RNA-seq of total and polysomal RNA of clk-1(qm30) mutants in Caenorhabditis elegans
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ABSTRACT:
INSTRUMENT(S): Illumina HiSeq 4000
ORGANISM(S): Caenorhabditis elegans
SUBMITTER: Marte Molenaars
PROVIDER: E-MTAB-7166 | biostudies-arrayexpress |
REPOSITORIES: biostudies-arrayexpress
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Project description:The mammalian circadian clock relies on the master genes CLOCK (CLK) and BMAL1 and drives rhythmic gene expression to regulate biological functions under circadian control. We recently uncovered a surprising disconnect between the rhythmic binding of CLK:BMAL1 on DNA and the transcription of its target genes, suggesting that they are regulated by as yet uncharacterized mechanisms. Here we show that rhythmic CLK:BMAL1 DNA binding promotes rhythmic chromatin opening. The underlying mechanisms include CLK:BMAL1 binding to nucleosomes and rhythmic chromatin modifications, including the incorporation of the histone variant H2A.Z. This rhythmic chromatin remodeling mediates the rhythmic binding of other transcription factors adjacent to CLK:BMAL1, suggesting that the activity and the tissue-specific expression of these other transcription factors contribute to the genome-wide CLK:BMAL1 heterogeneous transcriptional output. These data therefore indicate that the clock regulation of transcription relies on the rhythmic regulation of chromatin accessibility and suggest that the concept of pioneer function extends to acute gene regulation, well beyond the current confines of developmental/cell specification. Mouse liver CLK ChIP-Seq signal on Mnase-digested or sonicated chromatin, at 2 different time point (ZT22 and ZT06) from the same mice. Libraries were sequenced using Ilumina HiSeq2000. For Mnase-digested chromatin, libraries contained a mononucleosome insert (e.g., ~147bp), whereas the insert was ~150-300bp for sonicated chromatin.
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Project description:To discover new miRNA targets, we generated a C. elegans transgenic line expressing a functional N-terminally Tandem Affinity Purification (TAP) tagged ALG-1 protein (C. elegans strain WS4303). We crossed the TAP::ALG-1 transgene into the mir-58(n4640) mutant background to generate the strain WS5041. For simplicity, we will hereafter term the TAP::ALG-1 transgenic animals as “wild type” and the transgenic WS5041 animals as “mir-58”. In addition to immunopurifying the TAP::ALG-1 and associated RNAs from these strains, we also compared total mRNA levels between these two strains. Total RNA was isolated from the same WS4303 and WS5041 total extracts that was further used for the TAP::ALG-1 RIP. Three independent biological replicates were analyzed. Long-oligo whole-genome C. elegans arrays, produced by the Genome Sequencing Center at Washington University in St. Louis (http://genome.wustl.edu/genome/celegans/microarray/ma_gen_info.cgi), were used for these experiments. A total of 10 µg of total RNA was used for cDNA synthesis.
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Project description:RIP-chip-SRM : a New Combinatorial Large Scale Approach Identifies a Set of Translationally Regulated bantam/miR 58 Targets in C. elegans RNA binding protein immunopurification + microarray + targeted protein quantification via Selected Reaction Monitoring This SuperSeries is composed of the following subset Series: GSE32941: Purification of TAP::ALG-1 complexes and associated RNA from mixed-stage TAP::ALG-1 transgenic (WS4303) and wild-type (N2) animals GSE32942: Microarray analysis of TAP::ALG-1 associated RNAs isolated from synchronized 'wild-type' animals and 'mir-58' mutants GSE32943: Expression analysis of WS4303 (wild type) vs WS5041 (mir-58 mutants) total mRNA Refer to individual Series
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Project description:To discover new miRNA targets, we generated a C. elegans transgenic line expressing a functional N-terminally Tandem Affinity Purification (TAP) tagged ALG-1 protein (C. elegans strain WS4303). We crossed the TAP::ALG-1 transgene into the mir-58(n4640) mutant background to generate the strain WS5041. For simplicity, we will hereafter term the TAP::ALG-1 transgenic animals as wild typeand the transgenic WS5041 animals as mir-58. We compared the mRNA population that coimmunopurified with TAP::ALG-1 from synchronized L4 stage wild-type animals with that from synchronized L4 stage mir-58 mutant animals by one-color Affymetrix gene arrays. miR-58 target mRNAs should be specifically underrepresented in the latter samples. Strains WS4303 (wt) and WS5041 (mir-58) were used for TAP::ALG-1 IPs. All experiments were conducted in three independent replicates. For each replicate, WS4303 and WS5041 were grown in parallel. 150 ng of TAP::ALG-1 associated RNA isolated from synchronized late L4 animals were sent to the GeneCore facilty in Heidelberg, Germany (http://www.genecore.embl.de/index.cfm), and the microarray data were generated according to their standard protocol (Weinmann et al. 2009).
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Project description:This SuperSeries is composed of the SubSeries listed below. Refer to individual Series
2014-01-06 | E-GEOD-47145 | biostudies-arrayexpress
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