesis. This approach will be useful for identifying mutations that enhance lifespan in daf-2(e1370) mutants, as well as genes which are required to maintain the longevity conferred by altered insulin/igf signaling. Genes affecting lifespan in a daf-2(lf) background will also be assayed in wild-type animals. This will allow the identification of genes required for lifespan extension when insulin/igf signaling is perturbed, but may also uncover mutations with an independent effect on lifespan. Our initial screens revealed a high level of mid-lifespan lethality in daf-2(e1370) animals following mutagenesis. This suggests that many genes are required for increased adult longevity and not all will be specific DAF-16 targets. 4000 haploid genomes have been screened. 5 suppressors and 1 enhancer of daf-2(e1370) lifespan have been isolated. Preliminary results indicate none of the 5 suppressors significantly alter lifespan in N2. The single enhancer isolated also increases lifespan in wild-type worms. We are currently characterizing and mapping these mutations.

Mark A Wilson et al. (2002) East Coast Worm Meeting "Screens to identify longevity-control genes in C. elegans"

Catherine A Wolkow, Mark A Wilson

[East Coast Worm Meeting, 2002]

Lifespan in C. elegans is controlled by an insulin-like signaling pathway that includes DAF-2, an insulin-receptor like protein, and AGE-1, a homolog of the PI3K p110 catalytic subunit. Reduction-of-function mutations in daf-2 or age-1 extend adult lifespan, while loss-of-function mutations result in constitutive dauer arrest. Signaling via the DAF-2/AGE-1 pathway antagonizes the DAF-16/forkhead transcription factor, which is required for increased longevity and dauer arrest in daf-2 and age-1 mutants. A working hypothesis is that, in animals lacking DAF-2 signaling, DAF-16 is active and directs the expression of target genes that confer long lifespan and control dauer arrest.

David J Katz et al. (2005) International Worm Meeting "Investigating the link between NANOS and chromatin for germ cell maintenance in C. elegans"

William G Kelly, David J Katz

[International Worm Meeting, 2005]

In Drosophila, nanos is required for primordial germ cells (PGCs) to differentiate into a functional germ line. Lack of NANOS function causes defects in germ cell migration, differentiation, and quiescence and ultimately leads to sterility in both males and females. Similarly, in mice a lack of nanos3 results in the complete loss of germ cells in both sexes (Tsuda et al. 2003) and functional loss of two of the three nanos genes in C. elegans, nos1 and nos2, also results in sterility (Subramaniam and Seydoux, 1999). It has therefore been proposed that nanos is a conserved master regulator of the germ cell fate. In the C. elegans early embryo, the chromatin in all cells contain the active chromatin mark, histone H3 methylated on lysine 4 (H3meK4). However upon their birth, the primordial germ cells Z2 and Z3 dramatically lose this mark. This loss of the active H3meK4 mark has been suggested to be part of a chromatin-based transcriptional repression mechanism. Remarkably, absence of the H3meK4 mark has been shown to be dependent upon nanos activity (Schaner et al. 2003). Thus it is possible that nanos may be partially affecting its role in maintaining the undifferentiated germ cell fate through a chromatin-based mechanism. We aim to explore this potential link by further characterizing the role of C. elegans nanos in germ cell fate. To this end, we are taking both forward and reverse genetic approaches as well as conducting ectopic expression studies.

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.

John Peloquin et al. (2007) International Worm Meeting "Worms and Filthy Lucre: Applying C. elegans to natural product development in industry."

Stuart Reeves, John Peloquin

[International Worm Meeting, 2007]

Diamond V Mills is a medium sized Agricultural Biotechnology company with more than 63 years of success in producing dietary supplements and ingredients. Recently the company has moved into human nutrition through its formation of Embria Health Sciences. Thus, the product repertoire has increased and is increasing. Our products arise naturally as the result of subtle alterations of fermentative processes, which are still not completely understood. Furthermore, their effects on animal biology are pleotropic. This presents some serious economic challenges in that far more product prototypes can be produced than economically tested in vertebrate models to demonstrate efficacy. Because many biochemical and signal transduction pathways are conserved between C. elegans and other animals, we have begun to develop C. elegans-based medium through-put screens to identify new product prototypes. We present preliminary data on anti-aging effects of an Embria product as an example of our future goals. (this work was initiated with technical assistance from Mark A. Wilson and Cathy Wolkow of the NIA).

