Michael A Beer (2005) International Worm Meeting "Systematic genome-wide identification of transcriptional cis-regulatory logic in C. elegans."

Michael A Beer

[International Worm Meeting, 2005]

We have developed a systematic approach for inferring cis-regulatory logic from whole-genome microarray expression data.[1] This approach identifies local DNA sequence elements and the combinatorial and positional constraints that determine their context-dependent role in transcriptional regulation. We use a Bayesian probabilistic framework that relates general DNA sequence features to mRNA expression patterns. By breaking the expression data into training and test sets of genes, we are able to evaluate the predictive accuracy of our inferred Bayesian network. Applied to S. cerevisiae, our inferred combinatorial regulatory rules correctly predict expression patterns for most of the genes. Applied to microarray data from C. elegans[2], we identify novel regulatory elements and combinatorial rules that control the phased temporal expression of transcription factors, histones, and germline specific genes during embryonic and larval development. While many of the DNA elements we find in S. cerevisiae are known transcription factor binding sites, the vast majority of the DNA elements we find in C. elegans and the inferred regulatory rules are novel, and provide focused mechanistic hypotheses for experimental validation. Successful DNA element detection is a limiting factor in our ability to infer predictive combinatorial rules, and the larger regulatory regions in C. elegans make this more challenging than in yeast. Here we extend our previous algorithm to explicitly use conservation of regulatory regions in C. briggsae to focus the search for DNA elements. In addition, we expand the range of regulatory programs we identify by applying to more diverse microarray datasets.[3] 1. Beer MA and Tavazoie S. Cell 117, 185-198 (2004). 2. Baugh LR, Hill AA, Slonim DK, Brown EL, and Hunter, CP. Development 130, 889-900 (2003); Hill AA, Hunter CP, Tsung BT, Tucker-Kellogg G, and Brown EL. Science 290, 809812 (2000). 3. Baugh LR, Hill AA, Claggett JM, Hill-Harfe K, Wen JC, Slonim DK, Brown EL, and Hunter, CP. Development 132, 1843-1854 (2005); Murphy CT, McCarroll SA, Bargmann CI, Fraser A, Kamath RS, Ahringer J, Li H, and Kenyon C. Nature 424 277-283 (2003); Reinke V, Smith HE, Nance J, Wang J, Van Doren C, Begley R, Jones SJ, Davis EB, Scherer S, Ward S, and Kim SK. Mol Cell 6 605-616 (2000).

Van Buskirk, C.L. et al. (2019) International Worm Meeting "Identification of components of stress-induced sleep (SIS) in an undergraduate laboratory course."

Van Buskirk, C.L., Full Immersion Research Experience, Students of BIOL447\/FIRE:

[International Worm Meeting, 2019]

Animals from cnidarians to vertebrates engage in sleep - quickly reversible periods of behavioral quiescence that are associated with reduced sensory responsiveness. Though the cellular function of sleep is of debate, its benefit is inarguable, and sleep loss is associated with a wide range of adverse effects from impairments in cognitive function to death. Interestingly, sleep in C. elegans does not appear to fall under circadian regulation. This nematode sleeps at the end of each larval molt during a period known as developmentally-timed sleep (DTS) or lethargus (Raizen et al., 2008). C. elegans also sleeps during recovery from exposure to damaging conditions - a phenomenon referred to as stress-induced sleep (SIS) (Hill et al., 2014; Nelson et al., 2014). Despite their phenotypic similarity, DTS and SIS are regulated by largely independent genetic and neural circuits (Trojanowski and Raizen, 2015). DTS is linked to the molting cycle and depends on the release of sleep-promoting flp-11 neuropeptides from the RIS interneuron (Turek et al., 2016). By contrast, SIS is triggered by conditions that cause cellular damage and is dependent on EGF signaling within the peptidergic ALA neuron and the collective action of a distinct set of ALA-expressed neuropeptides (Hill et al., 2014, Nelson et al., 2014, Nath et al., 2016). Engagement in SIS appears to be beneficial, as sleep-defective mutants are impaired for recovery following exposure to damaging conditions (Hill et al., 2014; Fry et al., 2016). We posit that SIS reveals a deeply conserved phenomenon, and that a core function of sleep is to repair cellular damage that accrues during wakefulness. In support of this notion, recent studies in zebrafish have shown that the number of double-strand breaks (DSBs) in neuronal nuclei increases during the day and that sleep promotes chromosome dynamics that are required for DSB repair (Zada et al., 2019). We are therefore interested in identifying additional components of SIS in C. elegans, with the goal of characterizing this potentially deeply conserved phenomenon. To this end, we are performing forward genetic screens for SIS-defective mutants, and we are doing this within the context of an undergraduate laboratory course at CSUN called BIOL447: FIRE (Full Immersion Research Experience). We will present several SIS components that we have identified thus far through mapping and whole-genome sequencing of our SIS-defective mutants.

