Marion Thellmann et al. (2001) International C. elegans Meeting "Analysis of the Specification of the NSM Sister Cell"

BARBARA CONRADT, Julia Hatzold, Marion Thellmann

[International C. elegans Meeting, 2001]

Gain-of-function (gf) mutations in the gene ces-1 ( ces , ce ll death s pecification) block the death of the NSM sister cells and the I2 sister cells, four cells that normally undergo programmed cell death (PCD) (1). Genetically, ces-1 acts as a negative regulator of the cell-death activator gene egl-1 ( egl , eg g l aying defective), which is required for most if not all PCD events that occur during development (2). ces-1 , hence, is a candidate cell-type specific regulator of the central PCD pathway in C. elegans (3). ces-1 encodes a zinc finger DNA binding protein most similar to the Snail family of transcription factors (4) and egl-1 is transcriptionally regulated, at least in some cells (2). It has therefore been proposed that the CES-1 protein specifies the cell death fate of the NSM and I2 sister cells by acting as a repressor of egl-1 transcription (4). The egl-1 locus contains four Snail family consensus sites, which are conserved in the egl-1 locus of C. briggsae . To test whether CES-1 can bind to these sites in vitro , we performed gel mobility shift assays with bacterially expressed GST-CES-1 fusion protein. Using this assay, we could show that GST-CES-1 binds to the four Snail-binding sites in a specific and cooperative manner. To investigate whether CES-1 protein can bind to the egl-1 locus in vivo , we are using a methodology devised by Carmi et al ., which will allow us to determine whether CES-1 protein localizes to extrachromosomal arrays containing multiple copies of the egl-1 locus in nuclei of transgenic embryos (5). Loss-of-function (lf) mutations in egl-1 block most if not all PCD during development, including the death of the NSM sister cells. When injected into egl-1 (lf) animals, an 8 kb fragment that includes the egl-1 transcription unit, 1 kb of its upstream region and 5.6 kb of its downstream region ( egl-1 rescuing fragment) can rescue the cell death defect of the animals. To determine whether the region that spans the four CES-1-binding sites (region B) is required to specify the NSM sister cell death in vivo , we are analyzing whether region B is necessary and sufficient for the ability of the rescuing fragment to rescue the death of the NSM sister cells in egl-1 (lf) mutant animals. To score the survival of the NSM sister cells, we are using the tph-1::gfp reporter ( tph , t ry p tophan h ydroxylase gene), which is specifically expressed in serotonergic neurons including the NSMs and surviving NSM sister cells (6). Using this experimental approach, we found that region B is required for the specification of the NSM sister cell death in vivo . Since lf mutations in ces-1 have no obvious phenotype we suggest that its function is redundant. In order to identify additional factors that act with CES-1 to specify the cell death fate of the NSMs and their sister cells, we are therfore performing a screen for mutations that cause the NSMs to undergo PCD in a ces-1 (lf) mutant background. 1. R. E. Ellis and H. R. Horvitz. (1991). Development 112, 591-603 2. B. Conradt and H. R. Horvitz. (1999). Cell 98, 317-327 3. M. M. Metzstein et al. (1998). TIG 14, 410-416 4. M. M. Metzstein and H. R. Horvitz. (1999). Molecular Cell 4, 309-319 5. I. Carmi et al. (1998). Nature 396, 168-173 6. J. Y. Sze et al . (2000). Nature 403, 560-564

Marion Thellmann et al. (2002) European Worm Meeting "The Snail-like zinc finger DNA-binding protein CES-1 is a direct regulator of egl-1 transcription"

Julia Hatzold, BARBARA CONRADT, Marion Thellmann

[European Worm Meeting, 2002]

Genetic and molecular analyses performed in C. elegans have defined a genetic pathway for programmed cell death (PCD), an important physiological process of animal development, which has been conserved through evolution (1). Four genes, egl-1 (egl, egg laying defective), ced-9 (ced, cell death abnormal), ced-4, and ced-3, form the central pathway required for cell-death execution in C. elegans. Mutations in these genes can block most if not all somatic PCD events that occur during the development of C. elegans. A group of genes, the ces (ces, cell death specification) genes, appears to regulate the central pathway in a cell-type specific manner. Gain-of-function mutations in the gene ces-1, for example, specifically block the death of the NSM sister cells, the sisters of the NSMs, two serotonergic, neurosecretory motoneurons (2). ces-1 encodes a Snail-like zinc finger DNA- binding protein (3), and negatively regulates the cell-death activator gene egl-1. egl-1 represents the most upstream gene of the central cell-death pathway and is regulated at the transcriptional level at least in some cells, including the NSM sister cells (4, Hatzold, J. et al.; European C. elegans Meeting 2002, abstract). Moreover, the egl-1 promoter contains four Snail family consensus sites, which are conserved in the egl-1 locus of C. briggsae. Therefore, we propose that the CES-1 protein specifies the cell-death fate of the NSM sister cells by directly regulating egl-1 expression. To determine which part of the egl-1 locus is essential for the death of the NSM sister cells, we analysed subclones of the egl-1 locus for their potential to rescue this particular cell-death event in egl-1(lf) mutant animals. We could show that the region that spans the four Snail family consensus sites (Region B) is necessary and sufficient for the specification of the NSM sister cell death in vivo. Moreover, using gel mobility shift assays with bacterially expressed GST-CES-1 fusion protein, we could also show that GST-CES-1 binds to the four Snail family consensus sites in Region B in a specific and cooperative manner. These data suggest that in the surviving NSMs, CES-1 acts as a repressor of egl-1 transcription. In addition, we have in vivo evidence indicating that CES-1 and a potential NSM-specific transcriptional activator may specify the NSM sister cell fate by competing for binding to the Snail family consensus sites in Region B. Genetic and molecular data suggest that this activator is a heterodimer of C. elegans Daughterless and Achaete-Scute like proteins (Hatzold, J. et al.; European C. elegans Meeting 2002, abstract), which belong to the family of basic helix-loop-helix (bHLH) transcription factors (5, 6).

Julia Hatzold et al. (2002) European Worm Meeting "bHLH transcription factors are involved in specifying the cell-death fate of the NSM sister cells"

Julia Hatzold, BARBARA CONRADT, Marion Thellmann

[European Worm Meeting, 2002]

In the developing C. elegans embryo, the NSMs differentiate into pharyngeal, serotonergic neurosecretory motoneurons; their sisters, however, die shortly after they are born as a result of programmed cell death. The gene ces-1, which codes for a Snail-like transcription factor, plays a major role in this specific cell-death event. In ces-1 gain-of-function (gf) mutants the NSM sister cells fail to die but instead develop into NSM-like cells also producing serotonin (1). The function of CES-1 in cell-death regulation might be conserved, since its human homolog, SLUG, is involved in the cell-death specification of B cells (2). Genetically, ces-1 acts as a negative regulator of the cell-death activator gene egl-1. egl-1 is the first gene of the central cell-death pathway and codes for a BH3-only protein, which is transcriptionally regulated in the NSMs. Our data indicate that, in the surviving NSMs, CES-1 binds to Snail-binding sites in a conserved region of the egl-1 locus that is necessary for this specific cell-death event (Thellmann, M. et al., European C. elegans Meeting, abstract). We postulate that CES-1 thereby blocks a NSM-specific transcriptional activator of egl-1. In the NSM sister cells, CES-1 might be present at lower levels, which would allow the NSM-specific activator to induce egl-1 transcription, resulting in the death of these cells. Using two different approaches, we plan to identify the activator or activators of egl-1 transcription which are blocked by CES-1 in the NSMs. It has been shown in other organisms that Snail-like proteins can antagonize the action of basic helix-loop-helix (bHLH) transcription factors (in particular the action of heterodimers of Daughterless and Achaete-Scute-like proteins) by direct competition for DNA binding to Snail- binding sites/E-boxes (3,4,5). Using existing mutants and RNAi, we therefore tested wether bHLH proteins are involved in specifying the death of the NSM sister cells. We found that a weak loss-of-function (lf) mutation in the only C. elegans Daughterless-like gene hlh-2, hlh-2(bx108), leads to the survival of the NSM sister cells with a low frequency. Furthermore, hlh-3(RNAi) causes the NSM sister cells to survive at a low frequency in wild-type animals and enhances the hlh-2(bx108) phenotype. We were able to show that a HLH-2/HLH-3 heterodimer can bind to the Snail-binding sites of the egl-1 locus in vitro. These data and additional in vivo evidence (Thellmann, M. et al., European C. elegans Meeting, abstract) suggest that an HLH-2/HLH-3 heterodimer acts as an activator of egl-1 transcription in the NSM sister cells. In addition, we are performing a genetic screen for mutations that enhance the weak NSM sister cell survival phenotype of hlh-2(bx108) mutants in order to identify other factors that might act with HLH-2 and HLH-3 to activate egl-1 transcription.

Julia Hatzold et al. (2003) International Worm Meeting "bHLH transcription factors are involved in specifying the cell-death fate of the NSM sister cells"

Marion Thellmann, Julia Hatzold, BARBARA CONRADT

[International Worm Meeting, 2003]

