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Objectives: Estrogen-related-receptor α (ERRα) plays a critical role in the transcriptional regulation of cellular bioenergetics and metabolism, and perturbations in its activity have been associated with metabolic diseases. While several coactivators and corepressors of ERRα have been identified to date, a knowledge gap remains in understanding the extent to which ERRα cooperates with coregulators in the control of gene expression. Herein, we mapped the primary chromatin-bound ERRα interactome in mouse liver., Methods: RIME (Rapid Immuno-precipitation Mass spectrometry of Endogenous proteins) analysis using mouse liver samples from two circadian time points was used to catalog ERRα-interacting proteins on chromatin. The genomic crosstalk between ERRα and its identified cofactors in the transcriptional control of precise gene programs was explored through cross-examination of genome-wide binding profiles from chromatin immunoprecipitation-sequencing (ChIP-seq) studies. The dynamic interplay between ERRα and its newly uncovered cofactor Host cell factor C1 (HCFC1) was further investigated by loss-of-function studies in hepatocytes., Results: Characterization of the hepatic ERRα chromatin interactome led to the identification of 48 transcriptional interactors of which 42 were previously unknown including HCFC1. Interrogation of available ChIP-seq binding profiles highlighted oxidative phosphorylation (OXPHOS) under the control of a complex regulatory network between ERRα and multiple cofactors. While ERRα and HCFC1 were found to bind to a large set of common genes, only a small fraction showed their colocalization, found predominately near the transcriptional start sites of genes particularly enriched for components of the mitochondrial respiratory chain. Knockdown studies demonstrated inverse regulatory actions of ERRα and HCFC1 on OXPHOS gene expression ultimately dictating the impact of their loss-of-function on mitochondrial respiration., Conclusions: Our work unveils a repertoire of previously unknown transcriptional partners of ERRα comprised of chromatin modifiers and transcription factors thus advancing our knowledge of how ERRα regulates metabolic transcriptional programs., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Author(s). Published by Elsevier GmbH.. All rights reserved.)

mTOR is a serine/threonine kinase that controls prostate cancer cell growth in part by regulating gene programs associated with metabolic and cell proliferation pathways. mTOR-mediated control of gene expression can be achieved via phosphorylation of transcription factors, leading to changes in their cellular localization and activities. mTOR also directly associates with chromatin in complex with transcriptional regulators, including the androgen receptor (AR). Nuclear mTOR (nmTOR) has been previously shown to act as a transcriptional integrator of the androgen signaling pathway in association with the chromatin remodeling machinery, AR, and FOXA1. However, the contribution of cytoplasmic mTOR (cmTOR) and nmTOR and the role played by FOXA1 in this process remains to be explored. Herein, we engineered cells expressing mTOR tagged with nuclear localization and export signals dictating mTOR localization. Transcriptome profiling in AR-positive prostate cancer cells revealed that nmTOR generally downregulates a subset of the androgen response pathway independently of its kinase activity, while cmTOR upregulates a cell cycle-related gene signature in a kinase-dependent manner. Biochemical and genome-wide transcriptomic analyses demonstrate that nmTOR functionally interacts with AR and FOXA1. Ablation of FOXA1 reprograms the nmTOR cistrome and transcriptome of androgen responsive prostate cancer cells. This works highlights a transcriptional regulatory pathway in which direct interactions between nmTOR, AR and FOXA1 dictate a combinatorial role for these factors in the control of specific gene programs in prostate cancer cells., Implications: The finding that canonical and nuclear mTOR signaling pathways control distinct gene programs opens therapeutic opportunities to modulate mTOR activity in prostate cancer cells., (©2023 American Association for Cancer Research.)

