Liddell S et al. (1995) International C. elegans Meeting "EXTRACELLULAR AND CYTOPLASMIC Cu/Zn SUPEROXIDE DISMUTASES FROM HAEMONCHUS CONTORTUS, A NEMATODE PARASITE OF SHEEP"

Knox DP, Liddell S

[International C. elegans Meeting, 1995]

Antioxidant enzymes are postulated to play an important role in parasite defence against the cellular, oxygen-mediated killing mechanisms of the host. In particular, recent reports of the presence of an extracellular form of the enzyme superoxide dismutase (SOD) in several parasitic helminths have fuelled speculations that secreted SODs contribute to parasite survival by acting as both antioxidants and as anti-inflammatory agents. We have isolated full length cDNAs encoding two forms of Cu/Zn SOD from the gastrointestinal ovine nematode H. contortus. The predicted amino acid sequences suggest that one class represents a cytoplasmic enzyme (SODc), whilst the other class (SODe) contains an additional 33 amino acids at the N-terminus with the characteristics of a signal sequence and therefore may represent an extracellular form of the enzyme. The deduced amino acid sequences share a higher degree of homology with the C. elegans SODs than with any other reported SOD sequences; SODc is 78% identical to SOD-1, and SODe is 62% identical to the second C. elegans Cu/Zn SOD (F55H2.1). All of the residues involved in catalysis, metal ion binding and intrachain di-sulphide bonding are conserved in the H. contortus SODs. The entire coding sequence of each cDNA was cloned into the E. coli expression vector pET22b(+). In the case of SODc, a soluble active enzyme was obtained. An antiserum raised against purified recombinant SODc reacted with two proteins with estimated molecular masses of 19.5 and 22 kDa on Western blots of extracts from adult parasites.

CJ Ceol et al. (1999) International C. elegans Meeting "lin-55 DP and an E2F-like gene act in the lin-35 Rb pathway to antagonize let-6 ras signaling during vulval induction"

CJ Ceol, HR Horvitz

[International C. elegans Meeting, 1999]

The Ras signaling pathway of vulval induction is antagonized by the synthetic multivulva (synMuv) genes. On the basis of genetic interactions, the synMuv genes have been grouped into two classes, A and B. An animal is Muv if it has mutations in both a class A gene and a class B gene, whereas an animal has a wild-type vulva if a mutation is present in a gene or genes of a single class. lin-35 , a class B synMuv gene, encodes a protein similar to the mammalian tumor suppressor pRb and the related proteins p107 and p130 (Lu and Horvitz, 1998, Cell 95 : 981-991). A well-characterized binding partner of pRb is the DP/E2F heterodimeric transcriptional activator. pRb/DP/E2F complexes are thought to repress transcription of DP/E2F-responsive genes, including genes required for S phase cell-cycle progression. We are determining the roles of DP and E2F proteins in the specification of vulval cell fates. We cloned the class B synMuv gene lin-55 and have found that it encodes a protein similar to the DP family of transcription factors. To investigate the null phenotype of lin-55 , we isolated a deletion allele, n3316 . In addition to a synMuv phenotype, lin-55(n3316) causes maternal-effect lethality. We have also identified two C. elegans E2F-like genes. A deletion allele, n3318 , of a gene we are temporarily calling C.e. E2F-1 behaves like a class B synMuv mutation. n3318 also causes maternal-effect lethality. We are currently comparing the lethal phase of n3318 embryos with that of lin-55(n3316) embryos and are investigating the role of the other E2F-like gene in vulval development and embryogenesis. We propose that LIN-55 and C.e. E2F-1 form a complex with LIN-35 Rb and other class B synMuv proteins and that this complex represses transcription of genes that promote vulval development.

