Metabolomics,Unknown,Transcriptomics,Genomics,Proteomics

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Transcription profiling of human HepG2 cells transfected with PGC-1 variants to identify ERRalpha dependent PGC-1alpha target genes

ABSTRACT: PGC-1 transcription factor was customized to limit its interations to ERRalpha. This mutant (2x9) was used to dissect the transcription activation patterns that are attributable to the PGC1-ERR interaction and PGC-1 actions that are independent of ERR. Inactive mutant with the deleted LLXXL motifs (L2L3) and wt PGC-1 were used as negative and positive controls respectively. BGAL-expressing construct was used to control for non-specific effects of adenoviral infection. Experiment Overall Design: Expression constructs for BGAL control and three variants of PGC-1 were introduced into HepG2 cells via adenoviral infection. RNA was isolated 24 hrs post infection and the changes in gene expression were detected using GeneChip technology (Affymetrix). Hybridization was performed on the HG-133 plus 2 platform. The experiment was performed in three biological replicates. Raw expression data were normalized using MAS5.0 algorithm.

ORGANISM(S): Homo sapiens

SUBMITTER: Dmitri Kazmin

PROVIDER: E-GEOD-5968 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:Mitochondrial oxidative function is tightly controlled to maintain energy homeostasis in response to nutrient and hormonal signals. An important cellular component in the energy sensing response is the target of rapamycin (TOR) kinase pathway; however whether and how mTOR controls mitochondrial oxidative activity is unknown. Here, we show that mTOR kinase activity stimulates mitochondrial gene expression and oxidative function. In skeletal muscle cells and TSC2-/- MEFs, the mTOR inhibitor rapamycin largely decreased gene expression of mitochondrial transcriptional regulators such as PGC-1alpha and the transcription factors ERRalpha and NRFs. As a consequence, mitochondrial gene expression and oxygen consumption were reduced upon mTOR inhibition. Using computational genomics, we identified the transcription factor YY1 as a common target of mTOR and PGC-1alpha that controls mitochondrial gene expression. Inhibition of mTOR resulted in a failure of YY1 to interact and be coactivated by PGC-1alpha. Notably, knock-down of YY1 in skeletal muscle cells caused a significant decrease in mRNAs of mitochondrial regulators and mitochondrial genes that resulted in a decrease in respiration. Moreover, YY1 was required for rapamycin-dependent repression of mitochondrial genes. Thus, we have identified a novel mechanism in which a nutrient sensor (mTOR) balances energy metabolism via transcriptional control of mitochondrial oxidative function. These results have important implications for our understanding of how these pathways might be altered in metabolic diseases and cancer. Experiment Overall Design: Using Affymetrix MOE430 v2 gene chips, biological triplicates of each condition were analyzed: vehicle-treated, rapamycin-treated, gfp-infected, and pgc-1alpha-infected resulting in a total of 12 samples. Experiment Overall Design: Data were analyzed by RMA (with default settings) in BioConductor 1.2 -- one batch for the Rapamycin vs. Vehicle, and another batch for the PGC vs GFP.

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Transcription profiling of human HepG2 cells transfected with PGC-1 variants to identify ERRalpha dependent PGC-1alpha target genes

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