During C. elegans development the NSMs differentiate into pharyngeal neurosecretory motoneurons, their sisters, however, die shortly after they are born as a result of programmed cell death. Our results indicate that the death of the NSM sister cells is specified by the asymmetric expression of the cell-death activator gene egl-1 (egl, egg laying defective), which codes for a BH3-only protein. egl-1 is transcriptionally active in the NSM sister cells, which are destined to die, but not in the NSMs, which are destined to survive. We show that a weak loss-of-function mutation of the only C. elegans Daughterless-like bHLH-gene hlh-2 (hlh, helix-loop-helix),hlh-2(bx108), causes specifically the NSM sister cells to survive with a low frequency. A similar phenotype can be observed in animals treated with dsRNA of the achaete-scute-like bHLH-gene hlh-3 (hlh-3(RNAi)). In addition, hlh-3(RNAi) strongly enhances the NSM sister cell survival phenotype of hlh-2(bx108) mutants, suggesting that hlh-2 and hlh-3 act together to kill the NSM sister cells. A heterodimer composed of HLH-2 and HLH-3, HLH-2/HLH-3, can bind to four E-box motifs (the DNA binding sites for bHLH proteins) in the egl-1 promoter in vitro, which are required for egl-1 expression in the NSM sister cells in vivo. We therefore propose that HLH-2/HLH-3 acts as a direct activator of egl-1 transcription. The death of the NSM sister cells can also be prevented by a gain-of-function mutation (gf) in ces-1 (ces, cell-death specification), which is thought to cause overexpression of CES-1, a Snail-like transcription factor, in the NSM sister cells. We found that the ces-1(gf) mutation blocks this cell-death event by repressing egl-1 expression in the NSM sister cells. Furthermore, in vitro, CES-1 binds to four Snail-binding sites in the egl-1 promoter, which overlap with the four E-box motifs, and the capability of CES-1 to repress egl-1 expression in vivo is dependent on its ability to bind to these sites. We therefore suggest that in ces-1(gf) animals the NSM sister cells survive because CES-1 prevents an activator of egl-1 expression, most likely HLH-2/HLH-3, from binding to the egl-1promoter.Finally, we have identified additional enhancers of the weak NSM sister cell survival phenotype in hlh-2(bx108) mutants. Their characterisation may elucidate new factors required for the specification of the NSM sister cell death and reveal the mechanisms involved in this process.

Phillips, Carolyn M. et al. (2011) International Worm Meeting "Characterization of Transposon Silencing Pathways."

Ruvkun, Gary B., Phillips, Carolyn M.

[International Worm Meeting, 2011]

Small RNA mediated silencing of transposons is an important mechanism to prevent DNA damage and subsequent mutations in the germline. A subset of the exogenous and endogenous small RNA pathways genes have been demonstrated to be involved in transposon silencing. When these genes are mutated, transposons can be excised, leaving a double-strand break, which must be repaired by the cellular machinery, either through homologous recombination or non-homologous end joining. I have developed a strategy to visualize double-strand breaks in the germline generated by unregulated transposons in the transposon silencing mutants. This assay will allow me to look at transposon hopping in mutants that are sterile and possibly to perform a small-scale cytological screen for new genes in the transposon silencing pathway. Additionally, I am generating fluorescently tagged proteins to perform localization analysis of both new and previously characterized genes in this pathway. Localization may identify particular cells where RNA based immunity is important or sub-cellular structures to which these proteins concentrate. Finally, to identify direct binding partners of these proteins and potentially identify additional components of the transposon silencing machinery, I will biochemically purify the components of this pathway. Interacting proteins will be identified by mass spectrometry. Any components identified through this strategy can be further characterized by mutational and localization analysis. These experiments will examine the relationship between the small RNA pathways and genome integrity, and further elucidate the mechanisms of transposon silencing in the germline. CMP is the Marion Abbe Fellow of the Damon Runyon Cancer Research Foundation.

Genta Ohno et al. (2006) East Asia C. elegans Meeting "Visualization of developmentally regulated alternative splicing events by a GFP/RFP based splicing reporter system"

Masatoshi Hagiwara, Hidehito KUROYANAGI, Genta Ohno

[East Asia C. elegans Meeting, 2006]

Alternative splicing produces huge diversity of mRNAs in multicellular organisms. Recently, we have developed a splicing reporter system by utilizing C. elegans as a model organism, and identified trans-acting factors and a cis-acting element regulating tissue-specific expression of egl-15 exon 5s (1). In order to elucidate molecular mechanisms of developmentally regulated alternative splicing in vivo, we are generating alternative splicing reporter worms by utilizing let-2 gene as a model. let-2 encodes α2 (IV) collagen in C. elegans and its mutually exclusive exons were developmentally regulated. mRNA containing exon 9 is predominant in embryos, while mRNA with exon 10 is predominant in adults (2). In order to monitor expression patterns of exons 9 and 10, we amplified exons 8 through 11 of the let-2 genomic DNA, and fused to GFP and RFP. Termination codons were introduced into exon 9 (E9) or 10 (E10) of these constructs so that usage of E9 leads to expression of GFP-fusion protein (E9-GFP) and the usage of E10 results in the expression of RFP-fusion protein (E10-RFP). When expressed in body wall muscles where endogenous let-2 is primarily expressed, reporter expression patterns gradually shift from E9-GFP to E10-RFP along with the developmental stages, consistent with the splicing patterns of endogenous let-2. When expressed under unc-51 promoter, which drives expression in a variety of tissues, transgenic worms showed differential expression pattern of GFP and RFP. These observations suggest that ectopically expressed let-2 exon 9/10 reporters are differentially spliced in developmentally regulated as well as tissue-specific manners. Through EMS mutagenesis and color-based screening, we isolated several lines of mutants that remain to express E9-GFP in body wall muscles until the adult stage. We are currently mapping the gene and are also identifying cis-elements regulating developmental and tissue-specific property of the let-2 reporter. 1. Kuroyanagi H. et al. Nature Methods, (in press). 2. Marion H. et al. JCB, (1993).