Snider, Spencer et al. (2021) International Worm Meeting "DREAM interrupted: Using CRISPR/Cas9-targeted mutagenesis to assess DREAM complex formation and function"

Hurt, Garret, Richards, Alex, Snider, Spencer, Goetsch, Paul

[International Worm Meeting, 2021]

Formation of the highly conserved 8-subunit DREAM (Dp, Retinoblastoma-like, E2F, and MuvB) complex culminates in direct repression of cell cycle and developmental genes. We are interested in what determinants of DREAM complex formation are essential for transcriptional repression of target genes. Structural studies on mammalian proteins identified key interaction interfaces between the 3 major components of DREAM, the E2F-DP transcription factor heterodimer (EFL-1 and DPL-1), the Retinoblastoma-like pocket protein (LIN-35), and the 5-subunit MuvB subcomplex (LIN-9, LIN-37, LIN-52, LIN-53, and LIN-54). For example, MuvB interacts with LIN-35 via an LxCxE interacting motif on LIN-52. LIN-35 also interacts directly with the EFL-1 transactivation domain via its pocket domain. DNA-binding by the E2F-DP transcription factor heterodimer and the MuvB subunit LIN-54 coordinate DREAM complex localization to chromatin. We hypothesize that MuvB chromatin occupancy, aided and stabilized by its association with E2F-DP and the pocket protein, establishes and maintains target gene repression. Using CRISPR/Cas9-targeted mutagenesis, we disrupted the interaction between MuvB and the C. elegans pocket protein LIN-35. We expected that severing MuvB from the complex would destabilize DREAM component assembly on chromatin and its repression of target genes. Instead, we observed that disrupting LIN-35-MuvB association did not affect DREAM chromatin occupancy, but we did observe upregulation of DREAM target genes. To continue our evaluation of our model of DREAM complex formation, we identified the conserved amino acid residues that likely mediate the protein-protein and protein-DNA associations for C. elegans DREAM. This analysis lays the groundwork for future application of our CRISPR/Cas9 functional genomics pipeline to establish how the molecular events that drive DREAM complex function contribute to target gene repression.

BD Page et al. (1999) International C. elegans Meeting "A Muv connection to early embryogenesis"

BD Page, DA Waring, JR Priess

[International C. elegans Meeting, 1999]

In screens for maternal-effect lethal mutants defective in specifying early cell fates, we isolated one allele each of genes we call mex-2 and mex-4 . The mex-2 and mex-4 mutants have very similar early embryonic phenotypes. In both mex-2 and mex-4 mutant embryos, the AB blastomere produces tissues similar to those normally generated by the EMS blastomere. In wild-type embryos, an isolated AB produces neurons and hypodermal cells. In contrast, an isolated AB from a mex-4 embryo produces body wall muscle (90%), pharyngeal (90%) and intestine (10%) cells. Such a phenotype is suggestive of misregulation of SKN-1, a protein important for specifying these three tissue types. Indeed both mex-2 and mex-4 embryos have a higher than wild-type level of SKN-1 protein in AB and its daughters. This result suggests that mutations in these genes affect expression of maternal transcripts. The mex-4 gene encodes a homologue of the vertebrate DP-1 protein. DP-1 is a member of a transcription heterodimer complex and its dimerization partner is E2F. Multiple E2F homologues are encoded in the C. elegans genome; one of these genes is very close to the genetic position of the mex-2 gene. I have found that the mex-2 mutant has a missense mutation in the E2F gene. How do E2F and DP-1 affect early cell fate in the embryo? In other organisms, DP-1 and E2F-1 cooperate to link cell cycle progression with transcription. We have not observed any cell cycle defects in mex-4 mutant embryos, and the gonad of a mex-4 hermaphrodite appears normal. Thus, we think that these genes are not affecting cell fates by influencing the cell cycle. Both mex-2 and mex-4 genes have roles in postembryonic development. These genes were independently isolated by the Horvitz lab based on their role in the synthetic multivulva pathway. Since ras mutations can suppress the synMuv phenotype, I tested whether ras mutations could also suppress the embryonic defect of mex-4 mutants. Preliminary experiments indicate that suppression occurs. This result suggests that parallels exist between vulva induction and embryogenesis.

