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C. elegans L4 hypodermal RNA polymerase DamID

ABSTRACT: To adapt RNA polymerase DamID (RAPID) for FLP-mediated spatial control in Caenorhabditis elegans, we inserted a Dam::rpb-6 fusion gene downstream of a FRT-flanked mCh::his-58 cassette and under control of the hsp-16.41 promoter. We introduced a single copy of this construct into the C. elegans genome and crossed the resulting line with a dpy-7p::FLP driver to enable basal Dam::rpb-6 expression in the hypodermis. Nematodes were cultured at 20 degrees Celcius to ensure low levels of Dam::RPB-6 expression in the hypodermis and total genomic DNA was purified from L4 larvae. DNA from animals expressing GFP::Dam was used to control for unspecific methylation. The genome-wide association profile of Dam::RPB-6 was determined by deep sequencing, which revealed a list of 2331 protein coding genes with FDR < 0.05. Original RAPID reference: Gomez-Saldivar et al (2020) Tissue-Specific Transcription Footprinting Using RNA PoI DamID (RAPID) in Caenorhabditis elegans. Genetics 216, 931–945. doi:10.1534/genetics.120.303774

ORGANISM(S): Caenorhabditis elegans

PROVIDER: GSE214300 | GEO | 2022/09/30

REPOSITORIES: GEO

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C. elegans L4 hypodermal SLAM-ITseq

Project description:To adapt SLAM-ITseq for Caenorhabditis elegans, we inserted a codon-optimized version of the Toxoplasma gondii UPRT gene downstream of a FRT-flanked mCh::his-58 cassette and under control of the strong and ubiquitous eft-3 promoter. We introduced a single copy of this construct into the C. elegans genome and crossed the resulting line with a dpy-7p::FLP driver. FLP-dependent expression of UPRT was confirmed by Western blotting (strain BN1190). As a proof of concept, we exposed animals to 4-TU from L3 to L4 stage and purified total RNA. RNA from animals without FLP expression was used to control for UPRT-independent labeling strain BN1158). RNA was alkylated with iodoacetamide, which leads to a T>C conversion of 4-TU-labeled positions during cDNA synthesis, and subjected to deep sequencing (see Materials and Methods). We confirmed that the presence of FLP and UPRT did not affect gene expression. Comparing the T>C conversion rate per gene revealed a significant increase in nematodes expressing UPRT in the hypodermis. Next, we performed a beta-binominal test to identify genes that were significantly labelled across all replicas, which retrieved 197 genes (p<0.003; FDR<0.1 after adjustment for multiple comparisons). Original SLAM-ITseq reference: Matsushima W, Herzog VA, Neumann T, Gapp K, Zuber J, Ameres SL, Miska EA (2018) SLAM-ITseq: sequencing cell type-specific transcriptomes without cell sorting. Development 145 (13). doi:10.1242/dev.164640

2022-12-31 | GSE192419 | GEO

embryo hypodermal cells tiling array

Project description:modENCODE_submission_662 This submission comes from a modENCODE project of Robert Waterston. For full list of modENCODE projects, see http://www.genome.gov/26524648 Project Goal: Our experiments are designed to detect all C. elegans transcripts by hybridizing RNA to commerically available genome tiling arrays. To maximize the chances of detecting rare transcripts with limited expression in specific cells, we are extracting RNA from selected embryonic cells isolated by FACS and from postembryonic cells by use of the mRNA tagging method. For data usage terms and conditions, please refer to http://www.genome.gov/27528022 and http://www.genome.gov/Pages/Research/ENCODE/ENCODEDataReleasePolicyFinal2008.pdf Keywords: Transcript tiling array analysis EXPERIMENT TYPE: Transcript tiling array analysis. BIOLOGICAL SOURCE: Strain: DM8001 (engineered, target gene dpy-7 tagged by GFP); Tissue: hypodermis; Developmental Stage: Mixed stage of embryos 20dC; Genotype: raIs/[rol-6(SU1006)+Pdpy-7::GFP]; Sex: Hermaphrodite; NUMBER OF REPLICATES: 3; EXPERIMENTAL FACTORS: Strain DM8001 (engineered, target gene dpy-7 tagged by GFP); temperature 20; Developmental Stage Mixed stage of embryos 20dC; Tissue hypodermis

