Transcription profiling of human HepG2 cells transfected with PGC-1 variants to identify ERRalpha dependent PGC-1alpha target genes
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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.
Project description: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. Keywords: ERRalpha, PGC-1alpha, nuclear receptor, orphan nuclear receptor, coactivator, transcription factors
Project description:In order to identify targets for HDAC4, NRVM were infected with adenoviral vectors encoding beta-Galactosidase or Flag- HDAC4, and incubated in serum free or 10% fetal calf serum containing growth medium for 48 hrs. NRVM were infected with adenoviral vectors encoding beta-Galactosidase (control) or Flag- HDAC4 (experiment), and incubated in serum free or 10% fetal calf serum containing growth medium for 48 hrs. 2 biological samples of each condition were analyzed.
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.
Project description:We found that TLS can work as a PGC-1alpha cofactor and this assay was carried out to test the functional dependency of TLS on PGC-1alpha on a whole genome scale Three independently-isolated cultures of primary hepatocytes from PGC-1α+/+ and PGC-1α-/- mice were infected with shTLS or control adenovirus. RNA was extracted by Trizol extraction, re-purified with RNAeasy (Invitrogen), and checked for integrity and quantity with the Agilent Bio-Analyzer QC. RNA was amplified and labeled with the One-Color Microarray-Based Gene Expression Analysis Protocol (Agilent Technologies, Palo Alto, CA, USA). Samples were hybridized to a G4122F 4x44K whole mouse genome microarray (Agilent Technologies). Arrays were scanned at 5 mm resolution with a G2565BA DNA microarray scanner (Agilent Technologies) at the default settings for 4x44k format one-color arrays. Images were analyzed using Feature Extraction software v10.1.1.1 (Agilent Technologies). Raw signals were thresholded to 1 and normalized by quantile (Bolstad et al., 2003) was performed using GeneSpring software. Data were analyzed on the log2 scale. Default flags were considered as absent, except for saturated spots that were flagged as marginal.
Project description:The PGC-1a gene is expressed as several transcriptional coactivator variants that regulate numerous adaptive processes. These include thermogenesis, hepatic metabolism, neuroprotection, and skeletal muscle adaptation to exercise training. To support such diverse functions, PGC-1 isoform expression and activation are under tight tissue- and context-specific control. Notably, PGC-1 isoforms generated by alternative gene promoter usage, and splicing are highly induced in skeletal muscle upon exercise. Here we show that PGC-12 and 3 are PGC-11 dimerization partners that limit its activity. Skeletal muscle PGC-12 and PGC-13 transgenics have reduced exercise performance and strength, which partially overlaps with PGC-1 loss-of-function. Mechanistically, PGC-11/3 dimerization precludes ERR recruitment and coactivation, so their co-expression in skeletal muscle impairs the innate bioenergetic adaptations characteristic of PGC-11 transgenics and reduces oxidative metabolism gene expression and exercise capacity. Removing this break to PGC-1a1 activity may have therapeutic applications in metabolic diseases and increase responsiveness to training.
Project description:An alternative promoter of the PGC-1 alpha gene gives arrise to 3 new isofroms of the PGC-1 alpha gene refered to as PGC-1a2 (A2), PGC-1a3 (A3) and PGC-1a4 (A4). The proximal PGC-1 alpha promotor transcribes the canonical PGC-1 alpha which is refered to as PGC-1a1 (A1).G1/G2/G3 samples refer to the Green fluorescent protein (GFP) control samples used in this experiment. Forced expression of specifically PGC-1a4 isoform results in muslce hypertrophy associated with increased IGF-1 signaling and repression of myostatin signaling. Adnoviral over expression assays were performed on primary myoblasts from C57BL/6 mice for each of the PGC-1 alpha isoforms originating from the proximal or alternative promotor.
