Li X et al. (2010) Aging, Metabolism, Stress, Pathogenesis, and Small RNAs, Madison, WI "The Stress Protein SKN-1 Monitors Translation Elongation"

Matilainen O, Holmberg C I, Li X, Blackwell T

[Aging, Metabolism, Stress, Pathogenesis, and Small RNAs, Madison, WI, 2010]

SKN-1, the C. elegans ortholog of the conserved NF-E2-related factors (Nrf1/2/3), promotes oxidative stress resistance and longevity. In response to stress, SKN-1 induces genes involved in detoxification, metabolism, and protein homeostasis. Our lab recently identified 41 genes for which RNAi resulted in constitutive activation of a SKN-1 target. These genes correspond to several cellular processes, including translation initiation and elongation. Here we have examined how SKN-1 is influenced by RNAi inhibition of several translation elongation factors. We find that interference with translation elongation results in activation of p38 MAPK signaling, SKN-1 translocation into intestinal nuclei, induction of a restricted set of SKN-1 target genes, and skn-1-dependent oxidative stress resistance. Interestingly, interference with elongation does not induce multiple other stress responses, and SKN-1 target genes appear to be upregulated through different pathways in response to inhibition of translation initiation or elongation. The quality control systems of protein synthesis, folding and degradation are all required to maintain protein homeostasis. A substantial proportion of newly synthesized proteins is normally degraded because of translation errors or misfolding. Previous work indicated that translation elongation factors recruit the proteasome for this co-translational polypeptide degradation. Using a novel in vivo method we have found that interference with translation elongation but not initiation impairs proteasomal turnover of an intact protein in the intestine. The data reveal mechanistic connections between translation elongation, protein degradation, and SKN-1, and illustrate how SKN-1-mediated stress responses are coupled to many biological processes.

Tabuse Y (1997) International C. elegans Meeting "LITHIUM CAUSES DEVELOPMENTAL DISORDER IN C. ELEGANS"

Tabuse Y

[International C. elegans Meeting, 1997]

Lithium (Li) has long been known to have teratogenic effects on the development of many organisms, including sea urchin, Xenopus and Dictyostelium. In Li-treated Xenopus embryos, ventral blastmeres are respecified to develop into dorsal structures, leading to dorsalized embryos lacking ventral mesodermal tissues. In Dictyostelium, Li alters the fate of prespore cells to become prestalk cells instead. Besides teratogenic effects, Li is also known to be a most effective treatment of manic-depressive illness.!@Although several models have been proposed to explain Li action, the molecular mechanism has remained unclear. Recently, GSK (glycogen synthase kinase)-3b was proposed to be a target of Li, suggesting that Li affects the wnt signaling pathway. To understand the mechanism of Li action, I first examined the effect of Li on C. elegans embryogenesis. I inoculated N2 animals at the late L4 stage onto NG plates containing 20 mM LiCl, incubated them at 20 oC. The number of eggs produced by treated animals was reduced to about half of the untreated control. Although cell division seemed to proceed, no embryos hatched on Li plates. Treated-embryos developed to produce gut granules, but did not execute normal morphogenesis at later embryonic stages. To identify genes involved in the action of Li, I have begun to screen for Li-resistant mutants that propagated on Li-containing medium. Several mutants were isolated, and one of them was mapped on the left side of unc-42 on chromosome V. On Li plates, the hatching rate of mutant eggs cross-fertilized by wild-type males was essentially the same as that for self-fertilized mutant eggs. On the contrary, no wild-type eggs cross-fertilized by mutant males hatched on Li-containing plates. So, this mutation appeared to be maternal. Further genetic analyses of the mutants and the observation on the cellular phenotype of Li-treated embryos are underway.