2010-08-04 | E-GEOD-23286 | biostudies-arrayexpress

DamID of Dam::EMR-1 in Caenorhabditis elegans L1 larvae

Project description:DamID of Dam::EMR-1 in Caenorhabditis elegans L1 larvae

| PRJNA520793 | ENA

Heme utilization in the Caenorhabditis elegans hypodermal cells is facilitated by HRG-2

Project description:The roundworm Caenorhabditis elegans is a heme auxotroph that requires the coordinated actions of HRG-1 heme permeases to transport environmental heme into the intestine and HRG-3, a secreted protein, to deliver intestinal heme to other tissues including the embryo. Here we show that heme homeostasis in the extraintestinal hypodermal tissue is facilitated by the transmembrane protein HRG-2. Systemic heme deficiency upregulates hrg-2 mRNA expression over 200-fold in the main body hypodermal syncytium hyp 7. HRG-2 is a type I membrane protein which binds heme and localizes to the endoplasmic reticulum and apical plasma membrane. Cytochrome heme profiles are aberrant in HRG-2 deficient worms, a phenotype that is partially suppressed by heme supplementation. Heme-deficient yeast strain, ectopically expressing worm HRG-2, reveal significantly improved growth at submicromolar concentrations of exogenous heme. Taken together, our results implicate HRG-2 as a facilitator of heme utilization in the C. elegans hypodermis and provide a mechanism for regulation of heme homeostasis in an extraintestinal tissue. Two experimental groups: Group 1: three 4um Controls vs. three 4uM hrg-2 Mutants; Group 2: three 20um Controls vs. three 20uM hrg-2 Mutants

2013-01-01 | E-GEOD-34471 | biostudies-arrayexpress

Gene expression profiling of epidermal cell types in C. elegans using Targeted DamID

Project description:In this study we use targeted DamID to assay RNA polymerase occupancy across the genome specifically within the seam cells and hypodermis of C. elegans to identify genes expressed in these cell-types.

2021-08-31 | GSE164775 | GEO

Caenorhabditis elegans

Project description:DamID of Dam::LMN-1 and Dam::EMR-1 in Caenorhabditis elegans adult worms

| PRJNA189179 | ENA

Heme utilization in the Caenorhabditis elegans hypodermal cells is facilitated by HRG-2

2013-01-01 | GSE34471 | GEO

LMN-1-DamID in N2 and set-25(n5021) met-2(n4256) mutant early C. elegans embryos

Project description:We asked if the perinuclear position of chromosome arms in C. elegans depends on the histone methyltransferases MET-2 and SET-25. To this end, we performed LMN-1-DamID in wild-type (N2) and mutant (set-25 met-2) strains. LMN-1-DamID signal on chromosome arms was significantly reduced in the mutant. Three biological replicas were performed for each genotype. For one replica dyes were swapped. For each replica methylated DNA amplified from a strain expressing LMN-1-Dam was competitively hybridized against DNA amplified from a strain expressing GFP-Dam.

2012-09-04 | E-GEOD-37226 | biostudies-arrayexpress

Transcriptome analysis of N2 and flp-6 Caenorhabditis elegans at 25 °C

Project description:Temperature is a prominent environmental stimulus that influences life span. Previous studies indicate that in Caenorhabditis elegans, thermosensory perception in the AFD neuron maintains life span at warm temperatures. How thermosensation is translated into neuronal signals that shape aging remains elusive. We found that the Caenorhabditis elegans CREB crh-1, as well as several key genes in AFD thermosensory transduction, were specifically required for normal life span at warm temperatures. crh-1 acted in the AFD to increase transcription of the CRE-containing neuropeptide gene flp-6 in a temperature-dependent manner. Both crh-1 and flp-6 were necessary and sufficient for longevity at warm temperatures, and their effects depended on the AIY interneuron. Moreover, flp-6 signaling downregulated ins-7/insulin and several insulin pathway genes, whose activity compromised life span. We postulate that temperature experience is integrated in the thermosensory neurons to generate CREB-dependent neuropeptide signals that antagonize insulin signaling and promote temperature-specific longevity.

2018-05-01 | GSE68464 | GEO

Identifying targets of DSX with DamID-seq

Project description:We have adapted the DamID protocol for use with high throughput sequencing. We have used DamID to identify the positions within the Drosophila genome where the transcription factor DSX is bound. We sequenced DpnI-digested genomic DNA from fly tissues containing UAS-Dam (control) or UAS-Dam-DsxF or UAS-Dam-DsxM.

2014-10-30 | GSE27269 | GEO

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