Catherine A Mastroieni et al. (2004) West Coast Worm Meeting "ANALYSIS OF MAP KINASE PHOSPHORYLATION SITES IN THE C. ELEGANS TRANSCRIPTION FACTOR LIN-31"

Corey A Morris, Catherine A Mastroieni, Leilani M Miller

[West Coast Worm Meeting, 2004]

Complex signaling pathways are required for the proper specification of cell fates. In the C. elegans hermaphrodite, six cells of initially equivalent developmental potential respond differentially to a Ras/MAP kinase signaling pathway, correctly choosing their respective fates to form the adult vulva. Activation of the Ras/MAP kinase pathway culminates in the phosphorylation of one or more transcription factors by MAP kinase, resulting in the induction of fate-specific genes. One of the transcription factors phosphorylated by MAP kinase is LIN-31, a winged-helix transcription factor (Miller et al., Genes Dev. 7:933, 1993) that contains four consensus MAP kinase phosphorylation sites. Co-immunoprecipitation experiments by Tan et al. (Cell 93:569, 1998) showed that LIN-31 forms a heterodimer with the LIN-1 ETS transcription factor and that this heterodimer is disrupted upon phosphorylation of LIN-31 by MAP kinase. These results support the following model in which LIN-31 has two functions: 1) in its unphosphorylated, dimerized form LIN-31 promotes non-vulval cell fates, and 2) in its phosphorylated, undimerized form LIN-31 promotes vulval cell fates. Consistent with the model that phosphorylation of the MAP kinase consensus sites is necessary for LIN-31 function in promoting vulval cell fates, previous studies (Tan et al., 1998) have shown that deletion of all four phosphorylatable sites in LIN-31 results in defective vulval formation. Since the core sequence for MAP kinase consensus sites (S/T-P) consists of only two amino acids, many "consensus" sites identified by sequence alone may not actually be phosphorylated by MAP kinase. Although the first MAP kinase phosphorylation site in the LIN-31 protein is a "full" site (P-X-S/T-P) and Tan et al. (1998) showed that removal of this site resulted in a 50% decrease in overall phosphorylation of the LIN-31 protein, the other three sites contain only the "core" sequence. We are using site-directed mutagenesis, protein expression and purification, and phosphorylation experiments to investigate the phosphorylation pattern of the putative MAP kinase phosphorylation sites in the LIN-31 protein.

Ewbank JJ et al. (2000) European Worm Meeting "Using C. elegans to identify Serratia marcescens virulence factors"

Ewbank JJ, Kurz CL, Finlay BB, Celli J

[European Worm Meeting, 2000]

Tan and Ausubel, working with the enterobacterium Pseudomonasaeruginosa, have established C. elegans as a model for the study of pathogenesis and host defences. Clearly, the use of the worm as a model for studying pathogenicity will be limited to those pathogens that are able to infect the worm. Luckily, in this respect, its susceptibility to P. aeruginosaappears not to be an isolated case. A second opportunistic human pathogen, Serratia marcescens, is also capable of infecting C. elegans. Like P. aeruginosa, S. marcescens is able to infect a broad range of plant and animal hosts and has been used as a model pathogen in studies of Drosophila innate immunity. Using a strain of S. marcescensthat expresses GFP, we have been able to follow the infection process. The bacteria are able to survive within the usually hostile environment of the nematode intestine, proliferate and kill the host. Under standard assay conditions, the progression of the infection is highly reproducible. We have used a transposon mutagenesis system to create a library of insertion mutants of S. marcescens. We are currently screening these mutant bacterial clones individually for those showing reduced virulence.Of the first 2000 mutants screened, 18 showing markedly reduced virulence have been retained for further study. The molecular characterization of these mutants may reveal novel virulence factors that represent potential drug targets. Tan MW, Ausubel FM. (2000) Caenorhabditis elegans: a model genetic host to study Pseudomonas aeruginosa pathogenesis. Curr Opin Microbiol. S: 29-34.

C Leopold Kurz et al. (2001) International C. elegans Meeting "Identifying universal Serratia marcescens virulence factors using C. elegans."

