Blum, Elyse et al. (2015) International Worm Meeting "Mechanism of peel-1 toxicity."

Blum, Elyse, Cattin, Jerome, Ailion, Michael

[International Worm Meeting, 2015]

In C. elegans, hybrid incompatibility between the Bristol N2 and Hawaiian CB4856 strains is mediated by the peel-1/zeel-1 genetic element. peel-1 encodes a sperm-expressed toxin and zeel-1 encodes its zygotically-expressed antidote. We are interested in determining the cellular mechanism of PEEL-1 toxicity. To identify factors required for PEEL-1 toxicity, we conducted a screen for suppressors of the lethal phenotype caused by ectopic PEEL-1 expression driven by a heat-shock promoter (hs::peel-1). Thus far, we have isolated five suppressors. All five suppressors are healthy and have no obvious visible phenotypes. They still express a heat-shock driven GFP transgene at normal levels, indicating that they do not suppress hs::peel-1 due to failed induction of the heat-shock response. The two strongest suppressors fully suppress hs::peel-1 and are allelic. The other three suppressors are partial suppressors belonging to two complementation groups and preferentially suppress hs::peel-1 in younger larvae. Thus, it seems likely that only a small number of genes can be mutated to suppress hs::peel-1. We have mapped these suppressors to single chromosomes and performed whole genome sequencing to clone the genes. We also tagged heat-shock driven PEEL-1 with GFP on either its N or C terminal. In both cases, induction of heat-shock leads to death of the worms, indicating that the tagged PEEL-1 protein is still functional. GFP is localized to a perinuclear compartment that may be the endoplasmic reticulum. Previously, we showed that PEEL-1 kills either muscle or neurons cell autonomously when specifically expressed in these cell types. We now show that PEEL-1 is also toxic when expressed in the intestine. Endogenous PEEL-1 is transmitted paternally to kill zeel-1(-) embryos. We wondered whether maternal expression would also kill zeel-1(-) embryos. We expressed peel-1 in the maternal germline using the pie-1 promoter and found that it did not lead to death of zeel-1(-) progeny. Thus, it appears that paternal expression and transmission is critical for the embryonic lethal phenotype.

Blum, Elyse S. et al. (2009) International Worm Meeting "A polyQ-repeat protein promotes the novel, morphologically conserved death of the linker cell."

Shaham, Shai, Blum, Elyse S.

[International Worm Meeting, 2009]

Programmed cell death is an essential process during metazoan development. During wild-type C. elegans development, nearly all cells slated to die activate caspases and undergo stereotypical morphological changes including chromatin compaction and cell shrinkage. The male-specific linker cell (LC), however, is an exception. The LC leads the migration of the developing gonad, and once migration is complete at the L4-Adult transition, the LC dies. LC death is independent of ced-3, ced-4, ced-9, and egl-1, indicating that LC death is controlled by a novel program. Indeed, electron micrographs of dying LCs reveal non-apoptotic features, including nuclear crenellation and organelle swelling. Remarkably similar features are also seen in normally dying cells of the vertebrate spinal cord and ciliary ganglion, suggesting that LC death is morphologically conserved. To understand the molecular mechanism of LC death, we performed a genome-wide RNAi screen to identify genes whose loss prevents LC death. RNAi against pqn-41, a gene predicted to encode a polyQ-repeat protein, blocks LC death in 20-30% of animals examined. Similar defects are seen in pqn-41(ns294) mutants we generated. A transgene containing pqn-41 genomic DNA fused to GFP is nuclearly expressed in many cells in the animal. Strikingly, expression in the LC is only visible as the cell begins to die, and we identified a pqn-41 promoter region required for LC expression. PQN-41 protein is localized to nuclei and nuclear puncta in all cells, except for the dying LC, where PQN-41 is also cytoplasmic. Previous studies revealed that LC death requires the heterochronic genes let-7 and lin-29, and that these genes act within the LC to promote its death. We have now shown that a MAPK module containing the TIR-1 adapter protein and the SEK-1 MAPKK also regulate LC death: 30% tir-1(RNAi) and 49% sek-1(ag1) adult males exhibit LC survival. The heterochronic and MAPK pathways likely function in parallel to regulate LC death, as double-mutants exhibit additive effects, and expression of components of one pathway does not depend on the other. Since both pathways impinge on transcriptional regulators, it is possible that both pathways converge on the promoters of key LC death genes. We are testing this hypothesis with regard to pqn-41. Several human neurodegenerative disorders result from polyQ expansions within endogenous proteins, however, the mechanisms promoting cell death in these diseases is not known. Our studies raise the intriguing possibility that these aberrant proteins activate an endogenous cell death program similar to that driving the death of the LC.

