Chiang, Wei-Chung et al. (2009) International Worm Meeting "Regulation of longevity by two novel heat-shock factor-1 regulators, ddl-1 and ddl-2."

Lee, Hee-chul, Chiang, Wei-Chung, Hsu, Ao-lin, Ching, Tsui-ting

[International Worm Meeting, 2009]

Heat-shock transcription factor-1 (HSF-1) acts as a cellular survival factor against various stresses, including but not limited to the heat stress. The activation of HSF-1 increases thermotolerance and longevity in C. elegans. However, how HSF-1 response to stress and influence longevity at molecular level remains largely unclear. Recently, two genes, ddl-1 and ddl-2 (ddl, daf-16 dependent longevity), were shown to influence lifespan and thermotolerance in C. elegans when inactivated by RNAi. To confirm this observation, we analyzed the lifespan of animals overexpressing ddl-1 and ddl-2. Consistent with previous findings, overexpression of ddl-1 or ddl-2 shortens lifespan. It has been previously suggested by yeast-two hybrid results that DDL-1 may physically interacts with DDL-2 and heat-shock factor binding protein-1 (HSB-1), a known negative regulator of HSF-1. Therefore we asked whether ddl-1 and ddl-2 regulate longevity by modulating hsf-1 activity. Our result showed that ddl-1 and ddl-2 RNAi fails to increase lifespan in hsf-1 mutant (sy441), suggesting that hsf-1 functions downstream of ddl-1 and ddl-2. Our biochemical evidences also showed that knock-down of ddl-1 and ddl-2 by RNAi increases HSF-1 activity. Taken together, our data suggest that DDL-1 and DDL-2 might influence longevity by regulating HSF-1 activity. We hypothesize that DDL-1and DDL-2 regulate HSF-1 activity by forming a complex with HSF-1, and this complex is important for keeping HSF-1 in an inactive form. Our preliminary results have confirmed the interaction between DDL-1 and HSF-1; DDL-1 and HSB-1; DDL-1 and DDL-2 by co-immunoprecipitation. By understanding the role of DDL-1 and DDL-2 in regulating HSF-1, we will be able to reveal the underlying mechanism by which HSF-1 regulates heat-shock response and promotes longevity in C. elegans.

Chiang, Wei-Chung et al. (2011) International Worm Meeting "C. elegans SIRT6/7 homolog SIR-2.4 as a novel regulator of DAF-16 localization and function."

Eckersdorff, Mark, Yang, Bo, Hsu, Ao-lin, Chiang, Wei-Chung, Wang, Angela R., Lombard, David B., Tishkoff, Daniel X.

[International Worm Meeting, 2011]

FoxO transcription factors and sirtuin family deacetylases/ADP-ribosyl-transferase regulate diverse biological processes, including stress responses, metabolism and longevity. Here we show that the Caenorhabditis elegans sirtuin SIR-2.4 - homolog of the mammalian SIRT6 and SIRT7 proteins - is required for responses to multiple stress: heat shock, oxidative insult, and proteotoxicity in C. elegans. It is known that multiple stresses promote nuclear translocation and activation of FoxO transcription factor DAF-16, which directs the transcriptional program controlling metabolism, longevity and stress resistance. We found that SIR-2.4 is required for stress-induced DAF-16 nuclear localization and DAF-16 dependent gene expression, indicating that SIR-2.4 may play an important role in modulating DAF-16 function in response to stress. We investigated the mechanism in which SIR-2.4 may regulates DAF-16 activity. We found that SIR-2.4 promotes deacetylation of the DAF-16. Surprisingly, the deacetylase/ADP-ribosyltransferase catalytic activity of SIR-2.4 is not required for DAF-16 deacetylation and nuclear localization, suggesting that SIR-2.4 modulates DAF-16 activity via a mechanism involving other unknown deacetylase or acetyltransferase. These findings reveal a novel role of SIR-2.4 in modulating DAF-16 function and demonstrate that SIR-2.4 is a critical mediator of multiple stress responses through a DAF-16-dependent pathway.

Chiang, Wei-Chung et al. (2013) International Worm Meeting "C. elegans SIRT6/7 homolog SIR-2.4 promotes stress response and longevity via distinct mechanisms."

Hsu, Ao-lin, Tishkoff, Daniel X., Yang, Bo, Ching, Tsui-ting, Yu, Xiaokun, Chiang, Wei-Chung, Lombard, David B.

[International Worm Meeting, 2013]

Sirtuins comprise a unique class of NAD+-dependent enzymes that modify multiple protein substrates to execute diverse biological functions. The sirtuin family is evolutionarily conserved from bacteria to eukaryotes and has been shown to regulate a broad range of processes, including transcription, metabolism, cancer and neuron-degeneration. Here we report that Caenorhabditis elegans sirtuin SIR-2.4, a homolog of mammalian SIRT6 and SIRT7 proteins, promotes longevity under dietary restriction, and also promotes re-localization of DAF-16 under various stress conditions, including heat shock, oxidative insult, and proteotoxity. Upon stress, FoxO transcription factor DAF-16 moves to the nucleus to regulate gene expression to ensure organismal survival. We found that SIR-2.4 negatively modulates DAF-16 acetylation, which is associated with the nuclear localization of DAF-16. However, this modulation does not depend on the catalytic activity of SIR-2.4, suggesting the potential involvement of other deacetylase or acetyltransferase. And we confirmed that SIR-2.4 functions antagonistically with acetyltransferase CBP-1 to modulate DAF-16 acetylation. By contrast, SIR-2.4 is largely dispensable for DAF-16 nuclear activation in response to reduced insulin/IGF-1-like signaling, which is known to influence longevity in worms. Alterations in the expression levels of SIR-2.4 do not appear to influence longevity in the wild type background. Interestingly, we found that SIR-2.4 is required for the longer lifespan of dietary restriction, suggesting that SIR-2.4 may regulate stress response and longevity via distinct mechanisms.

Liang, Chung-Yi et al. (2015) International Worm Meeting "Functional regulation of the DAF-16/FoxO transcription factor by acetylation in response to stress."

Chiang, Wei-Chung, Yu, Xiaokun, Hsu, Ao-lin, Lai, Yi-Chun, Liang, Chung-Yi, Lombard, David B.

[International Worm Meeting, 2015]

All living organisms face a challenge to sense and respond appropriately to the environmental cues. In C. elegans, the FoxO transcription factor DAF-16 translocates into nucleus upon stress to regulate gene expression for organismal survival. The subcellular localization and activity of DAF-16 are tightly controlled by its post-translational modifications, including phosphorylation, acetylation, ubiquitination, methylation and glycosylation. In our previous study, we found that sirtuin family protein SIR-2.4, a homolog of mammalian SIRT6 and SIRT7, promotes DAF-16 nuclear translocation and DAF-16-dependent transcription under stress conditions. SIR-2.4 acts antagonistically with the acetyltransferase CBP-1 to negatively regulate DAF-16 acetylation. However, this modulation does not require the catalytic activity of SIR-2.4. We further identified four CBP-1-mediated acetylation sites on DAF-16 by mass spectrometry analysis. Mutations on some of these acetylation sites alter the kinetics of DAF-16 translocation upon stress, indicating a new nuclear localization signal of DAF-16. Together, our studies identify acetylations modulated by SIR-2.4 play a critical role in regulating DAF-16 functions in response to stress.

Crittenden, Sarah L. et al. (2013) International Worm Meeting "Progress on developing the Q system to study GSCs and their control."

