van der Linden, Alexander et al. (2017) International Worm Meeting "Global accumulation of circRNAs during aging in C. elegans."

Bauer, Matthew, Gruner, Matthew, Voda, Alexandru-Ioan, Miura, Pedro, Gruner, Hannah, Cortes-Lopez, Mariela, van der Linden, Alexander, Cooper, Daphne

[International Worm Meeting, 2017]

Circular RNAs (circRNAs) have recently been identified as a natural occurring family of wide-spread and diverse endogenous non-coding RNAs (reviewed in Cortes-Lopez and Miura, Yale J. Biol, 2016). They are remarkably stable RNA molecules mostly generated by backsplicing events from known protein-coding genes, and are abundantly expressed in many animals, from C. elegans (Ivanov et al, Cell Rep, 2015; Memczak et al, Nature 2013), to humans. Recent reports suggest functional roles for circRNAs, such as regulating transcription, binding proteins, and as microRNA sponges. CircRNAs accumulate during aging on a genome-wide level in Drosophila and mice (Westholm et al, Cell Rep, 2014; Gruner H. et al, Sci Rep, 2016), but their potential role and function in the aging process is not known. We profiled for the first time genome-wide circRNA changes during aging in C. elegans. Using total RNA-seq of whole C. elegans from L4-staged larvae, Day-1, Day-7 and Day-10 adults, we identified 1166 circRNAs including 575 novel annotations. Individual circRNAs were validated by Northern blot, qRT-PCR, and Sanger sequencing. We identified a massive and progressive accumulation of circRNAs during aging, which is independent of linear RNA changes from shared host genes. More specifically, 290 circRNAs were significantly upregulated in Day-10 compared to Day-1 adults, whereas only 6 were downregulated. This includes a >20-fold enrichment in a circRNA derived from the gene for the CREB homolog crh-1, as verified by qRT-PCR. Other genes in longevity-associated pathways produce age-accumulated circRNAs, including daf-16 and daf-2. Unlike in other species, the age-accumulation trend appears not to be confined to neuronally expressed genes. Given the progressive accumulation of circRNAs and their resistance to decay, we propose that stability in post-mitotic tissues is the main driver of the age-upregulation trend. We are currently examining whether circRNA accumulation patterns change in mutants with altered lifespan phenotypes. Taken together, our characterization of age-accumulated circRNAs lays the foundation for future investigations into the functions of circRNAs and how they might play a role in the aging process.

van der Linden, Alexander M. et al. (2009) International Worm Meeting "Temperature and light-entrained circadian rhythms in C. elegans."

Sengupta, Piali, van der Linden, Alexander M., Kadener, Sebastian, Rosbash, Michael

[International Worm Meeting, 2009]

Circadian control of feeding is critical for animals to properly adapt to their environment, and for optimal development. However, the nature of this interaction and its relationship to metabolism is poorly understood. C. elegans has proved to be an extremely powerful model for understanding the molecular basis of feeding behavior, but it is unknown whether C. elegans feeding is under circadian control. Although circadian locomotory rhythms were described in C. elegans previously (1), these observations have been difficult to replicate, and no rhythmically expressed clock or clock-output genes in C. elegans have been identified. We set out to not only investigate the yet unidentified circadian clock in C. elegans, but to also explore the interaction between circadian clocks, metabolism and feeding behaviors. Since circadian transcripts exhibit rhythmic oscillations, genome-wide expression profiling experiments are a powerful approach to identify clock and clock-output genes. We used microarrays and a series of data analysis algorithms to identify circadian-regulated genes under photo- and thermocycling conditions in C. elegans. We identified subsets of candidate clock-output genes that exhibit robust rhythmic expression with 24 hr periodicity under either light-dark or warm-cold conditions, as well as in constant dark-dark or cold-cold conditions following entrainment. Our results indicate that thermocycles directly evoke a global expression response broader than photocycles, and that a subset of genes undergo cycling in a circadian manner. Verification of microarray data via qRT-PCR suggests that these genes may represent bona fide cycling genes. These sets include molecules predicted to regulate metabolic processes, suggesting that feeding - an activity closely related to energy metabolism - may be under circadian control. Preliminary results suggest that tax-2, which is important for thermo- and phototaxis (2) is necessary for the cycling of these genes. We have also developed a reporter assay system to monitor rhythmic circadian gene expression, and we are planning to use this system in genetic screens to define the core clock components. Moreover, we are determining whether feeding behavior is under circadian control, and whether food signals can entrain the clock. Since it is likely that the molecular components of the C. elegans clock are distinct from those of other organisms, this work is also expected to provide information regarding the evolution of circadian clocks. 1) Saigusa et al, 2002; Simonetta & Golombek, 2007; 2) Coburn et al, 1996; Ward et al, 2008.

