Yin, Jianhua et al. (2015) International Worm Meeting "Identification and Characterization of a C. elegans Rab GTPase 2 Regulator in Phagosome and Endosome Maturation."

Yin, Jianhua, Huang, Yaling, Li, Meiyang, Wang, Xiaochen, Guo, Pengfei

[International Worm Meeting, 2015]

During apoptosis, apoptotic cells are quickly recognized and engulfed by phagocytes to prevent release of immunogenic contents. The internalized cell corpses are enclosed within intracellular vesicles called phagosomes, which undergo a series of maturation process, leading to the degradation of apoptotic cells in phagolysosomes. Previously, we found that two Rab GTPases, RAB-14 and UNC-108/RAB2, are recruited to apoptotic cell-containing phagosomes at a similar stage and they function in parallel to regulate phagosome maturation. To understand how RAB-14 and UNC-108 function in cell corpse clearance, we performed genetic screens to search for enhancers of rab-14 and unc-108. From such screen, we have identified a gene whose loss of function significantly enhances persistent cell corpse phenotype in rab-14(lf) and temporarily named it ehrf-1 (Enhancer of Rab GTPase fourteen). We found that loss of ehrf-1 causes accumulation of embryonic and germ cell corpses, a phenotype that is similar to and not enhanced by unc-108 mutation. Interestingly, ehrf-1 mutants also show similar phenotypes in endosome maturation as in unc-108(lf), which is not altered in unc-108; ehrf-1 double mutants. These results suggest that ehrf-1 may act together with unc-108 to regulate phagosome and endosome maturation. We are currently in the process of further characterizing ehrf-1's function in phagosome and endosome maturation and investigating its relationship with UNC-108/RAB2.

Ahmad W et al. (2020) Biochem Biophys Res Commun "Prediction of human tau 3D structure, and interplay between O--GlcNAc and phosphorylation modifications in Alzheimer's disease: C.elegans as a suitable model to study these interactions invivo."

Ahmad I, Ahmad W, Shabbiri K

[Biochem Biophys Res Commun, 2020]

Tau protein regulates, maintains and stabilizes microtubule assembly under normal physiological conditions. In certain pathological circumstances, tau is post-translationally modified predominantly via phosphorylation and glycosylation. Hyper-phosphorylation of tau in Alzheimer's disease (AD) resulted in aggregated neurofibrillary tangles (NFTs) formation. Unfortunately, absence of tau 3D structure makes difficult to understand exact mechanism involved in tau pathology. Here by using ab-initio modelling, we predicted a tau 3D structure that not only explains its binding with microtubules but also elucidates NFTs formation. O-linked -N-acetylglucosaminylation (O--GlcNAc) is thought to regulate tau phosphorylation on single or proximal Ser/Thr residues (called as Yin-Yang sites). In this study, we not only validate the previously described three-serine residues (208, 238 and 400) as Yin-Yang sites but also predicted 22 more possible Ser/Thr O-glycosylation sites. Among them seventeen residues were predicted as possible Yin-Yang sites and are proposed to mediate NFT formation in AD. These predicted Yin-Yang sites may act as attractive therapeutic targets for the drug development in AD. Predicted 3D structure of tau₄₄₁ was highly accessible for phosphorylation and hyperphosphorylation, and showed higher surface accessibility for interplay between O--GlcNAc and phosphorylation modifications. Kinases and phosphatases involved in tau phosphorylation are conserved in human and other organisms. Homology modelling revealed conserved catalytic domain for both human and C.elegans O-GlcNAc transferase (OGT), suggesting that transgenic C.elegans expressing human tau may be a suitable model system to study these modifications.

Xu W et al. (2019) Elife "Evolution of Yin and Yang isoforms of a chromatin remodeling subunit precedes the creation of two genes."

