Unknown,Transcriptomics,Genomics,Proteomics

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Systematic Analysis of Tissue-Restricted miRISCs Reveals a Broad Role for microRNAs in Suppressing Basal Activation of the C. elegans Pathogen Response

ABSTRACT: Gene regulation by microRNAs (miRNAs) under specific physiological conditions often involves complex interactions between multiple miRNAs and a large number of their targets, as well as coordination with other regulatory mechanisms, limiting the effectiveness of classical genetic methods to identify miRNA functions. We took a systematic approach to analyze the miRNA-induced silencing complex (miRISC) in individual tissues of C. elegans and found that mRNAs encoded by pathogen-responsive genes were dramatically over-represented in the intestinal miRISC, and that multiple miRNAs accumulated in the intestinal miRISCs upon infection. Inactivation of the miRISC or ablation of miRNAs from different families, resulted in over-expression of several pathogen-responsive genes under basal conditions and, surprisingly, enhanced worm survival on pathogenic Pseudomonas aeruginosa. These results indicate that much of the miRNA activity in the gut is dedicated to attenuating the activity of the pathogen response system, uncovering a novel and complex physiological function of the miRNA network. Each array was used for one biological replicate of a given experiment. One channel was used to probe Total RNA, the other was for IP RNA. Channels were swapped between biological replicates. 4 biological replicates for each asynchronous intestine IPs, 4 biological replicates for asynchronous muscle IP, 5 biolgoical replicates for L4 intestine IP, and two new biological replicates for the GFP control IP (previous biological replicates can be found in GSE9073: GSM230865-GSM2308657 and GSM230873-GSM230875).

ORGANISM(S): Caenorhabditis elegans

SUBMITTER: Min Han

PROVIDER: E-GEOD-36419 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:Identifying the physiological functions of microRNAs (miRNAs) is often challenging because miRNAs commonly impact gene expression under specific physiological conditions through complex miRNA::mRNA interaction networks and in coordination with other means of gene regulation, such as transcriptional regulation and protein degradation. Such complexity creates difficulties in dissecting miRNA functions through traditional genetic methods using individual miRNA mutations. To investigate the physiological functions of miRNAs in neurons, we combined a genetic M-bM-^@M-^\enhancerM-bM-^@M-^] approach complemented by biochemical analysis of neuronal miRNA-induced silencing complexes (miRISCs) in C. elegans. Total miRNA function can be compromised by mutating one of the two GW182 proteins (AIN-1), an important component of miRISC. We found that combining an ain-1 mutation with a mutation in unc-3, a neuronal transcription factor, resulted in an inappropriate entrance into the stress-induced, alternative larval stage known as dauer, indicating a role of miRNAs in preventing aberrant dauer formation. Analysis of this genetic interaction suggests that neuronal miRNAs perform such a role partly by regulating endogenous cyclic guanosine monophosphate (cGMP) signaling, potentially influencing two other dauer-regulating pathways. Through tissue-specific immunoprecipitations of miRISC, we identified miRNAs and their likely target mRNAs within neuronal tissue. We verified the biological relevance of several of these miRNAs and found that many miRNAs likely regulate dauer formation through multiple dauer-related targets. Further analysis of target mRNAs suggests potential miRNA involvement in various neuronal processes, but the importance of these miRNA::mRNA interactions remains unclear. Finally, we found that neuronal genes may be more highly regulated by miRNAs than intestinal genes. Overall, our study identifies miRNAs and their targets, and a physiological function of these miRNAs in neurons. It also suggests that compromising other aspects of gene expression, along with miRISC, can be an effective approach to reveal miRNA functions in specific tissues under specific physiological conditions. Each array was used for one biological replicate, where the red channel is used for IP RNA and the green channel is used for Total RNA. IP RNA indicates the transcripts associated with a neuronally expressed, GFP-tagged, miRISC. Five biological replicates were done on asynchronous worms. Please note the two different microarray platforms that were used.