van der Linden, Alexander M. et al. (2009) International Worm Meeting "Identification of targets of KIN-29 SIK signaling in the regulation of food-related behaviors and development."

Sengupta, Piali, van der Linden, Alexander M.

[International Worm Meeting, 2009]

The availability and quality of food fluctuates over time in an animal''s environment. When food is scarce, an animal must undergo changes in its behavior, metabolism and physiology to promote survival. An animal must also regulate its feeding habits depending on nutritional status in order to maintain physiological homeostasis. Salt-inducing kinases (SIKs) are important regulators of energy homeostasis, and feeding and fasting behaviors in mammals (1). We previously found that KIN-29, the C. elegans homolog of SIK, regulates the expression of a subset of chemoreceptor (CR) genes in response to changes in external and internal conditions, and that this regulation likely underlies the ability of animals to correctly transduce food-derived signals to modulate behaviors such as quiescence and foraging as well as body-size (2).To further dissect the KIN-29 signaling pathways by which food signals are integrated to modulate behavior and development, we are taking three parallel strategies to define new targets of KIN-29. In the first, we are determining the gene expression profiles of specific neurons in wild-type and kin-29 mutants using microarrays coupled with a mRNA-tagging strategy. Preliminary results indicate that a large subset of genes predicted to function in transcriptional regulation and phosphorylation events are up- and down-regulated, respectively, in wild-type and kin-29 mutants in chemosensory neurons. Secondly, we are examining a KIN-29-dependent CR gene whose expression in the ADL chemosensory neurons is acutely altered by the presence and absence of food in adults. We will take advantage of these observations to identify the components and neurons by which food signals alter CR gene expression using genetic screens. Finally, we are examining the function of Y20F4.2, which encodes the single C. elegans homolog of TORC/CRTC. TORC is phosphorylated by SIK members, and is an important regulator of metabolism in mammals and flies (1). Y20F4.2 is widely expressed, including in most neurons. As in mammals and flies, we found that subcellular localization of Y20F4.2 is regulated by phosphorylation and oxidative stress. Interestingly, expression of the daf-7 TGF-b gene is reduced in Y20F4.2 mutants, indicating that Y20F4.2 regulates daf-7 TGFb expression, and therefore may control metabolism and feeding behavior (3). Together these experiments will provide a detailed understanding of the remarkably complex effects of food on animal development and behavior. 1) Hietakangas, 2008; 2) Lanjuin and Sengupta, 2002; Van der Linden et al. 2007, 2008; 3) Greer et al. 2008.

Alexander Van Der Linden et al. (2008) C.elegans Neuronal Development Meeting "Temperature- and Light-entrained Circadian Rhythms in C. elegans"

Sebastian Kadener, Michael Rosbash, Alexander Van Der Linden, Piali Sengupta

[C.elegans Neuronal Development Meeting, 2008]