D Royal et al. (2001) International C. elegans Meeting "Towards the Cloning of Gene That Can Mutate to Confer Lithium Resistance In C. elegans"

J White, M Driscoll, E Hsieh, N Sirotin, M A Royal, D Royal, S Kwok, K O'Connell, B Bowerman

[International C. elegans Meeting, 2001]

Little is known about how ions permeate the C. elegans gut and cuticle. How various ions influence development and behavior is also not understood. One ion with considerable impact in human biology is Li + , which modulates bipolar disorder by an unknown mechanism. In particular, the targets of lithium that cause side effects remain to be identified. We are using C. elegans to genetically identify targets of lithium and to elaborate on mechanisms of transport, which may have an impact on human health. Li + has dosage-dependent effects on C. elegans embryos. When L4 animals mature on NGM plates with Li + added to the final concentrations of 10mM-20mM, they produce embryos that are unable to hatch. We demonstrated that this failure to hatch is due to defects in cytokinesis that result in multi-nucleated embryos and symmetrically partitioned cells. Li + also has a dosage-dependent effect on larval development. When adult hermaphrodites are permitted to lay embryos on 10mM-20mM Li + NGM plates, the resulting offspring experience a developmental delay proportional to the concentration of Li + . The offspring become progressively paralyzed as they reach adulthood. We demonstrated earlier that there is a delay in the entry into the S phase of the cell cycle larval stages. To learn more about the biology of Li + sensitivity, we screened for Li + resistant mutants. We developed three screens that took advantage of the embryonic arrest and the larval delay caused by Li + . We isolated one mutant bz71 , in a screen of 44,000 haploid genomes, that is resistant to both the larval and embryonic blocks of 16mM Li + . bz71 is dominant. Using classical mapping techniques, we positioned it on LGIII between unc-32 and dpy-18 . We are currently using SNP strategy to obtain a higher resolution map and we hope to report on the identification and cloning of this locus.

Hodgkin, Jonathan et al. (2009) International Worm Meeting "Analysis of breakage and meiotic instability in attached-X-chromosome strains."

Hodgkin, Jonathan, O'Rourke, Delia, Jethwa, Nirmal

[International Worm Meeting, 2009]

The isolation of an attached X chromosome (X^X), consisting of two apparently intact X chromosomes joined at their left telomeres, was previously reported (Hodgkin and Albertson 1995, Genetics 141: 527). In contrast to attached X chromosome constructs in other organisms, and also to X-autosome fusions in C. elegans, this X^X configuration is unstable, frequently breaking down to give an apparently normal X chromosome, or else an X chromosome with a deletion of the left end, or an X chromosome carrying a duplication of the left end. The extent of these deletions and duplications is variable, and they are generated at high frequency (in at least 5% of all X^X oogenic meioses). X^X strains therefore provide a useful source of aberrations in this region of the genome, which contains both a meiotic pairing site and numerator sites for sex determination. It seemed likely that X^X breakage was occurring at meiosis, and might be dependent on the meiotic recombination machinery. To test this hypothesis, X^X was crossed onto a series of meiosis-defective backgrounds (him-1, him-3, him-5, him-6, him-8, him-14, xnd-1, etc.). X^X breakage, as measured by the production of self-progeny males, was significantly or greatly reduced in most of these backgrounds, indicating that the breakage events are partly or wholly dependent on meiotic recombination. Previous FISH studies using YAC probes indicated that the end junction between the two X chromosomes was symmetrical and involved no deletion. We attempted to clone the junction fragment by single primer PCR, in order to determine its exact structure. However, PCR amplification was not successful, perhaps because the junction may include a substantial stretch of telomere repeats or local rearrangements. Further investigation of the structure and properties of the end junction is in progress.

Chun Yi Huang et al. (2005) International Worm Meeting "A new player in the C. elegans programmed cell death pathway"

Yi-Chun Wu, Tsung-Yuan Hsu, Ruey-Hwa Chen, Chun Yi Huang, Pei-ju Lu, Jia-Yun Chen

[International Worm Meeting, 2005]