Zhao Y, Stevens L, Munoz J, McGrath PT, Long L, Xu W, Felipe I, Ellis RE

[Elife, 2019]

Genes can encode multiple isoforms, broadening their functions and providing a molecular substrate to evolve phenotypic diversity. Evolution of isoform function is a potential route to adapt to new environments. Here we show that <i>de novo</i>, beneficial alleles in the <i>nurf-1</i> gene became fixed in two laboratory lineages of <i>C. elegans</i> after isolation from the wild in 1951, before methods of cryopreservation were developed. <i>nurf-1</i> encodes an ortholog of BPTF, a large (&gt;300kD) multidomain subunit of the NURF chromatin remodeling complex. Using CRISPR-Cas9 genome editing and transgenic rescue, we demonstrate that in <i>C. elegans</i>, <i>nurf-1</i> has split into two, largely non-overlapping isoforms (NURF-1.D and NURF-1.B, which we call Yin and Yang, respectively) that share only two of 26 exons. Both isoforms are essential for normal gametogenesis but have opposite effects on male/female gamete differentiation. Reproduction in hermaphrodites, which involves production of both sperm and oocytes, requires a balance of these opposing Yin and Yang isoforms. Transgenic rescue and genetic position of the fixed mutations suggest that different isoforms are modified in each laboratory strain. In a related clade of <i>Caenorhabditis</i> nematodes, the shared exons have duplicated, resulting in the split of the Yin and Yang isoforms into separate genes, each containing approximately 200 amino acids of duplicated sequence that has undergone accelerated protein evolution following the duplication. Associated with this duplication event is the loss of two additional <i>nurf-1</i> transcripts, including the long-form transcript and a newly identified, highly expressed transcript encoded by the duplicated exons. We propose these lost transcripts are non-functional side products necessary to transcribe the Yin and Yang transcripts in the same cells. Our work demonstrates how gene sharing, through the production of multiple isoforms, can precede the creation of new, independent genes.

Guo D et al. (2014) RNA Biol "RNAa in action: from the exception to the norm."

Lin SS, Guo D, Barry L, Li LC, Huang V

[RNA Biol, 2014]

Small RNA programmed Argonautes are sophisticated cellular effector platforms known to be involved in a diverse array of functions ranging from mRNA cleavage, translational inhibition, DNA elimination, epigenetic silencing, alternative splicing and even gene activation. First observed in human cells, small RNA-induced gene activation, also known as RNAa, involves the targeted recruitment of Argonaute proteins to specific promoter sequences followed by induction of stable epigenetic changes which promote transcription. The existence of RNAa remains contentious due to its elusive mechanism. A string of recent studies in C. elegans provides unequivocal evidence for RNAa's fundamental role in sculpting the epigenetic landscape and maintaining active transcription of endogenous genes and supports the presence of a functionally sophisticated network of small RNA-Argonaute pathways consisting of opposite yet complementary &quot;yin and yang&quot; regulatory elements. In this review, we summarize key findings from recent studies of endogenous RNAa in C. elegans, with an emphasis on the Argonaute protein CSR-1.

Brown, Rebecca et al. (2011) International Worm Meeting "The receptor tyrosine phosphatase clr-1 is required for axon regeneration in C. elegans."

Hammarlund, Marc, Strittmatter, Stephen, Brown, Rebecca

[International Worm Meeting, 2011]

Axons in the central nervous system display limited regeneration after injury. Recent expression studies in rodents have identified dozens of genes that are upregulated after CNS injury, however, confirming each gene's role in regeneration in a mouse model is cumbersome and labor intensive. C. elegans is a useful model for studying the genetics of axon regeneration. Individual GFP-labeled axons can be lesioned by laser microsurgery, and their regrowth can be monitored in vivo. C. elegans motor neurons display robust regeneration and can reestablish functional connections after laser axotomy. Genes identified as differentially expressed in microarray or RT-PCR analyses of mammalian CNS or PNS regeneration were compiled from the literature (Benowitz and Yin, 2007) and from preliminary studies in our lab. This list was refined to include about 30 genes with C. elegans homologues and further to include only genes for which loss of function alleles were viable and available. Using these genes, we conducted a candidate screen for genes that contribute to regeneration in the GABA motor neurons. We identified clr-1, which encodes a receptor tyrosine phosphatase related to the LAR family, as a potential mediator of regeneration. clr-1 loss of function mutants are capable of regeneration per se, as the percentage of fibers that regenerate after axotomy in clr-1 mutants is similar to that seen in wild-type worms. However, clr-1 mutant regenerating axons display extensive lateral branching and rarely cross the lesion site to reach their targets in the dorsal cord.

