Stuart K Kim et al. (2001) International C. elegans Meeting "A gene expression map for C. elegans"

Kyle Duke, Min Jiang, George S Davidson, Stewart Scherer, Moni Kiraly, Stuart K Kim, Brian N Wylie, Jim Lund

[International C. elegans Meeting, 2001]

The completion of the C. elegans genome sequence has identified nearly all of the genes in the genome (19,282 genes), but the function for most of these genes remains mysterious. A scant 6% of them have been studied using classical genetic or biochemical approaches (1135 genes), and only about 53% show homology to genes in other organisms (10,303 genes). The next challenge is to develop high-throughput, functional genomics procedures to study many genes in parallel in order to elucidate gene function on a global scale. One such approach is to use DNA microarrays to assay the relative expression of nearly every gene in the genome between two samples. Knowing when, where and under which conditions a particular gene is expressed can reveal the function of that gene. A consortium of laboratories has used C. elegans DNA microarrays to profile gene expression changes in a wide variety of experiments. In each experiment, RNA from one sample was used to generate Cy3-labelled cDNA, and RNA from another sample was used to prepare Cy5-labelled cDNA. The two cDNA probes were simultaneously hybridized to a single DNA microarray and the ratio of the Cy3 to Cy5 hybridization intensities was measured, revealing which genes were relatively enriched in either RNA sample. Thirty different laboratories have collectively performed 553 experiments using these C. elegans DNA microarrays, including 179 experiments with microarrays containing 11,917 genes (63% of the genome) and 370 experiments using microarrays that have 17,817 genes (94% of the genome). The experiments compare RNA between mutant and wild-type strains, or between worms grown under different conditions. Many experiments have been done to date, including experiments on wild-type development, heat shock, Ras signaling, aging, the dauer stage, sex regulation and germ line gene expression. Individual microarray experiments reveal sets of genes that change in one mutant strain or growth condition. We combine the data from all of the experiments to assemble a gene expression database, and then used this database to group together co-regulated genes and visualize them using a three dimensional expression map. By matching the expression profile of an unknown gene to those of genes with known functions, the expression map can be used to ascribe functions to the large fraction of genes in the genome whose functions were previously unknown.

Stuart K Kim et al. (1999) International C. elegans Meeting "Using DNA microarrays to identify targets of the Ras/MAP kinase signaling pathway"

Stuart K Kim, Rebecca R Begley, Carrie B Van Doren

[International C. elegans Meeting, 1999]

We are entering a new age in molecular genetics in which we can use sequence data from genome projects to dissect cell, developmental and disease pathways more completely and more sensitively than ever before. We need to develop new experimental approaches to analyze the vast amounts of data that are now available from genome projects. C. elegans is the only animal model system with a complete genome sequence, and thus will play a leading role in establishing approaches that make use of the full genome sequence. One key functional genomics approach is to use DNA microarrays to define changes in gene expression patterns during development and during the onset of disease. DNA microarrays could be used to identify genes that are regulated by specific transcription factors, by specific cell signaling pathways, by expression of homologs of human disease genes in transgenic animals or by addition of various pharmaceutical drugs. We are currently producing DNA microarrays that contain 11,401 genes from the C. elegans genome. To demonstrate that C. elegans microarrays are powerful tools to dissect developmental pathways, we are using these DNA microarrays to identify targets of the RTK/Ras/MAP kinase signaling pathway during vulval development. To globally detect differences in transcription patterns due to Ras signaling, a cDNA microarray is simultaneously hybridized with Cy3-labelled cDNA (red) (from reverse transcription of polyA mRNA) from let-23(lf) mutants, and Cy5-labelled cDNA (green) from let-60(gf) mutants. Genes that are expressed at similar levels in the two strains hybridize equally with both probes, and appear yellow on the microarray. Genes that are expressed at higher levels in the first strain appear red, and genes that are expressed at higher levels in the second strain appear green. The image intensities of the cDNA microarray are digitized, and the expression ratios between the two strains are calculated for every cDNA clone. The Ras target genes identified by the DNA microarrays will play a critical role in elucidating the molecular basis for Ras signaling. Once we have developed full genome C. elegans microarrays, we can make our DNA microarrays available to the entire C. elegans academic community.

Xu, Xiao et al. (2011) International Worm Meeting "A GATA transcriptional circuit guides aging in C. elegans."

Kim, Stuart, Xu, Xiao

[International Worm Meeting, 2011]

To unveil the mechanisms underlying aging in C. elegans, we have carried out microarray analysis to profile changes in gene expression between young and old worms. Bioinformatics analysis revealed that the promoters of the 1254 age-regulated genes are highly enriched for the GATA binding motif. We previously found that age-related changes in expression of many of these genes are caused by three GATA transcription factors (elt-3, elt-5 and elt-6)(Budovskaya et al., Cell 2008). Here, we identify an additional GATA transcription factor, egl-27, that plays an important role during the aging process. We found that loss-of-function and gain-of-function alleles of egl-27 have opposite effects on lifespan; specifically, overexpression of egl-27 extends lifespan and knockdown of egl-27 decreases daf-2 lifespan without affecting wild-type lifespan. These results indicate that levels of egl-27(+) play a critical role in promoting longevity. To determine how egl-27 promotes longevity, we performed ChIP-seq analysis to find EGL-27 targets. We found that EGL-27 targets are highly enriched for genes whose expressions decrease with age, suggesting that EGL-27 plays a direct role in regulating the aging process. Further, we found that slow aging daf-2 mutants exhibit increased egl-27 expression while fast aging elt-3 mutants display decreased egl-27 expression, suggesting that egl-27 acts downstream of insulin signaling and GATA transcriptional networks. In addition to the GATA DNA binding domain, EGL-27 is homologous to mammalian metastasis tumor antigen 1 (MTA1) family, which functions as part of the nucleosome remodeling and decacetylase (NuRD) complex. This raises the possibility that changes in EGL-27 in old worms affect not only the GATA transcriptional network but also chromatin structure in general.

Mann, Frederick G. et al. (2011) International Worm Meeting "A transgenerational effect on lifespan."

Kim, Stuart, Mann, Frederick G.

[International Worm Meeting, 2011]

We have found that the lifespan of progeny is affected by the age of the mother when the embryo is formed, which is an example of an age-dependent transgenerational effect on lifespan in Caenorhabditis elegans. Progeny produced during the second day of fertility survive 10-25% longer than those produced during the fourth day of fertility. Progeny produced during the fourth day of fertility have a 5-fold higher incidence of mortality during the first 9 days of adulthood. ced-3 mutant animals, which are incapable of programmed cell death, produce progeny of identical lifespan across all days of fertility.

Sagi, Dror et al. (2009) International Worm Meeting "An engineering approach to aging."

Kim, Stuart, Sagi, Dror

[International Worm Meeting, 2009]

We are taking a synthetic biology approach to aging by bio-engineering worms with increased lifespans. Our approach is to improve aging by over-expressing genes that strengthen various pathways such as stress resistance and damage repair. Worms are particularly well-suited for this project because they have the shortest life span among animal models, and thus their stress and damage repair pathways may be relatively inefficient compared to those from longer lived animals such as flies or mice. Furthermore, events that occur in old age are outside the force of natural selection, as animals in the wild die from predation and disease well before old age. Lack of natural selection in old worms may permit cellular and biochemical pathways to become dysfunctional or inefficient, and thus it seems reasonable that one could improve physiology in old worms using a directed engineering approach. To make worms live longer, we are expressing genes from the zebrafish, which lives up to 5 years (about 100 times more than C. elegans) and lives at a similar temperature. Our assumption is that stress and damage repair proteins from zebrafish need to preserve cell function for a longer time than proteins from worms, and thus zebrafish proteins may be more efficient or have higher function than their worm orthologs. We picked five pathways believed to play key roles in aging: Oxidative damage repair, mitochondria activity, protein turnover, the immune system and gene regulation. We then chose 1-4 genes known to work in each pathway, and expressed the zebrafish gene using the orthologous worm promoter. Overall, we have found six genes belonging to four pathways that increase lifespan when overexpressed. In two cases, we could extend worm lifespan by expressing proteins that are present in zebrafish, but are absent in worms. The first example is zebrafish UCP2, which is a mitochondrial uncoupling protein. Vertebrates have UCP2, whereas worms use another uncoupling protein called UCP4. We found that overexpression of zebrafish UCP2 extended lifespan, but worm UCP4 did not. One possibility is that vertebrate UCP2 extends worm lifespan because the vertebrate protein makes the mitochondria more efficient or less toxic. The second example is zebrafish lyzozyme. Vertebrates and worms use different types of lysozyme to combat bacterial pathogens. We found that zebrafish lysozyme but not worm lysozyme could extened lifespan, suggesting that the vertebrate protein may be more efficient in combating pathogenesis. We are currently trying to upgrade two (or more) pathways at the time to look for synergistic effects in extending lifespan. Our work shows that it is possible to rebuild weak links in an animal by adding parts from a longer-lived life form in order to extend lifespan.

Sanchez-Blanco, Adolfo et al. (2011) International Worm Meeting "Variable Pathogenicity Determines Individual Lifespan in C. elegans."

Sanchez-Blanco, Adolfo, Kim, Stuart

[International Worm Meeting, 2011]

Stochasticity is a common property of aging in all animals, as chronologically and genetically identical individuals age at different rates. To unveil mechanisms that influence aging variability, we identified markers of remaining lifespan for C. elegans. In transgenic lines, we expressed fluorescent reporter constructs from promoters of C. elegans genes whose expression change with age. The expression levels of aging markers in individual worms from a young synchronous population correlated with their remaining lifespan. We identified eight aging markers, with expression of the superoxide dismutase gene sod-3 being the best single predictor of remaining lifespan. Visualizing the physiological age of chronologically-identical individuals allowed us to show that a major source of lifespan variability involves different responses to pathogenicity from individual to individual. Ingestion of mildly pathogenic E. coli activates daf-16 FOXO activity via the insulin-like signaling pathway, which induces a beneficial stress response that is protective and extends lifespan. The pathogenicity from the ingested bacteria primarily affects the intestine.

Van Nostrand, Eric et al. (2011) International Worm Meeting "High Occupancy Target (HOT) regions in C. elegans."

Kim, Stuart, Van Nostrand, Eric

[International Worm Meeting, 2011]

As part of the C. elegans modEncode project, regulatory targets for all C. elegans transcription factors (TFs) are being identified by ChIP-seq. In initial analyses of ChIP-seq for 23 transcription factors, we unexpectedly discovered that transcription factors have a continuum of downstream targets, ranging from targets that are bound specifically by that factor to a new type of DNA domain that is bound by most or nearly all TFs. 304 regions of clustered TF binding (bound by 15 or more TFs) were observed, which we term HOT (High Occupancy Target) regions. Genes located near HOT regions are characterized by ubiquitous and high expression, and tend to be essential for viability. Thus, large-scale analysis of worm ChIP-seq data has led to the discovery of an unexpected gene regulatory mechanism for essential, ubiquitous housekeeping genes. In contrast to HOT targets, the factor-specific targets were significantly enriched for genes with an expression pattern similar to the bound transcription factor, and encoded proteins with functions similar to the biological function of the bound transcription factor. For example, HLH-1 is a key regulator of muscle development, and the factor specific targets in the HLH-1 ChIP-seq experiment were enriched for genes expressed specifically in muscle and were induced in previous HLH-1 over-expression experiments. By contrast, the HOT regions bound by HLH-1 showed no association with muscle functions. Thus, for interpretation of target genes with specific biological functions from ChIP-seq experiments of tissue-specific transcription factors, it may be necessary to remove HOT regions from the analysis and focus on factor -specific targets.

Xu, Xiao et al. (2009) International Worm Meeting "Developmental drift of GATA transcription factors during C. elegans aging."

Xu, Xiao, Kim, Stuart K.

[International Worm Meeting, 2009]

To unveil the mechanisms underlying aging in C. elegans, we have carried out microarray analysis to profile changes in gene expression between young and old worms. Bioinformatics analysis revealed that the promoters of the 1254 age-regulated genes are highly enriched for the GATA binding motif. We previously found that age-related changes in expression of these genes is caused by three GATA transcription factors (elt-3, elt-5 and elt-6)(Budovskaya et al., Cell 2008). Here, we identify two additional GATA transcription factors (egr-1 and egl-27) that may also play important roles during the aging process. We found that RNAi knockdowns of egr-1 and egl-27 specifically decrease daf-2 lifespan without affecting wild type lifespan. Additionally, overexpression of egl-27 extends lifespan, suggesting that egl-27 normally functions to promote longevity. Expression of both egr-1 and egl-27 is age-regulated. egr-1 expression decreases specifically in the head with age, while egl-27 expression increases as worms age. In addition to containing GATA DNA binding domain, EGL-27 and EGR-1 are homologous to mammalian metastasis tumor antigen 1 (MTA1) family, which functions as part of the nucleosome remodeling and decacetylase (NuRD) complex. This raises the possibility that changes in these proteins in old worms affect not only the GATA transcriptional network but also chromatin structure in general.

