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Nutritional control of mRNA isoform expression during developmental arrest and recovery in C. elegans.

ABSTRACT: Nutrient availability profoundly influences gene expression. Many animal genes encode multiple transcript isoforms, yet the effect of nutrient availability on transcript isoform expression has not been studied in genome-wide fashion. When Caenorhabditis elegans larvae hatch without food, they arrest development in the first larval stage (L1 arrest). Starved larvae can survive L1 arrest for weeks, but growth and post-embryonic development are rapidly initiated in response to feeding. We used RNA-seq to characterize the transcriptome during L1 arrest and over time after feeding. Twenty-seven percent of detectable protein-coding genes were differentially expressed during recovery from L1 arrest, with the majority of changes initiating within the first hour, demonstrating widespread, acute effects of nutrient availability on gene expression. We used two independent approaches to track expression of individual exons and mRNA isoforms, and we connected changes in expression to functional consequences by mining a variety of databases. These two approaches identified an overlapping set of genes with alternative isoform expression, and they converged on common functional patterns. Genes affecting mRNA splicing and translation are regulated by alternative isoform expression, revealing post-transcriptional consequences of nutrient availability on gene regulation. We also found that phosphorylation sites are often alternatively expressed, revealing a common mode by which alternative isoform expression modifies protein function and signal transduction. Our results detail rich changes in C. elegans gene expression as larvae initiate growth and post-embryonic development, and they provide an excellent resource for ongoing investigation of transcriptional regulation and developmental physiology.

SUBMITTER: Maxwell CS

PROVIDER: S-EPMC3460187 | biostudies-literature | 2012 Oct

REPOSITORIES: biostudies-literature

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Nutritional control of mRNA isoform expression during developmental arrest and recovery in C. elegans.

Maxwell Colin S CS Antoshechkin Igor I Kurhanewicz Nicole N Belsky Jason A JA Baugh L Ryan LR

Genome research 20120426 10

Nutrient availability profoundly influences gene expression. Many animal genes encode multiple transcript isoforms, yet the effect of nutrient availability on transcript isoform expression has not been studied in genome-wide fashion. When Caenorhabditis elegans larvae hatch without food, they arrest development in the first larval stage (L1 arrest). Starved larvae can survive L1 arrest for weeks, but growth and post-embryonic development are rapidly initiated in response to feeding. We used RNA- ...[more]

PMID: 22539650

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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.

Project description:BACKGROUND:Developmental physiology is very sensitive to nutrient availability. For instance, in the nematode Caenorhabditis elegans, newly hatched L1-stage larvae require food to initiate postembryonic development. In addition, larvae arrested in the dauer diapause, a non-feeding state of developmental arrest that occurs during the L3 stage, initiate recovery when exposed to food. Despite the essential role of food in C. elegans development, the contribution of food perception versus ingestion on physiology has not been delineated. RESULTS:We used a pharmacological approach to uncouple the effects of food (bacteria) perception and ingestion in C. elegans. Perception was not sufficient to promote postembryonic development in L1-stage larvae. However, L1 larvae exposed to food without ingestion failed to develop upon return to normal culture conditions, instead displaying an irreversible arrest phenotype. Inhibition of gene expression during perception rescued subsequent development, demonstrating that the response to perception without feeding is deleterious. Perception altered DAF-16/FOXO subcellular localization, reflecting activation of insulin/IGF signaling (IIS). The insulin-like peptide daf-28 was specifically required, suggesting perception in chemosensory neurons, where it is expressed, regulates peptide synthesis and possibly secretion. However, genetic manipulation of IIS did not modify the irreversible arrest phenotype caused by food perception, revealing that wild-type function of the IIS pathway is not required to produce this phenotype and that other pathways affected by perception of food in the absence of its ingestion are likely to be involved. Gene expression and Nile red staining showed that food perception could alter lipid metabolism and storage. We found that starved larvae sense environmental polypeptides, with similar molecular and developmental effects as perception of bacteria. Environmental polypeptides also promoted recovery from dauer diapause, suggesting that perception of polypeptides plays an important role in the life history of free-living nematodes. CONCLUSIONS:We conclude that actual ingestion of food is required to initiate postembryonic development in C. elegans. We also conclude that polypeptides are perceived as a food-associated cue in this and likely other animals, initiating a signaling and gene regulatory cascade that alters metabolism in anticipation of feeding and development, but that this response is detrimental if feeding does not occur.

Dataset Information

Nutritional control of mRNA isoform expression during developmental arrest and recovery in C. elegans.

Publications

Nutritional control of mRNA isoform expression during developmental arrest and recovery in C. elegans.

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