The integrated stress response (ISR) is critical for cell survival under stress. In response to diverse environmental cues, eIF2α becomes phosphorylated, engendering a dramatic change in mRNA translation. The activation of ISR plays a pivotal role in the early embryogenesis, but the eIF2-dependent translational landscape in pluripotent embryonic stem cells (ESCs) is largely unexplored. We employ a multi-omics approach consisting of ribosome profiling, proteomics, and metabolomics in wild-type (eIF2α +/+ ) and phosphorylation-deficient mutant eIF2α (eIF2α A/A ) mouse ESCs (mESCs) to investigate phosphorylated (p)-eIF2α-dependent translational control of naive pluripotency. We show a transient increase in p-eIF2α in the naive epiblast layer of E4.5 embryos. Absence of eIF2α phosphorylation engenders an exit from naive pluripotency following 2i (two chemical inhibitors of MEK1/2 and GSK3α/β) withdrawal. p-eIF2α controls translation of mRNAs encoding proteins that govern pluripotency, chromatin organization, and glutathione synthesis. Thus, p-eIF2α acts as a key regulator of the naive pluripotency gene regulatory network., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Objective: Estrogen related receptor α (ERRα) occupies a central node in the transcriptional control of energy metabolism, including in skeletal muscle, but whether modulation of its activity can directly contribute to extend endurance to exercise remains to be investigated. The goal of this study was to characterize the benefit of mice engineered to express a physiologically relevant activated form of ERRα on skeletal muscle exercise metabolism and performance., Methods: We recently shown that mutational inactivation of three regulated phosphosites in the amino terminal domain of the nuclear receptor ERRα impedes its degradation, leading to an accumulation of ERRα proteins and perturbation of metabolic homeostasis in ERRα 3SA mutant mice. Herein, we used a multi-omics approach in combination with physical endurance tests to ascertain the consequences of expressing the constitutively active phospho-deficient ERRα 3SA form on muscle exercise performance and energy metabolism., Results: Genetic heightening of ERRα activity enhanced exercise capacity, fatigue-resistance, and endurance. This phenotype resulted from extensive reprogramming of ERRα global DNA occupancy and transcriptome in muscle leading to an increase in oxidative fibers, mitochondrial biogenesis, fatty acid oxidation, and lactate homeostasis., Conclusion: Our findings support the potential to enhance physical performance and exercise-induced health benefits by targeting molecular pathways regulating ERRα transcriptional activity., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Authors. Published by Elsevier GmbH.. All rights reserved.)

Nonalcoholic steatohepatitis (NASH) is epidemiologically associated with obesity and diabetes and can lead to liver cirrhosis and hepatocellular carcinoma if left untreated. The intricate signaling pathways that orchestrate hepatocyte energy metabolism and cellular stress, intrahepatic cell crosstalk, as well as interplay between peripheral tissues remain elusive and are crucial for the development of anti-NASH therapies. Herein, we reveal E3 ligase FBXW7 as a key factor regulating hepatic catabolism, stress responses, systemic energy homeostasis, and NASH pathogenesis with attenuated FBXW7 expression as a feature of advanced NASH. Multiomics and pharmacological intervention showed that FBXW7 loss-of-function in hepatocytes disrupts a metabolic transcriptional axis conjointly controlled by the nutrient-sensing nuclear receptors ERRα and PPARα, resulting in suppression of fatty acid oxidation, elevated ER stress, apoptosis, immune infiltration, fibrogenesis, and ultimately NASH progression in male mice. These results provide the foundation for developing alternative strategies co-targeting ERRα and PPARα for the treatment of NASH., (© 2023. The Author(s).)

Dysregulation of mTOR signaling plays a critical role in promoting prostate cancer growth. HOXB13, a homeodomain transcription factor, is known to influence the androgen response and prostate cancer development. Recently, HOXB13 was found to complex with mTOR on chromatin. However, the functional crosstalk between HOXB13 and mTOR remains elusive. We now report that mTOR directly interacts with and hierarchically phosphorylates HOXB13 at threonine 8 and 41 then serine 31 to promote its interaction with the E3 ligase SKP2 while enhancing its oncogenic properties. Expression of HOXB13 harboring phosphomimetic mutations at the mTOR-targeted sites stimulates prostate cancer cellular growth both in vitro and in murine xenografts. Transcriptional profiling studies revealed a phospho-HOXB13-dependent gene signature capable of robustly discriminating between normal prostate tissues, primary and metastatic prostate cancer samples. This work uncovers a previously unanticipated molecular cascade by which mTOR directly phosphorylates HOXB13 to dictate a specific gene program with oncogenic implications in prostate cancer., Implications: Control of HOXB13 transcriptional activity via its direct phosphorylation by the mTOR kinase is a potential therapeutic avenue for the management of advanced prostate cancer., (©2023 The Authors; Published by the American Association for Cancer Research.)

