H3K23me2 is a new heterochromatic mark in Caenorhabditis elegans

Genome-wide analyses in Caenorhabditis elegans show that post-translational modifications (PTMs) of histones are evolutionary conserved and distributed along functionally distinct genomic domains. However, a global profile of PTMs and their co-occurrence on the same histone tail has not been described in this organism. We used mass spectrometry based middle-down proteomics to analyze histone H3 N-terminal tails from C. elegans embryos for the presence, the relative abundance and the potential cross-talk of co-existing PTMs. This analysis highlighted that the lysine 23 of histone H3 (H3K23) is extensively modified by methylation and that tri-methylated H3K9 (H3K9me3) is exclusively detected on histone tails with di-methylated H3K23 (H3K23me2). Chromatin immunoprecipitation approaches revealed a positive correlation between H3K23me2 and repressive marks. By immunofluorescence analyses, H3K23me2 appears differentially regulated in germ and somatic cells, in part by the action of the histone demethylase JMJD-1.2. H3K23me2 is enriched in heterochromatic regions, localizing in H3K9me3 and heterochromatin protein like-1 (HPL-1)-positive foci. Biochemical analyses indicated that HPL-1 binds to H3K23me2 and interacts with a conserved CoREST repressive complex. Thus, our study suggests that H3K23me2 defines repressive domains and contributes to organizing the genome in distinct heterochromatic regions during embryogenesis.     

Differential Localization and Independent Acquisition of the H3K9me2 and H3K9me3 Chromatin Modifications in the Caenorhabditis elegans Adult Germ Line

Histone methylation is a prominent feature of eukaryotic chromatin that modulates multiple aspects of chromosome function. Methyl modification can occur on several different amino acid residues and in distinct mono-, di-, and tri-methyl states. However, the interplay among these distinct modification states is not well understood. Here we investigate the relationships between dimethyl and trimethyl modifications on lysine 9 of histone H3 (H3K9me2 and H3K9me3) in the adult Caenorhabditis elegans germ line. Simultaneous immunofluorescence reveals very different temporal/spatial localization patterns for H3K9me2 and H3K9me3. While H3K9me2 is enriched on unpaired sex chromosomes and undergoes dynamic changes as germ cells progress through meiotic prophase, we demonstrate here that H3K9me3 is not enriched on unpaired sex chromosomes and localizes to all chromosomes in all germ cells in adult hermaphrodites and until the primary spermatocyte stage in males. Moreover, high-copy transgene arrays carrying somatic-cell specific promoters are highly enriched for H3K9me3 (but not H3K9me2) and correlate with DAPI-faint chromatin domains. We further demonstrate that the H3K9me2 and H3K9me3 marks are acquired independently. MET-2, a member of the SETDB histone methyltransferase (HMTase) family, is required for all detectable germline H3K9me2 but is dispensable for H3K9me3 in adult germ cells. Conversely, we show that the HMTase MES-2, an E(z) homolog responsible for H3K27 methylation in adult germ cells, is required for much of the germline H3K9me3 but is dispensable for H3K9me2. Phenotypic analysis of met-2 mutants indicates that MET-2 is nonessential for fertility but inhibits ectopic germ cell proliferation and contributes to the fidelity of chromosome inheritance. Our demonstration of the differential localization and independent acquisition of H3K9me2 and H3K9me3 implies that the trimethyl modification of H3K9 is not built upon the dimethyl modification in this context. Further, these and other data support a model in which these two modifications function independently in adult C. elegans germ cells.     

