Li, Yini et al. (2021) MicroPubl Biol

Li, Yini, Snyder, Matthew, Maine, Eleanor M.

[MicroPubl Biol, 2021]

Histone modifications influence gene expression and chromosome dynamics by altering chromatin structure and recruitment of nonhistone proteins. Dimethylation of histone H3 on lysine 9 (H3K9me2) is a conserved modification often found within heterochromatin. During first meiotic prophase when homologous chromosomes undergo pairing and synapsis, immunolabeling of C. elegans male germ cells detects a relatively high H3K9me2 level on the single X chromosome and a relatively low H3K9me2 level on synapsed autosomes. This H3K9me2 distribution is influenced by several components of the small RNA machinery, including: EGO-1 RNA-directed RNA polymerase (RdRP); DRH-3 helicase; EKL-1, a Tudor domain protein; CSR-1 Argonaute; and RRF-3 RdRP. EGO-1, DRH-3, and EKL-1 function together to generate/stabilize 22G RNAs in the germ line. A subset of these 22G RNAs function together with CSR-1 to ensure correct gene expression. RRF-3 RdRP functions in biogenesis of 26G RNAs that feed into two germline regulatory mechanisms mediated by ERGO-1 Argonaute and the redundant ALG-3 and ALG-4 Argonaute proteins. Here, we report that meiotic H3K9me2 distribution is influenced by ALG-3 and ALG-4, as well as by two other factors required for 26G RNA synthesis, ERI-1 and ERI-5. Moreover, meiotic H3K9me2 distribution is influenced by activity of the poly(U) polymerases, PUP-1 (aka CDE-1, CID-1) and PUP-2.

Li, Yini et al. (2015) International Worm Meeting "PUP-1 and PUP-2: two redundant poly(U) polymerases regulating C. elegans germline development."

Li, Yini, Maine, Eleanor, Snyder, Matt

[International Worm Meeting, 2015]

Poly(U) polymerases can catalyze 3' uridylation of RNAs, a modification that is implicated in regulating RNA stability and editing in multiple organisms. The targets of poly(U) polymerases are diverse, and include mRNAs, siRNAs, and unmethylated miRNAs. Here, we report phenotypic findings of a double knock-out of two poly(U) polymerases, pup-1 and pup-2. We used the CRISPR-Cas9 system to delete the pup-1 pup-2 gene region. The pup-1(0) pup-2(0) double mutant exhibits several temperature-sensitive defects, including an incompletely penetrant maternal effect sterility. Maternal effect sterility was not observed in the pup single mutants. In addition, we also observed a mortal germline phenotype in the pup-1(0) pup-2(0) double mutant, and the germline mortality appeared to occur more quickly at high temperature. Phenotypic analysis shows that a great majority of sterile adults contain no germ cells, and a minority of sterile adults have germ cells but do not produce functional gametes. Our preliminary results indicate that the most severe phenotype may be associated with germ cell death during larval development. Taken together, our findings suggest that pup-1 and pup-2 function redundantly in regulating germline development. We are currently investigating the impact of pup-1 and pup-2 on chromatin during meiosis, and working to identify their potential protein/RNA interactors.

Gopal E et al. (2015) J Pharmacol Exp Ther "Species-specific influence of lithium on the activity of SLC13A5 (NaCT): lithium-induced activation is specific for the transporter in primates."

Ganapathy V, Ramachandran S, Gopal E, Babu E, Prasad PD, Bhutia YD

[J Pharmacol Exp Ther, 2015]

NaCT (SLC13A5) is a Na(+)-coupled transporter for Krebs cycle intermediates and is expressed predominantly in the liver. Human NaCT is relatively specific for citrate compared with other Krebs cycle intermediates. The transport activity of human NaCT is stimulated by Li(+), whereas that of rat NaCT is inhibited by Li(+). We studied the influence of Li(+) on NaCTs cloned from eight different species. Li(+) stimulated the activity of only NaCTs from primates (human, chimpanzee, and monkey); by contrast, NaCTs from nonprimate species (mouse, rat, dog, and zebrafish) were inhibited by Li(+). Caenorhabditis elegans NaCT was not affected by Li(+). With human NaCT, the Li(+)-induced increase in transport activity was associated with the conversion of the transporter from a low-affinity/high-capacity type to a high-affinity/low-capacity type. H(+) was able to substitute for Li(+) in eliciting the stimulatory effect. The amino acid Phe500 in human NaCT was critical for Li(+)/H(+)-induced stimulation. Mutation of this amino acid to tryptophan (F500W) markedly increased the basal transport activity of human NaCT in the absence of Li(+), but the ability of Li(+) to stimulate the transporter was almost completely lost with this mutant. Substitution of Phe500 with tryptophan in human NaCT converted the transporter from a low-affinity/high-capacity type to a high-affinity/low-capacity type, an effect similar to that of Li(+) on the wild-type NaCT. These studies show that Li(+)-induced activation of NaCT is specific for the transporter in primates and that the region surrounding Phe500 in primate NaCTs is important for the Li(+) effect.

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.

Tabuse Y (1996) Worm Breeder's Gazette "Lithium arrests embryonic development of C. elegans"

Tabuse Y

[Worm Breeder's Gazette, 1996]

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 bea most effective treatment of manic-depressive illness. Although several models have been proposed to explain Li action, the molecular mechansm remains unclear. The most widely accepted model is the inositol depletion hypothesis, in which Li is thought to affect inositol phosphate turnover by inhibiting inositol monophosphatase, thus resulting in the depletion of endogenous inositol. 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 C and observed the laid embryos. 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, which propagated on Li-containing medium. So far, I obtained one mutant. The mutation was tentatively assigned to chromosome V. Preliminary genetic analysis showed that the mutation showed maternal effect. 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. I am now trying to isolate other mutants and also to identify early defects of embryogenesis caused by lithium treatment. I would like to thank J. Miwa for encouragement and discussions.

Inokuchi A et al. (2015) J Appl Toxicol "Effects of lithium on growth, maturation, reproduction and gene expression in the nematode Caenorhabditis elegans."

Matsusaki H, Yamamoto R, Takumi S, Morita F, Inokuchi A, Arizono K, Tominaga N, Ishibashi H

[J Appl Toxicol, 2015]

Lithium (Li) has been widely used to treat bipolar disorder, and industrial use of Li has been increasing; thus, environmental pollution and ecological impacts of Li have become a concern. This study was conducted to clarify the potential biological effects of LiCl and Li(2)CO(3) on a nematode, Caenorhabditis elegans as a model system for evaluating soil contaminated with Li. Exposure of C. elegans to LiCl and Li(2)CO(3) decreased growth/maturation and reproduction. The lowest observed effect concentrations for growth, maturation and reproduction were 1250, 313 and 10 000m, respectively, for LiCl and 750, 750 and 3000m, respectively, for Li(2)CO(3). We also investigated the physiological function of LiCl and LiCO(3) in C. elegans using DNA microarray analysis as an eco-toxicogenomic approach. Among approximately 300 unique genes, including metabolic genes, the exposure to 78m LiCl downregulated the expression of 36 cytochrome P450, 16 ABC transporter, 10 glutathione S-transferase, 16 lipid metabolism and two vitellogenin genes. On the other hand, exposure to 375m Li(2)CO(3) downregulated the expression of 11 cytochrome P450, 13 ABC transporter, 13 lipid metabolism and one vitellogenin genes. No gene was upregulated by LiCl or Li(2)CO(3). These results suggest that LiCl and Li(2)CO(3) potentially affect the biological and physiological function in C. elegans associated with alteration of the gene expression such as metabolic genes. Our data also provide experimental support for the utility of toxicogenomics by integrating gene expression profiling into a toxicological study of an environmentally important organism such as C. elegans.

McColl G et al. (2008) J Biol Chem "Pharmacogenetic analysis of lithium-induced delayed aging in Caenorhabditis elegans."

Melov S, McColl G, Lithgow GJ, Vantipalli MC, Killilea DW, Hubbard AE

[J Biol Chem, 2008]

Lithium (Li+) has been used to treat mood affect disorders, including bipolar, for decades (1;2). This drug is neuroprotective and has several identified molecular targets. However, it has a narrow therapeutic range and the underlying mechanism(s) of its therapeutic action is not understood. Here we describe a pharmacogenetic study of Li+ in the nematode Caenorhabditis elegans. Exposure to Li+ at clinically relevant concentrations throughout adulthood increases survival during normal aging (up to 46% median increase). Longevity is extended via a novel mechanism with altered expression of genes encoding nucleosome-associated functions. Li+ treatment results in reduced expression of the worm ortholog of LSD-1 (T08D10.2), a histone demethylase; knockdown by RNA interference (RNAi) of T08D10.2 is sufficient to extend longevity (~25% median increase), suggesting Li+ regulates survival by modulating histone methylation and chromatin structure.

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.

Kelley, Leanne et al. (2019) International Worm Meeting "Germline development and poly(U) polymerase activity."

Maine, Eleanor, Kelley, Leanne, McJunkin, Katherine, Li, Yini

[International Worm Meeting, 2019]

Poly(U) polymerases (PUPs) regulate RNA stability by addition of 3' uracil. 3' uridylation activity has been observed in many species, including Drosophila, S. pombe, humans, Arabidopsis, and C. elegans (Scott and Norbury 2013). We found that three enzymes with documented PUP activity, PUP-1/CDE-1, PUP-2, and PUP-3, are critical for germline development and survival (Li and Maine 2018). While single gene mutations reduce fertility, 100% of pup-1/-2 double mutants are sterile by the F3 generation at 25 deg C. In these animals, germ cells do not maintain their identity as distinct from soma and die during late larval development. Interestingly, germ cell viability is partially rescued in pup-3; pup-1/-2 triple mutants. Protein blots reveal that PUP-3 abundance is elevated in pup-1/-2 mutant gonads compared to wildtype controls. We propose that one way in which PUP-1 and PUP-2 promote germline development and viability is by limiting PUP-3 expression in the germ line. PUP-3 overexpression in the pup-1/-2 mutant germ line results in abnormal RNA regulation and improper germline development. We want to understand how the combination of PUP-1, -2, and -3 activity ensures fertility. Previous work showed that PUP-1 targets CSR-1-associated siRNAs (van Wolfswinkel et al. 2009) and PUP-2 targets let-7 miRNA in somatic tissues (Lehrbach et al. 2009). Human orthologs of PUP-1 and PUP-2 uridylate mRNAs (Lim et al. 2014), and we hypothesize that C. elegans PUPs may modify mRNAs, as well. To date, there has not been a complete analysis of PUP RNA targets in the C. elegans germline. We are identifying non-coding RNA targets using small RNA-seq and mRNA targets using TAIL-seq, a method that analyzes mRNA 3' ends. Preliminary data suggest that the relative distribution of small RNA classes is altered in pup-1/-2 double mutants compared to wildtype. MiRNAs as a class have reduced 3' uridylation in pup-1/-2 mutants. A subset of miRNAs, including members of the miR-35 family, have increased abundance, suggesting that PUP-1 and PUP-2 normally target these miRNAs for degradation.

Li WQ et al. (1992) Worm Breeder's Gazette "Characterization of the Axonal Guidance and Outgrowth gene Unc-33"

Shaw JE, Li WQ, Herman RK

[Worm Breeder's Gazette, 1992]

Characterization of the axonal guidance and outgrowth gene unc-33 W. Li, R. K. Herman and J. E. Shaw Department of Genetics and Cell biology, University of Minnesota, St Paul, MN 55108