Jonathan Ewbank, Jean Celli, Dominique Ferrandon, B Brett Finlay, Emmanuel Andres, Sophie Chauvet, C Leopold Kurz, Mitchell Uh

[International C. elegans Meeting, 2001]

Tan and Ausubel, working with the enterobacterium Pseudomonas aeruginosa , have established C. elegans a s a model for the study of pathogenesis and host defences . A second opportunistic human pathogen, Serratia marcescens , is also capable of infect ing C. elegan s . Like P. aeruginosa , S. marcescens is able to infect a broad range of plant and animal hosts. Using a strain of S. marcescens that expresses GFP, we have been able to follow the infection process. T he bacteria are able to survive within the usually hostile environmen t of the nematode intestine, proliferate and kill the host. Under standard assay conditions, the progression of the infection is highly reproducible. We have used a transposon mutagenesis system to create a library of insertion mutants of S. marcescens . We are currently screening these mutant bacterial clones individually for those showing reduced virulence . Of the first 2000 mutants screened, 9 showing markedly reduced virulence have been retained for further study. T he molecular characterization of these mutants has revealed novel virulence factors. In order to determine whether these virulence factors are specific to the infection of the nematode, we have also tested them in a Drosophila infection model and found that 4 of them are attenuated for their virulence. Tests in a mammalian model will reveal whether we have identified virulence factors that are important irrespective of the host. Tan MW, Ausubel FM. (2000) Caenorhabditis elegans : a model genetic host to study Pseudomonas aeruginosa pathogenesis. Curr Opin Microbiol. 3: 29-34.

Eva L Mora-Blanco et al. (2001) International C. elegans Meeting "SITE-DIRECTED MUTAGENESIS OF LIN-31, A WINGED-HELIX TRANSCRIPTION FACTOR INVOLVED IN CAENORHABDITIS ELEGANS VULVAL DEVELOPMENT"

Leilani M Miller, Eva L Mora-Blanco, David B Doroquez, Yvonne Yang, Jill Haggerty

[International C. elegans Meeting, 2001]

In order to understand cell fate specification during vulval development, we are conducting a structure/function analysis of LIN-31, a member of the winged-helix family of transcription factors, which is required for the proper specification of vulval cell fates in C. elegans (Miller et al. , Genes and Dev., 7:933, 1993). The LIN-31 protein contains a DNA-binding domain, an acidic region, four MAP kinase consensus phosphorylation sites, and a small region of homology conserved among other winged-helix proteins. We are using site-directed mutagenesis to create plasmids carrying specific mutations in the MAP kinase consensus phosphorylation sites, acidic domain, or the homology region of the LIN-31 protein. These plasmids are then injected into an animal with no functional LIN-31 protein to test for their ability to provide LIN-31 function. LIN-31 is phosphorylated by the MAP kinase MPK-1 in response to an inductive signaling event (Tan et al. , Cell 93: 569, 1998). Tan et al . have already shown that removing all four MAP kinase consensus phosphorylation sites inactivates one of LIN-31's functions, but the individual contribution of each site is not known. Four individual clones are being created, with each one containing a different disrupted MAP kinase consensus phosphorylation site. In addition, it is appealing to imagine that the small acidic domain (six consecutive aspartic acid residues) adjacent to the DNA-binding domain is functioning as a transcriptional activator, as is the case with acidic regions in some other transcription factors. There is no proof, however, that this rather small acidic domain in LIN-31 is even required for function. Five mutant clones, in which different portions of the acidic domain have been removed or replaced will address this question. Finally, the carboxy-terminus of LIN-31 contains a small region of homology that shows similarity to a subset of winged-helix proteins. The function of this homology region is also unknown and is being explored in this project. This structure/function study is especially appealing since the current model (Miller et al. , 1993; Tan et al. , 1998; and Miller et al. , Genetics 156: 1595, 2000) proposes that LIN-31 has two functions: 1) to activate vulval cell fates in P5.p, P6.p, and P7.p and 2) to repress vulval cell fates in P3.p, P4.p, and P8.p. Microinjection of these clones into lin-31(null) animals will allow us to test if each specific mutation disrupts one, both, or none of LIN-31's functions. Thus, this approach will allow us to identify the roles of specific domains or sites in the LIN-31 protein.