Novel heterochronic functions of the Caenorhabditis elegans period-related protein LIN-42

LIN-42, the Caenorhabditis elegans homolog of the Period (Per) family of circadian rhythm proteins, functions as a member of the heterochronic pathway, regulating temporal cell identities. We demonstrate that lin-42 acts broadly, timing developmental events in the gonad, vulva, and sex myoblasts, in addition to its well-established role in timing terminal differentiation of the hypodermis. In the vulva, sex myoblasts, and hypodermis, lin-42 activity prevents stage-specific cell division patterns from occurring too early. This general function of timing stage-appropriate cell division patterns is shared by the majority of heterochronic genes; their mutation temporally alters stage-specific division patterns. In contrast, lin-42 function in timing gonad morphogenesis is unique among the known heterochronic genes: inactivation of lin-42 causes the elongating gonad arms to reflex too early, a phenotype which implicates lin-42 in temporal regulation of cell migration. Three additional isoforms of lin-42 are identified that expand our view of the lin-42 locus and significantly extend the homology between LIN-42 and other PER family members. We show that, similar to PER proteins, LIN-42 has a dynamic expression pattern; its levels oscillate relative to the molts during postembryonic development. Transformation rescue studies indicate lin-42 is bipartite with respect to function. Intriguingly, the hallmark PAS domain is dispensable for LIN-42 function in transgenic animals.     

Caenorhabditis elegans dna-2 is involved in DNA repair and is essential for germ-line development

Caenorhabditis elegans germ cell proliferation and development were severely damaged in second generation dna-2 homozygotes. Even in the first generation, a much higher incidence of aberrant chromosomes in oocytes and resultantly higher embryonic lethality were found vs. wild type, when DNA breaks were induced by gamma-rays or camptothecin. The deficiency of dna-2 in combination with RNA interference on mre-11 gene expression synergistically aggravated germ-line development, especially oocyte formation. These results suggest that C. elegans Dna-2 is involved in a DNA repair pathway paralleling homologous recombination or non-homologous end joining with mre-11 participation.     

PAR-1 is required for morphogenesis of the Caenorhabditis elegans vulva

The Caenorhabditis elegans vulva provides a simple model for the genetic analysis of pattern formation and organ morphogenesis during metazoan development. We have discovered an essential role for the polarity protein PAR-1 in the development of the vulva. Postembryonic RNA interference of PAR-1 causes a protruding vulva phenotype. We found that depleting PAR-1 during the development of the vulva has no detectable effect on fate specification or precursor proliferation, but instead seems to specifically alter morphogenesis. Using an apical junction-associated GFP marker, we discovered that PAR-1 depletion causes a failure of the two mirror-symmetric halves of the vulva to join into a single, coherent organ. The cells that normally form the ventral vulval rings fail to make contact or adhere and consequently form incomplete toroids, and dorsal rings adopt variably abnormal morphologies. We also found that PAR-1 undergoes a redistribution from apical junctions to basolateral domains during morphogenesis. Despite a known role for PAR-1 in cell polarity, we have observed no detectable differences in the distribution of various markers of epithelial cell polarity. We propose that PAR-1 activity at the cell cortex is critical for mediating cell shape changes, cell surface composition, or cell signaling during vulval morphogenesis.     

Cathepsin L is essential for embryogenesis and development of Caenorhabditis elegans.

Cysteine proteases play critical biological roles in both intracellular and extracellular processes. We characterized Ce-cpl-1, a Caenorhabditis elegans cathepsin L-like cysteine protease. RNA interference with Ce-cpl-1 activity resulted in embryonic lethality and a transient delayed growth of larvae to egg producing adults, suggesting an essential role for cpl-1 during embryogenesis, and most likely during post-embryonic development. Cpl-1 gene (Ce-cpl-1:lacZ) is widely expressed in the intestine and hypodermal cells of transgenic worms, while the fusion protein (Ce-CPL-1::GFP) was expressed in the hypodermis, pharynx, and gonad. The CPL-1 native protein accumulates in early to late stage embryos and becomes highly concentrated in gut cells during late embryonic development. CPL-1 is also present near the periphery of the eggshell as well as in the cuticle of larval stages suggesting that it may function not only in embryogenesis but also in further development of the worm. Although the precise role of Ce-CPL-1 during embryogenesis is not yet clear it could be involved in the processing of nutrients responsible for synthesis and/or in the degradation of eggshell. Moreover, an increase in the cpl-1 mRNA is seen in the intermolt period approximately 4 h prior to each molt. During this process Ce-CPL-1 may act as a proteolytic enzyme in the processing/degradation of cuticular or other proteins. Similar localization of a related cathepsin L in the filarial nematode Onchocerca volvulus, eggshell and cuticle, suggests that some of the Ce-CPL-1 function during development may be conserved in other parasitic nematodes.     

