Metabolomics,Unknown,Transcriptomics,Genomics,Proteomics

Dataset Information

Next-generation sequencing for the fat mass and obesity-associated protein (FTO) binding mRNA

ABSTRACT: Purpose: The goals of this study are to investigate the mRNAs that bind to the FTO protein in the white fat tissue from mice. Methods: 1. White fat from CAG promoter driven transgenic Flag-tagged FTO mice were extracted with RNA protected. 2. Flag-FTO proteins were immunoprecipitated with control IP using IgG. 3. The immunoprecipitated RNAs were deep sequenced and analyzed. Results: We focused on the mRNAs which have functions related to lipid metabolism and homeostasis. Immunoprecipitated RNAs profiles from Flag-FTO IP and IgG IP were generated by deep sequencing.

ORGANISM(S): Mus musculus

SUBMITTER: Chao-Yung Wang

PROVIDER: E-GEOD-74255 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

ACCESS DATA

Publications

Loss of FTO in adipose tissue decreases Angptl4 translation and alters triglyceride metabolism.

Wang Chao-Yung CY Shie Shian-Sen SS Wen Ming-Shien MS Hung Kuo-Chun KC Hsieh I-Chang IC Yeh Ta-Sen TS Wu Delon D

Science signaling 20151215 407

A common variant of the FTO (fat mass- and obesity-associated) gene is a risk factor for obesity. We found that mice with an adipocyte-specific deletion of FTO gained more weight than control mice on a high-fat diet. Analysis of mice lacking FTO in adipocytes fed a normal diet or adipocytes from these mice revealed alterations in triglyceride metabolism that would be expected to favor increased fatty acid storage by adipose tissue. Mice lacking FTO in adipocytes showed increased serum triglyceri ...[more]

PMID: 26671148

Similar Datasets

Project description:HuR is a regulator of mRNA turnover or translation of inflammatory genes through binding to adenylate-uridylate-rich elements (ARE) and related motifs present in the 3’untranslated region (UTR) of mRNAs. We aimed to identify HuR targets in the human airway epithelial cell line BEAS-2B challenged with TNFa plus IFNg, a strong stimulus for inflammatory epithelial responses. Ribonucleoprotein (RNP) complexes from resting and cytokine-treated cells were immunoprecipitated (IP) using anti-HuR and isotype-control antibody, and eluted mRNAs were reverse-transcribed and hybridized to an inflammatory-focused gene array. The chemokines CCL2, CCL8, CXCL1 and CXCL2 ranked highest among 27 signaling and inflammatory genes significantly enriched in the HuR RNP-IP from stimulated cells over the control IP. Among these, 20 displayed published HuR binding motifs. Association of HuR with the four endogenous chemokine mRNAs was validated by single-gene RNP-IP, and shown to be 3’UTR-dependent by biotin pull-down assay. Cytokine treatment increased mRNA stability only for CCL2 and CCL8, and transient silencing and overexpression of HuR affected only CCL2 and CCL8 expression in primary and transformed epithelial cells. Cytokine-induced CCL2 mRNA was predominantly cytoplasmic; conversely, CXCL1 mRNA remained mostly nuclear and unaffected, as CXCL2, by changes in HuR levels. Increase in cytoplasmic HuR and HuR target expression partially relied on the inhibition of AMP-dependent kinase, a negative regulator of HuR nucleocytoplasmic shuttling. We postulate that HuR critically regulates the epithelial response, by associating with multiple adenylate-uridylate-rich elements (ARE)-bearing, functionally related inflammatory transcripts. On the basis of genome-wide studies probing the relationship between RNA-binding proteins and the functional profile of their associated transcripts, we combined the specific HuR immunoprecipitation of RNPs and the genome-scale microarray to profile the target mRNAs of HuR in the human airway epithelial cell line BEAS-2B challenged with very strong inflammatory stimulation, TNFa plus IFNG. The array platform we used is the Human Autoimmune and Inflammatory Response Gene Array (SuperArray Bioscience, Frederick, MD). To validate the association of HuR and its target mRNAs, we planned to apply biotin pull-down assay. HuR overexpression and knockdown assays were also planned to further verify the association of HuR and its target mRNAs. Overall, we did three biological replicates for the IP array experiments, which include both IgG1 control IP and HuR IP arrays.

