Howard Chen et al. (2005) International Worm Meeting "Natural sequence variation and quantitative aging measures"

Joe Mayerle, Aaron Quinlan, Howard Chen, Stephen Wicks

[International Worm Meeting, 2005]

Aging is a complex and fascinating phenomenon. Without a doubt, aging is a genetically regulated mechanism. Many single gene mutations, particularly genes along the insulin-signaling cascade, have been shown to regulate lifespan in diverse organisms. DAF-16, a forkhead transcription factor in C. elegans is an extensively studied regulator of lifespan extension. Using a genetics approach, we hope to unravel novel genes or novel regulatory loci that are involved in the process of aging. We have generated 400 Recombinant Inbred Lines (RILs) by crossing geographically isolated Bristol N2 and Hawaiian CB4856 strains. A computer model of recombination dynamics was used to facilitate a statistical estimation of the optimal number of lines and genotyping density required to detect discreet genetic interactions, as well as the minimal number of generations that each RIL was to be inbred for prior to analysis. Each F1 progeny was isolated and self-propagated for 20 generations to generate independent lines with mosaic genomic patches. High-density and evenly spaced Single Nucleotide Polymorphisms (SNPs) exist between the parental strains. A subset of those are snip-SNPs, SNPs that when digested with a restriction enzyme yield distinct patterns. Using this PCR-based approach followed by RFLP (Restriction Fragment Length Polymorphism) analysis, we are able to genotype rapidly and cost-effectively. Due to the density of snip-SNPs, fine-scale mapping is also possible. In a preliminary study, we observed significant variations in lifespan and body volume after maturity of a subset of these lines. Worms were synchronized through egg-laying and the final determination of mortality was non-responsiveness to touch. The variations in aging phenotypes are attributed to the natural sequence variations. A second separate lifespan study measured lifespan in a sensitized daf-16 knocked-down background using RNAi. A number of RILs thus far have demonstrated an affect on lifespan under these conditions. We are now determining he extent to which these effects are daf-16-dependent. The same approach is applicable to other polygenic, quantitative traits as well. The same genotyping data will be useful in identifying loci important for regulating other complicated and important biological processes.

Howard H. Chen et al. (2007) International Worm Meeting "Cloning natural allelic interactions responsible for a novel roller phenotype."

Stephen R. Wicks, Christina L. Kearney, John P. Wing, Howard H. Chen, Aimee K. Wessel

[International Worm Meeting, 2007]

We have been studying the extent to which natural sequence variation, more specifically single nucleotide polymorphisms (SNPs), contributes to phenotypic variation in C. elegans. To do so, we have mated two strains isolated from widely disparate geographic regions (Bristol England and Hawaii) thus have been evolving in relative isolation. Both strains are robust and healthy, and over the course of their evolution, have diverged to the point where the strains are roughly as distinct from each other as any two humans might be from each other. Offspring from this cross were isolated and bred through many generations to form a recombinant inbred line (RIL). Each RIL is genotypically unique and was characterized to determine what parts of its genome were contributed by each of the two parental strains. These RIL genomes are best visualized as a unique patchwork mosaic of the two parental genomes. Since the allelic variants that comprise each RIL genome did not evolve together, we expect that some of these variants will interact in ways such that, while both parents were perfectly healthy and normal, some of the progeny will be either impaired or superior to either parent: a reflection of an expected increase in phenotypic variation expected in these shuffled genomes. We noted a rare and unexpected phenotype in one of the 800 or so lines that we have created in the lab. One line WX948, for the most part, produced normal progeny, but rarely (1% of the time) produced a Rol progeny. Rol mutants locomote in a distinctive and unusual way—the worms add a helical twist to the locomotion while travelling in a tight circle. To investigate the nature of the allelic interactions causing the mutant phenotype, we performed introgression of the RIL with both wild-type strains in parallel, in order to purify the background of the animal, all while still selecting for the mutant phenotype. We present the state of our understanding of how this mutant phenotype arose from the interactions of non-mutant genomes. Our strategy, combining introgression, chromosomal mapping and transgenic studies, demonstrates a general approach to characterize a novel mutant phenotype caused by a multi-allelic interaction among naturally occurring alleles. This bridges the missing link between population genetics and the molecular characterization at a single-gene level. Furthermore, what is at stake, is an understanding of the essence of diversity, the very nature of individuality and the origins of complex disease states that cannot be ascribed to mutations in single identified genes.

