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[
J Cell Biol,
2006]
Vertebrates produce multiple chondroitin sulfate proteoglycans that play important roles in development and tissue mechanics. In the nematode Caenorhabditis elegans, the chondroitin chains lack sulfate but nevertheless play essential roles in embryonic development and vulval morphogenesis. However, assignment of these functions to specific proteoglycans has been limited by the lack of identified core proteins. We used a combination of biochemical purification, Western blotting, and mass spectrometry to identify nine C. elegans chondroitin proteoglycan core proteins, none of which have homologues in vertebrates or other invertebrates such as Drosophila melanogaster or Hydra vulgaris. CPG-1/CEJ-1 and CPG-2 are expressed during embryonic development and bind chitin, suggesting a structural role in the egg. RNA interference (RNAi) depletion of individual CPGs had no effect on embryonic viability, but simultaneous depletion of CPG-1/CEJ-1 and CPG-2 resulted in multinucleated single-cell embryos. This embryonic lethality phenocopies RNAi depletion of the SQV-5 chondroitin synthase, suggesting that chondroitin chains on these two proteoglycans are required for cytokinesis.
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[
International Worm Meeting,
2009]
The nematode eggshell is a rigid, impermeable structure that protects the embryo during early development, and is required for the first embryonic cell division. The essential function of the eggshell makes it an attractive drug target to combat parasitic nematode infection, but little is known about the composition of the eggshell or the process by which it forms. We have identified two genes redundantly required for eggshell formation.
cpg-1/cej-1 and
cpg-2 encode chondroitin proteoglycans, a class of extracellular glycoproteins modified with chondroitin sugar chains. Live imaging of fluorescent strains shows that CPG-1 and CPG-2 are secreted from caveolin-enriched cortical granules during meiosis I, while immuno-electron microscopy experiments show they associate with the inner layer (the permeability barrier) of the eggshell. To better understand the function of chondroitin proteoglycans in eggshell assembly, we depleted CPG-1, CPG-2, and SQV-5 (the enzyme that synthesizes chondroitin) by RNAi. In utero, embryos co-depleted of CPG-1/CPG-2 or chondroitin have identical phenotypes, exhibiting defects in osmotic integrity and early cortical events dependent on the actomyosin contractile network, including polar body extrusion during meiosis I and II, membrane ruffling, pseudocleavage formation, polarity establishment, and cytokinesis. However, when osmotic support is provided to dissected embryos, early embryonic events (including cytokinesis and polarity establishment) are robustly rescued in chondroitin depletions, but only partially rescued in CPG-1/CPG-2 co-depletions. Ultra-structural analysis shows embryos depleted of chondroitin are able to form all three eggshell layers, while embryos co-depleted of CPG-1/CPG-2 fail to form the inner layer. The correlation of structural and functional data suggests that CPG protein cores have a function separable from that of the chondroitin chains during eggshell assembly, and provides evidence for the first structural proteins required for eggshell formation. To expand on this work, we are conducting an RNAi-based screen to identify additional genes required for the enigmatic process of eggshell assembly.
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[
C. elegans: Development and Gene Expression, EMBL, Heidelberg, Germany,
2010]
Metazoan oocytes have an extracellular coating that governs fertilization. Following fertilization, this covering is altered to prevent polyspermy and protect the developing embryo. In C. elegans, a vitelline layer covers oocytes prior to fertilization. Fertilization initiates conversion of the vitelline layer into a trilaminar eggshell consisting of an outer vitelline layer, a middle chitin-containing layer, and an inner layer proposed to serve as a permeability barrier. Here, we characterize CPG-1 and CPG-2, functionally redundant chondroitin proteoglycans that are the first described protein eggshell components. We show that CPG-1 and CPG-2 are delivered to the extracellular space after formation of the chitin layer by cortical granule exocytosis during meiosis I. Although they contain multiple chitin binding domains, CPG-1 and CPG-2 localize to the inner eggshell layer, whereas chitin is confined to the middle eggshell layer. We show that the inner eggshell layer is not the permeability barrier for small molecular weight solutes. Instead, this function resides in a previously undescribed layer that resides between the eggshell and the plasma membrane. Disruption of the permeability barrier leads to solute permeability and osmotic stress. Disruption of the inner CPG-1/2 eggshell layer causes these phenotypes, as well as adhesion of the embryonic plasma membrane to the eggshell and cytokinesis failure. Interfering with chitin layer assembly results in the inner layer phenotypes, plus polyspermy and catastrophic eggshell rupture. We conclude that the eggshell layers and permeability barrier are laid down in a step-wise fashion from outermost to innermost, with later assembly events requiring successful completion of previous ones.
