Unknown,Transcriptomics,Genomics,Proteomics

Dataset Information

FoxO RNAi in C2C12 cells

ABSTRACT: C2C12 cells are mouse skeletal muscle cells. These cells were transfected with shRNA against FoxO1, FoxO3, and FoxO4. FoxO1, FoxO3, and FoxO4 are the known paralogues expressed in this cell line. C2C12 cells are transfected with shRNA against FoxO1, FoxO3, and FoxO4, respectively. Colonies were selected for the best depletion of the target FoxOs, respectively. Cells were grown in DMEM medium. Total RNA was extracted from each line. Microarray analysis was performed by following the Affymetrix protocols. The data were analyzed by GCOS system. The target genes that we are interested in were further confirmed by qRT-PCR, reporter assay, and other biochemical and molecular biology assays.

ORGANISM(S): Mus musculus

SUBMITTER: Ping Hu

PROVIDER: E-GEOD-13347 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Publications

Codependent activators direct myoblast-specific MyoD transcription.

Hu Ping P Geles Kenneth G KG Paik Ji-Hye JH DePinho Ronald A RA Tjian Robert R

Developmental cell 20081001 4

Although FoxO and Pax proteins represent two important families of transcription factors in determining cell fate, they had not been functionally or physically linked together in mediating regulation of a common target gene during normal cellular transcription programs. Here, we identify MyoD, a key regulator of myogenesis, as a direct target of FoxO3 and Pax3/7 in myoblasts. Our cell-based assays and in vitro studies reveal a tight codependent partnership between FoxO3 and Pax3/7 to coordinatel ...[more]

PMID: 18854138

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Project description:The mammalian FoxO transcription factors - FoxO1, FoxO3, FoxO4 - function in the nucleus to direct transcription of specific gene targets governing cellular survival, proliferation, metabolism, differentiation and oxidative defense. Activation of PI3K by extracellular growth factors leads to AKT-mediated phosphorylation of FoxO1, FoxO3 and FoxO4, resulting in their sequestration in the cytoplasm such that they are unable to regulate their gene targets. Our study identified FoxOs as novel tumor suppressors in kidney cancer (Gan et al, 2010, Cancer Cell). To understand the tumor suppression function of FoxOs in kidney cancer cells, we performed gene expression profiling in human kidney cancer cells upon FoxO1 or FoxO3 reactivation in order to identify the key transcriptomic alterations mediating FoxO tumor suppression function in kidney cancer cells. We generated RCC4 and UMRC2 cell lines (two human kidney cancer cells with low endogenous FoxO1 and FoxO3 expression) with stable expression of FoxO1(TA)ERT2 or FoxO3(TA)ERT2 construct, which expressed a fusion protein consisting of FoxO(TA) (containing three Ser/Thr AKT phosphorylation sites mutated to alanine) fused to the T2-modified estrogen receptor (ERT2) moiety. We documented that the FoxO(TA)ERT2 fusion protein sequestered FoxO(TA) in the cytoplasm and that 4OHT treatment resulted in rapid translocation of FoxO(TA)ERT2 into the nucleus. We also established stable cell lines with ERT2 expression as control cell lines. (For simplicity, ERT2, FoxO1(TA)ERT2 and FoxO3(TA)ERT2 cell lines will be referred to as EV (empty vector), FoxO1 and FoxO3, respectively, hereafter). We then conducted comparative transcriptome analysis (using the Human Genome U133 Plus 2.0 Array) of EV, FoxO1, or FoxO3-expressing RCC4 and UMRC2 cells at 12 hours with or without 100 nm 4OHT treatment (cultured in DMEM+10% FBS with puromycin selection). To enrich for more proximal actions of FoxO, we selected the 12 hour time point as time course studies revealed dramatic transcriptional changes of known FoxO targets (such as Cyclin D1), yet no discernable cellular phenotypes (apoptosis and cell cycle arrest). We generated 4 transcriptome datasets: FoxO1 RCC4, FoxO3 RCC4, FoxO1 UMRC2, and FoxO3 UMRC2 (by comparing transcriptome data with or without 4OHT treatment), and normalized these transcriptome data against 4OHT-treated EV cells, which show modest 4OHT-induced transcriptional changes.

Project description:FOXO transcription factors control cellular formation of reactive oxygen species (ROS), which critically contribute to cell survival and cell death in neuroblastoma. Here, we report that C10orf10, also named M-bM-^@M-^\Decidual Protein induced by Progesterone (DEPP)M-bM-^@M-^], is a direct transcriptional target of FOXO3 in human neuroblastoma. As FOXO3-mediated apoptosis involves a biphasic ROS accumulation, we analyzed cellular ROS levels in DEPP-knockdown cells by live-cell imaging. Knockdown of DEPP prevented the primary and secondary ROS accumulation during FOXO3 activation and attenuates FOXO3-induced apoptosis, whereas its overexpression raises cellular ROS levels and sensitizes to cell death. In neuronal cells, cellular steady state ROS are mainly detoxified in peroxisomes by the enzyme CAT/catalase. As DEPP contains a peroxisomal-targeting-signal-type-2 (PTS2) sequence at its N-terminus that enables protein import into peroxisomes, we analyzed the effect of DEPP on peroxisomal function by measuring the catalase enzyme activity. Catalase activity was reduced by conditional DEPP overexpression and significantly increased in DEPP-knockdown cells. Using live cell imaging and fluorescent peroxisomal and mitochondrial probes we demonstrate that DEPP localizes to peroxisomes and mitochondria in neuroblastoma cells. The combined data indicate that DEPP reduces peroxisomal activity and thereby impairs the cellular ROS detoxification capacity and contributes to death sensitization. SH-EP, NB15 neuroblastoma cells and CCRF-CEM-C7H2 acute lymphoblastic leukemia cells were infected with the retrovirus plasmid pLIB-FOXO3(A3)-Ertm-iresNeo. Gene expression measures of samples with activated FOXO3 transcription factor (3h OHT treated) have been compared to untreated samples (0h time point). To rule out gene regulations by estrogen samples treated for 3 hours with tamoxifen have been compared to the untreated samples. Only genes that were more than two-fold regulated in the first, but not in the second comparison were defined to be FOXO3 regulated.

Dataset Information

FoxO RNAi in C2C12 cells

Publications

Codependent activators direct myoblast-specific MyoD transcription.

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