Howard Chang et al. (2003) International Worm Meeting "Functional analysis of AMPA-type glutamate receptor sequences and their contribution to receptor localization at synapses"

Christopher Rongo, Howard Chang

[International Worm Meeting, 2003]

The modulation of AMPA-type glutamate receptor localization to central nervous system synapses is an important component of synaptic plasticity, and can be triggered by LTP (Long Term Potentiation) and CaMKII (Type II calcium-calmodulin-dependent protein kinase) activity. (1, 2). In mammalian hippocampal neurons, the different AMPA receptor subunits GluR1, GluR2, GluR3, and GluR4 are proposed to form heteromultimers, and individual subunits can confer localization specificity to the multimeric channels that they comprise (2,3). One likely explanation for such subunit specificity is that the targeting of AMPA receptors to the synapses is probably due to the interaction of PDZ domain-containing proteins and the receptor subunit cytosolic tail sequences (4). In C. elegans, there are four AMPA receptor subunits that are expressed in the command interneurons, including, GLR-1, GLR-2, GLR-4 and GLR-5 (5,6). GLR-1 has C-terminal sequence that contributes to channel turnover (7). We have identified a glutamate receptor subunit domain that appears to be instructive for channel localization. This new finding may shed a new light on the localization and regulation of AMPA-type glutamate receptors in response to plasticity at synapses. (1) Rongo et al. Nature (1999), vol. 402, p. 195 (2) Shi et al. Science (1999), vol. 284, p.1811 (3) Shi et al. Cell (2001), vol. 105, p. 331 (4) Rongo et al. Cell (1998), vol. 94, p. 751 (5) Brockie et al. J. Neuroscience (2001), vol. 21(5), p. 1510 (6) Mellem et al. Neuron 2002 Dec 5; 36:933-944 (7) Burbea M et al. Neuron 2002 Jul 3;35(1):107-20

Robyn M Howard et al. (2003) International Worm Meeting "EOR-1/PLZF and EOR-2 are probable transcription factors downstream of Ras and Wnt signaling pathways"

Meera V Sundaram, Robyn M Howard

[International Worm Meeting, 2003]

In C. elegans, the Ras and Wnt signaling pathways cooperate to specify vulval and P12 cell fates in a Hox-dependent manner. We have shown that mutations in eor-1 or eor-2 suppress mutations that activate the Ras and Wnt pathways. Since Ras pathway mutations do not suppress activated Wnt pathway mutations and since eor-1 or eor-2 mutations cause lethal phenotypes not associated with Wnt pathway mutations, we hypothesize that eor-1 and eor-2 do not act solely downstream of the Ras pathway or the Wnt pathway. Instead, we favor a model where eor-1 and eor-2 act at a convergence of the two pathways. eor-1 encodes a nuclear-localized BTB/zinc-finger protein similar to the human transcription factor PLZF, which is altered in acute promyelocytic leukemia. eor-2 encodes a novel nuclear-localized protein that functions closely with eor-1. Because eor-1 and eor-2 act downstream of the Ras and Wnt pathways where transcription factors function and because of the molecular identity of EOR-1, it seems likely that EOR-1 and EOR-2 act at the level of transcriptional regulation. They could act as transcriptional repressors, like PLZF, or they could act as transcriptional activators. To distinguish between these two models, I have made fusions between the zinc-finger domains of EOR-1 and the Engrailed repressor domain and the VP16 activation domain and will test the ability of the fusion proteins to cause a phenotype in worms. I am also looking at expression of EGL-5 Hox and NOB-1 Hox in eor-1 mutants to determine if Hox genes could be targets of EOR-1. The results of these experiments will be presented.

Howard Chang et al. (2004) East Coast Worm Meeting "Functional analysis of AMPA-type glutamate receptor tail sequences in C. elegans."

Chris Rongo, Howard Chang

[East Coast Worm Meeting, 2004]

The modulation of AMPA-type glutamate receptor localization to central nervous system synapses is an important component of synaptic plasticity, and can be triggered and regulated by LTP (Long-Term Potentiation) and CaMKII (Type II calcium-calmodulin-dependent protein kinase) activity. In mammalian hippocampal neurons, the different AMPA receptor subunits GluR1, GluR2 and GluR3 are proposed to form heterotetramers, and individual subunits can confer localization specificity to the tetrameric channels that they comprise. One likely explanation for such subunit specificity is that the targeting of AMPA receptors to the synapses is probably due to the interaction of PDZ domain-containing proteins and the receptor subunit cytosolic tail sequences. In C. elegans , there are four AMPA/kainate-like receptor subunits that are expressed in the command interneurons, including, GLR-1, GLR-2, GLR-4 and GLR-5. GLR-1 has C-terminal sequences that contributes to channel turnover. We have identified that the GLR-2 subunit cytosolic tail domain appears to be instructive for channel localization. We will perform a yeast-two-hybrid screen using GLR-2 tail as a bait. Hopefully, we can identify the direct binding partner of AMPA-type glutamate receptors in C. elegans . These results should increase our understanding of the localization and regulation of AMPA-type glutamate receptors in vivo.<-i>

Chang, Howard et al. (2013) International Worm Meeting "Natural polymorphisms in HECW-1 E3 ubiquitin ligase affect C. elegans pathogen avoidance behavior."

Kim, Dennis, Chang, Howard

[International Worm Meeting, 2013]

C. elegans elicits aversive behavior when encountering toxic microbes. This avoidance behavior is especially advantageous to C. elegans survival when high levels of pathogens are present. Our lab uses C. elegans and P. aeruginosa as a host and pathogen model to study pathogen avoidance behavior. Previously, we showed that neuropeptide receptor NPR-1 promotes P. aeruginosa avoidance and consequently improves C. elegans resistance to P. aeruginosa. Here, we report that HECW-1, a previously uncharacterized E3 ubiquitin ligase, inhibits C. elegans pathogen avoidance and makes C. elegans more susceptible to P. aeruginosa infection. We also discovered that HECW-1 functions in sensory neurons OLLR and OLLL. Both hecw-1 deletion mutant animals and animals lacking OLL neurons have nose touch defects. This suggests C. elegans may use mechanosensory circuits to detect the presence of P. aeruginosa. Finally, we found hecw-1 acts upstream of npr-1, since animals carrying both hecw-1 and npr-1 null mutations elicit similar phenotypes as animals carrying npr-1 alone. Intriguingly, both hecw-1 and npr-1 were identified via phenotypic variances contributed by polymorphisms among C. elegans wild strains. These naturally occurring polymorphisms may provide survival benefits for C. elegans in nature.

Robyn M Howard et al. (2002) East Coast Worm Meeting "eor-1 and eor-2 act downstream of the Ras and Wnt pathways during vulval and P12 development"

Meera V Sundaram, Robyn M Howard

[East Coast Worm Meeting, 2002]

In C. elegans, a Ras/ERK signaling pathway controls multiple cell fate decisions and cooperates with a Wnt/B-catenin signaling pathway to specify vulval and P12 cell fates in a Hox-dependent manner. eor-1 and eor-2 are two new positively-acting nuclear components of the Ras and Wnt pathways. Mutations in eor-1 and eor-2 suppress gain-of-function mutations in mpk-1 ERK and loss-of-function mutations in pry-1 Axin, suggesting that eor-1 and eor-2 act at the convergence of the Ras and Wnt pathways. eor-1 and eor-2 function redundantly with the Mediator complex gene sur-2 and the functionally-related gene lin-25, such that removal of both eor-1/eor-2 and sur-2/lin-25 mimics the removal of a main Ras pathway component. Furthermore, the eor-1 and eor-2 mutant backgrounds reveal a positive role for the Elk1-related gene lin-1, and suggest that this positive role of lin-1 involves sur-2 and lin-25. eor-1 encodes the ortholog of human PLZF, a BTB/zinc finger transcription factor that is fused to RARalpha in acute promyelocytic leukemia. eor-2 encodes a novel protein. eor-1 and eor-2 appear to function closely together and cooperate with Hox genes to promote the expression of Ras- and Wnt-responsive genes.

Howard Chai-hao Chang et al. (2005) International Worm Meeting "Cytosolic tail sequences and subunit interactions are critical for synaptic localization of glutamate receptors in C. elegans."