Mansfield, R. et al. (2015) International Worm Meeting "Disruption of cellular proteostasis drives sleep behavior in C. elegans."

Van Buskirk, C., Mansfield, R.

[International Worm Meeting, 2015]

Sleep is widely regarded to be a restorative state, and the physiological perturbations associated with sleep deprivation are extensive. Despite intensive study, none of these perturbations has in turn been shown to drive sleep. Recently our lab has shown that in C. elegans, environmental stressors such as heat, cold and toxins induce a sleep-like state (Hill et al., 2014). As noxious environmental stressors are known to interrupt normal proteostasis, the existence of a subsequent sleep state suggests that sleep serves to assist in the restoration of normal proteostasis. This idea is supported by our observation that sleepless animals have impaired survival following severe stress (Hill et al., 2014). Further, we have found that chaperone response defective mutants display exaggerated sleep responses. These mutants include animals lacking the stress-induced transcription factors hsf-1/HSF-1, daf-16/FOXO and a component of the endoplasmic reticulum stress-response pathway hsp-4/BiP. We are testing site-of-action for this effect by examining strains with tissue-specific rescue of HSF-1. In addition, we are using pharmacological inhibitors of the proteasome to test whether direct inhibition of the proteostasis machinery can trigger sleep. Last, we are performing RNAi against both positive and negative regulators of the heat shock transcriptional response, and screening for sleep phenotypes. The results of these efforts will be presented.Hill AJ, Mansfield R, Lopez JMG, Raizen DM, Van Buskirk C (2014) Cellular stress induces a protective sleep-like state in C. elegans. Curr Bio 24:1-7.Zimmerman JE, Naidoo N, Raizen DM, Pack AI (2008) Conservation of sleep: insights from non-mammalian model systems. Trends Neurosci 31:371-6.

David S Peal et al. (2001) International C. elegans Meeting "Chemical and Genetic Inhibition of Sulfation in C. elegans"

David S Peal, Laura L Kiessling

[International C. elegans Meeting, 2001]

In order to examine the process of sulfation in C. elegans, sulfation was inhibited chemically using sodium chlorate, and genetically using the process of RNA-mediated interference (RNAi). Sodium chlorate inhibition during early larval stages resulted in a dose-dependant developmental delay. BLAST searches of characterized sulfotransferases against the worm genome resulted in the identification of 4 putative sulfotransferases: C34F6.4 and F08B4.6 (previously identified: [1] and [2]), F40H3.5, and Y34B4A.e. RNAi of the putative N-deacetylase/N-sulfotransferase F08B4.6 resulted in "stacking" of eggs in the gonad, along with eggs laid at the 2- and 4-celled stage. RNAi of the putative hexuronic 2-O sulfotransferase C34F6.4 resulted in a shortened, bulbous gonad. These initial results indicate that sulfation may be important during development of C. elegans. [1] Shworak, NW, Liu, J, Fritze, LMS, Schwartz, JJ, Zhang, L, Logeart, D, Rosenberg, RD. JBC 272: 28008-19 (1997). [2] Kobayashi, M, Sugumaran, G, Liu, J, Shworak, NW, Silbert, JE, Rosenberg, RD. JBC 274: 10474-80 (1999).

Hill KL et al. (2000) East Coast Worm Meeting "Phenotypic and Genetic Analysis of spe-16, a Gene Required for C. elegans Spermatogenesis"

Dobbins CA, Hill KL, L'Hernault SW

[East Coast Worm Meeting, 2000]

C. elegans spermatogenesis has proven to be a useful model for studying the more general developmental processes of cellular differentiation and cell-cell interactions. Mutants in the spe-16 gene show sperm-based sterility and pleiotropic defects in spermatogenesis, including abnormal meiotic spindle positioning and orientation, defects in nuclear and chromosomal segregation and inappropriate cytoplasmic polarization and organelle maturation. These defects may be more severe in hermaphrodite-derived sperm than in male-derived sperm. spe-16 lies on LGIIIR, between the cloned genes tra-1 and dpy-18 (Hill, Harfe, Dobbins and LHernault, Genetics, in press). We have used polymorphism mapping to narrow the physical interval that must contain the spe-16(hc54ts) mutation. To date, spe-16(hc54ts) has been mapped to a ~ 27 kb region containing two primary candidate genes, an oxysterol binding protein and a ribosomal protein s6 kinase. Mapping continues with three additional polymorphisms located within the 27 kb interval, and will be followed by sequencing to identify the spe-16(hc54ts) mutation.

