Myers TR et al. (2005) Dev Cell "lin-35 Rb acts in the major hypodermis to oppose ras-mediated vulval induction in C. elegans."

Greenwald I, Myers TR

[Dev Cell, 2005]

Specification of vulval precursor cell (VPC) fates in C. elegans has served as an important signal transduction paradigm. Genetic studies have indicated that a large group of synthetic multivulva (SynMuv) genes, including the Rb ortholog lin-35, antagonizes the activity of the EGF receptor-Ras-MAP kinase pathway during VPC specification. A prevalent view has been that Rb-mediated transcriptional regulation and chromatin remodeling activities act in the VPCs to antagonize Ras activation through effects on promoters of target genes of the EGF receptor-Ras-MAP kinase pathway that promote vulval fates. Here, we have investigated the cellular focus of lin-35 using conventional genetic mosaic analysis and tissue-specific expression. Our results indicate that lin-35 activity is required in the major hypodermal syncytium and not in the VPCs to inhibit vulval fates. LIN-35 Rb may inhibit vulval fates by regulating a signal from hyp7 to the VPCs or the physiological state of hyp7.

Myers TR et al. (2018) Sci Rep "JMJD-1.2 controls multiple histone post-translational modifications in germ cells and protects the genome from replication stress."

Amendola PG, Lussi YC, Salcini AE, Myers TR

[Sci Rep, 2018]

Post-translational modifications of histones, constitutive components of chromatin, regulate chromatin compaction and control all DNA-based cellular processes. C. elegans JMJD-1.2, a member of the KDM7 family, is a demethylase active towards several lysine residues on Histone 3 (H3), but its contribution in regulating histone methylation in germ cells has not been fully investigated. Here, we show that jmjd-1.2 is expressed abundantly in the germline where it controls the level of histone 3 lysine 9, lysine 23 and lysine 27 di-methylation (H3K9/K23/K27me2) both in mitotic and meiotic cells. Loss of jmjd-1.2 is not associated with major defects in the germ cells in animals grown under normal conditions or after DNA damage induced by UV or ionizing irradiation. However, jmjd-1.2 mutants are more sensitive to replication stress and the progeny of mutant animals exposed to hydroxyurea show increased embryonic lethality and mutational rate, compared to wild-type. Thus, our results suggest a role for jmjd-1.2 in the maintenance of genome integrity after replication stress and emphasize the relevance of the regulation of histone methylation in genomic stability.

Myers TR et al. (2007) Proc Natl Acad Sci U S A "Wnt signal from multiple tissues and lin-3/EGF signal from the gonad maintain vulval precursor cell competence in Caenorhabditis elegans."

Myers TR, Greenwald I

[Proc Natl Acad Sci U S A, 2007]

The Caenorhabditis elegans vulva has been a valuable paradigm for defining components of signaling pathways and elucidating how signaling events are coordinated to generate a developmental pattern. Vulval precursor cells (VPCs) are induced to adopt vulval fates in the third larval stage by LIN-3, an EGF-like signal produced by the gonad. Competence to respond to the inductive signal requires that the VPCs do not fuse to the major hypodermal syncytium, hyp7. We found that two Wnt-encoding genes, cwn-1 and egl-20, play a major role in preventing fusion of VPCs with hyp7 in the second larval stage. By using tissue-specific rescue of mig-14/Wntless, which is required for the production of Wnt ligands, we found that Wnt signal produced by multiple tissues, including neurons and muscles, promotes or maintains VPC competence before vulval induction. In addition, through laser ablation and genetic analysis, we provide evidence that LIN-3 signal from the gonad also plays a significant role in preventing VPCs from fusing with hyp7. We propose that Wnt signaling plays a permissive role in preventing VPCs from fusing with hyp7 and reevaluate the roles of Wnt and LIN-3/EGF signaling in competence and induction.

Lussi, Y.C. et al. (2015) International Worm Meeting "The C. elegans KDM5 homolog RBR-2 promotes cell fate acquisition by modifying H3K4 methylation levels at regulatory elements."

Lussi, Y.C., Krag, C., Wong, G., Salcini, A.E., Myers, T.R., Mariani, L.

[International Worm Meeting, 2015]

Methylation levels of histone 3 lysine 4 (H3K4) play a crucial role in transcription regulation and are finely controlled by the action of specific histone methyltransferases and demethylases during development. Here, we address the role of the H3K4me2/3 demethylase RBR-2, the unique member of the KDM5 class of histone demethylases in C. elegans, in cell differentiation during postembryonic development. We found that the enzymatic activity of RBR-2 is required cell-autonomously to regulate the 2deg cell fate acquisition in vulva precursor cells and to achieve correct vulva formation. Genome-wide analysis indicates that RBR-2 is needed for the regulation of H3K4me levels in regulatory regions of the genome, including transcription start sites and enhancer regions. Loss of RBR-2 results not only in aberrant acquisition of H3K4me3 at transcription start sites, but also in loss of enhancer signatures in regulatory regions. Accordingly, RNA sequencing analysis shows both up- and down-regulation of gene transcription, suggesting a complex function of RBR-2 in regulating gene activity.

