Genome-wide identification of the occupancy of RNA polymerase II (POL II) at L4/young adult stage in C. elegans
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ABSTRACT: We acquied the RNA polymerase II binding profiles in ama-1 transgenic worms. The large subuint of POL II, AMA-1, was C-terminally tagged with GFP, the binding regions were determined using chromatin immunoprecipitation with anti-GFP and anti-polymerase II antibodies followed by illumina high-throughput sequencing (ChIP-seq). For data usage terms and conditions, please refer to http://www.genome.gov/27528022 and http://www.genome.gov/Pages/Research/ENCODE/ENCODEDataReleasePolicyFinal2008.pdf RNA polymerase II binding profiles at L4/young adult stage were acquied with anti-native protein antibody (8WG16) and anti-epitope (GFP) antibody
Project description:We identified the DNA binding sites of DAF-16 in transgenic line TJ356 (daf-16::gfp) that carries high-copy number daf-16. The expression pattern was examined through fluorescent microscopy. The binding sites were determined using chromatin immunoprecipitation with anti-GFP antibody and anti-POL II antibody followed by illumina high-throughput sequencing (ChIP-seq). For data usage terms and conditions, please refer to http://www.genome.gov/27528022 and http://www.genome.gov/Pages/Research/ENCODE/ENCODEDataReleasePolicyFinal2008.pdf Identification of DAF-16 binding sites at L4/Young adult stage when DAF-16 was highly overexpressed
Project description:We identified the DNA binding sites of MAB-5 in C. elegans. MAB-5 was C-terminally tagged with GFP and the expression pattern was examined through fluorescent microscopy. The binding sites were determined using chromatin immunoprecipitation with anti-GFP antibody followed by illumina high-throughput sequencing (ChIP-seq). For data usage terms and conditions, please refer to http://www.genome.gov/27528022 and http://www.genome.gov/Pages/Research/ENCODE/ENCODEDataReleasePolicyFinal2008.pdf Identification of MAB-5 binding sites at L3 stage
Project description:C. elegans nuclear pore protein NPP-13 associates with small RNA genes transcribed by RNA Polymerase III. To test if the nuclear pore-chromatin interactions play a role in large-scale chromatin organization, we determined nuclear membrane-genome interactions and RNA Polymerase II localization in C. elegans embryos depleted for NPP-13. Genome-wide ChIP-seq and ChIP-chip for nuclear membrane protein LEM-2, RNA Polymerase II (AMA-1) and H3K4me3 were performed in mixed-stage C. elegans embryos depleted for NPP-13. As a control, ChIP was also performed in wild-type embryos treated with empty vector.
Project description:We acquied the RNA polymerase II binding profiles in ama-1 transgenic worms. The large subuint of POL II, AMA-1, was C-terminally tagged with GFP, the binding regions were determined using chromatin immunoprecipitation with anti-GFP and anti-polymerase II antibodies followed by illumina high-throughput sequencing (ChIP-seq). For data usage terms and conditions, please refer to http://www.genome.gov/27528022 and http://www.genome.gov/Pages/Research/ENCODE/ENCODEDataReleasePolicyFinal2008.pdf
Project description:We identified the DNA binding sites of DPY-27 in C. elegans. DPY-27 was C-terminally tagged with GFP and the expression pattern was examined through fluorescent microscopy. The binding sites were determined using chromatin immunoprecipitation with anti-GFP antibody followed by illumina high-throughput sequencing (ChIP-seq). For data usage terms and conditions, please refer to http://www.genome.gov/27528022 and http://www.genome.gov/Pages/Research/ENCODE/ENCODEDataReleasePolicyFinal2008.pdf Identification of DPY-27 binding sites at embryonic stage
Project description:C. elegans nuclear pore protein NPP-13 associates with small RNA genes transcribed by RNA Polymerase III. To test if the nuclear pore-chromatin interactions play a role in large-scale chromatin organization, we determined nuclear membrane-genome interactions and RNA Polymerase II localization in C. elegans embryos depleted for NPP-13. Genome-wide ChIP-seq and ChIP-chip for nuclear membrane protein LEM-2, RNA Polymerase II (AMA-1) and H3K4me3 were performed in mixed-stage C. elegans embryos depleted for NPP-13. As a control, ChIP was also performed in wild-type embryos treated with empty vector.