Howard Chai-hao Chang, Christopher Rongo

[International Worm Meeting, 2005]

AMPA-type glutamate receptors mediate excitatory synaptic transmission in the nervous system. The receptor subunit composition and subcellular localization play an important role in regulating synaptic strength. GLR-1 and GLR-2 are the C. elegans subunits most closely related to the mammalian AMPA-type receptors. These subunits are expressed in overlapping sets of interneurons, and contain type-I PDZ binding motifs in their carboxy-terminal cytosolic tail sequences. We report that GLR-1 and GLR-2 may form a heteromeric complex, the localization of which depends on either GLR-1 or GLR-2 tail sequences. Subunit interactions alone can mediate synaptic localization as endogenous GLR-1 or GLR-2 subunits can rescue the localization defects of subunits lacking tail sequences. Moreover, GLR-2 cytosolic tail sequences are sufficient to confer synaptic localization on a heterologous reporter containing a single-transmembrane domain. The localization of this GLR-2 reporter requires both a PDZ-binding motif in the GLR-2 tail sequence, and sequences outside of this motif. The PDZ protein LIN-10 regulates the localization of the reporter through the sequences outside of the PDZ-binding motif. Our results suggest that multiple synaptic localization signals reside in the cytosolic tail sequence of the receptor subunits, and that channel assembly can rescue the synaptic localization defects of individual mutant subunits as long as there are also wild-type subunits in the receptor complex.

Howard Chang et al. (2002) East Coast Worm Meeting "Functional analysis of AMPA-type glutamate receptor cytosolic tail sequences and their contribution to receptor localization at synapses"

Christopher Rongo, Howard Chang

[East Coast Worm Meeting, 2002]

The modulation of AMPA-type glutamate receptor localization to central nervous system synapses is an important component of synaptic plasticity, and can be triggered by LTP (Long Term Potentiation) and CaMKII (Type II calcium-calmodulin-dependent protein kinase) activity. (1, 2). In mammalian hippocampal neurons, the different AMPA receptor subunits GluR1, GluR2, GluR3, and GluR4 are proposed to form heteromultimers, and individual subunits can confer localization specificity to the multimeric channels that they comprise (2,3). One likely explanation for such subunit specificity is that the targeting of AMPA receptors to the synapses is probably due to the interaction of PDZ domain-containing proteins and the receptor subunit cytosolic tail sequences (4). In C. elegans, there are four AMPA receptor subunits that are expressed in the command interneurons, including, glr-1, glr-2, glr-4 and glr-5 (5). We are using the molecular, genetic, and cell biological approaches to examine the delivering of AMPA receptors. We hope to identify the PDZ domain-containing proteins that interact with the tail sequences of AMPA receptors in C. elegans in order to understand the localization and regulation of these receptors in response to plasticity at synapses.

Howard H. Chen et al. (2007) International Worm Meeting "Perturbations of conserved aging processes in natural longevity mutants from a library of recombinant inbred lines (RILs)."

Howard H. Chen, Nikola Lekic, Michael J. Reardon, Stephen R. Wicks, Allison K. Sheffield, John P. Wing

[International Worm Meeting, 2007]

Single-gene mutants have elucidated genetic pathways regulating worm lifespan, and further provided both the general understanding of genetic components programming human aging as well as a tool set for the characterization of age-related disease states and their associated treatments. Variations in longevity phenotypes are expected and observed in our library of recombinant inbred lines (RILs) and are attributed to natural sequence variation, more specifically single nucleotide polymorphisms (SNPs). Here we present our investigation on how the novel longevity mutants with non-mutagenized genomes age, providing a unique perspective on the underlying mechanisms. At the previous International C. elegans Meeting, we reported the generation of 400 RILs by crossing the geographically isolated Bristol N2 and Hawaiian CB4856 strains, and inbreed each progeny for multiple generations to ensure a homozygous genome. To date, an additional set of 400 or so RILs have been generated and we genotypically characterized the entire library using evenly distributed SNP markers. As expected in shuffled genomes, the majority of RILs assayed age similarly to the parental strains. However, a small fraction of the RILs have significant variations in lifespan. Age-associated phenotypes are further characterized in these natural aging mutants, and we present here the status of using multi-factorial analysis to tease out the contributions of these seemingly distinct but related biological processes to the overall lifespan. Evolutionarily conserved genetic pathways that ensure successful aging have been well characterized in worms. Using a candidate gene approach in combination with transcriptional perturbation, we aim to determine how these pathways behave in a completely natural, yet novel genetic background. Each RIL has a unique mosaic genome with random combinations of N2 and CB4856 DNA. This provides a collection of novel genetic backgrounds that cannot be recreated by a single-gene mutation, thus is distinct from one created artificially via chemical mutagenesis. We expect unexpected changes in age-related processes, providing a unique perspective to understanding the molecular mechanisms of aging and a novel approach to studying human aging and age-associated diseases attributed to natural sequence variations among individuals.