Unknown,Transcriptomics,Genomics,Proteomics

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Molecular profiling of activated neurons by phosphorylated ribosome capture [Illumina BeadArray]

ABSTRACT: We report that phosphorylated ribosomes can be immunoprecipitated from mouse brain homogenates, enriching for mRNAs that were selectively expressed in activated neurons Mice were given an injection of concentrated salt solution (2M NaCl, 325 uL) or PBS, and then sacrificed 2h later and hypothalami dissected. Tissue homogenates were prepared from hypothalamus and phosphorylated ribosomes immunoprecipitated using pS6 244/247 antibodies. The immunoprecipitated RNA and total RNA from both control and salt treated animals were then purified and analyzed by Illumina Microarray.

ORGANISM(S): Mus musculus

SUBMITTER: Zachary Knight

PROVIDER: E-GEOD-40994 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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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.