NO-mediated transcriptional response in C. elegans
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ABSTRACT: We used next generation sequensing to determine C.elegans genes induced by Nitroc Oxide (NO) treatment Compare expression profile of NO treated and untreated control.
Project description:The human AdipoR2 protein and its C. elegans homolog PAQR-2 are multi-pass plasma membrane proteins that protect cells against membrane rigidification. We seek to better understand the mechanisms by which these proteins can promote the synthesis and incorporation of membrane-fluidizing unsaturated fatty acids into phospholipids. To this end, we performed immunoprecipitations of tagged AdipoR2 and PAQR-2 expressed in HEK293 cells or whole C. elegans, respectively, and identified co-immunoprecipitated proteins using mass spectroscopy. Among the conserved interaction partners, we identified many proteins likely important for the protein life cycles (e.g. ribosome, proteasome, vesicle trafficking components). Importantly, several proteins important for fatty acid synthesis, elongation and incorporation into phospholipids were also identified as evolutionarily conserved AdipoR2/PAQR-2 interaction partners. We experimentally verified several of these interactions, and propose that AdipoR2 and PAQR-2 can recruit an ER-bound membrane complex to promote the production and incorporation of unsaturated fatty acids into phospholipids.
Project description:Purpose: Understand the synergistic relationship between the methyltransferases Set1 and Set5 in the regulation of gene expression. Methods: Total mRNA was obtained from two independent biological replicates each of wildtype (WT), set1∆, set5∆, SET5 Y402A and set1∆set5∆ S. cerevisiae strains. Libraries were generated and sequenced using an Illumina HiSeq2000 platform. The sequence reads that passed quality filters were mapped using TopHat and expression levels were quantified using Cufflinks. Results: We generated FPKM expression values for each transcript and identified the differentially expressed genes using an FDR-adjusted p-value of 0.05. Subsequent data analysis was restricted to genes with fold-change greater than 1.7 relative to WT. Our results show that Set1 and Set5 have roles primarily in transcription repression. Moreover, lack of both Set1 and Set5 results in a synergistic exhacerbation of the transcriptional derepression observed in the single mutants. Further analysis revealed a specific enrichment of the Set5/Set1-repressed genes near repetitive DNA sequences of the genome. Conclusions: Our study uncovers an unexpected synergistic role of Set1 and Set5 in transcription repression of telomeric regions and Ty retrotransposons. mRNA profiles of wildtype (WT), set1∆, set5∆, SET5 Y402A and set1∆set5∆ were generated by sequencing using an Illumina HiSeq2000 platform. Two biological replicates of each strain were used.
Project description:How epigenetic information is transmitted from generation to generation remains largely unknown. Deletion of the C. elegans Histone H3 lysine 4 dimethyl (H3K4me2) demethylase spr-5 leads to inherited accumulation of the euchromatic H3K4me2 mark and progressive decline in fertility. Here we identified a genetic network of chromatin-modifying factors, including the H3K4me1/me2 methyltransferases SET-17 and SET-30, the H3K9me1/me2 methyltransferase MET-2, an H3K9me3 methyltransferase, SET-26, the H3K9me3 demethylase JMJD-2, and an H3K9me reader EAP-1, which regulate the trans-generational flow of epigenetic information. Importantly, genetic ablation of set-17, set-30, jmjd-2, or eap-1 suppresses the progressive transgenerational phenotypes, while loss of SET-26 or MET-2 accelerates the infertility of spr-5 mutant worms. We further show that loss of spr-5 also causes a trans-generational increase in lifespan, which is dependent on these chromatin regulators as well as DAF-36 and DAF-12, which control a germline to soma longevity signaling pathway. These findings suggest that the balance between the euchromatic H3K4 and the heterochromatic H3K9 methylation regulates trans-generational effects on longevity and fertility. EAP-1 binding ChIPseq libraries were prepared from 50 ul of packed young adult worms which were maintained at 16 degrees until the appropriate generation and then shifted to 25 degrees after birth. Two biological repeats were generated for wildtype and generation 20 spr-5(by101) mutant worms and a single repeat for generation 10. There were four input samples and eap-1 null mutant worms were also analyzed.
Project description:Small RNA libraries from total RNA isolated from adult animals Small RNA libraries were derived from genetically identical strains carrying a 21Usensor transgene in single copy. In one strain the transgene has become permanently silent and is not reactivated by RNAi against prg-1 (RNAe). In the other the transgene reactivates upon RNAi against prg-1.
