Lee, Siu Sylvia et al. (2019) International Worm Meeting "Links between altered fat metabolism and lifespan in different types of self-sterility in C. elegans."

Chaturbedi, Amaresh, Lee, Siu Sylvia

[International Worm Meeting, 2019]

We are interested in how reproduction impacts fat metabolism and longevity. It has been well known that the germline stem cells in C. elegans signal to modulate longevity and altered fat metabolism is likely part of the underlying mechanism. We are comparing different classes of mutations that cause self-sterility in C. elegans, and found that whereas these other mutants accumulate excess fat, they do not enjoy a lifespan benefit. Comparing these various sterile mutants may help to delineate the unique mechanism that germline stem cells have on fat regulation and longevity.

Siu Sylvia Lee et al. (2002) East Coast Worm Meeting "A critical role for mitochondria in C. elegans lifespan regulation"

Raymond Lee, Ravi Kamath, Julie Ahringer, Siu Sylvia Lee, Andrew Fraser, Gary Ruvkun

[East Coast Worm Meeting, 2002]

To identify new components that regulate C. elegans lifespan, we performed a genetic screen for long-lived mutants. To eliminate the isolation of components of the well-characterized daf-2 pathway, we screened for long life in a daf-16 null mutant background. In a pilot screen of 2000 haploid genome, we isolated mg312, which exhibited a dramatic increase in lifespan, up to two-fold greater than controls. mg312 also displayed other pleiotropies, including reduced pumping and defecation rate, slow growth and sterility. mg312 is a mutation in the mitochondrial leucyl-tRNA synthetase gene lrs-2. lrs-2(mg312) is a probable null mutant because it is a nonsense mutation in the second exon of the gene. Using a rescuing lrs-2::GFP fusion gene, we showed that LRS-2 is localized to the mitochondria of many different tissues, including neurons, intestine and body-wall muscle. To assess mitochondrial morphology, we used a GFP marker that is specifically expressed in the body-wall muscle mitochondria. In wild-type animals, this marker exhibits highly organized rod-like staining, but in lrs-2(mg312) animals, it was found in contorted and swollen patterns, suggesting that mitochondria were somehow compromised in these mutants. The mechanism contributing to the long-lived phenotype of lrs-2(mg312) is unclear. However, a prediction based on the oxidative theory of aging is that reduced reactive oxygen species (ROS) production will lead to reduced cellular damage and longer lifespan. To test this hypothesis, we subjected animals to the oxidative damage-inducing agent paraquat. We found that lrs-2(mg312) animals were more sensitive to paraquat treatment, suggesting that these mutants may in fact produce more endogenous ROS, perhaps due to a block in electron transport chain activity. We also utilized an RNA interference (RNAi) strategy to systematically analyze lifespan alterations following reduced expression of each of the predicted C. elegans gene. Inactivation of each gene of Chromosome I revealed that a large number (about 10%) of the RNAi candidates leading to a long-lived phenotype are annotated as mitochondrial genes. These results further indicate that mitochondria likely play an essential role in controlling C. elegans lifespan.

Siu Sylvia Lee et al. (2001) International C. elegans Meeting "A genetic screen to identify novel players in C. elegans lifespan regulation"

Gary Ruvkun, Siu Sylvia Lee, Raymond Lee

[International C. elegans Meeting, 2001]

Multiple genetic pathways play a role in worm lifespan control. Most notably, mutations leading to reduced daf-2 insulin-like signaling extend the lifespan of C. elegans up to 4-fold. The longevity phenotype associated with daf-2(lf) is completely suppressed by loss-of-function mutations in the forkhead transcription factor daf-16 , suggesting daf-16 likely regulates genes that control C. elegans lifespan. To identify daf-16 targets, as well as genes in other parallel pathways, which regulate lifespan, we performed a genetic screen to identify mutants that exhibit an extended lifespan phenotype in a daf-16 null background. From a pilot screen of 1600 haploid genomes, we recovered three independent mutants with lifespans significantly longer than the starting daf-16(mgDf47) strain. Among the three mutants, age-3(mg312) displayed the greatest lifespan extension, up to 3-fold that of control strain. age-3(mg312) is pleiotropic, including slight uncoordinated movement, arrested germ line development and sterility. These pleiotropies allowed rapid mapping of this mutation to a small region around +2 on chromosome I. Cosmid rescue and sequencing of genes in the region suggested that age-3(mg312) resulted from a nonsense mutation in the only C. elegans leucyl-tRNA synthetase gene. Single gene rescue experiments and further characterization of age-3 are underway. While it is not clear how such a fundamental player of gene expression specifically regulates lifespan, other basic cell machinery components have been previously implicated in longevity, including the WRN DNA helicase in human, the Indy Krebs cycle transporter in Drosophila , and the SIR2 histone deacetylase in worm and yeast. On the other hand, given the lifespan effects of germ line ablations, the sterility associated with age-3 could indirectly contribute to its lifespan phenotype. Phenotypic analysis and mapping of the other two longevity mutations is also in progress. In addition, a large-scale genetic screen to isolate more mutations with an extended lifespan phenotype is ongoing. Taking into account that mutations which affect global metabolism may modulate C. elegans lifespan, as well as other developmental processes, mutants with significant extension in lifespan, but no other pleiotropies, will be of the highest priority for mapping and cloning. Furthermore, we are also using the recently generated chromosome I RNAi library (A. Fraser, et. al ., Nature, 2000.) to survey for genes which, when inactivated, result in long lifespan.

Siu Sylvia Lee et al. (2003) International Worm Meeting "Insulin-signaling regulated DAF-16 target genes that control C. elegans lifespan and metabolism"

Scott Kennedy, Siu Sylvia Lee, Gary Ruvkun, Andrew C Tolonen

[International Worm Meeting, 2003]

Signaling from the daf-2/insulin receptor to the daf-16/FOXO transcription factor controls longevity, metabolism and development in disparate phyla. To identify genes that mediate the conserved biological outputs of daf-2/insulin-like signaling, we used comparative genomics to predict evolutionarily conserved DAF-16/FOXO transcriptional targets. We identified orthologous genes from Caenorhabditis and Drosophila that bear a DAF-16 binding site in the promoter region. We confirmed that the expression of some of these DAF-16 downstream candidate genes is regulated by daf-2/insulin-like signaling in a daf-16 dependent manner in C. elegans. Using RNAi, we found that several of them mediate distinct aspects of DAF-16 function, including longevity, metabolism, and development. The molecular identities of these genes illuminate the downstream pathways that mediate the diverse functions of daf-2/insulin-like signaling.

Sagulo, Seth et al. (2015) International Worm Meeting "Investigating the Role of Host Cell Factor 1 and Ribosomal S6 Kinase in Regulation of Longevity."

Sagulo, Seth, Lee, Siu Sylvia

[International Worm Meeting, 2015]

The Lee lab has identified Host Cell Factor 1 (HCF-1) as a transcriptional corepressor of DAF-16/FOXO, a major downstream effector of the highly conserved insulin/insulin-like (IIS) signaling pathway. C. elegans that are mutant in hcf-1 have an extended lifespan that is dependent on daf-16. Host Cell Factor 1 is a nuclear expressed protein that acts as a scaffolding protein as it does not have a clear DNA binding domain. Thus it likely plays a role in various protein-protein interactions, some of which may be important for regulating lifespan. Since many of the proteins that interact with HCF-1 have not yet been identified immunoprecipitation-mass spectrometry (IP-MS) was performed in collaboration with the Ruvkun lab and Ming Zhu, Meng-Qiu Dong of the Dong lab. This was done using a strain of C. elegans expressing HCF-1 tagged with GFP. Several chromatin related factors including the histone deacetylase SIN-3 which has been previously shown to interact with HCF-1 in addition to many metabolism proteins were found to interact with HCF-1. Surprisingly, the downstream kinase of the highly conserved target of rapamycin (TOR) pathway, ribosomal S6 kinase (RSKS-1/S6K1) was also identified which suggests it may play a role in the regulation of HCF-1 through phosphorylation. Interestingly, previously published data shows that rsks-1 mutants have an extended lifespan that is dependent on daf-16 which is similar to hcf-1 mutants. To support the IP-MS data, the genetic relationship of rsks-1 and hcf-1 was tested. The rsks-1;hcf-1 double mutant does not show an additive extension of lifespan compared to either of the rsks-1 or hcf-1 single mutants providing evidence that HCF-1 and RSKS-1 may be working through the same pathway to regulate longevity in C. elegans. Since HCF-1 and RSKS-1 are both highly conserved proteins understanding the novel biochemical and genetic connection between them will be relevant to aging in mammals.

Pu, Mintie et al. (2017) International Worm Meeting "Dynamic changes of gene-body H3K4me3 with age in C. elegans somatic cells."