Elyse Blum et al. (2008) Development & Evolution Meeting "TIR-1 and SEK-1/MAPKK Promote the Novel Morphologically Conserved Death of the Linker Cell"

Elyse Blum, Shai Shaham

[Development & Evolution Meeting, 2008]

Programmed cell death is an essential process during metazoan development. During wildtype C. elegans development, nearly all cells slated to die activate caspases and undergo stereotypical morphological changes including chromatin compaction and cell shrinkage. The male-specific linker cell (LC), however, is an exception. The LC leads the migration of the developing gonad to the cloaca, and once migration is complete at the L4-to-adult transition, the LC dies using a cell-autonomous death program. Strong loss-of-function alleles of canonical cell-death pathway genes, including ced-3, ced-4, and egl-1, as well as a ced-9(gf) mutation, do not block LC death, indicating that LC death must be controlled by a novel program (1). Indeed, electron micrographs of dying LCs reveal non-apoptotic features, including nuclear crenellation, absence of chromatin condensation, organelle swelling, and accumulation of cytoplasmic membrane-bound structures. Remarkably, similar features are seen during developmental deaths of neurons in the vertebrate spinal cord and ciliary ganglia, suggesting that LC death is morphologically conserved. Previous studies revealed that LC death requires the developmental timing genes let-7 and lin-29, and that these genes act within the LC to promote cell death. To identify additional components required for LC death, we performed a genome-wide RNAi screen aimed at identifying genes whose loss prevents LC death. We identified several candidate genes, and have begun to verify these. One of the genes we identified is tir-1: 30% of tir-1(RNAi) mutants exhibit LC survival. tir-1 encodes a Toll-like/interleukin-1 receptor adaptor protein that functions as a signaling protein in innate immunity and during neuronal cell-fate establishment. In these processes, tir-1 functions upstream of nsy-1/MAPKKK and sek-1/MAPKK. We reasoned that tir-1 could be activating a similar MAPK cascade in the LC and tested whether strong loss-of-function alleles of nsy-1 and sek-1 also block LC death. While the nsy-1(ky397) and nsy-1(ky542) alleles do not affect LC death, the sek-1(km4) allele prevents LC death in 20% of males. These results suggest that a MAPK cascade may control genes essential for LC death. Downstream targets of this cascade may emerge from studies of the other RNAi candidates we identified and/or from a genetic screen currently in progress (E.B. and R. Razi, unpublished). 1) Abraham et al., Dev. Cell 12:73-86, 2007.

Kinet, Maxime et al. (2010) C. elegans: Development and Gene Expression, EMBL, Heidelberg, Germany "A role for chromatin in linker cell death?"

Abraham, Mary, Shaham, Shai, Blum, Elyse, Kinet, Maxime

[C. elegans: Development and Gene Expression, EMBL, Heidelberg, Germany, 2010]