Sandra S Slutz et al. (2003) International Worm Meeting "Knowing your enemy: Pathogen specific responses in C. elegans"

Man-Wah Tan, Sandra S Slutz

[International Worm Meeting, 2003]

Caenorhabditis elegans is emerging as a new organism in which to study host-pathogen interactions. Its usefulness as a host-pathogen model is due to: 1) its general strengths as a genetically tractable organism and 2) its natural history as a soil-dwelling nematode which would have placed it in contact with pathogens and necessitated the evolution of defense mechanisms. Furthermore, studies in other invertebrate host-pathogen models, such as Drosophila, have elucidated pathways which are conserved in mammals thus leaving open the possibility that findings in worms will also contribute to the understanding of human-pathogen interactions.Previous screens for C. elegans strains with an enhanced susceptibility to pathogen (Esp) identified both the p38 MAP kinase pathway and the Sma/Mab TGF pathway as important for innate immunity against Pseudomonas aeruginosa. Further characterization of mutants in these pathways showed that these genetic cascades confer defense against multiple types of bacteria (Tan, 2001; Kim et al., 2002; Aballay et al., 2003). However, it has been shown that mammals and insects have both general and specific pathogen response pathways. Given the broad response range of the currently known C. elegans immunity pathways, the question remains whether C. elegans also has pathogen specific responses. In order to address this question we have conducted new Esp screens using both Enterococcus faecalis, a Gram-positive bacterium, and P. aeruginosa, a Gram-negative bacterium. Strains which die more rapidly than N2 when exposed to either E. faecalis or P. aeruginosa are considered to have an Esp phenotype. We have confirmed the Esp phenotype of our mutants and sorted them into classes based on whether they are susceptible to both Gram-positive and Gram-negative pathogens, Gram-positive bacteria only, or Gram-negative microbes only. All three classes of Esp mutants have been isolated. Here we report on our progress in cloning and mapping the corresponding genes. Aballay et al. (2003) Curr Biol 13:47-52; Kim et al. (2002) Science 297:623-626; Tan M-W (2001) Pediatric Pulmonology 32:96-97.

Catherine A Mastroieni et al. (2005) International Worm Meeting "Analysis of MAP kinase phosphorylation sites in the C. elegans transcription factor LIN-31."

Catherine A Mastroieni, Leilani M Miller, Corey A Morris

[International Worm Meeting, 2005]

Complex signaling pathways are required for the proper specification of cell fates. In the C. elegans hermaphrodite, six cells of initially equivalent developmental potential respond differentially to a Ras/MAP kinase signaling pathway, correctly choosing their respective fates to form the adult vulva. Activation of the Ras/MAP kinase pathway culminates in the phosphorylation of one or more transcription factors by MAP kinase, resulting in the induction of fate-specific genes. One of the transcription factors phosphorylated by MAP kinase is LIN-31, a winged-helix transcription factor (Miller et al., Genes Dev. 7:933, 1993) that contains four consensus MAP kinase phosphorylation sites. Previous studies by Tan et al. (Cell 93:569, 1998) have shown that deletion of all four putative phosphorylation sites in LIN-31 results in a vulvaless animal. Since the core sequence for MAP kinase consensus sites (S/T-P) consists of only two amino acids, many "consensus" sites identified by sequence alone may not actually be phosphorylated by MAP kinase. Although the first MAP kinase phosphorylation site in the LIN-31 protein is a "full" site (P-X-S/T-P) and Tan et al. (1998) showed that removal of this site resulted in a 50% decrease in overall phosphorylation of the LIN-31 protein, the other three sites contain only the "core" sequence. Using site-directed mutagenesis and microinjection, we are exploring the effect of these sites on LIN-31 function in vivo. Disruption of individual phosphorylation sites does not appear to have a dramatic effect on LIN-31 function in vivo, with the exception of site #4. Disruption of this site produced a multivulva phenotype. Because of this unexpected result, we are currently constructing multiple disruptions to determine what combination of sites are important for function. In addition, in order to investigate the actual phosphorylation pattern of the putative MAP kinase phosphorylation sites, we are also expressing wild-type and mutant LIN-31 in mammalian cells and performing in vitro phosphorylation experiments.

