Project description:Orphan nuclear receptor estrogen-related receptor alpha (ERRα) has recently been shown to carry negative prognostic significance in breast and ovarian cancers. The specific role of ERRα in tumor growth and progression, however, is yet to be fully understood. The significant homology between estrogen receptor alpha (ERα) and ERRα initially suggested that these receptors may share similar transcriptional targets. Using the well-characterized ERα-positive MCF-7 breast cancer cell line, we sought to gain a genome-wide picture of ERα-ERRα cross-talk using an unbiased microarray approach. Since a small molecule ligand has not been identified for ERRα, its transcriptional activity in these studies was induced using its known coactivator PGC-1α (peroxisome proliferator-activated receptor-γ coactivator-1α) as a protein ligand. Both wild-type PGC1, as well as ERRa-specific variant (PGC1 2x9) were used to activate ERRa. Non-NR-dependent activities of PGC-1α were controlled for using a variant PGC-1α in which the leucines within the NR-interacting domain had been mutated to alanines (L2L3M). Beta-galactosidase (BGAL) overexpression was used as a non-specific background control. Activation of endogenous ER was achieved by treatment with 10 nM estadiol. Keywords: Response to stimulus
Project description:We report that Zic family (Zic1/2/3) and orphan nuclear receptors family (Esrrb and Nr5a2) transcription factors (TFs) synergistically enhance the reprogramming efficiency when transduced with Oct4, Sox2 and Klf4 (OSK) into murine fibroblasts. To identify the molecular mechanisms underlying this synergy, we analyzed global gene expression at 6 days after introduction of reprogramming factors. As a result, we found that primary targets of these TFs are different when either of TFs was introduced with OSK, but a significant portion of genes including pluripotency makers such as Dppa2 was synergistically upregulated. Further analysis revealed that metabolic pathways are the important targets of these TFs for efficient reprogramming.
Project description:Peroxisome-proliferator activated receptor α (PPARα) activation reprograms liver gene expression to support fatty acid oxidation during fasting. How PPARα engages in transcriptional programs coping with catabolic fasting responses is insufficiently understood. By applying a protein-protein interaction methodology that also captures transient interactions, we revealed the orphan nuclear receptor estrogen-related receptor α (ERRα) as a novel interaction partner of liganded PPARα and found that this interaction is enhanced following cellular nutrient starvation. Among target genes affected by PPARα-ERRα transcriptional crosstalk in fasted murine livers, multiple components of the electron transport chain were identified. Using pharmacological tools to study hepatic gene subsets under dual PPARα and ERRα control and moving from short-term to prolonged nutrient deprivation, we found that ERRα can switch from being a PPARα target gene suppressor to a marked PPARα target gene activator. Mechanistically, ERRα may control PPARα transcriptional activity via binding onto PPARα’s coactivator interaction site and via facilitating cofactor relays. In sum, a variety of crosstalk mechanisms between PPARα and ERRα seems to co-ordinately drive essential gene regulatory changes in the starving hepatocyte.
Project description:Adrenergic stimulation of brown adipose tissue (BAT) activates thermogenesis, by uncoupling oxidative phosphorylation and enabling high rates of substrate oxidation. Moreover, adrenergic stimulation signals to the nucleus, inducing gene expression changes that increase BAT thermogenic and oxidative capacity. The purpose of this study was to determine the role of two orphan nuclear receptors, ERRα and ERRγ, in adipose tissue function in response to adrenergic stimulation. Here we identify ERRα and ERRγ as collectively critical effectors of the adrenergic induced transcriptional program in BAT. Mice lacking adipose ERRs (ERRαγAd-/-) have diminished oxidative and thermogenic capacity, and become rapidly hypothermic when exposed to cold. Notably, the ability of the β3-adrenergic agonist CL316,243 to expand BAT oxidative and thermogenic capacity, increase energy expenditure, promote weight loss, and improve glucose tolerance is lost in ERRαγAd-/-. At the transcriptional level, RNA sequencing experiments show that the bulk of the response of BAT to CL316,243 (>80% of CL316,243-induced genes) relies on ERRs. These findings establish ERRα and ERRγ as essential BAT regulators that act coordinately to relay adrenergic signals and expand the capacity for thermogenesis and energy expenditure.