The circadian clock is an internal timing mechanism that enables organisms to respond to, and even anticipate, changing environmental conditions associated with rotation of the earth. The output of these clocks results in circadian rhythms, which oscillate with circa 24-hr periods and control multiple aspects of behavior and physiology. Circadian rhythms can be entrained by daily environmental signals e.g. light/dark or temperature. Although circadian behaviors in C. elegans have been reported (1), no clock or clock-output genes have been identified. Homologs of animal clock genes are represented in the genome of C. elegans, but their only certain function is to regulate developmental timing. Thus, the nature, and existence of a C. elegans circadian clock remains uncertain. Since circadian transcripts exhibit rhythmic oscillations, genome-wide expression profiling is a powerful approach to identify clock and clock-output genes. We used microarrays to identify these cycling genes using temperature or light/dark as zeitgebers. Growth-synchronized populations of C. elegans were subjected to either 12:12 hr T-cycles (25:15degC) in constant darkness, or 12:12 hr photocycles at constant temperature. In order to identify 24-hr periodic gene expression, we determined the 24-hr spectral power and the probability of observing an equivalent or higher score from randomly permutated data. First, we find that in a 5-d dataset of temperature entrainment, T-cycles show a broad impact on global 24-hr periodicity (1900 transcripts, p0.02). This result can be explained by either a temperature-driven clock-independent effect or by a temperature-entrained clock-dependent effect. To distinguish between these two possibilities, we analyzed a 6-d data set combining 3-d of temperature entrainment and 3-d of subsequent constant free-running conditions, and find at least 148 temperature-entrained circadian transcripts (p0.02). Second, we identified a significant set of candidate light-entrained transcripts. Expression data from qRT-PCR suggests that these genes may represent bona fide cycling genes. Our results indicate that T-cycles directly evoke a global expression response broader than photocycles, and that a subset undergo cycling in a circadian manner. Since it is likely that the molecular components of the C. elegans clock are distinct from those of other organisms, C. elegans provides a new system for understanding circadian clocks as well as the evolution of circadian mechanisms.

Alexander van der Linden et al. (2003) International Worm Meeting "Genome-wide RNAi of C. elegans using the hypersensitive strain rrf-3."

Andrew Fraser, Celine Moorman, Ronald Plasterk, Ewart Kuijk, Ravi Kamath, Peter van der Berghe, Femke Simmer, Julie Ahringer, Alexander Van Der Linden

[International Worm Meeting, 2003]

Recently, an RNAi library was constructed that consists of bacterial clones expressing dsRNA corresponding to nearly 90% of the 19,427 predicted genes of C. elegans (1). Feeding of this RNAi library to N2 detected phenotypes for about 10% of the corresponding genes. In an attempt to increase the number of genes with a loss-of-function phenotype, we undertook a genome-wide RNAi screen using the rrf-3 strain. rrf-3 mutants are hypersensitive to RNAi; they show new and more severe RNAi phenotypes compared to those obtained with N2 (2).In a preliminary experiment, we fed the clones corresponding to chromosome I and systematically compared our phenotypic data with the data previously obtained using N2 (1). These data indicated that we could increase the amount of clones with a phenotype using rrf-3, but also that we miss phenotypes. To determine whether the differences were due to rrf-3 or other variations, we next screened chromosome I with N2 and rrf-3 in parallel. Using rrf-3, we found more clones with a phenotype compared to the data with N2 from this and previous studies (1). These data confirm that rrf-3 is more sensitive to RNAi. Furthermore, in comparing the results of the different data sets, we observed that the RNAi effect is in general variable and in particular that the frequency of false negatives can be high.The complete RNAi library was tested on rrf-3 mutant animals. In total, we obtained results for 16,401 clones and detected RNAi phenotypes for 2077 clones (12.7%). These include 623 clones for which previously no phenotype was found, increasing the number of clones with a phenotype by 36%. In addition, there are 286 clones for which only a phenotype was found using N2 (1). The 623 extra clones we found were broadly distributed over different phenotypic classes, suggesting that rrf-3 is in general more sensitive to RNAi. Taken together, we significantly increased the number of genes with a phenotype using rrf-3 and confirmed many RNAi phenotypes previously found. Finally, we are using the RNAi data to systematically clone unidentified genetic mutants, and we will give an update of the results. References: 1) Kamath et al. (2003). Nature 421, 231-237. 2) Simmer et al. (2002). Curr. Biol. 12, 1317-1319.