Tabuchi TM et al. (2014) G3 (Bethesda) "Opposing activities of DRM and MES-4 tune gene expression and X-chromosome repression in Caenorhabditis elegans germ cells."

Hagstrom KA, Rechtsteiner A, Strome S, Tabuchi TM

[G3 (Bethesda), 2014]

During animal development, gene transcription is tuned to tissue-appropriate levels. Here we uncover antagonistic regulation of transcript levels in the germline of Caenorhabditis elegans hermaphrodites. The histone methyltransferase MES-4 (Maternal Effect Sterile-4) marks genes expressed in the germline with methylated lysine on histone H3 (H3K36me) and promotes their transcription; MES-4 also represses genes normally expressed in somatic cells and genes on the X chromosome. The DRM transcription factor complex, named for its Dp/E2F, Retinoblastoma-like, and MuvB subunits, affects germline gene expression and prevents excessive repression of X-chromosome genes. Using genome-scale analyses of germline tissue, we show that common germline-expressed genes are activated by MES-4 and repressed by DRM, and that MES-4 and DRM co-bind many germline-expressed genes. Reciprocally, MES-4 represses and DRM activates a set of autosomal soma-expressed genes and overall X-chromosome gene expression. Mutations in mes-4 and the DRM subunit lin-54 oppositely skew the transcript levels of their common targets and cause sterility. A double mutant restores target gene transcript levels closer to wild type, and the concomitant loss of lin-54 suppresses the severe germline proliferation defect observed in mes-4 single mutants. Together, &quot;yin-yang&quot; regulation by MES-4 and DRM ensures transcript levels appropriate for germ-cell function, elicits robust but not excessive dampening of X-chromosome-wide transcription, and may poise genes for future expression changes. Our study reveals that conserved transcriptional regulators implicated in development and cancer counteract each other to fine-tune transcript dosage.

Haag, E.S. et al. (2019) International Worm Meeting "Evolution of sex ratio through gene loss."

Haag, E.S., Yin, D.

[International Worm Meeting, 2019]

The maintenance of males at intermediate frequencies is an important evolutionary problem. Several species of Caenorhabditis nematodes have evolved the androdioecious mating system, where selfing hermaphrodites and males coexist. They reproduce mostly through self-fertilization, but can also outcross. While selfing produces XX hermaphrodites, cross-fertilization produces 50% XO male progeny. Thus, male mating success dictates the sex ratio [1]. Here, we focus on the contribution of the male secreted short (mss) gene family to male mating success, sex ratio, and population growth. The mss family is essential for full sperm competitiveness in gonochoristic species, but has been lost in parallel in androdioecious species [2]. Using a transgene to restore mss function to the androdioecious C. briggsae, we examined how mating system and population subdivision influence the fitness of the mss+ genotype. Consistent with theoretical expectations, mss+ is sufficient to increase male frequency and depress population growth in genetically homogenous androdioecious populations. When mss+ and mss-null (i.e. wild-type) genotypes compete, mss+ is positively selected in both mixed-mating and strictly outcrossing situations, though more strongly in the latter. Thus, while sexual mode alone affects the fitness of mss+, it is insufficient to explain its parallel loss. We propose that the lack of inbreeding depression [3] and the strong subdivision that characterize natural Caenorhabditis populations [4] impose selection on sex ratio that makes loss of mss adaptive. By reducing, but not completely eliminating outcrossing, loss of the mss genes tunes the sex ratio to its new optimum after self-fertility is established. 1. Stewart AD, Phillips PC (2002) Genetics 160: 975 2. Yin et al. (2018) Science 359: 55 3. Dolgin et al. (2007) Evolution 61: 1339 4. Kiontke KC, et al. (2011) BMC Evol Biol 11: 339

