Mark R Engle et al. (2002) Mid-west Worm Meeting "CeGSTP2-2, an invertebrate glutathione S-transferase with activity for 4-hydroxynonenal: possible roles in stress resistance and aging"

Piotr Zimniak, Robert Shmookler Reis, Mark R Engle, Eva Liebau, Srinivas Ayyadevara, Rajani Ayyadevara, Sharda Singh

[Mid-west Worm Meeting, 2002]

Reactive oxygen species (ROS) and oxidative stress are the inevitable consequence of aerobic metabolism. Enhancing organismal defenses that confer resistance to ROS, the primary initiators of the oxidative stress cascade, have been shown to play a pivotal role in extending the life span of invertebrates. In Drosophila, overexpression in motor neurons of superoxide dismutase-1 (SOD-1) can extend longevity by up to 40%. Similarly, characterization of the age-1 mutation in the nematode Caenorhabditis elegans demonstrated that the long-lived mutants have increased late life expression of catalase and SOD. These enzymes are thought to attenuate oxidative damage by lowering the level of initiating ROS. The mechanism by which ROS exerts damaging effects that ultimately lead to a shortened life span is not completely understood. The OH radicals can react with proteins and DNA directly, although more detrimental maybe their ability to initiate the lipid peroxidation cascade. This occurs when ROS react with poly-unsaturated fatty acids on the membrane in a self-propagating mechanism that can amplify the original insult 100-1000 fold. The resulting lipid hydroperoxides are oxidants themselves but they can decompose to form toxic electrophiles such as 4-hydroxynonenal (4HNE). 4HNE has a relatively long half-life, is diffusible, and can react with proteins and DNA leading to cellular dysfunction. Glutathione S-transferases are a multifunctional family of phase II detoxification enzymes that catalyze the conjugation of glutathione to toxins in order to make them more soluble and consequently less toxic. We have recently cloned and characterized a pi-class (P) C. elegans GST with a high level of activity for 4-HNE. Although this enzyme accounts for less than 20% of the total 4-HNE activity of worm homogenate, it appears to be highly expressed in the neurons (figure 1). It therefore may play a significant role in neuronal defense against toxic electrophiles. Because of the electrophilic protection afforded to this organism by the 4-HNE-conjugating activity of CeGSTP2-2, it is our hypothesis that nematodes with increased levels of CeGSTP2-2 may have increased resistance to stress and aging. Consistent with this hypothesis, we found that long-lived daf-2 mutants show increased expression of CeGSTP2-2.

David J Katz et al. (2005) International Worm Meeting "Investigating the link between NANOS and chromatin for germ cell maintenance in C. elegans"

William G Kelly, David J Katz

[International Worm Meeting, 2005]

In Drosophila, nanos is required for primordial germ cells (PGCs) to differentiate into a functional germ line. Lack of NANOS function causes defects in germ cell migration, differentiation, and quiescence and ultimately leads to sterility in both males and females. Similarly, in mice a lack of nanos3 results in the complete loss of germ cells in both sexes (Tsuda et al. 2003) and functional loss of two of the three nanos genes in C. elegans, nos1 and nos2, also results in sterility (Subramaniam and Seydoux, 1999). It has therefore been proposed that nanos is a conserved master regulator of the germ cell fate. In the C. elegans early embryo, the chromatin in all cells contain the active chromatin mark, histone H3 methylated on lysine 4 (H3meK4). However upon their birth, the primordial germ cells Z2 and Z3 dramatically lose this mark. This loss of the active H3meK4 mark has been suggested to be part of a chromatin-based transcriptional repression mechanism. Remarkably, absence of the H3meK4 mark has been shown to be dependent upon nanos activity (Schaner et al. 2003). Thus it is possible that nanos may be partially affecting its role in maintaining the undifferentiated germ cell fate through a chromatin-based mechanism. We aim to explore this potential link by further characterizing the role of C. elegans nanos in germ cell fate. To this end, we are taking both forward and reverse genetic approaches as well as conducting ectopic expression studies.

Snyder, Matthew P. et al. (2013) International Worm Meeting "Turnover of the H3K9me2 Mark During Late Spermatogenesis."

Snyder, Matthew P., Xu, Xia, Maine, Eleanor

[International Worm Meeting, 2013]

Meiotic silencing is a conserved phenomenon targeting unpaired chromosomes and chromosomal regions during prophase of meiosis I. Meiotic silencing in animals typically occurs at the chromatin level and involves accumulation of histone modifications thought to promote a closed chromatin configuration. This chromatin structure may contribute to transcriptional repression and meiotic chromosomal events such as chromosome disjunction (Bean et al 2004, Jaramillo-Lambert and Engebrecht 2010). During meiosis in C. elegans, non-synapsed chromosomes are enriched for H3K9me2 relative to synapsed chromosomes (Kelly et al. 2002; Bean et al. 2004). Such non-synapsed chromosomes include the male X, homologous chromosomes that fail to synapse due to mutation, and chromosomal translocations/duplications. The pattern of H3K9me2 accumulation during meiosis depends on activity of the small RNA machinery, which may have a role in targeting the mark to unpaired chromosomes and ensuring that the mark does not persist abnormally (Maine et al. 2005; She et al. 2009). Taking a combined biochemical/genetic approach, we are identifying additional factors important for regulating H3K9me2 distribution during meiosis. Through this approach, we are identifying genes that appear to be important for turnover of the H3K9me2 mark during late spermatogenesis.