Programmed cell death plays a crucial role in development and disease; however, apoptosis and its variations may not account for all such cell deaths. Recently, our lab uncovered a new cell death program in the C. elegans male-specific linker cell. In contrast to most other cell deaths in C. elegans , which occur in undifferentiated young cells, the linker cell dies after a relatively long life spent elongating the male gonad. The linker cells death and removal connects the vas deferens to the cloaca, enabling sperm exit. Linker cell death is non-apoptotic by morphology and is independent of all known C. elegans cell death genes. Strikingly, ultrastructural features of this death, such as nuclear envelope indentation and open chromatin, have been described in developing vertebrate nervous systems and in polyglutamine -repeat neurodegenerative diseases like Huntingtons disease. A genome-wide RNAi screen (E. Blum and M. Abraham, unpublished) for genes required in this death uncovered the gene pqn-41 , which encodes a nuclear polyglutamine repeat protein, further suggesting a connection to neurodegenerative cell death. Another gene required for linker cell death, swd-2.2 , encodes a subunit shared by a histone H3K4 methyltransferase Set1 complex and an RNA cleavage and polyadenylation factor (CPF). We found that three different swd-2.2 RNAi clones blocked linker cell death but not its migration. Furthermore, promoter-GFP fusion studies suggest that swd-2.2 is expressed in the linker cell. We are testing whether swd-2.2 functions cell-autonomously to promote linker cell death using cell-specific RNAi, and aim to determine whether SWD-2.2 functions in the Set1 complex or RNA CPF or both to enable this death. Given that PQN-41 is found in nuclei and that polyQ-tract proteins can a ssociate with chromatin, it is possible that SWD-2.2 functions with PQN-41 to promote linker cell death.

Zuckerman, Jennifer A. et al. (2013) International Worm Meeting "let-70, an E2 ubiquitin-conjugating enzyme, promotes linker cell death in C. elegans."

Lu, Yun, Shaham, Shai, Zuckerman, Jennifer A.

[International Worm Meeting, 2013]

The C. elegans linker cell (LC) directs migration and development of the male gonad, dying in the L4-adult transition to allow vas deferens-cloaca fusion.We previously showed that the LC dies independently of apoptosis genes, displaying non-apoptotic features including nuclear membrane invagination, open chromatin, and organelle swelling.These features are conserved in dying cells in normal vertebrate development and in polyQ-expansion diseases. let-70 encodes an E2 ubiquitin-conjugating enzyme we identified in an RNAi screen for LC death genes.let-70(RNAi) males are defective in LC migration and death and LC-specific RNAi using rde-1 rescue blocks death but not migration. Thus, let-70 functions in the LC to promote cell death and non-autonomously to control migration. let-70 is transcriptionally induced in the LC as the cell begins to die. Similarly,we found that expression of a ubiquitin gene reporter, ubq-1::gfp,also increases, as does expression of PQN-41, a polyQ-repeat protein required for LC death (Blum et al, 2012).These results suggest that LC death is controlled by a transcriptional program. Inactivation of the MAPKK sek-1 or the histone methyltransferase set-16 blocks let-70 expression and LC death (Blum et al, 2012). nhr-67 RNAi induces early let-70::GFP expression in the LC; however, nhr-67(RNAi) blocks LC death and double RNAi against nhr-67 and let-70 synergistically increases cell survival.Thus, proper timing of expression and/or optimal levels of let-70 may be crucial for its cell death role.We screened for E3 ligases needed for LC death and found that RNAi against seven-in-absentia homolog siah-1 or the RING-box rbx-1 blocked LC death in 10% of animals. siah-1; rbx-1 double mutants show nearly 30% LC survival, suggesting that they act in parallel. Furthermore, 2-hybrid studies show that LET-70 and SIAH-1 interact. We suggest, therefore, that let-70 degrades proteins normally required to inhibit LC death. Supporting this notion, RNAi against proteasome regulatory components blocks LC death, as does RNAi against other SCF components.Our studies suggest a role for protein degradation in the control of a novel, conserved form of cell death with potential relevance to human neurodegeneration.