Webster, Rachel et al. (2021) International Worm Meeting "The role of CDK-4 in cell size and metabolism"

Auld, Douglas, Hilfinger, Andreas, Liu, Shixuan, Ginzberg, Miriam, Tan, Ceryl, Topisirovic, Ivan, Concannon, John, Derry, W. Brent, Iyengar, Seshu, Jenkins, Jeremy, Webster, Rachel, Rost, Hannes, Kafri, Ran, Wang, Yuan, Papadopoli, David, Patel, Nish

[International Worm Meeting, 2021]

The cell cycle is driven by the tightly-choreographed activity of cyclins and their catalytic partners, cyclin-dependent kinases (CDKs). While the roles of these complexes in cell cycle progression have been thoroughly characterized, more recent work has identified distinct functions of cyclins and CDKs in cellular processes such as differentiation, DNA repair, and metabolism. I am interested in understanding how cell size is regulated, and how a cell knows what size to be. In mammals, cell size is largely controlled through the coordination between growth rate and time spent in the G1 phase of the cell cycle. However, we have recently shown that CDK4, acting in G1, asserts a unique influence by which it dictates the size to which a cell aims to grow, or its target size, an ability not shared by other G1 CDKs. Inhibition of CDK4 results in a population of cells with uniformly increased size, while increased CDK4 activity causes cells to be uniformly small. To investigate the physiological implications of this phenomenon, we have turned to the tractability and wealth of genetic tools offered in C. elegans. Knockdown of the C. elegans CDK4 homolog cdk-4 or its conjugate cyclin cyd-1 is sufficient to increase the size of both the nucleus and nucleolus in seam cells, used here as a proxy for cell size due to the scaling of these organelles with cell size through development. Preliminary work also suggests that while overall metabolism is increased, fat stores are depleted in worms with reduced CDK-4 activity. We seek to understand the mechanisms behind this accumulation of cell mass, and the consequences of our observed changes in metabolism on the adult worm.

Corey A Morris et al. (2003) International Worm Meeting "Site-directed mutagenesis of the transcription factor LIN-31 reveals functional domains required in the regulation of vulval cell fates."

David B Doroquez, E Lorena Mora-Blanco, Leilani M Miller, Yvonne Yang, Corey A Morris

[International Worm Meeting, 2003]

In order to better understand cell fate determination in Caenorhabditis elegans, we are conducting a functional analysis of LIN-31, a winged-helix transcription factor (WH TF) that acts as a tissue-specific effector of the conserved Ras/MAP kinase signaling pathway to promote or suppress vulval cell fates in the development of the hermaphrodite vulva (Miller et al., Genes and Dev., 7:933, 1993). In addition to a DNA-binding domain (DBD), the LIN-31 protein contains several regions of interest: a small acidic-rich region, four MAP kinase consensus phosphorylation sites, and a small region at the C-terminus that displays homology with a subset of WH proteins. These regions could play a number of roles, from transcriptional activation to an interaction domain for LIN-1, which is known to heterodimerize with LIN-31 (Tan et al., Cell, 93:569,1998). Using site-directed mutagenesis techniques, specific mutations were introduced into the gene at these regions of interest. Stable transgenic lines were created through germline microinjection of mutant plasmids into animals with no functional LIN-31. Through phenotypic analysis of multiple transgenic lines, we are beginning to better understand the functional significance and contribution of each of these different sites to LIN-31 function. Our results thus far support the current model (Miller et al., 1993; Tan et al., 1998; Miller et al., Genetics, 156:1595, 2000), that LIN-31 has two functions: 1) to activate vulval cell fates in P5.p, P6.p and P7.p; and 2) to repress vulval cell fates in P3.p, P4.p, and P8.p.In addition, we are initiating an in vitro functional analysis of LIN-31 protein. We used a bacterial expression system to produce GST::LIN-31 fusion protein. Using electrophoretic mobility shift assays, we have determined that wild-type LIN-31 protein is able to specifically bind the promoter of another WH TF target. LIN-31's ability to interact with this promoter was disrupted when 1) LIN-31 carried a previously characterized point mutation in the DBD believed to disrupt its interaction with the target DNA (Miller et al., 2000) and 2) when the promoter sequence contained base substitutions. We are now in the process of creating, expressing, and purifying mutant GST::LIN-31 fusion proteins in order to investigate LIN-31 sequences required for heterodimerization with LIN-1.