The AP-3 clathrin-associated complex is essential for embryonic and larval development in Caenorhabditis elegans

The adaptor protein (AP) complexes are involved in membrane transport of many proteins. There are 3 AP complexes in C. elegans unlike mammals that have four. To study the biological functions of the AP-3 complexes of C. elegans, we sought homologues of the mouse and human genes that encode subunits of the AP-3 complexes by screening C. elegans genomic and EST sequences. We identified single copies of homologues of the m3, s3, b3 and d genes. The medium chain of AP-3 is encoded by a single gene in C. elegans but two different genes in mammals. Since there are no known mutations in these genes in C. elegans, we performed RNAi to assess their functions in development. RNAi of each of the genes caused embryonic and larval lethal phenotypes. APM-3 is expressed in most cells, particularly strongly in spermatheca and vulva. We conclude that the products of the C. elegans m3, s3, b3 and d genes are essential for embryogenesis and larval development.     

LIN-35/Rb Causes Starvation-Induced Germ Cell Apoptosis via CED-9/Bcl2 Downregulation in Caenorhabditis elegans

Apoptosis is an important mechanism for maintaining germ line health. In Caenorhabditis elegans, germ cell apoptosis occurs under normal conditions to sustain gonad homeostasis and oocyte quality. Under stress, germ cell apoptosis can be triggered via different pathways, including the following: (i) the CEP-1/p53 pathway, which induces germ cell apoptosis when animals are exposed to DNA damage; (ii) the mitogen-activated protein kinase kinase (MAPKK) pathway, which triggers germ cell apoptosis when animals are exposed to heat shock, oxidative stress, or osmotic stress; and (iii) an unknown mechanism that triggers germ cell apoptosis during starvation. Here, we address how starvation induces germ cell apoptosis. Using polysomal profiling, we found that starvation for 6 h reduces the translationally active ribosomes, which differentially affect the mRNAs of the core apoptotic machinery and some of its regulators. During starvation, lin-35/Rb mRNA increases its expression, resulting in the accumulation of this protein. As a consequence, LIN-35 downregulates the expression of the antiapoptotic gene ced-9/Bcl-2. We observed that the reduced translation of ced-9/Bcl-2 mRNA during food deprivation together with its downregulation drastically affects its protein accumulation. We propose that CED-9/Bcl-2 downregulation via LIN-35/Rb triggers germ cell apoptosis in C. elegans in response to starvation.     

Sphingosine phosphate lyase expression is essential for normal development in Caenorhabditis elegans

Sphingolipids are ubiquitous membrane constituents whose metabolites function as signaling molecules in eukaryotic cells. Sphingosine 1-phosphate, a key sphingolipid second messenger, regulates proliferation, motility, invasiveness, and programmed cell death. These effects of sphingosine 1-phosphate and similar phosphorylated sphingoid bases have been observed in organisms as diverse as yeast and humans. Intracellular levels of sphingosine 1-phosphate are tightly regulated by the actions of sphingosine kinase, which is responsible for its synthesis and sphingosine-1-phosphate phosphatase and sphingosine phosphate lyase, the two enzymes responsible for its catabolism. In this study, we describe the cloning of the Caenorhabditis elegans sphingosine phosphate lyase gene along with its functional expression in Saccharomyces cerevisiae. Promoter analysis indicates tissue-specific and developmental regulation of sphingosine phosphate lyase gene expression. Inhibition of C. elegans sphingosine phosphate lyase expression by RNA interference causes accumulation of phosphorylated and unphosphorylated long-chain bases and leads to poor feeding, delayed growth, reproductive abnormalities, and intestinal damage similar to the effects seen with exposure to Bacillus thuringiensis toxin. Our results show that sphingosine phosphate lyase is an essential gene in C. elegans and suggest that the sphingolipid degradative pathway plays a conserved role in regulating animal development.     