Functional oncogenic links between ErbB2 and ERRα in HER2+ breast cancer patients support a therapeutic benefit of co-targeted therapies. However, ErbB2 and ERRα also play key roles in heart physiology, and this approach could pose a potential liability to cardiovascular health. Herein, using integrated phosphoproteomic, transcriptomic and metabolic profiling, we uncovered molecular mechanisms associated with the adverse remodeling of cardiac functions in mice with combined attenuation of ErbB2 and ERRα activity. Genetic disruption of both effectors results in profound effects on cardiomyocyte architecture, inflammatory response and metabolism, the latter leading to a decrease in fatty acyl-carnitine species further increasing the reliance on glucose as a metabolic fuel, a hallmark of failing hearts. Furthermore, integrated omics signatures of ERRα loss-of-function and doxorubicin treatment exhibit common features of chemotherapeutic cardiotoxicity. These findings thus reveal potential cardiovascular risks in discrete combination therapies in the treatment of breast and other cancers., (© 2022. The Author(s).)

La-related protein 1 (LARP1) has been identified as a key translational inhibitor of terminal oligopyrimidine (TOP) mRNAs downstream of the nutrient sensing protein kinase complex, mTORC1. LARP1 exerts this inhibitory effect on TOP mRNA translation by binding to the mRNA cap and the adjacent 5'TOP motif, resulting in the displacement of the cap-binding protein eIF4E from TOP mRNAs. However, the involvement of additional signaling pathway in regulating LARP1-mediated inhibition of TOP mRNA translation is largely unexplored. In the present study, we identify a second nutrient sensing kinase GCN2 that converges on LARP1 to control TOP mRNA translation. Using chromatin-immunoprecipitation followed by massive parallel sequencing (ChIP-seq) analysis of activating transcription factor 4 (ATF4), an effector of GCN2 in nutrient stress conditions, in WT and GCN2 KO mouse embryonic fibroblasts, we determined that LARP1 is a GCN2-dependent transcriptional target of ATF4. Moreover, we identified GCN1, a GCN2 activator, participates in a complex with LARP1 on stalled ribosomes, suggesting a role for GCN1 in LARP1-mediated translation inhibition in response to ribosome stalling. Therefore, our data suggest that the GCN2 pathway controls LARP1 activity via two mechanisms: ATF4-dependent transcriptional induction of LARP1 mRNA and GCN1-mediated recruitment of LARP1 to stalled ribosomes., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

GCN2 (general control nonderepressible 2) is a serine/threonine-protein kinase that controls messenger RNA translation in response to amino acid availability and ribosome stalling. Here, we show that GCN2 controls erythrocyte clearance and iron recycling during stress. Our data highlight the importance of liver macrophages as the primary cell type mediating these effects. During different stress conditions, such as hemolysis, amino acid deficiency or hypoxia, GCN2 knockout ( GCN2 -/- ) mice displayed resistance to anemia compared with wild-type ( GCN2 +/+ ) mice. GCN2 -/- liver macrophages exhibited defective erythrophagocytosis and lysosome maturation. Molecular analysis of GCN2 -/- cells demonstrated that the ATF4-NRF2 pathway is a critical downstream mediator of GCN2 in regulating red blood cell clearance and iron recycling.