Human CED-X was identified as a strong interacting protein with the death domain of DAPK (death associated protein kinase) in a yeast two-hybrid screen. To investigate the potential involvement of CED-X in apoptosis, we study the function of its C. elegans homolog ced-x. Inactivation of ced-x by either RNAi or a deletion mutation resulted in decreased numbers of cell corpses during embryogenesis. Ectopic killing caused by transcriptional overexpression of egl-1 or ced-4 but not ced-3 in touch cells was suppressed by the ced-x(0) mutation. Therefore, ced-x genetically acts downstream of or in parallel to egl-1 and ced-4 and upstream of or in parallel to ced-3 in the programmed cell death pathway. In addition, the observation that the ced-x(0) mutation partially suppressed ectopic cell deaths caused by the icd-1(RNAi) mutation indicates that ced-x genetically acts downstream of or in parallel to icd-1. To further explore CED-X function, we generated antibodies against CED-X. The immunostaining signal revealed a filamentous CED-X localization pattern in the cytosol during embryogenesis. We are currently investigating if CED-X is associated with organelles or cytoskeleton. In summary, our results show that ced-x is important for promoting or proper execution of programmed cell death in C. elegans. As human ced-x can functionally substitute C. elegans ced-x in a ced-x mutant, the function of ced-x in apoptosis is likely conserved in evolution.

Lau, Alyssa et al. (2015) International Worm Meeting "Deciphering the mechanism of X-upregulation in C. elegans dosage compensation."

Lau, Alyssa, Csankovszki, Gyorgyi, Zhu, Kevin

[International Worm Meeting, 2015]

In C. elegans dosage compensation is required to balance X-linked gene expression to autosomes. It is hypothesized that an unknown mechanism causes X upregulation in both sexes. This mechanism balances the X to autosomal expression in males, but creates X overexpression in hermaphrodites. Therefore, to restore the balance, hermaphrodites downregulate gene expression two-fold on both X chromosomes. While many studies have focused on X chromosome downregulation, the mechanism of X upregulation is not known.Using 3D FISH microscopy to measure the volume of chromosome territories we found that the X chromosome, but not chromosome 1, territories in males are unexpectedly decondensed. We found that this X chromosome decondensation requires the activity of the histone acetyltransferase, MYS-1 (homologous to human TIP60). Interestingly, MYS-1 acetylates H4K16, the key factor responsible for male-specific X-upregulation in Drosophila melanogaster dosage compensation. This suggests that H4K16ac may be responsible for higher gene expression levels on the male X in both species. Depleting other members of a putative C. elegans TIP60-like complex led to similar X phenotypes as MYS-1 depletion. We hypothesize that a TIP60-like complex decondenses the X chromosome in C. elegans males, and this decondensation contributes to upregulation of gene expression on the chromosome. By analyzing X chromosome volumes in young male embryos we determined that the developmental time window for this male X chromosome decondensation occurs around the 30-to-50-cell stage, surprisingly the same stage as the downregulation process in hermaphrodites. Overall, our data indicates that histone acetylation by the MYST family HAT MYS-1 may play an important role in the highly debated X upregulation mechanism in C. elegans. .

Albritton, Sarah et al. (2011) International Worm Meeting "Regulation of X chromosome transcription in Caenorhabditis species."

Albritton, Sarah, Ercan, Sevinc

[International Worm Meeting, 2011]

Animals with different numbers of X chromosomes in males and females possess mechanisms to compensate for the difference in the X-linked gene dose between the two sexes. In addition to the X chromosome dose difference between the sexes, the presence of a single-copy X chromosome per two-copy diploid autosomes creates an important problem for males, because all X-linked genes are haploinsufficient compared to the autosomal genes. In C. elegans, hermaphrodites (XX) contain two Xes, whereas males (XO) contain a single X, therefore facing X haploinsufficiency. By performing microarray analysis of RNA abundance in XX and XO worms, we observed that the overall transcript levels from the X chromosome in both XX and XO animals is similar to that of overall expression from autosomes. This suggests that transcription from the single X in XO L3 hermaphrodites (TY2205, her-1(e1520) sdc-3(y126) V; xol-1(y9) X) is increased approximately two-fold. The mechanism of this upregulation is unclear. We had shown that the X chromosome promoters have higher GC content compared to the autosomes (Ercan et al 2010), suggesting a DNA-encoded mechanism of transcriptional regulation. We will study X upregulation by comparing transcription of orthologus genes that are on the X versus autosomes in four Caenorhabditis species. Ercan S, Lubling Y, Segal E, Lieb JD. High nucleosome occupancy is encoded at X-linked gene promoters in C. elegans. Genome Res. 2011 Feb;21(2):237-44. PMID:21177966.

Satoru Uzawa et al. (2007) International Worm Meeting "Visualization of the Spreading of the Dosage Compensation Complex on X by Microscopy."