Srinivas Ayyadevara et al. (2005) International Worm Meeting "Life Span and Stress Resistance of C.elegans Are Increased by Expression of Gluthathione Transferase capable of Metabolizing the Lipid peroxidation Product 4-Hydroxynonenal."

Robert J Shmookler Reis, Srinivas Ayyadevara, Abhijit Dandapat, Piotr Zimniak, Sharda P Singh, Mark R Engle, Helen Benes, Cheryl F Lichti, Eva Liebau

[International Worm Meeting, 2005]

Ceanorhabditis elegans expresses a glutathione-S-transferase (GST-10) belonging to the Pi class, that has the ability to conjugate the lipid peroxidation product 4-hydroxynonenal (4 HNE). Transgenic C. elegans strains were generated in which the 5' flanking region and promoter of gst-10 were placed upstream of gst-10 and mGsta4 cDNAs, respectively. mGsta4 encodes the murine mGSTA4-4, an enzyme with high catalytic efficiency for 4 HNE. The localization of both transgenes, determined by immunostaining, was predominantly in a few cells in head and tail. 4 HNE-conjugating activity, assessed in worm lysates, was increased in worms transgenic for mGsta4 or gst-10, relative to controls. Conversely, 4 HNE-protein adducts decreased in the transgenics relative to controls, indicating that the transgenic enzymes were active and effective in limiting electrophilic damage by 4 HNE. Stress resistance and life span were increased in transgenic animals (5 independent lines each), compared to two independent control strains. Our results suggest that electrophilic damage by 4 HNE contributes to C. elegans aging and stress responses.

Gyoergyi Csankovszki et al. (2002) West Coast Worm Meeting "Mapping chromosomal targets of the dosage compensation complex"

Barbara J Meyer, Gyoergyi Csankovszki

[West Coast Worm Meeting, 2002]

The worm C. elegans compensates for the difference in X-linked gene dosage between XX hermaphrodites and XO males by reducing gene expression two-fold in XX animals. Downregulation of gene expression is achieved by the dosage compensation complex (DCC), a large multiprotein complex that binds the two X chromosomes in hermaphrodites. However, the cis-acting chromosomal elements that recruit and bind the complex are not known. Using free and attached X chromosomal duplications we are attempting to identify regions of the X chromosome that are sufficient to recruit the complex. By analyzing the volume and number of DCC-bound chromosomal bodies, a large duplication of the right 30% of X, mnDp10, was shown earlier to be competent to bind the DCC (1). Using combined fluorescent in situ hybridization (to mark X chromosomal territories) and immunofluorescence (to mark chromosomes that bind the dosage compensation complex) we confirmed these results. By analyzing smaller duplications that span the entire chromosome we hope to map chromosomal targets of the DCC.

Pokhrel, Bharat et al. (2017) International Worm Meeting "Characterising C. elegans COMPASS complex in gene expression."

Chen, Ron, Appert, Alex, Pokhrel, Bharat, Clifford, Rosamund, Chen, Yannic, Ahringer, Julie, Dong, Yan, Stempor, Przemyslaw

[International Worm Meeting, 2017]

Trimethylation of histone lysine 4 (H3K4me3) is regarded as an active promoter mark across all eukaryotes. H3K4me3 is mainly deposited by an evolutionarily conserved COMPASS complex that contains two key subunits: SET-2, H3K4me3/2 methyltransferase, and CFP-1, a conserved CpG binding protein. Perturbation of this mark has been found to be associated with different diseases, development defects, and incorrect cell fate specification. However, it is unclear whether H3K4me3 play an instructive role in transcription or just a consequence of gene expression. To address this question, we phenotypically characterised and compared cfp-1(tm6369) mutants with a reported loss-of-function set-2(bn129) mutant allele. We observed dramatic loss of H3K4me3, poor fertility, slow growth and ectopic gene expression in both cfp-1 and set-2 mutants. We found that the role of H3K4me3 is to fine tune gene induction, likely by modulating other histone modifications that play an essential role in gene activation. Our results shed light on the role of H3K4me3 in chromatin regulation and function.