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[
International Worm Meeting,
2007]
Chondroitin is a long unbranched sugar polymer synthesized on a protein core that is found either on the cell surface or in the extracellular matrix. Roles for chondroitin in C. elegans embryogenesis and vulval morphogenesis were established based on identification of the squashed vulva (sqv) genes as enzymes required for chondroitin biosynthesis. Subsequently, we reported the identification of nine novel chondroitin proteoglycan (CPG) core proteins that harbor chondroitin chains, none of which are homologous to vertebrate chondroitin sulfate proteoglycans. Two of these proteoglycans, CPG-1(CEJ-1) and CPG-2(B0280.5), are translationally regulated in the germline by GLD-1, which suggested a role in the early embryo. While RNAi depletions of either CPG-1 or CPG-2 alone showed no phenotype, double depletions resulted in penetrant embryonic lethality. Early cortical events dependent on the actomyosin contractile network, including polar body extrusion during meiosis I and II, membrane ruffling, pseudocleavage formation, polarity establishment, and cytokinesis, were absent in doubly depleted embryos. However, nuclear events and spindle dynamics appeared to occur with normal morphology and kinetics. CPG-1 and -2 both contain functional chitin binding domains, suggesting they interact with the chitinous eggshell. We are therefore using a number of approaches to more thoroughly understand the enigmatic process of how the C. elegans eggshell is formed, what role CPGs play in this process, and why it is required for actomyosin contraction. To visualize formation of the eggshell, we are performing live imaging of transgenic lines expressing GFP-tagged versions of CPG-1 and -2, and are developing an assay to monitor, in real time, extrusion of the chitinous layer following fertilization. We are currently conducting immunoprecipitation experiments with CPG-1 and -2 antibodies and using chondroitin affinity columns to identify interacting proteins that may be involved in eggshell formation. While the eggshell has been reported to be dispensable for embryogenesis between the 2-cell stage and hatching, our studies suggest that the eggshell is essential for the actomyosin-driven cortical events immediately following fertilization prior to the first cell division.
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[
International Worm Meeting,
2011]
Metazoan oocytes have an extracellular coating that governs fertilization. Following fertilization, this covering is altered to prevent polyspermy and protect the developing embryo. In C. elegans, a vitelline layer covers oocytes prior to fertilization. Fertilization initiates conversion of the vitelline layer into a trilaminar eggshell consisting of an outer vitelline layer, a middle chitin-containing layer, and an inner layer proposed to serve as a permeability barrier. Here, we characterize CPG-1 and CPG-2, functionally redundant chondroitin proteoglycans that are the first described protein eggshell components. We show that CPG-1 and CPG-2 are delivered to the extracellular space after formation of the chitin layer by cortical granule exocytosis during meiosis I. Although they contain multiple chitin binding domains, CPG-1 and CPG-2 localize to the inner eggshell layer, whereas chitin is confined to the middle layer. We show that the inner eggshell layer is not the permeability barrier for small molecular weight solutes. Instead, this function resides in a previously undescribed layer that assembles between the eggshell and the plasma membrane following meiosis II. Disruption of the permeability barrier leads to solute permeability and osmotic stress. Disruption of the inner CPG-1/2 eggshell layer causes these phenotypes, as well as adhesion of the embryonic plasma membrane to the eggshell and cytokinesis failure. Interfering with chitin layer assembly results in the inner layer phenotypes, plus polyspermy and catastrophic eggshell rupture. We conclude that the eggshell layers and permeability barrier are laid down in a step-wise and cell cycle-dependent fashion, with later assembly events requiring successful completion of previous ones. To build on this work, we also conducted an RNAi screen to identify additional genes that regulate eggshell and permeability barrier assembly. Several screen hits rendered the eggshell permeable, with minimal deleterious effects on early embryonic development. We therefore developed a reliable method to permeabilize and immobilize embryos to allow temporally-controlled and acute drug/inhibitor treatment to study early embryonic processes with live imaging.