Satoru Uzawa, Barbara Meyer

[International Worm Meeting, 2007]

For proper development of XX and XO animals, the dosage of X-chromosome-linked genes have to be compensated. In free-living soil nematode C. elegans, the dosage compensation of the X-linked genes is accomplished by reducing the gene expression from the X chromosomes in XX hermaphrodites by half. This process is done by the molecular machinery known as the dosage compensation complex (DCC), which resembles condensin complex. DCC loads specifically onto the X chromosome in early embryos (~20 cell stage) and remains on the X chromosome throughout the life of the hermaphrodite. The DCC is recruited onto the X chromosome through specific DNA sequences on the X chromosomes, rex (recruitment element on X). The X chromosome regions that lack a rex site receive DCC through spreading from other rex sites in cis. The nature and molecular mechanism of the spreading of the DCC on the X chromosomes is unknown. We are trying to visualize this spreading process through various microscopic methods using FISH and IF double staining for high resolution images and live cell imaging for time domain analysis.

LaMunyon CW et al. (1995) International C. elegans Meeting "SPERM COMPETITION IN HERMAPHRODITIC CAENORHABDITIS SPECIES"

Ward S, LaMunyon CW

[International C. elegans Meeting, 1995]

As in C. elegans, male sperm take precedence over hermaphrodite sperm after C. briggsae worms mate. However, in C. briggsae the initial outcross progeny are principally hermaphrodites and become male-biased with time. Thus, X-bearing male sperm have a striking fertilization advantage over non-X-bearing male sperm. Results from controlled matings show that the X-bearing sperm neither outnumber nor activate faster than their non-X-bearing counterparts. The advantage (meiotic drive) of the X-bearing sperm is therefore due to a competitive edge over non-X-bearing sperm. Thus, in C. briggsae, male X-bearing sperm outcompete male non-X-bearing sperm which themselves outcompete hermaphrodite sperm. While we do not know the mechanism underlying the competitive effect of the X chromosome, we do have evidence from artificial insemination that the precedence of male sperm over hermaphrodite sperm in C. elegans is independent of both seminal fluid and the mode of sperm activation (male or hermaphrodite). Male sperm precedence in hermaphroditic Caenorhabditis maximizes outcrossing after mating, and the X-bearing sperm advantage in C. briggsae delays the associated cost of making males which are by themselves incapable of reproduction.

Albritton, Sarah E. et al. (2013) International Worm Meeting "Gene movement between X and autosomes and its effect on transcription."

Kranz, Anna-Lena, Ercan, Sevinc, Albritton, Sarah E.

[International Worm Meeting, 2013]

In Caenorhabditis species, dosage compensation acts to reduce transcriptional output of both female/hermaphrodite X chromosomes by one half. This balances X expression between the sexes, but potentially gives females the same problem faced by males: functional monosomy of the X chromosome. It has been proposed that a mechanism evolved to upregulate X expression to balance X and autosomal transcription, thereby overcoming male monosomy. However, owing to biased gene content and tissue-specific regulation of the X, direct comparison of X and autosomal transcription is difficult. In order to more directly compare X and autosomal transcription we looked at expression of 1:1 orthologs that are differentially located on the X or an autosome between two nematode species. Our work focused on four species: C. elegans, C. briggsae, C. remanei, and Pristionchus pacificus. The C. elegans and C. briggsae genomes are well assembled and annotated. The genomes of C. remanei and P. pacificus have been sequenced, but their genes have not yet been assigned to chromosomal locations. Since our analysis depends on comparing differentially located orthologs, we first needed to map genes to either the X or autosomes. We took a read-depth-variation approach. We performed genomic DNA-seq in males and females/hermaphrodites of C. brenneri, C. remanei and P. pacificus. C. briggsae males and hermaphrodites were also sequenced as controls. Genes located on the X chromosome were expected to have a 1:2 ratio of sequencing coverage between males and hermaphrodites (X:XX) and all autosomal genes a 1:1 ratio. Our analysis yielded a list of X and autosomal genes for each of the four nematode species and allowed the identification of differentially located 1:1 orthologs. Comparison of X and autosomal transcription showed no bias towards male upregulation of X-located orthologs.