Zuckerman, Jennifer et al. (2010) C. elegans: Development and Gene Expression, EMBL, Heidelberg, Germany "Uncovering the role of LET-70 in linker cell death in C. elegans"

Zuckerman, Jennifer, Shaham, Shai

[C. elegans: Development and Gene Expression, EMBL, Heidelberg, Germany, 2010]

Cell death is an essential process, providing a means to direct tissue morphogenesis. The C. elegans linker cell is an important regulator of male gonad morphogenesis. It directs migration of the gonad, and its death allows fusion of the vas deferens with the cloacal exit channel. Unlike most dying cells, which die shortly after they are generated, the linker cell survives for two days before its death during the L4/adult transition. Studies from our lab showed that linker cell death is independent of known C. elegans cell death genes and is characterized by nonapoptotic features, including nuclear membrane invagination and lack of chromatin condensation. These features have been described in cell death that accompanies normal development of the vertebrate nervous system and in neurodegenerative diseases promoted by p olyglutamine expansions. Interestingly, an RNAi screen for genes important in linker cell death identified the gene pqn-41, a polyglutamine repeat protein, as a regulator of linker cell death (E Blum and S Shaham, unpublished). Additional genes were also identified, including let-70 , a putative E2 ubiquitin-conjugating enzyme. Wild-type males fed E. coli expressing let-70 dsRNA have surviving linker cells. A block in the final stage of linker cell migration is also seen, but is separable from the death defect. The let-70 defect is not enhanced by mutations in sek-1 /MAPKK but is slightly enhanced by mutations in the Zn-finger transcriptional regulator lin-29 . sek-1 and lin-29 seem to act in parallel to promote linker cell death. Linker cell survival is enhanced by a mutation in pqn-41 . Like pqn-41 , expression of let-70 promoter::GFP transgenes is induced in the linker cell as it begins to die. Several studies suggest that polyglutamine proteins promoting neuodegeneration are ubiquitinated. We are interested in testing the possibility that PQN-41 or other proteins implicated in linker cell death are modified by LET-70.

Kinet, Maxime J et al. (2011) International Worm Meeting "An MLL-type H3K4 methyltransferase complex is required for linker cell death."

Kinet, Maxime J, Shaham, Shai

[International Worm Meeting, 2011]

Programmed cell death plays a crucial role in development and disease; however, apoptosis and its variations may not account for all such cell deaths. Recently, our lab uncovered a new cell death program in the C. elegans male-specific linker cell. In contrast to most other cell deaths in C. elegans, which occur in undifferentiated young cells, the linker cell dies after a relatively long, differentiated life spent elongating the male gonad. The linker cell's death and removal connects the vas deferens to the cloaca, enabling sperm exit. Linker cell death is non-apoptotic by morphology and is independent of all known C. elegans cell death genes. Ultrastructural features of this death, such as nuclear envelope indentation and open chromatin, have been described in developing vertebrate nervous systems and in polyglutamine-repeat neurodegenerative diseases. Strikingly, pqn-41, a polyglutamine-repeat gene, promotes linker cell death (Blum et al, unpublished). Here we present evidence that a highly conserved histone H3K4 methyltransferase complex is also involved in linker cell death. Targeting several predicted subunits of this complex by RNAi results in a death defect phenotype. RNAi against core structural and regulatory subunits swd-2.2, rbbp-5, wdr-5 and ash-2, as well as a potential targeting mediator, pis-1, resulted in the survival of 10-17% of linker cells examined. Importantly, targeting an MLL-type catalytic H3K4 methyltransferase gene, set-16, but not the SET1-like gene set-2, blocked linker cell death in 50% of animals. Finally, knockdown of utx-1, encoding a catalytic histone H3K27 demethylase that in other systems associates with MLL-type complexes, also blocked linker cell death (18%). To determine how this putative complex might act in linker cell death, we used cell-specific RNAi to show that at least two genes, swd-2.2 and set-16 can act cell-autonomously in linker cell death. Consistent with a cell-autonomous role for a set-16 complex, both genes are expressed in the linker cell. While set-16 genetic mutants die embryonically, mosaic analysis of mutants rescued with a genomic set-16 clone confirms that set-16 acts in the linker cell to promote its death. Interestingly, set-16 is homologous to human MLL3, a gene found mutated in a variety of cancers. MLL-type H3K4 methyltransferase complexes function as transcriptional coactivators controlling a finite set of targets. In light of this role, we are currently undertaking cell-specific transcriptome studies to identify genes that are regulated by set-16 and may contribute to linker cell death.