Wang, Minghui, Lee, Siu Sylvia, Pu, Mintie

[International Worm Meeting, 2017]

Recent studies demonstrate that altered histone modifications can profoundly impact longevity. However how histone modification changes modulate aging and how aging alters histone modifications are still largely unclear. To gain insights into the histone modification pattern during aging, we monitored the genomic locations and abundance of several histone modifications involved in gene transcriptional regulation in the somatic cells of C. elegans at different ages. Previously we found that the genome-wide pattern of H3K36me3 is largely stably maintained during aging and that H3K36me3 marking plays an important role in maintaining gene expression stability and lifespan. We have now surveyed the genome-wide pattern of H3K4me3 through similar aging time points. We identified specific regions of H3K4me3 marking that show dynamic change with age and that these H3K4me3 changes are highly correlated with gene expression alterations. Interestingly, the H3K4me3 regions that change with age and are accompanied by RNA expression alterations largely mark gene-bodies, contrary to the canonical marking of H3K4me3 around transcriptional start sites (TSS). Comparison with modENCODE data suggest that the gene-body marking of H3K4me3 that tend to change with age are not marked by H3K4me3 during early development but become apparent during late larval and/or adult stages, whereas the H3K4me3 marking around TSS are clearly established during early development and remain largely stable with age. Our study suggests an intriguing possibility of adult-specific H3K4me3 marking that are more susceptible to age-dependent change, which may have important implications for age-dependent RNA expression dynamics.

Pu, Mintie et al. (2013) International Worm Meeting "Epigenetic Mechanism of Longevity Regulation in C. elegans."

Pu, Mintie, Ni, Zhuoyu, Wang, Xiujuan, Yu, Haiyuan, Lee, Siu Sylvia

[International Worm Meeting, 2013]

Recent studies demonstrate longevity is affected and controlled by histone modifications. The link between histone modification and aging was first demonstrated by the observation that RNAi knockdown or genetic inactivation of histone modification enzymes resulting in extended longevity in model organisms, including C. elegans. However how histone modification changes impact aging and how aging impacts histone modifications are not clear. To gain insights into the histone modification pattern during aging, we monitored the genomic locations and abundance of several histone modifications involved in gene transcriptional regulation at different ages using ChIP-seq. Here we surveyed the genomic H3K36me3 profile in C. elegans somatic cells during aging. We found H3K36me3 pattern is largely stably maintained during aging. Importantly, we found H3K36me3 level is negatively correlated with gene expression variation during aging. Our data indicate that genes that are lowly methylated are significantly more likely to show greater changes of mRNA levels with age. Our analyses indicate that this negative correlation between H3K36me3 modification level and gene expression variation is independent of gene expression level, gene length and tissue specificity, suggesting an interesting function of H3K36me3 in maintaining the stability of gene transcription during aging.

Rizki, Gizem et al. (2009) International Worm Meeting "HCF-1 modulates lifespan and stress responses in C. elegans by regulating the transcriptional activities of DAF-16 and SIR-2.1."

Jan, Max, Rizki, Gizem, Li, Ji, Lee, Siu Sylvia, Murphy, Coleen T.

[International Worm Meeting, 2009]

DAF-16, the C. elegans ortholog of the evolutionarily conserved FOXO transcription factors, is a key mediator of longevity. DAF-16 acts as a central integrator of diverse environmental and cellular stimuli, and mounts a transcriptional response to modulate stress responses and lifespan. Although multiple pathways and factors that affect the function of DAF-16 have been identified, the precise molecular mechanisms determining the specific transcriptional responses by DAF-16 have yet to be discovered. We recently identified the C. elegans host cell factor 1 (hcf-1) as a novel longevity determinant and as a transcriptional co-repressor of DAF-16 (Li J. et al, 2008, PLoS Biol). While not much is known about C. elegans HCF-1, the mammalian ortholog of HCF-1 is known to regulate the activities of several transcription factors by assembling appropriate transcriptional complexes. Loss of C. elegans hcf-1 results in a robust lifespan extension and resistance to oxidative and UV-stress that is dependent on daf-16. HCF-1 antagonizes the transcriptional activity of DAF-16 in the nucleus by physically associating with DAF-16 and limiting its access to select target gene promoters. To test if hcf-1 also requires the activity of sir-2.1, another key longevity determinant that acts as a daf-16 cofactor, we performed epistasis analysis between hcf-1 and sir-2.1. Our preliminary results indicate that loss of sir-2.1 suppressed the longevity and UV-stress-resistance phenotypes of hcf-1 mutants. In addition, sir-2.1 was necessary for the elevated expression of superoxide-dismutase 3 (sod-3), a DAF-16 target gene, in hcf-1 mutants. Co-immunoprecipitation experiments revealed that HCF-1 and SIR-2.1 proteins physically associate. Using gene-expression microarray analysis, we identified genes regulated by DAF-16 in response to HCF-1. In agreement with our hypothesis that HCF-1 modulates specific functions of DAF-16, we found that the HCF-1/DAF-16 target genes constituted only a subset of known DAF-16 target genes altered in response to insulin/IGF signaling (Shaw WM et al, 2007, Curr Biol). Interestingly, comparison of daf-16-dependent gene expression changes in long-lived hcf-1 mutants to those in long-lived sir-2.1 overexpressing strains (CTM, unpublished results) revealed a >90% overlap of similarly regulated genes. Therefore, our results suggest that HCF-1 modulates longevity and stress responses through antagonizing the transcriptional activities of DAF-16 and SIR-2.1.

