Metabolomics,Unknown,Transcriptomics,Genomics,Proteomics

Dataset Information

MH003B:FOXO3 in control of circadian rhythms via direct regulation of Clock. Part 2: knockdown of FoxO3.

ABSTRACT: Circadian rhythms are responsive to a variety of external cues, light and metabolism being the most important. In mammals, the light signal is sensed by the retina and transmitted to the SCN master clock, where it is translated into the molecular oscillator via regulation of clock gene transcription. The signalling pathways governing the molecular translation from metabolic signals to circadian output in peripheral oscillators, in contrast, are less understood. FOXO transcription factors are known to translate external metabolic cues to internal transcriptional programs. In the past couple of years it has become evident that both FOXO transcription factors and the circadian clock are of key importance in the underlying mechanisms of ageing and the regulation of metabolism. We now show FOXO3 to be a crucial modulator of circadian rhythmicity via direct transcriptional regulation of Clock, a core component of the molecular oscillator, and identify FOXO3 as a novel link in the circadian feedback loop, which is required for circadian rhythms in liver. We propose that FOXO3 directly feeds back into the circadian oscillator in response to metabolic cues. We performed a microarray study on synchronized NIH 3T3 cells upon transient knock-down of FoxO3 (siO3). Cells were harvested for RNA isolation 24h (time1), 30h(time2), 36h(time3) and 42h(time4) after synchronization. Experimental samples were hybridized against a reference pool of cRNA, which was derived from unsynchronized NIH 3T3 cells. AS controlgroup a scrambled siRNA was transfected. Experiments were performed 4 times, of each sample group two samples were labeled with cy5 and co-hybridized with reference RNA labeled with cy3, and two samples were labeled and hybridized in the opposite way. Microarrays used were Mouse Whole Genome Gene Expression Microarrays V1 (Agilent Technologies, Belgium)

ORGANISM(S): Mus musculus

SUBMITTER: Marian Groot Koerkamp

PROVIDER: E-GEOD-41555 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Publications

Insulin-FOXO3 signaling modulates circadian rhythms via regulation of clock transcription.

Chaves Inês I van der Horst Gijsbertus T J GT Schellevis Raymond R Nijman Romana M RM Koerkamp Marian Groot MG Holstege Frank C P FC Smidt Marten P MP Hoekman Marco F M MF

Current biology : CB 20140522 11

Circadian rhythms are responsive to external and internal cues, light and metabolism being among the most important. In mammals, the light signal is sensed by the retina and transmitted to the suprachiasmatic nucleus (SCN) master clock [1], where it is integrated into the molecular oscillator via regulation of clock gene transcription. The SCN synchronizes peripheral oscillators, an effect that can be overruled by incoming metabolic signals [2]. As a consequence, peripheral oscillators can be un ...[more]

PMID: 24856209

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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.

Dataset Information

MH003B:FOXO3 in control of circadian rhythms via direct regulation of Clock. Part 2: knockdown of FoxO3.

Publications

Insulin-FOXO3 signaling modulates circadian rhythms via regulation of clock transcription.

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