Nikooei, Tatiana et al. MicroPubl Biol

Nikooei, Tatiana, M. van der Linden, Alexander, McDonagh, Aja

[MicroPubl Biol, 2020]

kin-29 encodes the C. elegans homolog of mammalian Salt-Inducible Kinases (SIKs). kin-29 mutants are small, have increased propensity to develop into non-reproductive dauer larvae, have reduced chemoreceptor gene expression (Lanjuin and Sengupta, 2002; van der Linden et al.., 2007), have reduced cellular ATP despite increased fat stores, and show reduced sleep (Grubbs et al., 2020). KIN-29 phosphorylates and inhibits the class II histone deacetylase 4 homolog HDA-4 to regulate gene expression in sensory neurons (van der Linden et al., 2007). The longevity phenotype of kin-29 mutants is suppressed by mutations in daf-16 (Lanjuin and Sengupta, 2002), which encodes a forkhead box protein O (FOXO) transcription factor (Kenyon et al., 1993). These results indicated that the increased lifespan of kin-29 mutants may be due to reduced insulin signaling. To determine whether HDA-4 is required for the KIN-29 regulation of lifespan, we performed a survival analysis. As previously reported (Lanjuin and Sengupta, 2002), kin-29(oy38) mutants are long-lived. In contrast, hda-4(oy57) mutants are short lived (Fig. 1). The kin-29hda-4 double mutant longevity phenotype was similar to the phenotype of hda-4 single mutants (Fig. 1), suggesting that hda-4 acts downstream of kin-29 to regulate lifespan. Together with our previous findings (van der Linden et al., 2007), these genetic results suggest that KIN-29 may regulate lifespan via the action of HDA-4. Since SIKs can inhibit FOXO activity via HDAC4 to regulate gene expression (Mihaylova et al., 2011; Wang et al., 2011), it would be interesting in future studies to test the model that KIN-29/HDA-4 signaling converges on DAF-16/FOXO to modulate gene expression associated with longevity.

Alexander M van der Linden et al. (2002) European Worm Meeting "Novel downstream targets of G-protein -subunits in C. elegans"

Edwin Cuppen, Celine Moorman, Ronald H A Plasterk, Alexander M van der Linden

[European Worm Meeting, 2002]

Heterotrimeric G-proteins transduce signals from various serpentine transmembrane receptors to intracellular effectors. Several G-proteins have been identified in C. elegans: 21 G -subunits, including one homologue of each of the four mammalian classes Gs, Gi/o, Gq and G12, two G-subunits and at least two G -subunits. To identify novel downstream targets of G-protein signaling pathways, we are following two different approaches: second-site suppressors screens of G-protein mutants and two-hybrid interaction screens. Animals that lack both gpb-2 (G 5) and goa-1 (Go) function, arrest during larval development, whereas single gpb-2 and goa-1 mutants are viable. This synthetic larval lethal phenotype is rescued when EGL-30 (Gq ) activity is reduced. While this result suggests that EGL-30 activity causes the gpb-2 goa-1 double mutant synthetic lethality, no suppression is observed when EGL- 8 (a potential downstream effector of EGL-30) activity is reduced. Thus, we expected to find as yet unidentified mediators of EGL-30 signaling in a screen for suppressors of the synthetic lethal phenotype of gpb-2 goa-1 double mutants. So far, we have isolated 47 suppressors of this synthetic lethality, and found that most suppressors are alleles of egl-30. At present, we are mapping the remaining suppressors that are likely to disrupt novel downstream effectors of EGL- 30. Secondly, we screened for components in G12-mediated signaling. Overexpression of activated GPA-12 (G12 /G13) results in larvae that arrest their growth at an early stage. We found that mutations in tpa-1, encoding two isoforms (TPA-1A and TPA-1B) of a protein kinase C

