Transcriptomic analysis of thoraces after overexpression of an active allele of REPTOR in muscle
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ABSTRACT:
PROVIDER: PRJNA781995 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR16989402.fastq.gz | Fastqsanger.gz | |||
| SRR16989403.fastq.gz | Fastqsanger.gz | |||
| SRR16989404.fastq.gz | Fastqsanger.gz | |||
| SRR16989405.fastq.gz | Fastqsanger.gz | |||
| SRR16989406.fastq.gz | Fastqsanger.gz |
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Project description:Purpose: identify global changes in gene expression in thorax tissues caused by an increase in activity of REPTOR in muscles. Note that the thorax of adult flies is composed mainly by muscle tissue but fat body is also present. Changes in gene expression do not exclusively reflect the muscle transcriptome Methods: To extract total RNAs for RNA-Seq experiment, we used 5-6 thoraces dissected out from both tub-Gal80ts/+ ; dMef2-GAL4/+ (Con) and tub-Gal80ts/UAS-REPTOR[ACT] ; dMef2-GAL4/+ (REPTOR), making sure the gut of these flies was completely removed. Crosses were kept at 18°C to avoid expression of REPTOR during development. Adult males were collected every 24-48 hours and incubated 3-4 days at 18°C before being shifted to 29°C. Flies were then incubated for 4 days at 29°C. After assessing RNA quality with Agilent Bioanalyzer, mRNAs were enriched by poly-A pull-down. Then, sequencing libraries constructed with Illumina TruSeq RNA prep kit were sequenced using. We multiplexed samples in each lane, which yields targeted number of single-end 75 bp reads for each sample, as a fraction of 180 million reads for the whole lane. Sequence reads were mapped back to the Drosophila genome (flybase genome annotation version r6.30) using STAR. With the uniquely mapped reads, we quantified gene expression levels using Cufflinks (FPKM values). Next, differentially expressed genes between experimental and control data were analyzed with DESeq2. Results: Gene list enrichment analysis of the downregulated thoracic transcriptome by REPTOR overexpression revealed a striking enrichment of multiple metabolic processes impinging on carbohydrate metabolism, mitochondria, glycolysis and oxidative metabolism. Also, Thor, a well-characterized target of REPTOR was upregulated and it was validated with qPCR. Conclusions: Our study indicates that REPTOR is a strong regulator of muscle metabolism in adult flies.
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Project description:Purpose: REPTOR and FoxO are two transcription factors that regulate muscle metabolism. However, these transcription factors share around 40% of target genes. Furthermore, the thorax of adult flies is composed of several tissues including muscle and fat body, making it difficult to discover direct target genes of REPTOR and FoxO specifically in muscle tissue. This experimental approach allows the identification of REPTOR-specific and FoxO-specific target genes in muscle clusters Methods: snRNA-seq analysis of dissected adult fly thoraces, when an active allele of REPTOR or an active allele of FoxO are overexpressed using a muscle-specific driver (dMef2-Gal4) Results: Identification of the transcriptional signature of each tissue present in the thorax of adult flies when REPTOR or FoxO are overexpressed in muscle. Discovery of potential direct target genes of REPTOR and FoxO in muscle tissue, as well as metabolic pathways regulated by each transcription factor. Conclusions: REPTOR and FoxO modulate distinct gene signatures in muscle tissue to regulate metabolism in adult flies.
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Project description:Lambs that inherit a callipyge allele from their dam have an up-regulation of maternally imprinted transcripts near the callipyge mutation but do not exhibit muscle hypertrophy. It is not clear what effects these maternally expressed transcripts have in the muscle or how the inheritance of a maternal callipyge allele prevents the expression of the callipyge phenotype which is seen paternal heterozygotes only. This experiment utilized bovine gene expression arrays to profile the gene expression of the semimembranosus muscle of 30 day old half-sibling lambs of all four possible genotypes with respect to the callipyge allele (+/+, C/+, +/C, and C/C). Experiment Overall Design: 10 lambs were used for this study. N=3 biological reps for C/C genotype, N=3 biological reps for Cmat/+ genotype, N=2 biological reps for +/Cpat genotype, and N=2 biological reps for +/+ genotype. Pairwise contrasts were performed to analyze the presence of a maternally inherited callipyge allele and the effect on a homozygous animal (+/C vs CC; +/+ vs C/+; and C/+ vs C/C). Experiment Overall Design: These data make up two experiments. Experiment Overall Design: [1] The maternal allele (4 genotype) experiment compares all four possible genotypes of a particular allele at one age and one tissue (30 days semimembranosus). Experiment Overall Design: [2] The paternal allele (2 genotype) experiment compares only the NC and the NN genotypes in one tissue (SM) across 4 ages (10, 20, 30, and 80 days). Experiment Overall Design: Images were interpreted with Microarray Suite version 5.0 (MAS5.0) in GCOS with no scaling or normalization (i.e., Samples GSM298143-GSM298164). Images were also interpreted by RMA using Bioconductor package of R (i.e., Samples GSM298165-GSM298186).
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