Project description:In the murine system, Oct4, Sox2, c-Myc and Klf4 are sufficient to convert fibroblasts to induced pluripotent stem (iPS) cells that exhibit many characteristics of embryonic stem (ES) cells. Herein, we show that the orphan nuclear receptor Esrrb works in conjunction with Oct4 and Sox2 to mediate reprogramming of mouse embryonic fibroblasts (MEFs) to iPS cells. Esrrb reprogrammed cells share similar expression and epigenetic signatures as ES cells. These cells are also pluripotent and can differentiate in vitro and in vivo into the three major embryonic cell lineages. Furthermore, these cells contribute to mouse chimeras and are germline transmissible. In ES cells, Esrrb targets many genes involved in selfrenewal and pluripotency. This suggests that Esrrb may mediate reprogramming through the up-regulation of ES cell-specific genes. Our findings also indicate that it is possible to reprogram MEFs without exogenous Klf transcription factors and link a nuclear receptor to somatic cell reprogramming. Global gene expression effects of silencing the Esrrb gene. We used microarrays to detail the global programme of gene expression after silencing the Esrrb gene. Keywords: time-course Three biological replicates each for control GFP and Esrrb RNAi. The global gene expression profiles of the Esrrb knockdown cells were compared to control GFP knockdown cells for days 2, 4 and 6.
Project description:mTOR and ERRα are key regulators of common metabolic processes. However, the extent of functional overlap between these two factors has not been investigated. ChIP-sequencing analyses on mouse liver reveal mTOR recruitment to regulatory regions of genes on a genome-wide scale including enrichment at genes shared with ERRα that are involved in the TCA cycle and lipogenesis pathway. A total of 9469 and 23226 peaks were identified for mTOR and ERRα ChIP-seq datasets, respectively. mTOR and ERRα mouse liver ChIP-seq datasets obtained from pooling 8 individual ChIPs from a chromatin pool of 26 livers.
Project description:In the murine system, Oct4, Sox2, c-Myc and Klf4 are sufficient to convert fibroblasts to induced pluripotent stem (iPS) cells that exhibit many characteristics of embryonic stem (ES) cells. Herein, we show that the orphan nuclear receptor Esrrb works in conjunction with Oct4 and Sox2 to mediate reprogramming of mouse embryonic fibroblasts (MEFs) to iPS cells. Esrrb reprogrammed cells share similar expression and epigenetic signatures as ES cells. These cells are also pluripotent and can differentiate in vitro and in vivo into the three major embryonic cell lineages. Furthermore, these cells contribute to mouse chimeras and are germline transmissible. In ES cells, Esrrb targets many genes involved in selfrenewal and pluripotency. This suggests that Esrrb may mediate reprogramming through the up-regulation of ES cell-specific genes. Our findings also indicate that it is possible to reprogram MEFs without exogenous Klf transcription factors and link a nuclear receptor to somatic cell reprogramming. This SuperSeries is composed of the SubSeries listed below.
Project description:Orphan nuclear receptor Esrrb is vital in maintaining ES cells and like Oct4, Sox2 and Nanog is essential for self-renewal and pluripotency. Esrrb functions in somatic cells via LBD/AF-2-dependent coactivator recruitment to target genes. Here we show that in ES cells coactivator recruitment is similarly required and identify Ncoa3 as the Esrrb coactivator needed for activation of its target genes. Ncoa3 is essential for self-renewal and the induction of pluripotency in reprogramming, and genome-wide analysis of Ncoa3 binding reveals extensive overlap with Esrrb and pluripotency factors along with marks of active genes. Mechanistically, we show Ncoa3 is specifically required to bridge RNApol2 to Esrrb. We thus identify a new member of the ES pluripotency network and describe Esrrb and Ncoa3 as key factors linking core pluripotency factors to the general transcription machinery. ChIP experiments were carried out with chromatin prepared from E14 cells as previously described (Stock et al., 2007), using 8-10 ug primary antibody for NcoA3 and 600 ug pre-cleared chromatin per IP. Antibody for NcoA3 was from Santacruz (sc-9119) .
Project description:Orphan nuclear receptor Esrrb is vital in maintaining ES cells and like Oct4, Sox2 and Nanog is essential for self-renewal and pluripotency. Esrrb functions in somatic cells via LBD/AF-2-dependent coactivator recruitment to target genes. Here we show that in ES cells coactivator recruitment is similarly required and identify Ncoa3 as the Esrrb coactivator needed for activation of its target genes. Ncoa3 is essential for self-renewal and the induction of pluripotency in reprogramming, and genome-wide analysis of Ncoa3 binding reveals extensive overlap with Esrrb and pluripotency factors along with marks of active genes. Mechanistically, we show Ncoa3 is specifically required to bridge RNApol2 to Esrrb. We thus identify a new member of the ES pluripotency network and describe Esrrb and Ncoa3 as key factors linking core pluripotency factors to the general transcription machinery. Three biological replicates each for control scrambled shRNA and Ncoa3 shRNA transfected E14 mouse ESCs. The global gene expression profiles of Ncoa3 knockdown cells were compared to control scrambled shRNA knockdown cells 4 days post-transfection.