Crittenden, Sarah L., Kimble, Judith, Mohanty, Ipsita

[International Worm Meeting, 2013]

The Q system (Potter et al., 2010) permits inducible gene expression in C. elegans (Wei et al., 2012) and is similar in principal to the widely used GAL4-UAS system. The QF transcriptional activator directs transcription via an upstream activating sequence (QUAS). But in addition, another protein, QS, can inhibit QF activation and QS repression can be relieved by a small molecule, quinic acid or QA. QA is non-toxic and can be fed to worms (Wei et al., 2012). This inducible system has the potential to control target genes in both space and time. A chemically inducible method to control gene expression in specific cells at specific times would be tremendously useful for analyses of germline stem cells (GSCs) and their niche, the somatic distal tip cell (DTC). To this end, we are generating mosSCI insertion transgenes that rely on DTC-specific and GSC-specific regulatory sequences to drive QF expression. We are also generating a QUAS driven nuclear GFP (fused to H2B). Once we have the QF/QUAS pair working well for spatial regulation, we will add the QS/QA pair for temporal regulation. Preliminary experiments are promising and results will be shared at the meeting.

Driesschaert, Brecht et al. (2022) MicroPubl Biol

Driesschaert, Brecht, Temmerman, Liesbet

[MicroPubl Biol, 2022]

The Q system is a genetic tool developed to deliver spatiotemporal control over gene expression (Giles et al. 1991; Potter et al. 2010; Wei et al. 2012). Although it has already been adapted for use in C. elegans by Wei et al. in 2012, to date, the Q system has not been applied extensively in this nematode. In the relatively few available reports, it is mainly used to constitutively restrict gene expression in a spatial manner (e.g. Schild et al. 2014; Schild and Glauser 2015; Jee et al. 2016; Tolstenkov et al. 2018; Chiyoda et al. 2021), while but a handful of studies also explore the temporal aspect of the system (Matus et al. 2015; Yuan et al. 2016; Cottee et al. 2017; Hoang and Miller 2017). We aimed to apply this tool in the C. elegans nervous system to gain both spatial and temporal control over expression of a gene encoding a reporter protein that is targeted to the secretory pathway. Despite our efforts, we here report that in our hands, the Q system is not suitable for application in the neurons due to a lack of dynamic range.

Clovis Y et al. (2016) MicroPubl Biol "Mutations in KCNQ potassium channels cause pharyngeal pumping defects in C. elegans."

Roberts B, Webb A, Clovis Y, Turner C

[MicroPubl Biol, 2016]

Pumps were stimulated with 10mM 5HT in M9 recorded as electropharyngeograms (EPGs) for 2 minutes in a NemaMetrix ScreenChip, and analyzed using NemAnalysis software (NemaMetrix). The null mutant strains kqt-1(aw3) and kqt-3 (aw1) were kindly donated by Dr. Aguan Wei (Wei et al., 2002) A) Pump frequency in kqt-1(aw3) animals was significantly lower than in N2s, while kqt-3(aw1) worms showed an increase in pump frequency (*p<0.05; ***p<0.01; 1-tailed Mann-Whitney U-test; n = 21-23 worms in each strain). B) Microfluidic EPG recordings show that pumping pattern in kqt-1(aw3) mutants is arrhythmic, with frequent drops in frequency. C) Overlay of first 50 pumps of recordings show that pump duration is higher in kqt-1(aw3) and kqt-3(aw1) animals than in N2s. Pumps are showed aligned on E spikes, which occur when the pharynx is fully contracted. D) Duration histogram illustrating the probability of occurrence of inter-pump interval (E to E duration) for each mutant strain. Histograms were binned to 4 ms width and normalized to reach an area underneath the curve equal to 1 (duration 100% likelihood to occur). In kqt-1(aw3)) animals, the time between two pumps is significantly increased compared to N2s (p < 0.01).

Huang CG et al. (2006) Mol Biotechnol "Isolation of C. elegans deletion mutants following ENU mutagenesis and thermostable restriction enzyme PCR screening."

Lamitina T, Agre P, Strange K, Huang CG

[Mol Biotechnol, 2006]

The ability to generate null mutants is essential for studying gene function. Gene knockouts in Caenorhabditis elegans can be generated in a high throughput manner using chemical mutagenesis followed by polymerase chain reaction (PCR) assays to detect deletions in a gene of interest. However, current methods for identifying deletions are time and labor intensive and are unable to efficiently detect small deletions. In this study, we expanded the method pioneered by Wei et al., which used the thermostable restriction enzyme PspGI and tested the usefulness of other thermostable restriction enzymes including BstUI, Tsp45I, ApeKI, and TfiI. We designed primers to flank one or multiple thermostable restriction enzymes sites in the genes of interest. The use of multiple enzymes and the optimization of PCR primer design enabled us to isolate deletion in 66.7% of the genes screened. The size of the deletions varied from 330 bp to 1 kb. This method should make it possible for small academic laboratories to rapidly isolate deletions in their genes of interest.