Project description:Impaired mitochondrial function has been implicated in the pathogenesis of type 2 diabetes, heart failure and neurodegeneration as well as during aging. Studies with the PGC-1 transcriptional coactivators have demonstrated that these factors are key components of the regulatory network that controls mitochondrial function in mammalian cells. Here we describe a genome-wide coactivation assay to globally identify the transcriptional partners for PGC-1α. These analyses revealed a molecular signature of the PGC-1α transcriptional network, and identified BAF60a (Smarcd1), a subunit of the SWI/SNF chromatin-remodeling complex, as a critical regulator of lipid homeostasis. Adenoviral-mediated expression of BAF60a stimulates fatty acid β-oxidation in cultured hepatocytes and reduces hepatic triglyceride levels in diet-induced obese mice. BAF60a physically interacts with PGC-1α and is recruited to PPARα target genes in the fasted liver. Liver-specific RNAi knockdown of BAF60a impairs fatty acid oxidation and results in severe hepatic steatosis following starvation. These results define a role for the SWI/SNF complexes in the regulation of hepatic lipid metabolism, and reveal a potential target for therapeutic intervention. Keywords: Adenoviral transduction.
Project description:Skeletal muscle tissue shows an extraordinary cellular plasticity, but the underlying molecular mechanisms are still poorly understood. Here we use a combination of experimental and computational approaches to unravel the complex transcriptional network of muscle cell plasticity centered on the peroxisome proliferator-activated receptor M-NM-3 coactivator 1M-NM-1 (PGC-1M-NM-1), a regulatory nexus in endurance training adaptation. By integrating data on genome-wide binding of PGC-1M-NM-1 and gene expression upon PGC-1M-NM-1 over-expression with comprehensive computational prediction of transcription factor binding sites (TFBSs), we uncover a hitherto underestimated number of transcription factor partners involved in mediating PGC-1M-NM-1 action. In particular, principal component analysis of TFBSs at PGC-1M-NM-1 binding regions predicts that, besides the well-known role of the estrogen-related receptor M-NM-1 (ERRM-NM-1), the activator protein-1 complex (AP-1) plays a major role in regulating the PGC-1M-NM-1-controlled gene program of hypoxia response. Our findings thus reveal the complex transcriptional network of muscle cell plasticity controlled by PGC-1M-NM-1. We used microarrays to detect changes in gene expression in C2C12 cells following PGC-1alpha over-expression or GFP (control) over-expression. We used 3 biological replicates for each condition.
Project description:As an oncogene, use of HER2 vaccines in humans requires the development of HER2 immunotherapies with maximal immunologic potential, but minimal oncologic potential. To address these issues, we developed a recombinant adenoviral vector expressing a mutated HER2 inactivated for kinase function (Ad-HER2-ki). Ad-HER2-ki was highly expressed, but non-phosphorylated and elicited minimal transcription dysregulation in primary cells. In contrast, Ad-HER2-wt elicited a strong oncogenic signature associated with tumorigenesis. Experiment Overall Design: Early Passage Human Mammary Epithelial cells (HMECs) were serum starved for 36hrs. and infected at a MOI of 150 with either Ad-GFP, Ad-HER2-wt, or Ad-HER2-ki vectors. At 18 hpi, RNA was extracted and transcriptomes evaluated by microarray. Five samples were infected per virus treatment, each a completely separate biologic replicate.
Project description:E47 is a basic Helix Loop Helix (bHLH) transcription factor that has important roles in cell fate determination and differentiation of many cell types. In the nervous system E47 heterodimerizes with tissue-specific, pro-neural bHLH transcription factors and activates downstream target genes. To identify the relevant target genes of bHLH transcription factors in neural cells, we performed gene expression profiling of the human neuroblastoma cell line SK-N-SH engineered to acutely express ectopic E47 by an adenoviral vector. The experiments were done at two time points following adenoviral infection, 8 hours and 20 hours. Genes induced by E47 after 8 hours are likely to be direct targets of this transcription factor.