The Caenorhabditis elegans innexin INX-3 is localized to gap junctions and is essential for embryonic development

Innexins are the proposed structural components of gap junctions in invertebrates. Antibodies that specifically recognize the Caenorhabditis elegans innexin protein INX-3 were generated and used to examine the patterns of inx-3 gene expression and the subcellular sites of INX-3 localization. INX-3 is first detected in two-cell embryos, concentrated at the intercellular interface, and is expressed ubiquitously throughout the cellular proliferation phase of embryogenesis. During embryonic morphogenesis, INX-3 expression becomes more restricted. Postembryonically, INX-3 is expressed transiently in several cell types, while expression in the posterior pharynx persists throughout development. Through immuno-EM techniques, INX-3 was observed at gap junctions in the adult pharynx, providing supporting evidence that innexins are components of gap junctions. An inx-3 mutant was isolated through a combined genetic and immunocytochemical screen. Homozygous inx-3 mutants exhibit defects during embryonic morphogenesis. At the comma stage of early morphogenesis, variable numbers of cells are lost from the anterior of inx-3(lw68) mutants. A range of terminal defects is seen later in embryogenesis, including localized rupture of the hypodermis, failure of the midbody to elongate properly, abnormal contacts between hypodermal cells, and failure of the pharynx to attach to the anterior of the animal.     

G9a/GLP catalyzes H3K14me1 and H3K14me2 in vivo and in vitro

Science China Life Sciences -     

Determination of the parental pronuclear origin in bovine zygotes: H3K9me3 versus H3K27me2-3

To study the dynamics of 5-methylcytosine and 5-hydroxymethylcytosine in zygotes, the parental origin of the pronuclei needs to be determined. To this end the use of the asymmetric distribution of histone modifications in pronuclei is becoming more popular. Here, we demonstrated that histone 3 lysine 27 di-tri-methylation shows a stable pattern being present in the maternal but not in the paternal pronucleus of bovine zygotes, even in late stages of pronuclear development. In contrast, the pattern of histone 3 lysine 9 tri-methylation is very variable, and therefore cannot be used to reliably determine the parental origin of bovine pronuclei.     

Modulation of Caenorhabditis elegans infection sensitivity by the LIN-7 cell junction protein

In Caenorhabditis elegans, the LIN‐2/7/10 protein complex regulates the activity of signalling proteins. We found that inhibiting lin‐7 function, and also lin‐2 and lin‐10, resulted in enhanced C. elegans survival after infection by Burkholderia spp., implicating a novel role for these genes in modulating infection outcomes. Genetic experiments suggested that this infection phenotype is likely caused by modulation of the DAF‐2 insulin/IGF‐1 signalling pathway. Supporting these observations, yeast two‐hybrid assays confirmed that the LIN‐2 PDZ domain can physically bind to the DAF‐2 C‐terminus. Loss of lin‐7 activity also altered DAF‐16 nuclear localization kinetics, indicating an additional contribution by hsf‐1. Unexpectedly, silencing lin‐7 in the hypodermis, but not the intestine, was protective against infection, implicating the hypodermis as a key tissue in this phenomenon. Finally, consistent with lin‐7 acting as a general host infection factor, lin‐7 mutants also exhibited enhanced survival upon infectionby two other Gram‐negative pathogens, Pseudomonas and Salmonella spp.     