Project description:We recently showed that the mammalian genome encodes more than a thousand large intergenic non-coding RNAs (lincRNAs) that are clearly conserved across mammals and thus functional. Gene expression patterns have implicated these lincRNAs in diverse biological processes including cell cycle regulation, immune surveillance, and embryonic stem cell pluripotency. However, the mechanism by which these lincRNAs function is unknown. Here, we expand the catalog of human lincRNAs to ~3300 by analyzing chromatin-state maps of various human cell types. Inspired by the observation that the well-characterized lincRNA HOTAIR bind the Polycomb Repressive Complex 2 (PRC2), we tested whether many lincRNAs are physically associated with PRC2. Remarkably, we observe that ~20% of lincRNAs expressed in various cell types are bound by PRC2, and that additional lincRNAs are bound by other chromatin-modifying complexes. Moreover, we show that siRNA-mediated depletion of certain lincRNAs associated with PRC2 leads to changes in gene expression and that the upregulated genes are enriched for those normally silenced by PRC2. We propose a model in which some lincRNAs guide chromatin–modifying complexes to specific genomic loci to regulate gene expression. siRNA-mediated lincRNA knockdown siRNAs targeting specific lincRNAs were transected into human fibroblasts. Non-targeting siRNAs were used as controls. Both of these experiment types were hybridized to Affymetrix gene expression arrays. PRC2 RIP-Chip The PRC2 complex was immunoprecipitated using antibodies targetting Suz12 and the associated RNA was hybridized to Nimblegen tiling arrays representing lincRNAs. In parallel an IgG IP was performed and hybridized to the same NG arrays. RIP was performed for PRC2 and hybridized using a Cy3 label. In parallel, RIP was performed using IGG and labelled with Cy5 PRC2 RIP-Chip (dye swap) The PRC2 complex was immunoprecipitated using antibodies targetting Suz12 and the associated RNA was hybridized to Nimblegen tiling arrays representing lincRNAs. In parallel an IgG IP was performed and hybridized to the same NG arrays. RIP was performed for PRC2 and hybridized using a Cy5 label. In parallel, RIP was performed using IGG and labelled with Cy3 PRC2 RIP-Chip (no proteins) The PRC2 complex was immunoprecipitated using antibodies targetting Suz12 and the associated RNA was hybridized to Nimblegen tiling arrays representing lincRNAs. In parallel an IgG IP was performed and hybridized to the same NG arrays. RIP was performed for PRC2 and hybridized using a Cy3 label. In parallel, RIP was performed using IGG and labelled with Cy5 COREST RIP-Chip The COREST complex was immunoprecipitated using antibodies targetting COREST and the associated RNA was hybridized to Nimblegen tiling arrays representing lincRNAs. In parallel an IgG IP was performed and hybridized to the same NG arrays. RIP was performed for COREST and hybridized using a Cy3 label. In parallel, RIP was performed using IGG and labelled with Cy5 H3K27me3 and H3K4me2 modified histones RIP-Chip The H3K27me3 and H3K4me2 modified histones were immunoprecipitated using antibodies targetting COREST and the associated RNA was hybridized to Nimbelgen tiling arrays representing lincRNAs. In parallel an IgG IP was performed and hybridized to the same NG arrays. RIP was performed for COREST and hybridized using a Cy3 label. In parallel, RIP was performed using IGG and labelled with Cy5 Human lincRNA expression Human lincRNAs were profiled in HeLa, Lung FB, and Foot FB. In addition, nuclear and cytoplasmic fractions were taken in HeLa cells Total RNA was extracted from HeLa, Foot, and Lung cells and hybridized to NG tiling array. In addition, the nucleus was fractionated and RNA was hybridized to an array.

Dataset Information

Next-generation sequencing for the fat mass and obesity-associated protein (FTO) binding mRNA

Publications

Loss of FTO in adipose tissue decreases Angptl4 translation and alters triglyceride metabolism.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure