Thomas A McCloskey et al. (2005) International Worm Meeting "An RNAi screen for apoptotic suppressors in C. elegans"

Thomas A McCloskey, Joel Rothman

[International Worm Meeting, 2005]

Programmed cell death (PCD, aka. apoptosis) is a well-conserved, genetically defined process of cellular suicide used across metazoan phylogeny. During C. elegans development, PCD features in organogenesis, refinement of cell lineages, and formation of sexually dimorphic structures. Nearly all developmental apoptosis in the worm results from activation of the same molecular program, involving the pro-apoptotic regulators EGL-1, CED-3, and CED-4, and the anti-apoptotic protein CED-9. Despite extensive knowledge of this core cell death machinery, the pathways that mediate this event downstream of, or in parallel to, CED-3 are not clearly defined, as underscored by the recent discovery of the apoptotic suppressor ICD-1. Inhibition of icd-1 results in widespread CED-4-dependent PCD that is apparently independent of the CED-3 caspase and that requires the CSP-1 caspase (see abstract by Bloss and Rothman). Based on these observations, we performed an RNAi screen for similar apoptotic suppressors by identifying genes that when inactivated result in apoptotic cell corpses in a ced-3(-) background. This screen identified ~60 genes, each of which has been genetically classified based on epistasis analysis in N2, ced-3(-), and ced-4(-) backgrounds. These studies revealed that the identified genes appear to act at several distinct stages in the cell death pathway. Bayesian network algorithms that mine information from publicly available data sources (e.g., the C. elegans interactome and microarray data) are being applied to the gene candidates to map putative functional networks. Informatics studies suggest that the candidates from the screen fall into distinct structural and/or functional categories. For example, the combined results from the literature and our screen suggest that AAA ATPases are important for repressing apoptosis and that these activities may reveal the pathogenesis of certain human disease states, including neurodegenerative diseases and cancers.

Thomas A McCloskey et al. (2003) International Worm Meeting "An RNAi Screen for Programmed Cell Death Suppressors"

Thomas A McCloskey, Andrew Fraser, Ravi Kamath, Joel Rothman, Julie Ahringer

[International Worm Meeting, 2003]

The machinery that triggers programmed cell death (PCD) is present in many, if not all cells and it is critical that the activity of pro-apoptotic regulators is maintained in a quiescent state in surviving cells. Among the known cell death regulators in C. elegans, CED-9/Bcl-2 is a pivotal component that protects surviving cells from undergoing apoptosis by inhibiting the function of the pro-apoptotic protein, CED-3. Although the core PCD machinery has been well described, the molecular processes that act downstream of, or in parallel to, CED-3, to mediate apoptosis are poorly understood. We have undertaken a genome-wide RNAi screen to identify additional genes required to suppress PCD. The basis for our screen derives from studies on ICD-1 (see abstract by Bloss et al.). Debilitating the function of this apoptotic suppressor results in widespread apoptotic death, which, like normal developmental PCD, requires CED-4/Apaf-1. However, unexpectedly, this apoptotic death occurs in the absence of CED-3 caspase function. These observations suggest that ICD-1 acts downstream of CED-3 to suppress PCD, that this cell death is promoted by a redundantly acting caspase, or perhaps both. It also encouraged us to screen for additional anti-apoptotic factors by identifying mutants in which cell death occurs in a ced-3(-) genetic background. For the screen, ced-1; ced-3 worms were grown on each feeder strain from the RNAi library and progeny scored for the presence of embryonic PCD by Nomarski microscopy. The ced-3 mutation eliminates nearly all PCD and the ced-1 engulfment mutation causes cell corpses to persist, thereby facilitating their identification. This approach is exquisitely sensitive for discovering genes that, when debilitated, result in inappropriate cell death. From an initial screen of 800 genes that show embryonic lethality by RNAi and another 1200 viable genes, we have identified >80 candidates that exhibit from a few fold to >1000-fold elevation in PCD. Included in the set of genes we identified were icd-1 and icd-2 (see abstract by Bloss et al.). Some of the candidates share homology with proteins from other organisms that are implicated in suppression of PCD (e.g., thioredoxin-2 and the benzodiazepine receptor, both mitochondrial proteins whose inactivation results in apoptosis in mammals).

Thomas A McCloskey et al. (2002) West Coast Worm Meeting "Identification of candidate genes that function in CED-3-independent programmed cell death"

Joel H Rothman, Marc Dorfman, Thomas A McCloskey

[West Coast Worm Meeting, 2002]

Among the known cell death regulators in C. elegans, the caspase CED-3 (Ellis et al., 1986) is a key member of the pathway, both as the terminal component in the core pathway and as the trigger for chromatin condensation, cytoskeletal degradation, and intracellular vacuolization accompanying apoptotic death. In the absence of functional CED-3, cells that would normally undergo programmed cell death instead develop, differentiate, and integrate into the animal's tissues. Despite the knowledge provided by the discovery of the core cell death machinery, the molecular processes that mediate the biochemical and cellular events downstream of CED-3 remain poorly understood. Based on the recent finding in our laboratory that an apparent cell death suppressor, icd-1, leads to CED-3-independent cell death when debilitated (Bloss et al., 2002), we are analyzing candidate genes that may protect cells from dying and that function downstream, or independently, of ced-3. To this end, we are studying previously identified zygotic embryonic lethal mutations from the laboratory collection (Rothman, 1991) that lead to widespread cell death in the absence of ced-3 function. We are also using an RNAi library of bacterially expressed dsRNAs (Kamath et al., 2001) to identify genes whose loss of function results in the occurrence of apoptotic cell death in ced-1;ced-3 worms. Candidate genes that show RNAi-induced apoptosis in ced-3 mutant worms will be further characterized for their role in programmed cell death.

Thomas A McCloskey et al. (2004) West Coast Worm Meeting "Suppression of CED-3-Independent Programmed Cell Death In C. elegans"

Thomas A McCloskey, Peter Bowers, Ambuj Singh, Chiou-Wei Tsai, Joel Rothman, Vebjorn Ljosa

[West Coast Worm Meeting, 2004]

Programmed cell death (PCD) is a well conserved, genetically controlled process of cell suicide that occurs naturally during the life span of many organisms. Among the core molecular components that mediate apoptosis in C. elegans , the caspase CED-3 is a necessary activator of nearly all developmental PCD. Strong ced-3(-) mutants show nearly no PCD and all cells that would normally die instead develop, differentiate, and integrate into the host organism's tissues. Despite extensive knowledge of the core cell death machinery, the pathways that mediate this event downstream of, or in parallel to, CED-3 are not clearly defined. Debilitating the function of the apoptotic suppressor ICD-1 (Bloss et al., Nature 424: 1066 (2003)) results in widespread cell death, which, like developmental PCD, requires the essential cell death activator CED-4. However, this apoptotic death does not require CED-3 activity. Based on these observations, we performed an RNAi screen for additional suppressors of ced-3 -independent PCD which has revealed a set of ~130 candidate genes. Our investigation of these newly identified apoptotic suppressor candidates consists of three principal objectives: (1) We are currently categorizing the newly identified genes into genetic and phenotypic groups to establish classes of PCD repression. Genetic interactions with the core PCD machinery are being examined by performing candidate gene RNAi on ced-3(-) and ced-4(-) mutants. (2) We are applying Bayesian networking methods of bioinformatics to predict functional networks through which these genes may act in the organism. (3) Based on these studies, we are developing a refined list of prominent candidates that we intend to pursue by detailed mechanistic studies.

Thomas JH et al. (1994) Worm Breeder's Gazette "lin-36, a Class B Synthetic Multivulva Gene, Encodes a Novel Protein"

Thomas JH, Horvitz HR

[Worm Breeder's Gazette, 1994]

lin-36, a Class B Synthetic Multivulva Gene, Encodes a Novel Protein Jeffrey H. Thomas and H. Robert Horvitz, HHMI, Dept. Biology, MIT, Cambridge, MA 02139, USA

Chao-I Chen et al. (2004) West Coast Worm Meeting "A Bayesian approach toward analyzing the network of genes involved in C. elegans programmed cell death"

Chao-I Chen, Vebjorn Ljosa, S Rao Jammalamadaka, Chiou-Wei Tsai, Ambuj K Singh, Thomas A McCloskey, Xiaofang Lei, Joel H Rothman