Bejsovec AM et al. (1988) Worm Breeder's Gazette "antimorphic mutations in the head region of the unc-54 myosin heavy chain gene."

Bejsovec AM, Anderson P

[Worm Breeder's Gazette, 1988]

We are investigating the molecular basis of dominant antimorphic mutations at the unc-54 locus. These dominant-uncoordinated, recessive lethal mutations disrupt the assembly of thick filaments in the body wall muscle. By genetic and ultrastructural criteria, the mutant gene product interacts with both wild-type myosin heavy chain B, the unc-54 gene product, and myosin heavy chain A, the minor heavy chain class found in body wall muscle thick filaments. The strong disruptive effect is mediated by very low levels of full length protein - depending on the allele the amount ranges from less than 2% to approximately 20% of wild-type levels. Following EMS mutagenesis, these antimorphic missense alleles arise at a frequency equal to that of recessive null alleles at the unc-54 locus. To determine what portions of the myosin heavy chain are affected by these mutations, we have employed a technique developed by Myers et al. (Science 230: 1242, 1985) for detecting single base changes in genomic DNA. Base changes have been localized for 10 independently isolated EMS-induced alleles; all 10 show changes in the region of the gene encoding the globular head. [See Figure 1] One allele, unc-54(r342), carries 2 separate point mutations - one in the head and one in the rod portion of the gene. Both mutations have been sequenced and only the one located in the head creates an amino acid substitution. We have used the polymerase chain reaction ( PCR) technique (Saiki et al., Science 239: 487, 1988) to amplify and then sequence without cloning the affected region from mutant genomic DNA. Mutant sequences confirm the accuracy of mismatch positions estimated by the Myers technique. The head mutations sequenced so far create non-conservative substitutions for amino acids that are highly conserved among many myosin heavy chain sequences (including the yeast heavy chain). Thus we have mutationally identified parts of the globular head that are important in thick filament assembly. [See Figure 1]

McArdle K et al. (1998) J Cell Biol "Ca2+-dependent muscle dysfunction caused by mutation of the Caenorhabditis elegans troponin T-1 gene."

Allen TS, Bucher EA, McArdle K

[J Cell Biol, 1998]

We have investigated the functions of troponin T (CeTnT-1) in Caenorhabditis elegans embryonic body wall muscle. TnT tethers troponin I (TnI) and troponin C (TnC) to the thin filament via tropomyosin (Tm), and TnT/Tm regulates the activation and inhibition of myosin-actin interaction in response to changes in intracellular [Ca2+]. Loss of CeTnT-1 function causes aberrant muscle trembling and tearing of muscle cells from their exoskeletal attachment sites (Myers, C.D., P.-Y. Goh, T. StC. Allen, E.A. Bucher, and T. Bogaert. 1996. J. Cell Biol. 132:1061-1077). We hypothesized that muscle tearing is a consequence of excessive force generation resulting from defective tethering of Tn complex proteins. Biochemical studies suggest that such defective tethering would result in either (a) Ca2+-independent activation, due to lack of Tn complex binding and consequent lack of inhibition, or (b) delayed reestablishment of TnI/TnC binding to the thin filament after Ca2+ activation and consequent abnormal duration of force. Analyses of animals doubly mutant for CeTnT-1 and for genes required for Ca2+ signaling support that CeTnT-1 phenotypes are dependent on Ca2+ signaling, thus supporting the second model and providing new in vivo evidence that full inhibition of thin filaments in low [Ca2+] does not require TnT.

Eto K et al. (1999) J Biol Chem "Ca(2+)/Calmodulin-dependent protein kinase cascade in Caenorhabditis elegans. Implication in transcriptional activation."

Kimura Y, Eto K, Tokumitsu H, Arai K, Takahashi N, Muramatsu M, Masuho Y

[J Biol Chem, 1999]

We have recently demonstrated that Caenorhabditis elegans Ca(2+)/calmodulin-dependent protein kinase kinase (CeCaM-KK) can activate mammalian CaM-kinase IV in vitro (Tokumitsu, H., Takahashi, N., Eto, K., Yano, S., Soderling, T.R., and Muramatsu, M. (1999) J. Biol. Chem. 274, 15803-15810). In the present study, we have identified and cloned a target CaM-kinase for CaM-KK in C. elegans, CeCaM-kinase I (CeCaM-KI), which has approximately 60% identity to mammalian CaM-KI. CeCaM-KI has 348 amino acid residues with an apparent molecular mass of 40 kDa, which is activated by CeCaM-KK through phosphorylation of Thr(179) in a Ca(2+)/CaM-dependent manner, resulting in a 30-fold decrease in the K(m) of CeCaM-KI for its peptide substrate. Unlike mammalian CaM-KI, CeCaM-KI is mainly localized in the nucleus of transfected cells because the NH(2)-terminal six residues ((2)PLFKRR(7)) contain a functional nuclear localization signal. We have also demonstrated that CeCaM-KK and CeCaM-KI reconstituted a signaling pathway that mediates Ca(2+)-dependent phosphorylation of cAMP response element-binding protein (CREB) and CRE-dependent transcriptional activation in transfected cells, consistent with nuclear localization of CeCaM-KI. These results suggest that the CaM-KK/CaM-KI cascade is conserved in C. elegans and is functionally operated both in vitro and in intact cells, and it may be involved in Ca(2+)-dependent nuclear events such as transcriptional activation through phosphorylation of CREB.