Project description:modENCODE_submission_2623 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: OP201(made_by : S Kim description : This strain's transgene was constructed by Mihail Sarov at the Max Planck Institute for Cell Biology in Tubiginen using Tony Hyman's recombineering pipeline. The resulting plasmid was used for biolistic transformation of an unc-119(ed3) strain. The PQM-1::EGFP fusion protein is expressed in the correct pqm-1 spatio-temporal expression pattern. This strain was used for ChIP-seq experiments to map the in vivo binding sites for the PQM-1 transcription factor. tags : GFP::3xFlag mutagen : Bombard outcross : 3 genotype : unc119(ed3);wgIs201(pqm-1::TY1 EGFP FLAG C;unc119) official name : OP201 ); Developmental Stage: L3; Genotype: unc119(ed3);wgIs201(pqm-1::TY1 EGFP FLAG C;unc119); Sex: Hermaphrodite; EXPERIMENTAL FACTORS: Developmental Stage L3; Target gene pqm-1; Strain OP201(made_by : S Kim description : This strain's transgene was constructed by Mihail Sarov at the Max Planck Institute for Cell Biology in Tubiginen using Tony Hyman's recombineering pipeline. The resulting plasmid was used for biolistic transformation of an unc-119(ed3) strain. The PQM-1::EGFP fusion protein is expressed in the correct pqm-1 spatio-temporal expression pattern. This strain was used for ChIP-seq experiments to map the in vivo binding sites for the PQM-1 transcription factor. tags : GFP::3xFlag mutagen : Bombard outcross : 3 genotype : unc119(ed3);wgIs201(pqm-1::TY1 EGFP FLAG C;unc119) official name : OP201 ); temp (temperature) 20 degree celsius Series_type: CHIP-seq

Project description:modENCODE_submission_2610 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: OP184(made_by : S Kim description : This strain's transgene was constructed by Mihail Sarov at the Max Planck Institute for Cell Biology in Tubiginen using Tony Hyman's recombineering pipeline. The resulting plasmid was used for biolistic transformation of an unc-119(ed3) strain. The LIN-15B::EGFP fusion protein is expressed in the correct lin-15B spatio-temporal expression pattern. This strain was used for ChIP-seq experiments to map the in vivo binding sites for the LIN-15B transcription factor. tags : GFP::3xFlag mutagen : Bombard outcross : 3 genotype : unc119(ed3);wgIs184(lin-15B::TY1 EGFP FLAG;unc119) official name : OP184 ); Developmental Stage: L3; Genotype: unc119(ed3);wgIs184(lin-15B::TY1 EGFP FLAG;unc119); Sex: Hermaphrodite; EXPERIMENTAL FACTORS: Developmental Stage L3; Target gene lin-15B; Strain OP184(made_by : S Kim description : This strain's transgene was constructed by Mihail Sarov at the Max Planck Institute for Cell Biology in Tubiginen using Tony Hyman's recombineering pipeline. The resulting plasmid was used for biolistic transformation of an unc-119(ed3) strain. The LIN-15B::EGFP fusion protein is expressed in the correct lin-15B spatio-temporal expression pattern. This strain was used for ChIP-seq experiments to map the in vivo binding sites for the LIN-15B transcription factor. tags : GFP::3xFlag mutagen : Bombard outcross : 3 genotype : unc119(ed3);wgIs184(lin-15B::TY1 EGFP FLAG;unc119) official name : OP184 ); temp (temperature) 20 degree celsius Series_type: CHIP-seq

Project description:modENCODE_submission_2612 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: OP109(made_by : R Waterston description : This strain's transgene was constructed by Mihail Sarov at the Max Planck Institute for Cell Biology in Tubiginen using Tony Hyman's recombineering pipeline. The resulting plasmid was used for biolistic transformation of an unc-119(ed3) strain. The BLMP-1::EGFP fusion protein is expressed in the correct blmp-1 spatio-temporal expression pattern. This strain was used for ChIP-seq experiments to map the in vivo binding sites for the BLMP-1 transcription factor. tags : GFP::3xFlag mutagen : Bombard outcross : 3 genotype : unc-119 (ed3) III; wgIs109 [blmp-1::TY1::EGFP::FLAG; unc-119 (+)] official name : OP109 ); Developmental Stage: fed L1; Genotype: unc-119 (ed3) III; wgIs109 [blmp-1::TY1::EGFP::FLAG; unc-119 (+)]; Sex: Hermaphrodite; EXPERIMENTAL FACTORS: Developmental Stage fed L1; Target gene blmp-1; Strain OP109(made_by : R Waterston description : This strain's transgene was constructed by Mihail Sarov at the Max Planck Institute for Cell Biology in Tubiginen using Tony Hyman's recombineering pipeline. The resulting plasmid was used for biolistic transformation of an unc-119(ed3) strain. The BLMP-1::EGFP fusion protein is expressed in the correct blmp-1 spatio-temporal expression pattern. This strain was used for ChIP-seq experiments to map the in vivo binding sites for the BLMP-1 transcription factor. tags : GFP::3xFlag mutagen : Bombard outcross : 3 genotype : unc-119 (ed3) III; wgIs109 [blmp-1::TY1::EGFP::FLAG; unc-119 (+)] official name : OP109 ); temp (temperature) 20 degree celsius Series_type: CHIP-seq