Ceol CJ et al. (1998) East Coast Worm Meeting "lin-55 DP AND A C. ELEGANS E2F-LIKE GENE ACT IN A PATHWAY WITH lin-35 Rb TO NEGATIVELY REGULATE VULVAL INDUCTION"

Ceol CJ, Horvitz HR

[East Coast Worm Meeting, 1998]

We are studying the mechanisms by which multiple signaling pathways specify the fates of the P(3-8).p vulval precursor cells. The synthetic multivulva (synMuv) genes affect P(3-8).p fate determination by negatively regulating the activity of a receptor tyrosine kinase/Ras pathway that controls vulval induction. The synMuvs are grouped into two classes, A and B. AB double mutants are Muv, whereas AA and BB double mutants and A and B single mutants have wild-type vulvae. Recently, lin-35, a class B synMuv gene, has been shown to encode a protein similar to the pocket proteins pRb, p107 and p130 (see abstract by X. Lu and B. Horvitz). Mammalian cell culture studies have shown that pocket proteins form complexes with the DP and E2F transcriptional activators and that, for most promoters containing E2F binding sites, pocket protein/E2F/DP complexes are transcriptionally inert, suggesting that these complexes may function to repress transcription. We sought to determine if DP and E2F proteins play a role in P(3-8).p cell fate determination. A single allele of lin-55, n2994, was isolated in a screen for class B synMuvs, and was subsequently mapped between unc-4 and rol-6 on LGII (J. Thomas, personal communication). In transformation rescue experiments, a subclone of cosmid C32F7 that contains a single predicted gene rescues the Muv phenotype of lin-55; lin-15A double mutants. The sequence of this gene in n2994 mutants contains a G-to-A splice-acceptor mutation preceding the fifth exon. The predicted gene encodes a protein that is similar to the DP family of transcription factors. Deficiency studies indicate that n2994 is likely a partial loss-of-function allele and that the null phenotype of lin-55 is lethal. We have found that a C. elegans E2F-like gene also inhibits vulval induction. We obtained cDNA clones of this gene, which was identified by the C. elegans genome consortium, and found that the gene encodes a predicted protein that is 20-25% identical to mammalian E2F family members. There is striking conservation in the putative DNA-binding domain, in the domain that interacts with DP family members and in the domain that interacts with pocket proteins. RNAi studies indicate this gene functions as a class B synMuv. Specifically, injection of C. elegans E2F antisense RNA into lin-15A mutants yielded Muv progeny, whereas injection into lin-15B and N2 animals yielded phenotypically wild-type progeny. We cloned the genomic locus of C. elegans E2F and are currently screening for deletion alleles. We propose that LIN-55 and C. elegans E2F form a complex with other class B synMuv proteins and that this complex represses transcription of genes required for P(3-8).p to adopt vulval cell fates.

McKim KS et al. (1991) International C. elegans Meeting "GENETIC ANALYSIS OF SPONTANEOUS DUPLICATION LOSS AND BREAKAGE."

Rose AM, McKim KS

[International C. elegans Meeting, 1991]

Duplications spontaneously break at a frequency which is related to their mitotic stability. We have analyzed the structure and mitotic stability of 32 duplications resulting from spontaneous breakage events. Most of these duplications were derived from pre-existing chromosome I duplications, although three arose from spontaneous breakage of an X-chromosome into which a large piece of chromosome I ( hDp14) had inserted. Where it has been possible to observe a cluster of spontaneous shortening events, only a single exceptional duplication was produced by any one worm. Thus, most of the spontaneous duplications may have formed during meiosis. The structures of the spontaneous duplications were determined by genetic complementation. In general there were no hot spots for breakage. Derivatives from four of the duplications had complex structures. Two of these four progenitors were of spontaneous origin. The structures were most simply resolved by proposing the progenitor duplications were ring chromosomes with a superimposed inversion. Similar analysis of most of the other duplications were consistent with a linear structure. Most of the proposed ring chromosomes were mitotically unstable, suggesting ring structures increase the frequency of chromosome loss. Unlike the other duplications, the spontaneous breakage events of hDp14 were clustered; they all appeared to occur close to or at the site of the chromosome I insertion. Thus, the insertion appeared to create a type of fragile site. Most of the spontaneous duplications remained free. Three, however, had become linked to other autosomes. Like free duplications of chromosome 1, these linked duplications (Dp(I;A)) tended to segregate from the X- chromosome in males. Interestingly, only a subset of males carrying these linked duplications demonstrated Dp(I;A) - X segregation patterns. Half of the males demonstrated random segregation of the Dp( I;A) and the X-chromosome. This was not observed with the free duplications. It is possible that in each individual male, premeiotic events determine if the Dp(I;A) and X-chromosome segregate at meiosis. The mitotic stabilities of the spontaneous free duplications were variable, indicating that they acquired different structures during their formation. Supported by an MRC studentship to K.S. McKim and an NSERC grant to A. M. Rose.

Gal, C et al. (2015) International Worm Meeting "Regulation of gene expression by the DREAM complex."

Latorre, I, Appert, A, Chesney, M, Ahringer, J, Gal, C

[International Worm Meeting, 2015]

The conserved eight subunit DREAM (DP, Retinoblastoma [Rb]-like, E2F, and MuvB) complex is a regulator of cell cycle and quiescence genes and plays an important role in development and disease. The C. elegans DREAM members are LIN-35/Rb-like, EFL-1/E2F, DPL-1/DP1, LIN-8, LIN-37, LIN-52, LIN-53, and LIN-54. Recent work has demonstrated that C. elegans direct DREAM targets are de-repressed in the absence of the Rb-like protein LIN-35 and that high gene body levels of the histone variant H2A.Z are linked with target repression1. However, the mechanism that generates gene body H2A.Z enrichment, and the relationship between DREAM and H2A.Z are not currently understood. Furthermore, the functions and regulatory interactions of individual complex members are not clear. For example, LIN-35 has been demonstrated to be a transcriptional repressor by a number of groups, whilst the DP and E2F proteins have previously been reported to act as activators of transcription2. In addition, mutants of individual DREAM complex members do not all have the same phenotype, indicating different functions. Therefore, we are investigating the contribution of DPL-1 (DP) and EFL-1 (E2F) to DREAM target gene repression. We are also carrying out a GFP reporter screen to identify additional components required for DREAM-mediated repression, to elucidate the mechanisms behind DREAM mediated developmental control.1. Latorre, I., Chesney, M.A., Garrigues, J.M., Stempor, P., Appert, A., Francesconi, M., Strome, S., and Ahringer, J. (2015) 'The DREAM complex promotes gene body H2A.Z for target repression', Genes Dev., 29, 495-5002. Chi, W & Reinke, V. (2006) 'Promotion of oogenesis and embryogenesis in the C. elegans gonad by EFL-1/DPL-1 (E2F) does not require LIN-35 (pRB)', Development, 133, 3147-57.

Craig Ceol et al. (2001) International C. elegans Meeting "IDENTIFICATION AND CHARACTERIZATION OF lin-35 Rb PATHWAY GENES THAT ANTAGONIZE RAS SIGNALING DURING VULVAL INDUCTION"

Bob Horvitz, Craig Ceol

[International C. elegans Meeting, 2001]

The synthetic multivulva (synMuv) class A and class B genes define two functionally redundant pathways that antagonize Ras signaling during vulval induction. Cloned class B synMuv genes encode proteins similar to the mammalian tumor suppressor pRB and the pRB-binding heterodimeric transcription factor DP/E2F. We propose that the class B synMuv proteins act as transcriptional repressors to negatively regulate Ras signaling. Specifically, we propose that a DPL-1 DP/EFL-1 E2F heterodimer binds DNA and recruits LIN-35 Rb and other synMuv proteins involved in chromatin modification, including LET-418 Mi-2, LIN-53 RbAp48 and HDA-1 HDAC, to repress the transcription of genes required for vulval cell-fate specification. To further investigate this RB signaling pathway, we cloned additional known class B synMuv genes and performed a genetic screen to identify new class B synMuv genes. The class B synMuv genes lin-52 and lin-54 were identified by Chip Ferguson and Jeff Thomas, respectively, two former graduate students in our laboratory. We cloned both genes and found that each encodes a protein of unknown function. We obtained candidate null alleles of each gene and are using these alleles to characterize the multivulva, sterile and other abnormalities caused by lin-52 or lin-54 loss of function. We are focusing additional functional studies on lin-54 . We are currently assessing whether a human lin-54 - like gene can functionally substitute for lin-54 and are conducting a range of protein-protein interaction studies with LIN-54 and the human LIN-54-like protein. Together with graduate students Frank Stegmeier and Melissa Harrison we screened for additional class B synMuv genes. Using a lin-15A background, we screened 6,500 mutagenized haploid genomes and obtained at least 51 class B synMuv mutations. Our analyses to date have identified at least five new candidate class B synMuv genes. We will describe our genetic and molecular characterizations of these genes.