Project description:RNAi targeting a conserved C. elegans cyclophilin, sig-7, causes defective development and embryonic arrest consistent with a global defect in transcription. The goal of this study was to compare the location of RNA Pol II in sig-7(RNAi) embryos to the localization in L4440/RNAi control embryos to examine the global effect of loss of sig-7 on RNA Pol II regulation and its distribution within gene bodies anti-AMA-1 ChIP in 2 replicates each of sig-7(RNAi) and RNAi control early stage embryos
Project description:Spatiotemporal control of gene expression is crucial for development and subject to evolutionary changes. Although proteins are the final product of most genes, the developmental proteome of an animal has not yet been comprehensively defined, and the correlation between RNA and protein abundance during development is largely unknown. Here, we globally measured and compared protein and mRNA expression changes during the life cycle of the nematodes C. elegans and C. briggsae, separated by ~30 million years of evolution. We observed that developmental mRNA and protein changes were highly conserved, to a surprisingly similar degree, but poorly correlated within a species, suggesting important and widespread post-transcriptional regulation. Post-transcriptional control was particularly well conserved if mRNA fold changes were buffered on the protein level, indicating a predominant repressive function. Finally, among divergently expressed genes, we identified insulin signaling, a pathway involved in life span determination, as a putative target of adaptive evolution. Samples of C. elegans and C. briggsae were collected at major developmental stages throughout the nematode life cycle. These stages comprise a population of mixed embryonic stages (E), populations of all four larval stages (L1, L2, L3, L4), late L4 larvae (LL4), young adults (YA), and a reference sample consisting of a mixture of all stages. To obtain synchronized worm populations, embryos were extracted by bleaching gravid adults and synchronized by starvation. Later stages were picked at fixed timepoints after determining the developmental stages by microscopic observation. For all stages, at least a single poly(A)-extracted mRNA library was sequenced on a single lane of an Illumina Genome Analyzer IIx.
Project description:We applied a middle-down proteomics strategy for large scale protein analysis during in vivo development of Caenorhabditis elegans. We characterized post-translational modifications (PTMs) on histone H3 N-terminal tails at eight time points during the C. elegans lifecycle, including embryo, larval stages (L1 to L4), dauer and L1/L4 post dauer. Histones were analyzed by our optimized middle-down protein sequencing platform using high mass accuracy tandem mass spectrometry. This allows quantification of intact histone tails and detailed characterization of distinct histone tails carrying co-occurring PTMs. We measured temporally distinct combinatorial PTM profiles during C. elegans development. We show that the doubly modified form H3K23me3K27me3, which is rare or non-existent in mammals, is the most abundant PTM in all stages of C. elegans lifecycle. The abundance of H3K23me3 increased during development and it was mutually exclusive of the active marks H3K18ac, R26me1 and R40me1, suggesting a role for H3K23me3 in to silent chromatin. We observed distinct PTM profiles for normal L1 larvae and for L1-post dauer larvae, or L4 and L4 post-dauer, suggesting that histone PTMs mediate an epigenetic memory that is transmitted during dauer formation. Collectively, our data describe the dynamics of histone H3 combinatorial code during C. elegans lifecycle and demonstrate the feasibility of using middle-down proteomics to study in vivo development of multicellular organisms.
Project description:modENCODE_submission_2439 This submission comes from a modENCODE project of Michael Snyder. For full list of modENCODE projects, see http://www.genome.gov/26524648 Project Goal: We are identifying the DNA binding sites for 300 transcription factors in C. elegans. Each transcription factor gene is tagged with the same GFP fusion protein, permitting validation of the gene's correct spatio-temporal expression pattern in transgenic animals. Chromatin immunoprecipitation on each strain is peformed using an anti-GFP antibody, and any bound DNA is deep-sequenced using Solexa GA2 technology. For data usage terms and conditions, please refer to http://www.genome.gov/27528022 and http://www.genome.gov/Pages/Research/ENCODE/ENCODEDataReleasePolicyFinal2008.pdf EXPERIMENT TYPE: CHIP-seq. BIOLOGICAL SOURCE: Strain: N2(genotype : wild type genotype : DR subclone of DB original (Tc1 pattern I) official name : N2 ); Developmental Stage: L3; Genotype: wild type; Sex: Hermaphrodite; EXPERIMENTAL FACTORS: Developmental Stage L3; Target gene ama-1; Strain N2(genotype : wild type genotype : DR subclone of DB original (Tc1 pattern I) official name : N2 ); temp (temperature) 20 degree celsius Series_type: CHIP-seq