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[
Genome Res,
2014]
Most vertebrate promoters lie in unmethylated CpG-dense islands, whereas methylation of the more sparsely distributed CpGs in the remainder of the genome is thought to contribute to transcriptional repression. Nonmethylated CG dinucleotides are recognized by CXXC finger protein 1 (CXXC1, also known as CFP1), which recruits SETD1A (also known as Set1) methyltransferase for trimethylation of histone H3 lysine 4, an active promoter mark. Genomic regions enriched for CpGs are thought to be either absent or irrelevant in invertebrates that lack DNA methylation, such as C. elegans; however, a CXXC1 ortholog (CFP-1) is present. Here we demonstrate that C. elegans CFP-1 targets promoters with high CpG density, and these promoters are marked by high levels of H3K4me3. Furthermore, as for mammalian promoters, high CpG content is associated with nucleosome depletion irrespective of transcriptional activity. We further show that highly occupied target (HOT) regions identified by the binding of a large number of transcription factors are CpG-rich promoters in C. elegans and human genomes, suggesting that the unusually high factor association at HOT regions may be a consequence of CpG-linked chromatin accessibility. Our results indicate that nonmethylated CpG-dense sequence is a conserved genomic signal that promotes an open chromatin state, targeting by a CXXC1 ortholog, and H3K4me3 modification in both C. elegans and human genomes.
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Down, T., Chen, R., Egelhofer, T., Ahringer, J., Chen, Q., Hillier, L., Stempor, P., Jeffers, T., Zeiser, E.
[
International Worm Meeting,
2013]
RNA PolII transcription initiation sites are largely unknown in C. elegans. The initial 5' end of most protein-coding transcripts are removed by trans-splicing, and non-coding initiation sites have not been investigated. We identify 73,500 distinct clusters of initiation. Bidirectional transcription is frequent, with a peak of transcriptional pairing at 120 bp. We assign transcription initiation sites to 7691 protein-coding genes and find that they display features typical of eukaryotic promoters. Strikingly, the majority of initiation occurs in intergenic regions with enhancer-like chromatin signatures. Remarkably, productive transcription elongation across enhancers is predominantly in the same orientation as that of the nearest downstream gene. This oriented transcription at upstream enhancers could potentially deliver RNA Pol II to a downstream proximal promoter, or alternatively might function as a distal promoter. CG dinucleotides (CpG islands) are enriched in mammalian promoters. CpG density is thought to be irrelevant in invertebrates that lack DNA methylation such as C. elegans. We find that CpG enrichment at worm promoters shares features of mammalian CpG islands. CpG clusters are found at protein-coding promoters showing nucleosome depletion. In mammals, non-methylated CpGs are bound by Cfp1/CXXC1, which leads to H3K4me3 marking at promoters through recruitment of Set1. Interestingly, a worm Cfp-1 ortholog was reported to be required for global H3K4me3 levels. We found that the worm Cfp-1 is enriched at high H3K4me3 promoters containing high density of CpGs. Moreover, we find that highly occupied target (HOT) regions bound by multiple transcription factors are CpG-rich promoters in worm and human genomes, suggesting that the HOT regions may be caused by CpG-induced nucleosome depletion. Our results suggest that non-methylated CpG-dense sequence is a conserved genomic signal dictating an open chromatin state and marking by the H3K4me3 modification.
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[
Proc Natl Acad Sci U S A,
2010]
CpG dinucleotides contribute to epigenetic mechanisms by being the only site for DNA methylation in mammalian somatic cells. They are also mutation hotspots and approximately 5-fold depleted genome-wide. We report here a study focused on CpG sites in the coding regions of Hox and other transcription factor genes, comparing methylated genomes of Homo sapiens, Mus musculus, and Danio rerio with nonmethylated genomes of Drosophila melanogaster and Caenorhabditis elegans. We analyzed 4-fold degenerate, synonymous codons with the potential for CpG. That is, we studied "silent" changes that do not affect protein products but could damage epigenetic marking. We find that DNA-binding transcription factors and other developmentally relevant genes show, only in methylated genomes, a bimodal distribution of CpG usage. Several genetic code-based tests indicate, again for methylated genomes only, that the frequency of silent CpGs in Hox genes is much greater than expectation. Also informative are NCG-GNN and NCC-GNN codon doublets, for which an unusually high rate of G to C and C to G transversions was observed at the third (silent) position of the first codon. Together these results are interpreted as evidence for strong "pro-epigenetic" selection acting to preserve CpG sites in coding regions of many genes controlling development. We also report that DNA-binding transcription factors and developmentally important genes are dramatically overrepresented in or near clusters of three or more CpG islands, suggesting a possible relationship between evolutionary preservation of CpG dinucleotides in both coding regions and CpG islands.