Ahmed, Rina et al. (2011) International Worm Meeting "Multi-platform sequencing identifies novel mirtron with an embryonic lethal phenotype in Caenorhabditis elegans."

Dieterich, Christoph, Ahmed, Rina, Sar, Funda, Gunsalus, Kristin, Miska, Erik, Chang, Zisong

[International Worm Meeting, 2011]

MicroRNA genes (miRNAs) are post-transcriptional regulators of mRNA stability and/or translation and are regulated themselves on the level of gene transcription, processing and decay. MiRNAs regulate many biological processes and aberrant miRNA expression has been implicated in several disease states. Mirtrons are a special class of miRNAs since they originate from properly sized introns (~ 70 nt) of protein-coding genes. Precursor miRNAs (pre-miRNAs) of mirtrons are excised by the splicing machinery from the host gene, debranched, and directly processed by Dicer. The expression pattern of a mirtron is consequently similar if not identical to its host gene's expression pattern. Only a few mirtrons have been identified in vertebrates and invertebrates since their discovery in 2007. None of them has been linked to any phenotype so far. We revisited the repertoire of miRNAs in Caenorhabditis elegans with a multi-platform sequencing approach (ABI SOLiD and Illumina GA II) to screen for novel miRNA gene candidates. Both platforms differ in sequencing bias, which is usually expressed in divergent normalized cross-platform read counts for any given miRNA. Consequently, both platforms complement one another in the gene discovery process. With this approach, we were able to extend the known set from four to six mirtrons. The modENCODE consortium (Chung et al., 2011) has independently confirmed our discovery. However, one novel mirtron caught our attention and we started a functional characterization of this candidate gene. At the time of writing, we are certain that a knockout of the host gene has an embryonic lethal phenotype and shows greatly reduced levels of hatching worms. The knockout phenotype can be, at least partially, rescued by a mirtron-expressing transgene. Most surprisingly, this mirtron has been acquired recently and is not present in any of the other available Caenorhabditis genomes. We will give an update of our experimental findings at the International Worm Meeting. References: Chung, W.J. et al. Computational and experimental identification of mirtrons in Drosophila melanogaster and Caenorhabditis elegans. Genome Res. 21, 286-300 (2011).

Stroeher VL et al. (1991) International C. elegans Meeting "POSSIBLE LINEAGE-SPECIFIC ACTIVATORS OF THE C. elegans ges-1 GENE."

Stroeher VL, McGhee JD

[International C. elegans Meeting, 1991]

The ges-1 gene of Caenorhabditis elegans encodes a nonspecific carboxylesterase that is expressed exclusively in the intestinal lineage. Deletion and transformation analyses show that this restricted expression is due to lineage specific activators and repressors that bind to upstream promoter sequences (see abstract by E. J. Aamodt, M.A. Chung and J.D.M.). As a first step to clone these regulatory factors, we are identifying their binding sites using gel retardation assays (bandshifts) and DNasel footprinting experiments. At least seven regions of protein interaction have been found in the sequences 935 to 1309 basepairs (bp) upstream of the translation start site. One of these sites, which may bind a putative 'gut activator' molecule is being examined more closely. A doublestranded, 30 bp oligomer (30mer) representing this region has been synthesized. In gel retardation assays with crude nuclear extract from FUdR-blocked embryos, this 30mer gives a single major shifted species, which is effectively competed with unlabelled double-strand 30mer. DNA-protein binding reactions have been exposed to UV irradiation to crosslink the bound protein(s) to the labelled, doublestrand 30mer; a major 80 kd protein is detected. We are now investigating when this protein appears in development.