Alexander M van der Linden et al. (2005) International Worm Meeting "Regulation of chemoreceptor gene expression by MEF-2 and class II HDACs in C. elegans"

Katie Nolan, Alexander M van der Linden, Piali Sengupta

[International Worm Meeting, 2005]

C. elegans is able to modulate the expression of chemoreceptors in response to neuronal activity and environmental cues. This provides a simple mechanism by which C. elegans can rapidly alter its sensory behaviors in response to changing conditions. How is the expression of chemoreceptor genes regulated? Previously, we showed that the Ser/Thr-kinase KIN-29 regulates the efficient expression of a subset of chemoreceptors. kin-29 mutants exhibit reduced expression of str-1 and sra-6 in the AWB olfactory and ASH sensory neurons, respectively. In addition, kin-29 mutants exhibit a reduced body-size and are hypersensitive to dauer pheromone, indicative of alterations in the ability to correctly sense environmental conditions. All phenotypes can be rescued by kin-29 expression in chemosensory neurons, suggesting that modulation of receptor expression may be crucial for regulating body-size and pheromone responses. However, the mechanisms through which KIN-29 functions to regulate chemoreceptor gene expression are unknown. In order to identify targets of KIN-29, we identified suppressor mutations in the MADS-box transcription factor mef-2 and the class II histone deacetylase hda-4. MEF2 and class II HDACs have previously been shown to regulate gene expression in response to intracellular signaling and electrical activity in neurons. Cell-specific expression indicates that mef-2 and hda-4 function in chemosensory neurons to regulate the KIN-29-mediated phenotypes. To further investigate MEF-2/HDA-4 function, we dissected the cis-regulatory sequences driving str-1 expression. We compared promoter sequences of str-1 and its ortholog in C. briggsae and carried out promoter deletion analyses. We found that sequences required for developmentally regulated str-1 expression in AWB are distinct from sequences required for KIN-29-mediated modulation of expression levels. Within the sequences required for conferring KIN-29-mediated regulation, we identified a putative MEF-2 binding site. Interestingly, loss of either this MEF-2 site but also a conserved GATA factor binding site resulted in alteration of str-1 expression, suggesting that KIN-29 may function through a MEF-2/GATA/HDA-4 complex to modulate expression. We have identified these sites upstream of a subset of chemoreceptor genes and are determining whether they are also regulated by KIN-29. Finally, preliminary results suggest that neuronal activity may play a role in regulating the KIN-29-mediated modulation of str-1 expression. Taken together, our findings suggest a role for chromatin remodeling in the regulation of chemoreceptor gene expression, and reveal an unexpected complexity in the mechanisms required for correct receptor expression levels.

Alexander M van der Linden et al. (2004) East Coast Worm Meeting "Regulation of chemoreceptor gene expression by MEF-2 and class II HDACs in C. elegans"

Piali Sengupta, Katie Nolan, Alexander M van der Linden

[East Coast Worm Meeting, 2004]