Jie Ding et al. (2002) Mid-west Worm Meeting "LIN-44 misexpresion confers defects in the development of the vulval secondary lineage."

Russell J Hill, Jie Ding

[Mid-west Worm Meeting, 2002]

The Wnt pathway is a conserved signaling pathway that regulates many developmental processes in animals. Our research group is investigating the possibility that the Wnt pathway regulates the development of the C. elegans vulva. The vulva develops from precursors that adopt either a primary or secondary fate. These precursors then divide to produce descendants that differentiate into one of seven distinct vulval cell types. Several lines of evidence suggest that the Wnt signaling pathway could regulate the cell type decisions made by these descendants. First, mutants in the gene lin-17, which encodes a putative Wnt receptor, display possible cell type transformations among the descendants of the vulval precursor P7.p. Second, these descendant cells express POP-1, a transcription factor that functions in the Wnt signaling pathway, in a manner that correlates with their cell type (Hill et al., I.W.M., 1999). We have been using a genetic, gain-of-function approach to determine if any of the C. elegans Wnt signaling molecules are capable of regulating the cell type decisions made by these descendant cells. Transgenes have been produced that should express C. elegans Wnt signals under the control of the inducible hsp16-2 or hsp16-41 promoters. We observe that expression of high levels of the Wnt signal LIN-44 during early vulval development results in defects in vulval anatomy during the mid-L4 stage. By examining the pattern of cell fusions that occur among the vulval cells, we have obtained preliminary evidence that at least some of these anatomical defects are due to cell fate transformations among the descendants of P5.p and P7.p (the approach of using cell fusions to detect possible cell fate transformations is described in an abstract by Browning and Hill, this meeting). An alternative interpretation of these results is that ectopic expression of LIN-44 alters the cell fusion behavior of the vulval cells. We intend to examine the expression of cell-type specific markers in the LIN-44 transgenic animals to help distinguish between these two interpretations.

Van Buskirk, Cheryl (2017) International Worm Meeting "A forward genetic screen for mutants defective in recovery sleep."

Van Buskirk, Cheryl

[International Worm Meeting, 2017]

In response to damaging conditions such as noxious heat or UV exposure, C. elegans enters a period of behavioral quiescence known as stress-induced sleep or recovery sleep (RS), during which sensory responses are dampened and feeding and movement cease1. Recovery sleep is mediated by activation of EGF receptors on the peptidergic ALA interneuron and subsequent release of a collection of neuropeptides1-3. At this time it is not known how cellular damage leads to the initiation of EGF signaling, and gaps remain in our understanding of signal transduction events within ALA as well as in the target tissues affected by ALA peptides. In order to uncover genes required for recovery sleep we have initiated an EMS screen for sleepless F2 animals, using the pore-forming toxin Cry5B as our damage-inducing agent. Progeny of sleepless candidates are tested for responses to other known sleep-inducing stressors, such as heat and UV light. Mutants that are found to be generally defective in recovery sleep are kept for SNP mapping, complementation as needed, and eventual whole-genome sequencing. We will present our detailed methods, some obstacles that have been overcome in our mapping of sleep mutants, and information on candidate identity if available. We also describe how this project has been implemented within the context of an undergraduate laboratory course called BIOL447 FIRE: Full Immersion Research Experience. 1. Hill AJ, Mansfield R, Lopez JMNG, Raizen DM, Van Buskirk C. 2014. Cellular Stress Induces a Protective Sleep-like State in C. elegans. Curr. Biol. 24, 2399-2405. 2. Nelson MD, Lee KH, Churgin MA, Hill AJ, Van Buskirk C, Fang-Yen C, Raizen DM. 2014. FMRFamide-like FLP-13 neuropeptides promote quiescence following heat stress in Caenorhabditis elegans. Curr. Biol. 24:2406-2410. 3. Nath RD, Chow ES, Wang H, Schwarz EM, Sternberg PW. 2016. C. elegans stress- induced sleep emerges from the collective action of multiple neuropeptides. Curr. Bio.l 26:2446-2455.