Project description:modENCODE_submission_2621 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: OP177(made_by : S Kim description : This strain's transgene was constructed by Mihail Sarov at the Max Planck Institute for Cell Biology in Tubiginen using Tony Hyman's recombineering pipeline. The resulting plasmid was used for biolistic transformation of an unc-119(ed3) strain. The EGL-27::EGFP fusion protein is expressed in the correct egl-27 spatio-temporal expression pattern. This strain was used for ChIP-seq experiments to map the in vivo binding sites for the EGL-27 transcription factor. tags : GFP::3xFlag mutagen : Bombard outcross : 3 genotype : unc119(ed3);wgIs177(egl-27::TY1 EGFP FLAG;unc119) official name : OP177 ); Developmental Stage: fed L1; Genotype: unc119(ed3);wgIs177(egl-27::TY1 EGFP FLAG;unc119); Sex: Hermaphrodite; EXPERIMENTAL FACTORS: Developmental Stage fed L1; Target gene egl-27; Strain OP177(made_by : S Kim description : This strain's transgene was constructed by Mihail Sarov at the Max Planck Institute for Cell Biology in Tubiginen using Tony Hyman's recombineering pipeline. The resulting plasmid was used for biolistic transformation of an unc-119(ed3) strain. The EGL-27::EGFP fusion protein is expressed in the correct egl-27 spatio-temporal expression pattern. This strain was used for ChIP-seq experiments to map the in vivo binding sites for the EGL-27 transcription factor. tags : GFP::3xFlag mutagen : Bombard outcross : 3 genotype : unc119(ed3);wgIs177(egl-27::TY1 EGFP FLAG;unc119) official name : OP177 ); temp (temperature) 20 degree celsius Series_type: CHIP-seq

Project description:modENCODE_submission_2620 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: OP120(made_by : R Waterston description : This strain's transgene was constructed by Mihail Sarov at the Max Planck Institute for Cell Biology in Tubiginen using Tony Hyman's recombineering pipeline. The resulting plasmid was used for biolistic transformation of an unc-119(ed3) strain. The CEH-30::EGFP fusion protein is expressed in the correct ceh-30 spatio-temporal expression pattern. This strain was used for ChIP-seq experiments to map the in vivo binding sites for the CEH-30 transcription factor. tags : GFP::3xFlag mutagen : Bombard outcross : 3 genotype : unc119(ed3);wgIs120(ceh-30::TY1 EGFP FLAG;unc119) official name : OP120 ); Developmental Stage: late embryo; Genotype: unc119(ed3);wgIs120(ceh-30::TY1 EGFP FLAG;unc119); Sex: Hermaphrodite; EXPERIMENTAL FACTORS: Developmental Stage late embryo; Target gene ceh-30; Strain OP120(made_by : R Waterston description : This strain's transgene was constructed by Mihail Sarov at the Max Planck Institute for Cell Biology in Tubiginen using Tony Hyman's recombineering pipeline. The resulting plasmid was used for biolistic transformation of an unc-119(ed3) strain. The CEH-30::EGFP fusion protein is expressed in the correct ceh-30 spatio-temporal expression pattern. This strain was used for ChIP-seq experiments to map the in vivo binding sites for the CEH-30 transcription factor. tags : GFP::3xFlag mutagen : Bombard outcross : 3 genotype : unc119(ed3);wgIs120(ceh-30::TY1 EGFP FLAG;unc119) official name : OP120 ); temp (temperature) 20 degree celsius Series_type: CHIP-seq