C. elegans is able to regulate the expression of chemoreceptors in response to environmental cues. This provides a simple mechanism by which C. elegans can rapidly respond to changing environmental conditions. How is the expression of chemoreceptor genes regulated, both during development and in response to environmental signals? Previously, we identified a role for the Ser/Thr-kinase KIN-29 in regulating the efficient expression of a subset of candidate olfactory receptors in the chemosensory system [1]. kin-29 mutants exhibit reduced expression of the str-1 receptor in the AWB olfactory neurons and the sra-6 receptor in the ASH sensory neurons. In addition, kin-29 mutants exhibit a reduced body-size and are hypersensitive to dauer pheromone, indicative of alterations in the ability to correctly sense environmental conditions. All phenotypes can be rescued by kin-29 expression in chemosensory neurons, suggesting that modulation of receptor expression may be crucial for regulating body-size and pheromone responses. However, the mechanisms through which KIN-29 functions to regulate gene expression are unknown. In order to identify targets of KIN-29, we identified suppressor mutations in the MADS box transcription factor mef-2 (MEF2) and the class II histone deacetylase hda-4 . All phenotypes of kin-29 mutants are suppressed by mutations in mef-2 and hda-4 . MEF2 and class II HDACs have previously been shown to regulate gene expression in response to intracellular signaling and electrical activity in muscles and neurons. Cell-specific expression indicates that mef-2 functions in the nervous system to regulate both str-1::gfp expression and body-size. Promoter deletion experiments suggest that sequences required for developmentally regulated str-1 expression in the AWB neurons are distinct from sequences required for KIN-29-regulated modulation of expression levels. There are several potential MEF2 binding sequences within the str-1 proximal promoter, indicating that KIN-29 may function via MEF-2 to modify the direct transcription of str-1 . All together, our findings suggest an intriguing model for a remodeling of chromatin structure in regulating chemoreceptor gene expression in response to environmental signals in C. elegans . [1] Lanjuin & Sengupta (2002), Regulation of chemosensory receptor expression and sensory signaling by the KIN-29 Ser/Thr kinase, Neuron, Vol. 33, 369-381.

Alexander M van der Linden et al. (2001) International C. elegans Meeting "Novel downstream targets of GOA-1, EGL-30, GPA-12 and the C. elegans specific G-proteins"

Ronald H A Plasterk, Edwin Cuppen, Femke Simmer, Celine Moorman, Alexander M van der Linden

[International C. elegans Meeting, 2001]

In a recent study, we have shown a functional role for the second G-protein b subunit, GPB-2, in GOA-1 (mammalian homologue of Go a ) and EGL-30 (mammalian homologue of Gq a ) signaling. GPB-2 is most related to the divergent mammalian G b 5 subunit that, unlike the other mammalian G b subunits (G b 1-4), has the ability to interact with the G g subunit-like (GGL) domain of a subset of RGS proteins . Using both genetic and yeast two-hybrid analysis, we found that GPB-2 interacts with the regulator of G-protein signaling (RGS) proteins EAT-16 and EGL-10 in C. elegans , and that GPB-2 regulates the functions of these proteins in the Go a / Gq a signaling network (1). Two interesting outcomes of this study were that: first, animals lacking both gpb-2 and goa-1 function have a synthetic larval lethal phenotype, which is rescued when EGL-30 activity is reduced. While this result suggests that Gq a activity causes the gpb-2 goa-1 double mutant synthetic lethality, no suppression is observed when EGL-8 (a potential downstream effector of EGL-30) activity is reduced. Thus, we expected to find as yet unidentified mediators of EGL-30 signaling in a screen for suppressors of the synthetic lethal phenotype of gpb-2 goa-1 double mutants. To date, we have isolated 28 suppressors of this synthetic lethality, and found that 4 of these are alleles of egl-30 . Thus, some of the remaining are likely to disrupt novel downstream effectors of EGL-30. Secondly, we have shown that members of a G-protein signaling pathway can be identified using yeast two-hybrid analysis. Thus, we have performed two-hybrid interaction screens using all 20 C. elegans G a subunits. We have identified 11 different proteins that interact with 4 different G a subunits. Some of these proteins suggest novel signaling pathways downstream of G-proteins. Currently, we are investigating the biological significance of these interactions. Furthermore, we have screened for novel downstream targets of GPA-12 (mammalian homologue of G12). Overexpression of dominant active GPA-12 under control of a heat-shock promoter results in larvae that arrest at the L1 stage but eventually recover. We have found 42 mutations that suppress this phenotype, and are in the progress of characterizing these suppressors. Thus, we will present novel targets of several different G-proteins identified using diverse techniques. Reference: Van der Linden AM, Simmer F, Cuppen E and Plasterk RHA. (2001) The G protein b subunit GPB-2 in Caenorhabditis elegans regulates the Go a and Gq a signaling network through interactions with the regulator of G-protein signaling proteins EGL-10 and EAT-16. Genetics (in press)