[West Coast Worm Meeting, 2004]

From an RNAi screen for genes that repress apoptosis, we have identified a set of about 130 candidates (see abstract by McCloskey et al.). We are using computational methods in an effort to predict functional networks through which these genes may act and to identify additional candidates and their relationships within the postulated networks. We are initially working with a number of datasets as the basis for our predictions: microarray expression data, the physical orientation of genes within the worm genome, phylogenetic profile data, and PSORT predictions of protein localization. The datasets are normalized according to their overlap with the worm interactome and Wormbase subcellular localization data. They are then combined using a nave Bayesian network, to yield a set of potential functionally interacting protein pairs along with a likelihood score for each pair. Finally, we construct network maps of the protein pairs that fall above a desired likelihood ratio. Our initial efforts suggest a cluster of functional interactions that are predicted from the gene set identified by the RNAi screen; refinements of the computational methods are in progress.

Tax FE et al. (1994) Worm Breeder's Gazette "Some Notes on Mapping Contigs Using Molecular Analysis of Deficiences"

Thomas JH, Tax FE

[Worm Breeder's Gazette, 1994]

Some notes on mapping contigs using molecular analysis of deficiencies Frans Tax and Jim Thomas, Dept. of Genetics, University of Washington, Seattle WA 98195

Burglin TR et al. (1993) Worm Breeder's Gazette "ceh-6 Expression and Generation of a Knock-Out"

Plasterk RHA, Ruvkun GB, Burglin TR

[Worm Breeder's Gazette, 1993]

ceh 6 expression and generation of a knock-out Thomas Burglin, Ron Plasterk*, Gary Ruvkun Dept. of Molecular Biology, Wellman 8, Massachusetts General Hospital, Boston, MA, 02114, USA, and *Netherlands Cancer Institute, Plesmanlaan 121, 1066CX Amsterdam, The Netherlands.

Reilly, Molly et al. MicroPubl Biol

Hobert, Oliver, Reilly, Molly

[MicroPubl Biol, 2020]

We have previously described the expression patterns of all but one of the 102 homeobox genes present in the C. elegans genome, using either fosmid-based reporter transgenes or CRISPR/Cas9-engineered gfp reporter alleles (Reilly et al., 2020). The last remaining homeobox gene for which we were initially not able to generate a reporter reagent for is ceh-84 (Reilly et al., 2020). As previously recognized by Thomas Brglin (Hench et al., 2015), ceh-84 codes for an unusual homeodomain protein with two divergent homeodomains (Fig.1D). It likely is a tandem duplicate of the ceh-85 gene, which has been classified as a pseudogene (Fig.1B). ceh-84 has no recognizable homologs in other Caenorhabditis species (Hench et al., 2015). Together with its duplicate, ceh-85, localizes to a large cluster of extensively duplicated genes on chromosome II, described by James Thomas (Thomas, 2006). Most of the duplicated genes code for MATH and BTB type proteins (Fig.1A). The entire cluster seems to be C. elegans-specific (Thomas, 2006) and, apart from a number of pseudogenes, it also contains the C. elegans-specific ceh-81, ceh-82 and ceh-83 homeobox genes (Fig.1A).

Tissenbaum HA et al. (1995) International C. elegans Meeting "GENETIC AND MOLECULAR ANALYSIS OF daf-2 AND daf-16."

Gottlieb S, Lee L, Tissenbaum HA, Ruvkun GB, Lam F

[International C. elegans Meeting, 1995]

We have been studying the genetic interactions between the genes daf-2, daf-23 and daf-16 (Gottlieb and Ruvkun 1994). These genes either act in a branch separate from the daf-11/daf-21 and daf-7 group of the genetic epistasis pathway for dauer formation or further downstream in the pathway ( Riddle et al. 1981; Vowels and Thomas 1992; Thomas et al. 1993; Gottlieb and Ruvkun 1994). Molecular analysis of daf-2, daf-16 as well as daf-23 (see abstract by Morris et al. this meeting) will help to elucidate function (e.g. neurons vs. target tissues) as well as shed light on the complex genetic interactions of these genes.