Project description:When C. elegans larvae hatch in the absence of food they persist in a stress resistant, developmentally arrested state (L1 arrest) for weeks or until food becomes available. We characterized growth, mRNA expression, and RNA Polymerase II activity genome-wide during L1 arrest and recovery. RNA Pol II binding data resulting from ChIP-Seq experiments using the Illumina Genome Analyzer are included in this GEO submission. Complementary mRNA expression data from the Affymetrix microarray platform can be found at GEO accession# GSE11055. The goals of the Pol II ChIP-Seq compononent of this project were to use Pol II antibodies 1) to investigate patterns of transcription in developmentally arrested larvae, when mRNA expression levels have reached steady state; 2) to investigate patterns of transcription in immediate response to feeding, when mRNA expression levels change dramatically; and 3) to investigate accumulation of Pol II at promoters during arrest and recovery. We started our ChIP studies with the S2 antibody (Abcam ab5095) since it was raised against a Ser2 phosphorylated peptide from the C-terminal domain of Pol II and should therefore be relatively specific for active, elongating Pol II, and it worked well for the first two goals above. In order to investigate accumulation of Pol II at promoters, we used the S2 antibody and complemeted it with antibody 4H8 (Abcam ab5408), which according to the manafacturer recognizes phosphorylated and non-phosphorylated Pol II, and antibody 8WG16 (Abcam ab817), which binds primarily to non-phosphorylated Pol II but also relatively weakly to phosphorylated Pol II. We were somewhat surprised to find that the results obtained with each of these antibodies were very similar, though upon deeper analysis we discovered relatively subtle differences consistent with the relative specificities of each antibody and existing models regarding phosphorylation state of Pol II and its activity and location. In summary, we find that while Pol II continues transcribing starvation genes, it is M-bM-^@M-^XpausedM-bM-^@M-^Y accumulates on the promoters of growth and development genes during L1 arrest. Consistent with it poising arrested larvae for recovery, pausingpromoter accumulation decreases in response to feeding, while elongation and mRNA levels increase. These results demonstrate that Pol II pausing is widespread in C. elegans and that it is nutritionally controlled during development.These results demonstrate that accumulation of Pol II at promoters of growth and development genes is common in C. elegans and that promoter accumulation anticipates nutritionally controlled gene expression during development. RNA Pol II binding was examined with three different antibodies (S2, 4H8, and 8WG16) during either L1 arrest (starvation) or after 1 hr recovery by feeding. For the S2 antibody two different time points during L1 arrest were examined (6 and 12 hr). 14 total samples are included: 4 independent control samples (Input), a pair biological replicates with the S2 antibody at 6 hr L1 arrest, a pair of biological replicates with the 4H8 antibody at 12 hr L1 arrest and at 1 hr recovery, and singletons for the S2 and 8WG16 antibodies at 12 hr L1 arrest and at 1 hr recovery. "GSE13973_PolII_ChIP-Seq.xls.gz" contains 5 worksheets with the following contents: "geneDensity" includes the number of reads per million mapping to each gene model normalized to gene length. Units are reads per million per kilobase. "TSSdensity" includes the number of reads per million mapping to a 200 bp window spanning the most upstream transcription start site for each gene and normalized by length (200 bp). Units are reads per million per kilobase. "geneEnrichment" includes the fold-enrichment of read density over each gene model relative to input. Where input values were below the median of all input values then the median was used. Units are fold-enrichment. "TSSenrichment" includes the fold-enrichment of read density over the 200 bp TSS window relative to input. Where input values were below the median of all input values then the median was used. Units are fold-enrichment. "5' bias" includes 5' bias calculation for each gene and each antibody in each condition. "GSE13973_CelegansWS190_GeneCoordinates.xls.gz" contains the gene coordinates based on version WS190 of the C. elegans genome.

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Systematic Analysis of Tissue-Restricted miRISCs Reveals a Broad Role for microRNAs in Suppressing Basal Activation of the C. elegans Pathogen Response

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