Akwasi Anyanful et al. (2005) International Worm Meeting "Paralysis and killing of C. elegans by enteropathogenic E. coli requires the bacterial tryptophanase gene"

Daniel Kalman, Jennifer Dolan-Livengood, Lisa Kalman, Melanie Sherman, Mark DeZalia, Taiesha Lewis, Guy Benian, Akwasi Anyanful, Seema Sheth

[International Worm Meeting, 2005]

Enteropathogenic E. coli (EPEC), a major cause of food and water-borne disease causes high morbidity and mortality in developing countries. We have developed a model of EPEC virulence based on our observation that these bacteria paralyze and kill C. elegans. Paralysis and killing of C. elegans by EPEC did not require direct contact, suggesting mediation by a secreted toxin. Virulence however did require tryptophan and bacterial tryptophanase because lack of tryptophan in growth media or deletion of tryptophanase gene failed to paralyze or kill C. elegans. While known tryptophan metabolites failed to complement an EPEC tryptophanase mutant when presented extracellularly, complementation was achieved with the enzyme itself expressed either within the pathogen or within a co-cultured K12 strains. Thus, an unknown metabolite of tryptophanase, derived from EPEC or from commensal nonpathogenic strains, appears to directly or indirectly regulate toxin production within EPEC. EPEC strains containing mutations in the locus of enterocyte effacement (LEE), a pathogenicity island required for virulence in humans, also displayed attenuated capacity to paralyze and kill the nematodes. Furthermore, tryptophanase activity was required for full activation of LEE promoters, and for efficient formation of actin-filled membranous protrusions (attaching and effacing lesions) that form on the surface of mammalian epithelial cells following attachment and which depends on LEE genes. Finally, several C. elegans genes, including daf-2, age-1, hif-1 and egl-9, rendered C. elegans less susceptible to EPEC when mutated, suggesting their involvement in mediating toxin effects. Other genes, including daf-16, mev-1, mek-1, pgp-1,3 and vhl-1, rendered C. elegans more susceptible to EPEC effects when mutated, suggesting their involvement in protecting the worms. Together, these data suggest that this C. elegans/EPEC system will be valuable in elucidating novel factors relevant to human disease that regulate virulence in the pathogen or susceptibility to infection in the host.

Lee, Inhwan et al. (2013) International Worm Meeting "Epigentic regulation of L1 longevity."

You, Young-jai, Lee, Inhwan

[International Worm Meeting, 2013]

Animals encounter food infrequently and starvation is a common physiological state in their natural circumstance [1]. Hatching in the absence of food, worms arrest their development at the L1 stage and survive starvation more than two weeks [2]. It was shown that adult longevity is regulated epigenetically by chromatin alterations and the genes that regulate epigenetics [3]. Here, we investigate whether L1 longevity is also regulated epigenetically. When we measured various histone modifications using Western blot, we found histone 3 lysine 4 tri-methylation, known to be a modification to activate gene transcription, is increased in L1 starvation. Moreover, mutants of set-2, which encodes a histone 3 lysine 4 tri-methyl transferase that functions in adult longevity [3], has reduced L1 longevity. There are several genes essential for normal L1 longevity, such as aak-2 and daf-16. Based on our data, we hypothesize that chromatin remodeling through histone 3 lysine 4 tri-methylation regulates transcription of genes involved in L1 longevity. Currently we are testing this hypothesis by measuring expression levels these genes by ChIP-qPCR during L1 starvation in set-2 mutant. References [1] Brian H. Lee, Plus Genetics, 2008 [2] Inhwan Lee, Plus One, 2012 [3] Eric L. Greer, Nature, 2010.