Transcriptome profiling of mutant ASH2,RBBP5,WDR5 and WT(N2 or wild type) of C.elegans by RNASeq
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ABSTRACT:
INSTRUMENT(S): Illumina HiSeq 2500
ORGANISM(S): Caenorhabditis elegans
SUBMITTER:
PROVIDER: E-MTAB-5500 | biostudies-arrayexpress |
SECONDARY ACCESSION(S): ERP114093
REPOSITORIES: biostudies-arrayexpress
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Project description:The two major methods for growing C. elegans are liquid and plate culture. Differences in oxygenation and other environmental variables could affect many experimentally relevant biological processes, such as metabolism, cellular structure, and gene regulation. Worms on solid support move with deliberate sinusoidal movements, while worms in liquid thrash rapidly and almost constantly, often appearing elongated. We investigated differential gene expression under these two commonly used growth conditions. Embryos were isolated from adults grown on plates. After hatching, animals were placed either on plates or in liquid under standard conditions, and grown until young adulthood, approximately 48 hours later. RNA was isolated independently from each growth, differentially labeled with a fluorescent dye, and mixed directly for comparative hybridization to NimbleGen DNA microarrays, which contained probes designed to measure expression from all predicted C. elegans coding genes. The experiment was performed in triplicate. Analysis of the expression data yielded surprisingly few genes with differential expression, about 1%. Overall, 176 genes were downregulated significantly (p<0.01; Student's t) in liquid culture relative to plates, but of these only 102 genes were down two-fold or more. A total of 121 genes were upregulated (p<0.01. Student's t) in liquid; of these, RNA corresponding to 50 increased by at least two-fold. Surprisingly few genes directly associated with a stress response were differentially regulated in this experiment. However, the single gene most strongly upregulated in liquid is nnt-1, which encodes a mitochondrial NADP transhydrogenase, which protects against oxidative stress (Arkbladt et al., 2005 PMID: 15890626). Other molecules associated with reducing oxidation are also upregulated in liquid culture, including thioredoxin, and two glutathione-S-transferases. As a class, however, the group of genes most differentially regulated encode collagens. A total of 12 collagen genes were downregulated in liquid culture relative to plates, while none are upregulated. Other genes affected include those encoding proteins involved in ubiquitin-mediated proteolysis, Ras signaling, transcriptional regulation, ribosome function, and multiple membrane-associated proteins. The limited changes in relative RNA levels under different culture conditions provide increased confidence in comparisons made between worms grown in liquid and solid culture, and more generally between datasets obtained in different labs under slightly different culture conditions. Knowing which genes are most responsive to different culture conditions inform us about how environment affects the animal, and should be of interest for comparison to other stress and longevity global datasets. Total RNA was extracted from young adult hermaphrodite C. elegans grown either in s-basal liquid cultures or nematode growth medium plates under standard conditions.
2010-05-04 | E-GEOD-21526 | biostudies-arrayexpress
Project description:Genomic response of C. elegans after infection with Microbacterium nematophilum.<br><br>The interaction between the nematode Caenorhabditis elegans and a Gram-positive bacterial pathogen, Microbacterium nematophilum, provides a model for an innate immune response in nematodes. This pathogen adheres to the rectal and post-anal cuticle of the worm, causing slowed growth, constipation, and a defensive swelling response of rectal hypodermal cells. To explore the genomic responses that the worm activates after pathogenic attack we used microarray analysis of transcriptional changes induced after 6 hr infection, comparing virulent with avirulent infection.
2005-01-01 | E-MEXP-696 | biostudies-arrayexpress
Project description:Our aim was to identify the genome-wide direct LET-418 binding sites in early C.elegans embryos. a FLAG-tagged version of LET-418 was expressed in let-418(ts) mutants synchronised as early mixed stage embryos, and processed for ChIP followed by deep-sequencing. The obtained binding profile was compared to non-enriched input DNA and significant binding sites were identified.
2016-09-12 | E-MTAB-4716 | biostudies-arrayexpress
Project description:Here we exploit the essential process of X-chromosome dosage compensation to elucidate basic mechanisms that control the assembly, genome-wide binding, and function of gene regulatory complexes that act over large chromosomal territories. We demonstrate that a subunit of C. elegans MLL/COMPASS, a gene-activation complex, acts within the dosage compensation complex (DCC), a condensin complex, to target the DCC to both X chromosomes of hermaphrodites and thereby reduce chromosome-wide gene expression. The DCC binds to two categories of sites on X: rex sites that recruit the DCC in an autonomous, sequence- dependent manner, and dox sites that reside primarily in promoters of expressed genes and bind the DCC robustly only when attached to X. We find that DCC mutants that abolish rex-site binding do not eliminate dox-site binding, but instead reduce it to the level observed at autosomal binding sites in wild-type animals. Changes in DCC binding to these non-rex sites occur throughout development and correlate with transcriptional activity of adjacent genes. Moreover, autosomal DCC binding is enhanced by rex-site binding in cis in X-autosome fusion chromosomes. Thus, dox and autosomal sites exhibit similar binding properties. Our data support a model for DCC binding in which low-level DCC binding at dox and autosomal sites is dictated by intrinsic properties correlated with high transcriptional activity. Sex-specific DCC recruitment to rex sites then greatly elevates DCC binding to dox sites in cis, which lack intrinsically high DCC affinity on their own. We also show here that the C. elegans DCC achieves dosage compensation through its effects on transcription. The processed data files available here were used in Figure 6 of the paper which references this data. ChIP-chip experiments using antibodies against DPY-27 in wild type mixed embryos and fed L1 larvae.