Julia Grabitzki et al. (2006) European Worm Meeting "Identification of O-linked N-acetylglucosamine Modified Proteins in Caenorhabditis elegans"

Gunter Lochnit, Rudolf Geyer, Michael Ahrend, Brigitte Schmitz, Julia Grabitzki

[European Worm Meeting, 2006]

Julia Grabitzki1, Michael Ahrend2, Brigitte Schmitz2, Rudolf Geyer1 and Gnter Lochnit1. The posttranslational modification N-acetylglucosamine O-glycosidically linked (O-GlcNAc) to serine and threonine residues of proteins has been shown to be ubiquitous amongst eukaryotic proteins of the nucleus, cytoskeleton, cytoplasm, and has also been detected on cytosolic tails of membrane proteins [1]. O-GlcNAcylated proteins can form reversible multimeric complexes with other polypeptides or structures. The modification is often accompanied by phosphorylation/ dephosphorylation. O-GlcNAc can act either simultaneously or in a reciprocal fashion with phosphorylation. According to the Yin-Yang hypothesis, the phosphorylation/ dephosphorylation regulates O-GlcNAc-modified protein function (z.B. signal transduction and protein-protein interaction) in concert with phosphorylation [2-4]. The addition of O-GlcNAc to and the removal from the protein backbone is dynamic with rapid cycling in response to cellular signals or cellular stages.. Despite the fact, that Caenorhabiditis elegans is the best studied model organism, there have been no studies on O-GlcNAcylation in this organism so far. Therefore, to elucidate the role of O-GlcNAcylation, we investigated the proteome of a C. elegans mixed-stage population by two-dimensional gelelectrophoresis and subsequent western-blotting with the O-GlcNAc-specific antibody CTD 110.6 for the occurrence of this modification and identified the modified proteins by mass-spectrometry. We detected and identify several O-GlcNAc-modified proteins in C. elegans. Most of the identified proteins are involved in metabolic pathways. The prediction of the cellular localisation of the identified proteins revealed a predominant cytosolic occurrence of the O-GlcNAc modification.. References:. [1]. Rex-Mathes, M., J. Koch, Werner, S., Griffith, L. S and B. Schmitz. 2002. Methods Mol Biol 194: 73-87.. [2] Zachara, N.E. and G.W. Hart, Chem Rev, 2002. 102(2): p.431-8.. [3]. Griffith, L. S. and B. Schmitz. 1999. Eur J Biochem 262(3): 824-31.. [4] Wells, L. and G. W. Hart. 2003. FEBS Lett 546(1): 154-8.

R. Gatsi et al. (2006) European Worm Meeting "The Role of IP3 Signalling in the Regulation of Rhythms and Rates in C. elegans"

HA. Baylis, R. Gatsi

[European Worm Meeting, 2006]

R. Gatsi and HA. Baylis. . Rhythmic processes are an integral part of life of most organisms. Biological rhythms of less than a day are called ultradian rhythms. In C. elegans, ultradian rhythms include pharyngeal pumping, defecation, and ovulation. There is a growing body of evidence which supports the hypothesis that these rhythmic functions share a common molecular mechanism1. A central component of this pathway is the second messenger IP3. IP3 is produced by the action of phospholipase C in response to external stimuli. IP3 binds to IP3R located in the ER and regulates the release of calcium. IP3Rs are regulated by a broad range of molecules and proteins including Ca2+ itself and ATP. Signalling through IP3, itr-1 (IP3R) and Ca2+ are clearly involved in the control of rhythms in pumping, defecation, and ovulation 2,3,4,5. Interestingly, genetic analysis also suggests that ultradian rhythms are linked to mechanisms that control the rates of growth and ageing. In particular clk and gro genes regulate both types of processes 6,7.. We are currently dissecting the role of IP3 signalling in the regulation of timing in C. elegans by analysing the relationship between rhythmic processes, such as pharyngeal pumping, and rates such as the rate of growth and ageing. We have evidence which suggests that IP3 signalling might play a central role in these processes. We have shown that, in addition to changes in ultradian rhythms, mutants of itr-1 show alteration in growth rate (see also Dal Santo et al, 1999) and ageing. We have characterised these changes in more detail. In particular our results indicate that the ATP-binding regulatory site of the IP3Rs might play a significant role in these processes. In addition, phenotypic characterization of suppressor mutants of itr-1 (sa73) (ts l.o.f. mutant) suggest that growth rate may be regulated independently of other phenotypes. 1. Baylis HA. (2005) Cell. 123, 5-7. 2. Clandinin TR, DeModena JA & Sternberg PW (1998) Cell. 92, 523-533.. 3. Dal Santo P et al (1999) Cell. 98, 757-767.. 4. Walker DS., et al. (2002) Mol Biol Cell. 13, 1329-1337.. 5. Yin X, Gower NJ, Baylis HA & Strange K. (2004) Mol Biol Cell. 15, 38-49.. 6. Branicky R, Benard C and Hekimi S (2000). Bioessays 22, 48-56.. 7. Hekimi S, Lakowski B, Barnes TM, Ewbank J (1998) J. Trends Genet. 14, 14-20.

Marc Colosimo et al. (2004) East Coast Worm Meeting "Using enriched populations of single neuron types and microarrays to identify sensory neuron-specific genes"

Piali Sengupta, Anne Lanjuin, Saikat Mukhopadhyay, Marc Colosimo, Adam Brown

[East Coast Worm Meeting, 2004]

To gain a better understanding of the gene sets that are expressed in and required for the functions of specific sensory neuron types, our lab utilized a previously described C. elegans primary cell culture technique (1) coupled with FACS sorting to obtain enriched collections of single neuron types. We extracted RNA from these populations and hybridized to the C. elegans Affymetrix Genechips to compare the gene expression profiles of the aversive olfactory neuron type, AWB, and the primary thermosensory neuron type, AFD. We identified a list of approximately 250 genes with at least a 2 fold change in one neuron type as compared to the other. The expression predictions were further validated via real-time PCR and in vivo expression analyses using gfp fusion constructs. Expression patterns of several genes were confirmed by green fluorescent protein (gfp) fusion constructs. Genes analyzed represent a diverse array of gene families encoding sensory receptors, kinases, and transcription factors. We have further characterized a subset of genes identified in these experiments (also see abstract by Brown et al). In particular, we have shown that dac-1 , the C. elegans dachshund homolog, is expressed in AFD, as well as weakly in the neurons ASK, ASE, and AWC. dachshund belongs to the SKI/SNO family of proteins, and encodes a nuclear factor involved in the development of several organ types, including the Drosophila eye. Interestingly, a dac-1 knockout allele, dac-1(gk211) , exhibits a cryophilic phenotype when placed on a linear temperature gradient, similar to ttx-1 , an Otd/otx transcription factor required for AFD development. This implicates dac-1 in the development and/or function of the AFD thermosensory neurons. These experiments validate the use of cell culture and microarrays in C. elegans to identify new genes required for development and function of specific neuronal subtypes. Current experiments are aimed at further analyzing the functions of genes identified by these experiments, and application of this technology to identify genes expressed in additional sensory neuron types. 1.) Christensen M, Estevez A, Yin X, Fox R, Morrison R, McDonnell M, Gleason C, Miller DM 3rd, Strange K. (2002). A primary culture system for functional analysis of C. elegans neurons and muscle cells. Neuron 33, 503-14.