The Caenorhabditis elegans ortholog of human PHF5a shows a muscle-specific expression domain and is essential for C. elegans morphogenetic development

We have recently described a novel human and murine multigene-family that is highly conserved during evolution and shows a PHD-finger-like domain present in the deduced protein sequences. Here, we describe the cloning and characterization of the Caenorhabditis elegans ortholog of human PHF5a. Transgenic phf-5::yfp-reporter techniques in C. elegans identified temporal C. elegans phf-5 expression being restricted to late C. elegans development. The phf-5::yfp expression starts within the morphogenetic phase of embryonic development and lasts to the stage of adult worms. Spatial phf-5 expression is muscle-specific with an expression in the developing pharynx, in body wall muscular structures, and in the anal muscles. By phf-5 RNAi we further demonstrated that PHF-5 is essential in the morphogenetic phase of C. elegans embryonic development as well as in young larvae. In contrast, phf-5 RNAi does not show an evident phenotype to adult worms. Taken together, this is the first report providing evidence for a tissue and stage-specific expression of a PHF5a ortholog, named phf-5, in C. elegans while our data further suggest an essential role of the encoded PHF-5 protein in morphogenetic development and muscle function.     

Metformin induces S‐adenosylmethionine restriction to extend the Caenorhabditis elegans healthspan through H3K4me3 modifiers

Metformin, a widely prescribed first‐line drug for the treatment of type II diabetes mellitus, has been shown to extend lifespan and delay the onset of age‐related diseases. The precisely mechanisms by which these effects are realized remain elusive. We find that metformin exposure is restricted to adults, which is sufficient to extend lifespan. However, limiting metformin exposure to the larvae has no significant effect on Caenorhabditis elegans longevity. Here, we show that after metformin treatment, the level of S‐adenosylmethionine (SAM) is reduced in adults but not in the larvae. Potential mechanisms by which reduced SAM might increase lifespan include altering the histone methylation. However, the molecular connections between metformin, SAM limitation, methyltransferases, and healthspan‐associated phenotypes are unclear. Through genetic screening of C. elegans, we find that metformin promotes the healthspan through an H3K4 methyltransferase/demethylase complex to downregulate the targets, including mTOR and S6 kinase. Thus, our studies provide molecular links between meformin, SAM limitation, histone methylation, and healthspan and elucidate the mode action of metformin‐regulated healthspan extension will boost its therapeutic application in the treatment of human aging and age‐related diseases.     

The hedgehog-related gene wrt-5 is essential for hypodermal development in Caenorhabditis elegans

The Caenorhabditis elegans genome encodes a series of hedgehog-related genes, which are thought to have evolved and diverged from an ancestral Hh gene. They are classified into several families based on their N-terminal domains. Here, we analyze the expression and function of a member of the warthog gene family, wrt-5, that lacks the Hint/Hog domain. wrt-5 is expressed in seam cells, the pharynx, pharyngeal-intestinal valve cells, neurons, neuronal support cells, the excretory cell, and the reproductive system. WRT-5 protein is secreted into the extracellular space during embryogenesis. Furthermore, during larval development, WRT-5 protein is secreted into the pharyngeal lumen and the pharyngeal expression changes in a cyclical manner in phase with the molting cycle. Deletion mutations in wrt-5 cause embryonic lethality, which are temperature sensitive and more severe at 15 degrees C than at 25 degrees C. Animals that hatch exhibit variable abnormal morphology, for example, bagging worms, blistering, molting defects, or Roller phenotypes. We examined hypodermal cell junctions using the AJM-1Colon, two colonsGFP marker in the wrt-5 mutant background and observed cell boundary abnormalities in the arrested embryos. AJM-1Colon, two colonsGFP protein is also misplaced in pharyngeal muscle cells in the absence of WRT-5. In conclusion, we show that wrt-5 is an essential gene that - despite its lack of a Hint domain - has multiple functions in C. elegans and is implicated in cell shape integrity.