Aroian RV et al. (1996) West Coast Worm Meeting "ACTIN BINDING PROTEINS AND EARLY EMBRYONIC DEVELOPMENT"

Field CM, Aroian RV, Alberts BM

[West Coast Worm Meeting, 1996]

The actin cytoskeleton plays an important role in early C. elegans development. The experiments of Hill and Strome (1988, 1990) showed that disruption of actin filaments in the one cell embryo results in a mis-positioning of both developmental molecules and the mitotic spindle. To study the role of the actin cytoskeleton during early embryogenesis, we have adopted an approach little used in C. elegans-- biochemistry. We have isolated 17 potential actin binding proteins from C. elegans oocytes using actin filament affinity chromatography. These C. elegans actin column binding proteins (CABPs) bind tightly to columns containing actin filaments, do not bind control albumin columns, and are highly enriched relative to their abundance in whole cell extract. We have made antibodies to some of these CABPs by excising them individually from SDS polyacrylamide gels and injecting them into individual mice. For six CABPs, we have polyclonal sera that recognize predominantly one band of the correct molecular weight as judged by protein immunoblotting. Using immunofluorescence microscopy, four of these, CABP1, CABP14, CABP11, and CABP13, show localization to subsets of the actin cytoskeleton in fixed early embryos. CABP1 staining co-localizes with actin in the cortex of early embryonic cells, but, unlike actin, is noticeably weaker at cell-cell boundaries. CABP14 staining associates with the nucleus of cells in prophase, the entire cortex and cytoplasm of cells in metaphase, but exclusively with the cleavage furrow of cells in telophase. The most dramatic asymmetries are seen with CABP11 and CABP13 staining. Unlike actin, which surrounds the entire cortex of the one cell embryo, CABP11 transiently associates with the anterior cortex of the early one-cell embryo. CABP13, on the other hand, associates asymmetrically with the lateral cortex (dorsal-ventral or left-right) of one-cell embryos, providing the first evidence that there is lateral asymmetry this early in C. elegans embryogenesis. CABP11 has been cloned and shows some homology to a known actin binding protein. Preliminary experiments in which CABP11 maternal function was eliminated using antisense RNA indicates that it an essential gene important for early development. Our data support the results of Hill and Strome suggesting that the actin cytoskeleton is intimately involved with generating asymmetry in the one-cell embryo.

Hill, Sarah et al. (2017) International Worm Meeting "Autophagosome biogenesis at the synapse."

Colon-Ramos, Daniel, Hill, Sarah

[International Worm Meeting, 2017]

Autophagy removes bulk cytoplasm or organelles from cells by forming double-membrane autophagosomes that fuse to lysosomes for degradation. Regulation of autophagosome formation and trafficking is especially important in neurons, which have long polarized processes distant from the cell body. We and others have observed that autophagosomes form at presynaptic sites and then undergo retrograde transport towards the cell body, which contains the lysosomes (Stavoe and Hill et al , 2016). However, the function of autophagy and regulation of autophagosome transport at the synapse is not completely understood. We used neurons from the C. elegans thermotaxis circuit as a model to study autophagy at the synapse. This system allows us to visualize autophagosome biogenesis and trafficking in vivo, control temperature-dependent synaptic firing, and simultaneously monitor potential autophagic regulators and targets at single-cell resolution. Using this system, we found that loss of synaptic activity results in decreased autophagosomes at the synapse and that JIP3/UNC-16, an adaptor protein known to regulate early endosome and lysosome transport, also plays an important role in regulating autophagosome transport in C. elegans neurons.

Melissa Ann Browning et al. (2002) Mid-west Worm Meeting "POP-1 function within the vulval secondary lineage."

Melissa Ann Browning, Russell J Hill

[Mid-west Worm Meeting, 2002]

The C. elegans vulva is produced from the descendants of the primary and secondary vulval lineages. These descendant cells differentiate into distinct vulval cell types in a precise pattern. The mechanisms which regulate the cell type decisions occurring among these descendant cells is currently not well understood. The transcription factor POP-1 is a component of the C. elegans Wnt pathway and is expressed during vulval development in a pattern that correlates with the cell type decisions made by these descendants (Hill et al., I.W.M., 1999). For example, after the precursor P7.p has undergone two rounds of cell division (P7.pxx), its descendants are of cell types D, C, B, and A (from anterior to posterior). These descendant cells express POP-1 in the pattern Low, High, Low, High (from anterior to posterior). Thus, each P7.pxx cell expresses a different level of POP-1 than its sister cell and adopts a cell type distinct from its sister. Therefore, we tested whether sister P7.pxx cells would produce the same cell type if they had the same level of POP-1 activity.