Sujata Bhattacharyya et al. (2008) Development & Evolution Meeting "The Role of Histone Deacetylation in the Maintenance of Transcriptional Repression in Primordial Germ Cells"

Sujata Bhattacharyya, William Kelly

[Development & Evolution Meeting, 2008]

Experimental data from a number of model organisms suggests that transcriptional quiescence is essential for safe-guarding totipotency of the embryonic germ line. In C.elegans, global repression in germline blastomeres is mediated by a maternal protein, PIE-1. Subsequent to its degradation, chromatin-based mechanisms accompanied by epigenetic erasure events suppress transcription in the primordial germ cells, Z2 and Z3. In particular, genome-wide H3K18 acetylation is deleted from Z2 and Z3 within 30 minutes of their birth.The rapidity with which this mark is lost suggests an enzymatic mode of removal. This hypothesis is corroborated by evidence which indicates that loss of H3K18Ac occurs on a normal schedule in the psa-1 mutant which is defective in nucleosome remodeling. Consequently, we are analyzing mutants in each of nine histone deacetylases to determine which in particular is responsible for this erasure event. RNA interference will be used if genetic redundancy is suspected. The functional consequences of inappropriately retaining this mark on the totipotency of the germ line will be elaborated in the appropriate loss-of-function mutant(s).

O'Meara, M. Maggie et al. (2009) International Worm Meeting "MYST family member lsy-12 is required for correct specification of the ASE chemosensory neurons."

Hobert, Oliver, O'Meara, M. Maggie

[International Worm Meeting, 2009]

A large-scale genetic screen for mutants that effect the bilateral specification of the ASE chemosensory neurons revealed six mutants falling into the same complementation group that are required for correct specification of ASEL fate. Acting upstream of a bistable feedback loop required for initiation of ASEL/R fate, lsy-12 also appears to act as an ASEL inducer, having the ability to drive ASEL cell fate. Mapping, cloning, rescue, and RT-PCR experiments confirm that lsy-12 is a single gene product coded from R07B5.8 and R07B5.9 locus. The combine gene is a member of the MYST histone acetyltransferase family. This implicates a role in chromatin modification, which may be necessary early in development as a mark of the lineage distinction necessary for the bilaterally asymmetric cell fates of the ASEL and ASER neurons.

Rand JB et al. (2000) East Coast Worm Meeting "UNC-13 in the C. elegans Nervous System"

Kohn RE, Rand JB, McManus JR, Moulder GL, Duke A, Barstead RJ, Duerr JS

[East Coast Worm Meeting, 2000]

C. elegans unc-13 and its homologues in vertebrates and Drosophila are involved in neurotransmitter release. UNC-13 has several regions homologous to PKC regulatory domains; these domains confer it with calcium, phorbol ester and phospholipid binding properties (Maruyama and Brenner, PNAS 88, 1991). A 5.9kb transcript coding for a 200kDa protein was initially identified (now designated L-R for left and right regions). We have identified two additional types of transcripts. One transcript includes a 1kb novel exon (L-M-R, M for middle region) and another transcript lacks the 5' region included in the other two transcripts (M-R). All three transcripts are identical at the 3' end (R). C. elegans with mutations in the 5' end of the gene (L) alter two types of transcripts (L-R and L-M-R) resulting in an uncoordinated coily phenotype and resistance to the anti-cholinesterease, aldicarb. A 2.7kb deletion near the 3' end (R) (identified by Bob Barstead using PCR analysis) affects all unc-13 transcripts and results in a lethal phenotype. Antibodies recognizing the N-terminal region of UNC-13 (L) label synapses, but not synaptic vesicles, of most or all neurons; many mutations in L and R remove staining with this antibody. Supported by grants from the NIH and OCAST.

Murthy K et al. (2010) Neuronal Development, Synaptic Function and Behavior, Madison, WI "In vivo imaging of retrogradely transported synaptic vesicle proteins in C. elegans neurons"

Bhat J M, Murthy K, Koushika S P

[Neuronal Development, Synaptic Function and Behavior, Madison, WI, 2010]

Axonal transport is an essential process that carries cargoes in the anterograde direction to the synapse and in the retrograde direction back to the cell body. Currently there is no in vivo method to exclusively mark retrogradely moving compartments carrying specific proteins, as opposed to anterogradely moving compartments. We have developed a method in the C. elegans model to mark retrogradely moving compartments carrying Synaptobrevin-1 in neurons. This method is based on the uptake of a fluorescently labeled anti-GFP antibody delivered in an animal expressing Synaptobrevin-1::GFP in neurons. We show that this method largely labels only retrogradely moving compartments. Very little labeling is observed if there is a block of vesicle exocytosis or if the synapse is physically separated from the cell body. The extent of labeling is also dependent on the dyenin-dynactin complex. These data support the interpretation that the labeling of Synaptobrevin-1::GFP largely occurs after vesicle fusion and the major labeling likely takes place at the synapse. This method is very general and can be readily adapted to any transmembrane protein on synaptic vesicles with a GFP tag inside the vesicle. It can also be easily extended to other model systems such as Drosophila.