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[
Parasitol Res,
2019]
The recombinant heavy chain myosin of Brugia malayi (Bm-Myo) has earlier been reported as a potent vaccine candidate in our lab. Subsequently, we further enhanced its efficacy employing heterologous DNA prime/protein boost (Myo-pcD+Bm-Myo) immunization approach that produced superior immune-protection than protein or DNA vaccination. In the present study, we evaluated the efficacy of heterologous prime boost vaccination in combination with CpG, synthetic oligodeoxynucleotides (ODN) adjuvant in BALB/c mice. The results showed that CpG/Myo-pcD+Bm-Myo conferred 84.5+/-0.62% protection against B. malayi infective larval challenge which was considerably higher than Myo-pcD+Bm-Myo (75.6+/-1.10%) following immunization. Although, both the formulations of immunization elicited robust production of specific IgG antibody and their isotypes (IgG1, IgG2a, IgG2b, and IgG3); however, CpG/Myo-pcD+Bm-Myo predominantly enhanced the level of IgG2a suggesting Th1 biased immune response in presence of CpG. Furthermore, spleen isolated from mice that immunized with CpG/Myo-pcD+Bm-Myo had greater accumulation of CD4+, CD8+, and CD19+ B cells and there was an augmented expression of co-stimulatory molecules CD40, CD86 on host dendritic cells (DCs). In contrast to Myo-pcD+Bm-Myo group, the splenocytes of CpG/Myo-pcD+Bm-Myo immunized mice developed comparatively higher pro-inflammatory cytokines IL-2 and IFN- leaving anti-inflammatory cytokine levels unchanged. Moreover, CpG formulation also upregulated the RNA expression of IL-12 and TNF- in spleenocytes. The current findings suggest that the use of CpG would be more advantageous as an adjuvant predominantly in DNA/protein prime boost vaccine against Bm-Myo and presumably also for filarial infection.
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Gemma, Carolina, Dong, Yan, Janes, Jurgen, Stempor, Przemyslaw, Ahringer, Julie, Chen, Ron, Zeiser, Eva, Down, Thomas, Feuer, Sky
[
International Worm Meeting,
2015]
A hallmark of most vertebrate promoters is their association with non-methylated CG-dense sequences (CpG islands), while methylation of the more sparsely distributed CpGs in the remainder of the genome is thought to contribute to transcriptional repression. Non-methylated CG dinucleotides are recognized by the CXXC finger protein 1 (CXXC1/CFP1) which is part of the COMPASS complex. In the complex, the Set1 methyltransferase (SET-2 in C. elegans) generates H3K4me3, a mark of active promoters. Promoter CpGs were thought to be either absent or irrelevant in invertebrates that lack DNA methylation, such as C. elegans. However, a C. elegans CXXC1 ortholog (CFP-1) is present and required for the generation of H3K4me3 (1,2). We found that C. elegans CFP-1 targets promoters with high CpG density and that these promoters are marked by high levels of H3K4me3. Furthermore, as for mammalian promoters, high promoter CpG content in C. elegans is associated with nucleosome depletion irrespective of transcriptional activity. We further show that highly occupied target (HOT) regions identified by the binding of a large number of transcription factors are CpG-rich promoters in C. elegans and human genomes, suggesting that the unusually high factor association at HOT regions may be a consequence of CpG-linked chromatin accessibility. Our results indicate that non-methylated CpG-dense sequence is a conserved genomic signal that promotes an open chromatin state, and is targeted by a CXXC1 ortholog and H3K4me3 modification in both C. elegans and human genomes. To understand the function of CFP-1 and COMPASS/H3K4me3, we are studying a
cfp-1 deletion mutant. This mutant has nearly undetectable H3K4me3 and has defects in fertility and the repression of somatic genes in the germline, similar to the phenotype of
set-2 mutants (3). Profiling RNA and chromatin in
cfp-1 mutants uncovered alterations in histone modifications, chromatin accessibility, and patterns of gene expression. Our results shed light on the mechanism of action and role of CFP-1/H3K4me3 in chromatin regulation and function.References: (1) Simonet et al., 2007, Dev. Biol. 312, 367-83. (2) Li and Kelly, 2011, PLoS Genet. 7,
e1001349 (3) Robert et al., 2014, Cell Reports 9, 443-450.