Hunt, Piper R. et al. (2009) International Worm Meeting "Effects of Naphthoquinone Derivatives on Gene Expression and Aging in C. elegans."

Yu, Quian-sheng, Grieg, Nigel H., Wolkow, Catherine A., Hunt, Piper R., Wilson, Mark A.

[International Worm Meeting, 2009]

Macromolecular damage caused by the accumulation of reactive oxygen species (ROS) is thought to underlie many of the deleterious changes associated with aging. In numerous studies in C. elegans, resistance to oxidative stress has been linked to extended lifespan. Therefore, treatments that reduce cellular levels of ROS may also extend lifespan. The term hormesis refers to the beneficial effects on health of low doses of compounds, often plant derived, that are toxic at higher doses. ROS may be the modulators of hormesis; if concentrations of ROS are high enough to stimulate an adaptive response at the cellular level, yet low enough not to cause irreparable damage, increased health and longevity at the organismal level may result. Fourteen commercially available phytocompounds were screened for hormetic effects in C. elegans. In this initial screen, three compounds had beneficial effects at low doses. For further study, we chose to focus on plumbagin, a naphthoquinone present in the Plumbago and Juglans plant genera. Species from these plant families are used in the traditional medicines of various cultures around the world to treat ailments ranging from infections and ringworm to acne and inflammatory diseases. Plumbagin treatment induces the generation of ROS and is toxic at high doses. However, low doses of plumbagin have been shown to have anti-microbial, anti-malarial, anti-atherosclerotic, and anticarcinogenic effects. We generated a panel of plumbagin (5-hydroxy-2-methyl-1,4-napthoquinone) analogs with the goal of potentiating plumbagin''s beneficial effects on morbidity while minimizing its toxicity. Here we show that, in C. elegans, expression of the Phase II reporter gene pgst4::gfp responds in a dose dependent manner to plumbagin. Toxic concentrations of menadione (2-methyl-1,4-napthoquinone) and 5,8-dihydroxy-1,4-napthoquinone also induce increases in pgst4::gfp expression of 15% or more over background. For the non-toxic compounds in our panel, excessive environmental concentrations of 500uM and above were required to consistently see even a 10% increase in pgst4::gfp expression, indicating that pgst4::gfp expression levels of 15% or more over background may act as a biosensor for potentially toxic compounds.

Pokhrel, Bharat et al. (2017) International Worm Meeting "Characterising C. elegans COMPASS complex in gene expression."

Chen, Ron, Appert, Alex, Pokhrel, Bharat, Clifford, Rosamund, Chen, Yannic, Ahringer, Julie, Dong, Yan, Stempor, Przemyslaw

[International Worm Meeting, 2017]

Trimethylation of histone lysine 4 (H3K4me3) is regarded as an active promoter mark across all eukaryotes. H3K4me3 is mainly deposited by an evolutionarily conserved COMPASS complex that contains two key subunits: SET-2, H3K4me3/2 methyltransferase, and CFP-1, a conserved CpG binding protein. Perturbation of this mark has been found to be associated with different diseases, development defects, and incorrect cell fate specification. However, it is unclear whether H3K4me3 play an instructive role in transcription or just a consequence of gene expression. To address this question, we phenotypically characterised and compared cfp-1(tm6369) mutants with a reported loss-of-function set-2(bn129) mutant allele. We observed dramatic loss of H3K4me3, poor fertility, slow growth and ectopic gene expression in both cfp-1 and set-2 mutants. We found that the role of H3K4me3 is to fine tune gene induction, likely by modulating other histone modifications that play an essential role in gene activation. Our results shed light on the role of H3K4me3 in chromatin regulation and function.