Budovskaya, Yelena V. et al. (2011) International Worm Meeting "Molecular aging driven by Wnt signaling in C.elegans."

Budovskaya, Yelena V., Kim, Stuart K.

[International Worm Meeting, 2011]

We are using a system biology approach to reveal the molecular basis for aging in nematodes C. elegans by characterizing gene expression differences between young and old animals and then determining at a molecular level how these changes contribute to aging. This analysis revealed that gene expression differences between young and old animals are under control of a relatively simple gene regulatory network that involves the elt-3, elt-5, and elt-6 GATA transcription factors. Expression of elt-5 and elt-6 increases in old age, leading to decreased expression of elt-3, thus causing changes in the expression of the many downstream target genes. We found no evidence that it is caused by cellular damage or environmental stresses. Rather, we found that elt-3 expression in the adult is controlled by increased expression of the repressors elt-5 and elt-6, which also guide elt-3 expression during development. These results suggest that age-regulation of elt-3 is caused by age-related drift of an intrinsic developmental program that becomes imbalanced in old age. This elt-3/elt-5/elt-6 transcriptional circuit as the first genetic mechanism that is responsible for some of the age-related changes that occur as the worm grows old. A key unanswered question is what causes elt-3/elt-5/elt-6 transcriptional drift during aging? In C. elegans, Wnt/Wingless signaling pathways activate elt-5 and elt-6 expression during development. Here we demonstrated that Wnt signaling is responsible for the increased expression of elt-5 and elt-6 GATA transcription factors during aging. Mutations in one of the b-catenins, wrm-1, decreases expression levels of the elt-5 and elt-6 GATA transcription factors throughout life, leading to increased elt-3 GATA expression. We demonstrated that mutation in wrm-1/ b-catenin, extends lifespan of other wise wild type animals by ~50%. These results indicate that changes in Wnt signaling - a regulator of the elt-3/elt-5/elt-6 GATA transcriptional circuit during normal development - plays an important role in the age-related regulation of the elt-3/elt-5/elt-6 transcriptional circuit and possibly many others.

Van Nostrand, Eric L et al. (2009) International Worm Meeting "Transcriptional cascades regulating C. elegans aging."

Van Nostrand, Eric L, Budovskaya, Yelena, Kim, Stuart K

[International Worm Meeting, 2009]

Recent work in the Kim lab has identified the GATA transcription factors elt-3, elt-5 & elt-6 as a developmental circuit that becomes mis-regulated during aging, regulating lifespan. Microarray analysis of gene expression in young and old C. elegans populations led to the identification of a highly significant enrichment for a GATA sequence motif in promoters of genes with differential expression during aging. Further work determined that a developmental circuit, in which elt-5 and elt-6 act to repress elt-3 during normal development, inappropriately continues to drive elt-3 expression down with age, causing elt-3 regulatory targets to further decrease and negatively regulating lifespan. Using ChIP-Seq technology, we will identify the regulatory targets of elt-3 in order to determine whether elt-3 is chiefly responsible for age-regulated expression changes, or if the expression changes are due to the combination of a number of other regulators. In addition, a comparison of ChIP-Seq targets in young and old worms will determine whether decreased expression of elt-3 during aging affects specific targets or causes a general loss of regulation across all targets. In addition, microarray profiling identified an additional 30 age-regulated transcription factors, with a variety of functions and expression patterns. In an approach parallel to that described for elt-3, the roles of these additional regulators during aging will be determined. In particular, we will determine whether these regulators function as part of known aging regulatory circuits, or if they present additional examples of circuits that become mis-regulated during aging. Identification of regulatory targets for these additional age-regulated transcription factors by ChIP-Seq will enable the construction of a full aging expression regulatory network, allowing us to better understand the key regulatory factors controlling aging and age-dependent gene expression in C. elegans.