Hsu M et al. (1997) International C. elegans Meeting "CHARACTERIZING A LOCUS REQUIRED FOR STRIATED MUSCLE DIFFERENTIATION"

Ahnn JH, Hsu M, Fire A

[International C. elegans Meeting, 1997]

To find embryonically expressed genes with central roles in striated muscle formation, we have previously carried out a deficiency-based screen covering >80% of the genome [1]. Partial or complete striated muscle differentiation was seen in deficiencies for all-but-one of the regions tested. The remaining "myogenic" region, on chromosome V, is defined by five deficiencies, of which sDf33 is the smallest. Homozygote sDf33 embryos made little or none of the striated muscle markers tested, but do express hypodermal, pharyngeal and neural markers. Data from the genome project and a limited set of genetic markers in the region, although useful, were insufficient to directly identify the myogenic locus. To facilitate molecular analysis, we constructed our own physical map by probing pulsed field southern blots with cosmids, YACs, and other donations from the region [2]. This data has been used (tentatively) to narrow the putative myogenic locus to a single telomere-containing restriction fragment. For phenotypic analysis and transgene rescue assays, we needed a balanced sDf33 strain with a minimum of alternate embryonic arrest phenotypes. The commonly used eT1 and nT1 translocation balancers yield high fractions of spurious arrested embryos; hence, we created a set of simpler duplication balancers for the region. Frequent loss of these duplication balancers from sDf33;Dp parents results in a high fraction of sDf33 progeny. We have used a body muscle specific myo-3::gfp construct (thanks to S.Kostas) and antibody staining to further characterize myogenesis in the sDf33 embryos. The arrested embryos express a myo-3::gfp in a small number of cells (0-4). We are currently investigating the derivation of these cells (e.g., are they the myo-3 positive [but nonstriated] intestine-associated muscles). In the future, we hope to use injection rescue assays with the sDf33;Dp strains and cosmid/YAC clones in the region to assign a molecular identity (i.e. a gene) to this myogenic locus. 1. Ahnn & Fire, Genetics 137:483; 2. McKim et al., Genetics 124:115; E.Gilchrist & D.Baillie [unc-60 ] D.Weinshenker & J.Thomas [egl-2 ]; C.Wicky, F.Muller & A.Rose for a VL telomere probe

Manser J (1991) International C. elegans Meeting "mig-10: IN OR OUT?"

Manser J

[International C. elegans Meeting, 1991]

mig-l O III mutants exhibit partially defective embryonic migration of CAN, ALM and HSN neurons and have shortened posterior excretory canals (pec's). Genetic mosaic analysis suggests that mig-lO(+) activity within the excretory cell is neither necessary nor sufficient to effect normal pec outgrowth. Thus, mig-10 may encode a cell- extrinsic component involved in cell movement. Patterns of mosaic Dp loss are consistent with a hypodermal focus, although certain other tissues have not been ruled out. Because the cells whose movements are affected in mig-10 mutants develop in contact with the body wall, a hypodermal focus would not be surprising. mig-l O has been cloned by transformation rescue. Germline microiniection of either of two overlapping cosmid clones, FlOE9 or F49A3, can completely rescue mig-l O(ct41 ) phenotypes. Both cosmids have been used as probes to isolate a set of at least 22 genomic lambda phage clones containing sequences from the overlap. The lambdas are currently being used in transformation rescue experiments designed to narrow mig-10 to a physical interval of manageable size for molecular analysis.