2011-03-01 | E-GEOD-25877 | biostudies-arrayexpress
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.
2015-11-02 | PXD002525 | Pride
Project description:The overall goal of this investigation was to investigate X-content of sex-biased genes in several nematode species. The following species of nematode were investigated: *C. elegans, *N2; *C. brenneri, *PB2801; *C. briggsae, *AF16; *C. remanei*, PB4641; *P. **pacificus, *PS312*.* Genomic DNA sequencing data was used to assign X and autosomal - linkage to unassembled sequencing contigs. Male and female RNA seq data was then generated and used to determine sex-biased expression. For both DNA and RNA experiments, 50bp paired-end (DNA) or single-end (RNA) reads were generated on the Illumina HiSeq 2500. Sequencing lanes were multiplexed. Genomic DNA was isolated from 50-100 hand-picked young adult worms. At least two replicates for each sex were prepared. DNA was sheared via sonication and 350-500 bp sequencing libraries were prepared following the Illumina protocol. Total RNA was isolated from at least 1000 hand-picked L4/young adult worms (*C. **elegans, *N2) or J4/young adult worms (*P. pacificus, *PS312*). *PolyA beads were used to enrich for mRNA. Stranded RNAseq libraries were prepared via incorporation of dUTPs during cDNA synthesis, following the protocol detailed in Parkhomchuk et al, 2009. DNAseq and RNAseq reads were aligned to the appropriate WS228 reference genomes. DNA sequencing data (2-3 replicates) from male and female YA worms are included along with RNAseq data from C.elegans YA hermaphrodites and P.pacificus YA males and hermaphrodites.
2014-04-17 | E-GEOD-53144 | biostudies-arrayexpress
Project description:In many species, a dosage compensation complex (DCC) is targeted to X chromosomes of one sex to equalize levels of X gene products between males (1X) and females (2X). Here we identify cis-acting regulatory elements that target the C. elegans X chromosome for repression by the DCC. The DCC binds to discrete, dispersed sites on X of two types. rex sites recruit the DCC in an autonomous, DNA sequence-dependent manner using a 12 bp consensus motif that is enriched on X. This motif is critical for DCC binding, is clustered in rex sites, and confers much of X-chromosome specificity. Motif variants enriched on X by 3.8-fold or more are highly predictive (95%) for rex sites. In contrast, dox sites lack the X-enriched variants and cannot bind the DCC when detached from X. dox sites are more prevalent than rex sites and, unlike rex sites, reside preferentially in promoters of some expressed genes. These findings fulfill predictions for a targeting model in which the DCC binds to recruitment sites on X and disperses to discrete sites lacking autonomous recruitment ability. To relate DCC binding to function, we identified dosage-compensated and non-compensated genes on X. Unexpectedly, many genes of both types have bound DCC, but many do not, suggesting the DCC acts over long distances to repress X gene expression. Remarkably, the DCC binds to autosomes, but at far fewer sites and rarely at consensus motifs. DCC disruption causes opposite effects on expression of X and autosomal genes. The DCC thus acts at a distance to impact expression throughout the genome. Keywords: dosage compensation, condensin, X chromosome, gene expression, epigenetics, C. elegans ChIP-chip experiments included: biological duplicates of SDC-3 and MIX-1 IPs, a single DPY-27 IP, and 3 mock IPs.
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Project description:Here we exploit the essential process of X-chromosome dosage compensation to elucidate basic mechanisms that control the assembly, genome-wide binding, and function of gene regulatory complexes that act over large chromosomal territories. We demonstrate that a subunit of C. elegans MLL/COMPASS, a gene-activation complex, acts within the dosage compensation complex (DCC), a condensin complex, to target the DCC to both X chromosomes of hermaphrodites and thereby reduce chromosome-wide gene expression. The DCC binds to two categories of sites on X: rex sites that recruit the DCC in an autonomous, sequence- dependent manner, and dox sites that reside primarily in promoters of expressed genes and bind the DCC robustly only when attached to X. We find that DCC mutants that abolish rex-site binding do not eliminate dox-site binding, but instead reduce it to the level observed at autosomal binding sites in wild-type animals. Changes in DCC binding to these non-rex sites occur throughout development and correlate with transcriptional activity of adjacent genes. Moreover, autosomal DCC binding is enhanced by rex-site binding in cis in X-autosome fusion chromosomes. Thus, dox and autosomal sites exhibit similar binding properties. Our data support a model for DCC binding in which low-level DCC binding at dox and autosomal sites is dictated by intrinsic properties correlated with high transcriptional activity. Sex-specific DCC recruitment to rex sites then greatly elevates DCC binding to dox sites in cis, which lack intrinsically high DCC affinity on their own. We also show here that the C. elegans DCC achieves dosage compensation through its effects on transcription. ChIP-chip experiments using antibodies against DPY-27, SDC-3, DPY-30, DPY-26, MIX-1, SMC-4, ASH-2 in wild-type embryos. ChIP-chip experiments using antibodies against SDC-3, DPY-27, DPY-30, ASH-2, and IgG in different DCC mutants. ChIP-chip experiments using antibodies against RNA Pol II (hypophosphorylated and S2 and S5) in wild type and sdc-2 partial loss of function mutants.
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