Corey A Morris et al. (2002) West Coast Worm Meeting "SITE-DIRECTED MUTANTS OF THE C. elegans TRANSCRIPTION FACTOR LIN-31 REVEAL DISTINCT FUNCTIONS."

Corey A Morris, E Lorena Mora-Blanco, Yvonne Yang, David B Doroquez, Leilani M Miller

[West Coast Worm Meeting, 2002]

In order to better understand cell fate determination in Caenorhabditis elegans, we are conducting a functional analysis of LIN-31, a winged-helix transcription factor (WH TF) that acts as a tissue-specific effector of the conserved Ras/MAP kinase signaling pathway to promote or suppress vulval cell fates in the development of the hermaphrodite vulva (Miller et al., Genes and Dev., 7:933, 1993). In addition to a DNA-binding domain (DBD), the LIN-31 protein contains several regions of interest: a small acidic-rich region, four MAP kinase consensus phosphorylation sites, and a small region at the C-terminus that displays homology with a subset of WH proteins. These regions could play a number of roles, from transcriptional activation to an interaction domain for LIN-1, which is known to heterodimerize with LIN-31 (Tan et al., Cell, 93:569,1998). Using site-directed mutagenesis techniques, specific mutations were introduced into the gene at these regions of interest. Stable transgenic lines were created through germline microinjection of mutant plasmids into animals with no functional LIN-31 protein. Through phenotypic analysis of multiple transgenic lines, we are beginning to better understand the functional significance and contribution of each of these different sites to LIN-31 function. Our results thus far support the current model (Miller et al., 1993; Tan et al., 1998; Miller et al., Genetics, 156:1595, 2000), that LIN-31 has two functions: 1) to activate vulval cell fates in P5.p, P6.p and P7.p; and 2) to repress vulval cell fates in P3.p, P4.p, and P8.p. In addition, we are initiating a functional analysis of LIN-31 protein using two assays: ability to bind a putative DNA target sequence and ability to heterodimerize with LIN-1. We used a bacterial expression system to produce GST::LIN-31 fusion protein. Using gel-shift assays, we confirmed function of wild-type protein by demonstrating its ability to bind the transthyretin (TTR) promoter, a consensus sequence recognized by HNF-3, another WH TF sharing DBD sequence homology (Costa et al., Mol. Cell. Biol., 9:1415, 1989). We are now in the process of creating, expressing, and purifying GST::LIN-31 fusion proteins carrying specific mutations, including two point mutations in the DBD believed to disrupt interaction of the LIN-31 with its target DNA (Miller et al., 2000). These mutant proteins will allow us to test in vitro their ability to bind the TTR promoter and to heterodimerize with LIN-1.

Christensen S et al. (1995) International C. elegans Meeting "lag-1 APPEARS TO BE A TRANSCRIPTION FACTOR IN THE glp-1 SIGNALING PATHWAY.S"

Friedman LC, Christensen S, Kimble JE, Bosenberg M, Kodoyianni V

[International C. elegans Meeting, 1995]

lin-12 and glp-1 are members of a receptor family that includes Drosophila Notch and human Tan-1. Loss of function mutations in lag-1 mimic the lin-12 glp-1 double mutant phenotype suggesting that these genes function in a common signal transduction pathway. Genomic clones that rescue the lag-1 homozygous phenotype contain a gene spanning > 15 kb. To verify that this locus is lag-1 we have identified a nonsense mutation which eliminates the conserved C-terminus of the predicted protein in a strong allele, lag-1(q385). By in situ hybridization, lag-1mRNA is strongly expressed in the hermaphrodite germline, consistent with the idea that lag-1 acts downstream of lin-12 and glp-1 during signal transduction. The lag-1 transcript is 2.6 kb and encodes a protein homologous to Drosophila Supressor of Hairless and mammalian RBPJk. Proteins in this gene family bind to a highly conserved DNA sequence and function as transcriptional regulators. There are multiple copies of the SU(H)/RBPJk consensus binding site in lin-12, glp-1 and lag-1 itself, but not in lag-2, suggesting that lag-1 may mediate feedback regulation in this pathway. As a first step in characterizing the regulatory role of lag-1, we are testing the DNA binding activity of the in vitro translated protein by gel mobility shift assay.