Cheryl Van Buskirk et al. (2004) West Coast Worm Meeting "A physiological role for let-23/EGFR in C. elegans growth"

Paul Sternberg, Cheryl Van Buskirk

[West Coast Worm Meeting, 2004]

The single EGF receptor homolog in C. elegans, LET-23, has multiple functions during development. LET-23 signals through the ras/MAPK pathway to specify cell fates in the ventral cord, vulva, male tail, and excretory system(1). LET-23 also signals through a PLC/IP3 pathway to regulate ovulation(2). We have observed that overexpression of the EGFR ligand LIN-3 from a heat shock inducible promoter, in addition to affecting the expected cell fates, also slows down growth of the animals as compared to heat-shocked controls. This growth defect, which affects time to adulthood but not the final size of the animal, occurs in response to overexpression of LIN-3 at any developmental stage, and thus is unlikely to be due to a cell fate specification defect. The growth defect is suppressible by mutations in let-23, and we are currently testing known downstream targets of LET-23 for suppression to determine which pathway is involved. We are also investigating possible underlying causes of this growth phenotype and we will present these data along with our genetic analysis of this previously undescribed physiological role for let-23 signaling. 1. Moghal, N.M. and P.W. Sternberg. 2003. The Epidermal Growth Factor System in Caenorhabditis elegans. Exp. Cell Res. 284, 150-159. 2. Clandinin, T.R, DeModena, J.A., and P.W. Sternberg. 1998. Inositol Trisphosphate mediates a RAS-Independent Response to LET-23 Receptor Tyrosine Kinase Activiation in C. elegans. Cell 92, 523-533.

Allen TS et al. (1997) International C. elegans Meeting "Phenotypes of C. elegans Transformed with Mutant Troponin T"

Allen TS, Bucher EA, Kuk E, McArdle K

[International C. elegans Meeting, 1997]

Troponin T (TnT), in combination with tropomyosin, troponin I (TnI), and troponin C, confers Ca2+-sensitivity upon force production in striated muscle. Three loci code for TnT in C. elegans. TnT-1 is defined genetically (mup-2) and encodes the major embryonic body wall muscle isoform (Myers et al. JCB 132: 1061, 1996). TnT-2 (on cosmid F53A9) and TnT-3 (on cosmid C14F5) were uncovered by the genome sequencing project. TnT-2 is expressed in body wall muscle, mainly during larval and adult stages; TnT-3 is expressed mainly in pharyngeal muscle. In common with all other known TnTs, the products of these genes have a motif of four heptad repeats of leucine (putative leucine zippers) that is directly followed by the sequence QxYz, where x and z are charged residues. This small region is believed important for the interaction between TnT and TnI (Tanokura et al. J. Biochem. 93: 331, 1983). We analyzed the phenotype of N2 and mup-2 (e2346ts) worms transformed with mutant TnT-1 either lacking the small region (delQDYD) or having alanines in place of charged residues in the region (QAYA). Both constructs, as high copy number transgenes, cause progressive development of dumpiness. We hypothesize that the dumpy phenotype is caused by hypercontracting muscles (cf. Reiner et al. Genetics 141: 961, 1995). This phenotype is consistent with a function of the conserved QxYz region in inhibiting contraction, possibly through the proposed interactions with TnI.

Patel, R.S. et al. (2019) International Worm Meeting "Effect of Triclosan on Odorant Avoidance."

Patel, R.S., Boyle, D., Myers, E.M.

[International Worm Meeting, 2019]

Triclosan (TCS) is an antimicrobial present in some hygiene and dental products. TCS has been shown to disrupt the SKN-1 oxidative stress response in C. elegans. Paraquat, another chemical that induces oxidative stress, has been shown to increase C. eleganssensitivity to glycerol and octanol. High doses of H2O2, however, have been shown to decrease glycerol avoidance. We wanted to determine whether and how TCS exposure alters octanol sensitivity. C. elegans were cultivated on agar plates with various concentrations (0-10mg/L) of triclosan for 24 hours. After triclosan exposure, an octanol avoidance assay was performed on individual adult worms. The strength of avoidance was measured as the amount of time it took individual worms to move backwards, away from an octanol stimulus. We predicted that if triclosan had similar effects as paraquat, the time it took a worm to avoid octanol should decrease, suggesting an increased sensitivity to octanol. We observed decreased sensitivity to octanol after triclosan exposure, however. This increased response time does not appear to be due to an effect of TCS on movement, since the same TCS treatment causes and increase in body bends (Myers lab, unpublished). A population assay was conducted as well where groups of worms were exposed to triclosan. Avoidance of the population was measured after 30 minutes. A similar decrease in octanol sensitivity was observed. This suggests that TCS, like high doses of H2O2, compromises octanol sensitivity.