Project description:The aim of this experiment was to identify changes in gene expression in muscle and liver between pigs that were divergent in feed efficiency. The animals were selected from two different farms of origin. 48 animals were utilized in this study. Following slaughter RNA Total RNA was extracted from liver and muscle ((25 mg) tissue using Trizol Reagent (Sigma-Aldrich, Arklow, Ireland) according to the manufacturer’s instructions, the crude RNA extract was further purified using the GenElute Mammalian Total RNA Miniprep Kit (RTN70, Sigma-Aldrich). Library construction and sequencing was performed by the next generation sequencing facility at the Institute of Molecular Medicine, University of Leeds, United Kingdom. The RNA-seq libraries were constructed using the Illumina TruSeq RNA Sample Preparation Kit v2 (Illumina, San Diego, CA) per the manufacturer’s instructions. One hundred base paired-end sequencing was run on an Illumina HiSeq2000 platform with each pool ran on two lanes on a flow cell.
Project description:Comparision of mRNA abundance and translation efficiency mediated by cytoplasmic poly(A) polymerases in C. elegans comparision of different adults treated with RNAi
Project description:The goal of this study is to compare gene expression data for a well known model organism (Escherichia coli) using different technologies (NGS here, microarray from GSE48776). mRNA profiles of Wild Type and two Mutant Strains (ydcR (b1439) MUTANT and yjiR (b4340) MUTANT), growth in minimal medium, were generated by deep sequencing, in triplicate, using Illumina MiSeq.
Project description:Investigation of whole genome gene expression level changes in C. elegans genes daf-9 and daf-12 mutant,compared to the wild-type strain NimbleScan softwareM-bM-^@M-^Ys implementation of RMA offers quantile normalization and background correction. There are three steps of RMA: Firstly, background subtraction was performed using a local background estimator. Secondly, quantile normalization (Bioinformatics 2003; 19:185) at probe level was done to make the expression distributions the same for all arrays. Last, probe set summarization was performed using Robust Multichip Average (RMA) algorithm as described by Irizarry, et al. (Nucleic Acids Res. 2003; 31:e15 and Biostatistics 2003; 4:249). A nine chip study using total RNA recovered from three separate wild-type cultures of C.elegans and three separate cultures of daf-9 mutantM-oM-<M-^Lthree separate cultures of daf-12 mutant.The C. elegans 12 x 135K Gene Expression Array was manufactured by Roche NimbleGen. This array enables you to conveniently and simultaneously hybridize 12 samples on each slide. About 13,187 genes are collected from the authoritative data source including Wormbase.
Project description:Genome-wide expression analysis in C. Elegans grown in axenic media with low to toxic selenium concentrations We performed Affymetrix micorarray-based transcriptional profiling on wild-type C. Elegans Bristol N2 grown in low Se axenic media supplemented with five concentrations of selenium, from low to toxic, and harvested at the L4-larva stage. RNA was prepared for hybridization to Affy microarrays from synchronized cultures of wild-type C. elegans seeded in low Se axenic media, supplemented with graded 0, 0.05, 0.1, 0.2, and 0.4 mM Se added as sodium selenite, and harvested at the L4-larva stage (1 culture/sample per Se concentgration).
Project description:Purpose: The goals of this study are to compare transcriptomes using RNA-seq of mouse myoblasts (C2C12 cell line) in undifferentiated and differentiated states and with siRNA-mediated knock down of the RNA binding proteins, Rbfox1 (only expressed in differentiated state) and Rbfox2 (expressed in both undifferentiated and differentiated states). Methods: Differentiated and undifferentiated C2C12 cultures treated with Rbfox1 (differentiated only) or Rbfox2 siRNAs or a mock siRNA transfection were used for RNA-Seq analysis using Illumina HiSeq2000. 101x2 paired-end RNA-seq reads were first uniquely aligned to the mouse genome (mm9) using TopHat 1.4.1. RSEM was used to count the number of reads mapped to genes using UCSC database, followed by edgeR to call differentially expressed genes with false discovery rate less than 0.01. Cufflinks was used to reconstruct isoforms and analyze alternative splicing and percent spliced in (PSI) was calculated. PSI values were validated by RT-PCR. Results: 58-88% of the RNA-seq reads from technical and biological replicates mapped uniquely to the mouse genome. Analysis of gene expression and alternative splicing changes are published in Singh et al. Molecular Cell (2014). Conclusions: Our study has identified gene expression and alternative splicing transitions that occur during myoblast differentiation, demonstrate that 30% of the splicing transitions are regulated by Rbfox2, demonstrated that Rbfox2 is required for a late step of myoblast differentiation and identified two Rbfox2-regulated splicing transitions that are required for differentiation. Undifferentiated and differentiated C2C12 cultures with Rbfox2 depletion or Rbfox1 depletion (differentiated only) in at least duplicate samples analyzed by deep sequencing on Illumina HiSeq2000.