Sujata Bhattacharyya et al. (2008) Development & Evolution Meeting "The Role of Histone Deacetylation in the Maintenance of Transcriptional Repression in Primordial Germ Cells"

Sujata Bhattacharyya, William Kelly

[Development & Evolution Meeting, 2008]

Experimental data from a number of model organisms suggests that transcriptional quiescence is essential for safe-guarding totipotency of the embryonic germ line. In C.elegans, global repression in germline blastomeres is mediated by a maternal protein, PIE-1. Subsequent to its degradation, chromatin-based mechanisms accompanied by epigenetic erasure events suppress transcription in the primordial germ cells, Z2 and Z3. In particular, genome-wide H3K18 acetylation is deleted from Z2 and Z3 within 30 minutes of their birth.The rapidity with which this mark is lost suggests an enzymatic mode of removal. This hypothesis is corroborated by evidence which indicates that loss of H3K18Ac occurs on a normal schedule in the psa-1 mutant which is defective in nucleosome remodeling. Consequently, we are analyzing mutants in each of nine histone deacetylases to determine which in particular is responsible for this erasure event. RNA interference will be used if genetic redundancy is suspected. The functional consequences of inappropriately retaining this mark on the totipotency of the germ line will be elaborated in the appropriate loss-of-function mutant(s).

Gyoergyi Csankovszki et al. (2002) West Coast Worm Meeting "Mapping chromosomal targets of the dosage compensation complex"

Barbara J Meyer, Gyoergyi Csankovszki

[West Coast Worm Meeting, 2002]

The worm C. elegans compensates for the difference in X-linked gene dosage between XX hermaphrodites and XO males by reducing gene expression two-fold in XX animals. Downregulation of gene expression is achieved by the dosage compensation complex (DCC), a large multiprotein complex that binds the two X chromosomes in hermaphrodites. However, the cis-acting chromosomal elements that recruit and bind the complex are not known. Using free and attached X chromosomal duplications we are attempting to identify regions of the X chromosome that are sufficient to recruit the complex. By analyzing the volume and number of DCC-bound chromosomal bodies, a large duplication of the right 30% of X, mnDp10, was shown earlier to be competent to bind the DCC (1). Using combined fluorescent in situ hybridization (to mark X chromosomal territories) and immunofluorescence (to mark chromosomes that bind the dosage compensation complex) we confirmed these results. By analyzing smaller duplications that span the entire chromosome we hope to map chromosomal targets of the DCC.

Sambongi Y et al. (1998) European Worm Meeting "Physiological roles of C. elegans copper transport ATPase"

Wakabayashi T, Sambongi Y, Yoshimizu T, Futai M

[European Worm Meeting, 1998]

Copper is essential for cuproenzymes, but its excess causes cell damage through oxidizing proteins and lipids. Therefore, intracellular copper ion concentration must be tightly regulated. Two human genetic disorders, Menkes and Wilson diseases, are defective in copper metabolism. Genes responsible for both disorders encode homologous heavy metal P-type ATPases. To understand the physiological roles of the ATPase, we cloned a homologous cDNA from C. elegans (CUA-1, a 1,238 amino acid protein) that has sequence identities of 46% and 43% to Menkes and Wilson disease proteins, respectively. We showed that the CUA-1 cDNA could rescue the yeast Dccc2 mutant (copper ATPase gene disruptant), suggesting that CUA-1 functions as a copper transporter. The upstream region of cua-1 was fused with the reporter genes (b-galactosidase or GFP) and introduced into the N2 worms. High level expression of the reporter gene was observed specifically in pharyngeal terminal bulb muscle pm6 and most anterior intestinal cells in the transgenic adult worms, suggesting that CUA-1 functions primarily in the digestive organs. In addition to these cells, prominent expression was found in hypodermal cells in larval stages. We also report subcellular localization of CUA-1 and discuss physiological role(s) of this ATPase in C. elegans. We thank Yuji Kohara for providing yk29a9 clone and Andy Fire for GFP and lacZ vectors.