Project description:To understand the mechanism by which HLH-26 regulated protection against S. enterica infection after exposure to E. faecium, we used RNA sequencing to focus on transcriptional changes induced by E. faecium in hlh-26(ok1453) compared with wild-type animals.
Project description:Mammalian TFEB and TFE3, as well as their ortholog in C. elegans HLH-30, play an important role in mediating cellular response to a variety of stress conditions, including nutrient deprivation, oxidative stress and pathogen infection. In this study we identify a novel mechanism of TFEB/HLH-30 regulation through a cysteine-mediated redox switch. Under stress conditions, TFEB-C212 undergoes oxidation, allowing the formation of intermolecular disulfide bonds that result in TFEB oligomerization. TFEB oligomers display increased resistance to mTORC1-mediated inactivation and are more stable under prolonged stress conditions. Mutation of the only cysteine residue present in HLH-30 (C284) significantly reduced its activity, resulting in developmental defects and increased pathogen susceptibility. Therefore, cysteine oxidation represents a new type of TFEB post-translational modification that functions as a molecular switch to link changes in redox balance with expression of TFEB/HLH-30 target genes.
Project description:In Caenorhabditis elegans, the six proteins that make up the REF-1 family are HES homologs that act in both Notch dependent and Notch-independent pathways to regulate embryonic events. To further our understanding of how the REF-1 family works to coordinate post-embryonic cellular events, we performed transcriptome analysis of HLH-25 and HLH-29 mutant strains. Gene expression microarray, including probe preparation, hybridization, fluidics run and chip scan, was performed by Georgia State University DNA/Protein Core Facility. Global gene expression in synchronized young adult populations of VC1220 (hlh-25 mutant) and tm284 (hlh-29 mutant) animals were compared to expression in N2 (wild-type) animals using GeneChip C. elegans Genome Array (Affymetrix). Data collection was carried out using GCOS 1.4 software (Affymetrix). Probe intensity values were normalized to a target value of 500 with normalization factor equal to 1. Data analysis was performed using GeneSpring GX 11 Software (Aglient, Palo Alto,CA).
Project description:In Caenorhabditis elegans, the six proteins that make up the REF-1 family are HES homologs that act in both Notch dependent and Notch-independent pathways to regulate embryonic events. To further our understanding of how the REF-1 family works to coordinate post-embryonic cellular events, we performed transcriptome analysis of HLH-25 and HLH-29 mutant strains.
Project description:The hlh-30 gene encodes a C. elegans basic-helix-loop-helix (bHLH) transcription factor; We compared RNA from wild type worms and worms mutant for the hlh-30 gene to identify putative target genes of the HLH-30 transcription factor.
Project description:To investigate the structure of the bodywall muscle differentiation network, we sought to both identify what role HLH-1 binding has in this regulation. We mapped the relationship between the regulatory targets and HLH-1 binding, as measured by ChIP-seq in animals enriched for bodywall muscle tissue through RNAi feeding. While regulatory targets are enriched for HLH-1 binding, the majority of HLH-1 binding actually occurs away from genes that are affected by hlh-1 loss of function. Nevertheless, HLH-1 binding is associated with both the expected E-box and, near genes belonging to specific regulatory subgroups, novel motifs that may serve as binding sites for cooperative transcription factors. HLH-1 binding genome-wide in the early embryo of C. elegans
Project description:The hlh-30 gene encodes a C. elegans basic-helix-loop-helix (bHLH) transcription factor; We compared RNA from wild type worms and worms mutant for the hlh-30 gene to identify putative target genes of the HLH-30 transcription factor. Experiment Overall Design: Total RNA from three biological replicates were isolated from embryos, L1 larvae, and L2 larvae from both wild type and hlh-30(tm1978) mutant worms
Project description:E. faecium is inherantly resistant to cephalosporins. Resistance is lost in Class A penicillin binding protein PbfF PonA mutants, but is reversible by pencillin exposure. E. faecium Affymetrix GeneChips were used to compare E. faecium expression properties of pbfF ponA mutant cells in the absence or presence of penicillin exposure. Significant differences were observed between the expression properties of mock and penicillin treated E. faecium CV571 (pbfF ponA double mutant) cells.
Project description:modENCODE_submission_2431 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: OP64(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 HLH-1::EGFP fusion protein is expressed in the correct hlh-1 spatio-temporal expression pattern. This strain was used for ChIP-seq experiments to map the in vivo binding sites for the HLH-1 transcription factor. tags : GFP::3xFlag mutagen : Bombard outcross : 3 genotype : unc-119(ed3) III; wgIs64 [unc-119(+) hlh-1::TY1::EGFP::3xFLAG] official name : OP64 ); Developmental Stage: embryo; Genotype: unc-119(ed3) III; wgIs64 [unc-119(+) hlh-1::TY1::EGFP::3xFLAG]; Sex: Hermaphrodite; EXPERIMENTAL FACTORS: Developmental Stage embryo; Target gene hlh-1; Strain OP64(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 HLH-1::EGFP fusion protein is expressed in the correct hlh-1 spatio-temporal expression pattern. This strain was used for ChIP-seq experiments to map the in vivo binding sites for the HLH-1 transcription factor. tags : GFP::3xFlag mutagen : Bombard outcross : 3 genotype : unc-119(ed3) III; wgIs64 [unc-119(+) hlh-1::TY1::EGFP::3xFLAG] official name : OP64 ); temp (temperature) 20 degree celsius Series_type: CHIP-seq
Project description:The enterococci comprise a genus of 49 low-GC content Gram-positive commensal species within the Firmicutes phylum that are known to occupy diverse habitats, notably the gastrointestinal core microbiota of nearly every phylum, including human. Of particular clinical relevance are two rogue species of enterococci, Enterococcus faecalis and the distantly related Enterococcus faecium, standing among the nefarious multi-drug resistant and hospital-acquired pathogens. Despite increasing evidence for RNA-based regulation in the enterococci, including regulation of virulence factors, their transcriptome structure and arsenal of regulatory small sRNAs (sRNAs) are not thoroughly understood. Using dRNA-seq, we have mapped at single-nucleotide resolution the primary transcriptomes of E. faecalis V583 and E. faecium AUS0004. We identified 2517 and 2771 transcription start sites (TSS) in E. faecalis and E. faecium, respectively. Based on the identified TSS, we created a global map of s70 promoter motifs. We also revealed features of 5’ and 3’UTRs across the genomes. The transcriptome maps also predicted 150 and 128 sRNA candidates in E. faecalis and E. faecium, respectively, some of which have been identified in previous studies and many of which are new. Finally, we validated several of the predicted sRNAs by Northern Blot in biologically relevant conditions. Comprehensive TSS mapping of two representative strains will provide a valuable resource for the continued development of RNA biology in the Enterococci.