Rapid immunoprecipitation mass spectrometry of endogenous protein (RIME) is a technique to study protein complexes on chromatin. The protocol below describes specific steps for RIME analysis of the male human-derived prostate cancer cell line LNCaP. This approach can also be applied to other prostate cancer cell lines such as 22Rv1, DU145, and PC3. For other cell types, we recommend optimizing the number of cell culture plates to ensure adequate sample for mass spectrometry protein detection. For complete details on the use and execution of this protocol, please refer to Mohammed et al. (2016) and Dufour et al. (2022)., Competing Interests: The authors declare no competing interests., (© 2022 The Author(s).)

Insulin resistance, a harbinger of the metabolic syndrome, is a state of compromised hormonal response resulting from the dysregulation of a wide range of insulin-controlled cellular processes. However, how insulin affects cellular energy metabolism via long-term transcriptional regulation and whether boosting mitochondrial function alleviates insulin resistance remains to be elucidated. Herein we reveal that insulin directly enhances the activity of the nuclear receptor ERRα via a GSK3β/FBXW7 signaling axis. Liver-specific deletion of GSK3β or FBXW7 and mice harboring mutations of ERRα phosphosites (ERRα 3SA ) co-targeted by GSK3β/FBXW7 result in accumulated ERRα proteins that no longer respond to fluctuating insulin levels. ERRα 3SA mice display reprogrammed liver and muscle transcriptomes, resulting in compromised energy homeostasis and reduced insulin sensitivity despite improved mitochondrial function. This crossroad of insulin signaling and transcriptional control by a nuclear receptor offers a framework to better understand the complex cellular processes contributing to the development of insulin resistance., (© 2022. The Author(s).)

A growing number of studies support a direct role for nuclear mTOR in gene regulation and chromatin structure. Still, the scarcity of known chromatin-bound mTOR partners limits our understanding of how nuclear mTOR controls transcription. Herein, comprehensive mapping of the mTOR chromatin-bound interactome in both androgen-dependent and -independent cellular models of prostate cancer (PCa) identifies a conserved 67-protein interaction network enriched for chromatin modifiers, transcription factors, and SUMOylation machinery. SUMO2/3 and nuclear pore protein NUP210 are among the strongest interactors, while the androgen receptor (AR) is the dominant androgen-inducible mTOR partner. Further investigation reveals that NUP210 facilitates mTOR nuclear trafficking, that mTOR and AR form a functional transcriptional module with the nucleosome remodeling and deacetylase (NuRD) complex, and that androgens specify mTOR-SUMO2/3 promoter-enhancer association. This work identifies a vast network of mTOR-associated nuclear complexes advocating innovative molecular strategies to modulate mTOR-dependent gene regulation with conceivable implications for PCa and other diseases., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Non-alcoholic fatty liver disease (NAFLD) is the most frequent liver disease worldwide and can progress to non-alcoholic steatohepatitis (NASH), which is characterized by triglyceride accumulation, inflammation, and fibrosis. No pharmacological agents are currently approved to treat these conditions, but it is clear now that modulation of lipid synthesis and autophagy are key biological mechanisms that could help reduce or prevent these liver diseases. The folliculin (FLCN) protein has been recently identified as a central regulatory node governing whole body energy homeostasis, and we hypothesized that FLCN regulates highly metabolic tissues like the liver. We thus generated a liver specific Flcn knockout mouse model to study its role in liver disease progression. Using the methionine- and choline-deficient diet to mimic liver fibrosis, we demonstrate that loss of Flcn reduced triglyceride accumulation, fibrosis, and inflammation in mice. In this aggressive liver disease setting, loss of Flcn led to activation of transcription factors TFEB and TFE3 to promote autophagy, promoting the degradation of intracellular lipid stores, ultimately resulting in reduced hepatocellular damage and inflammation. Hence, the activity of FLCN could be a promising target for small molecule drugs to treat liver fibrosis by specifically activating autophagy. Collectively, these results show an unexpected role for Flcn in fatty liver disease progression and highlight new potential treatment strategies., (© 2021. The Author(s).)

The estrogen-related receptor alpha (ERRα) is a primary regulator of mitochondrial energy metabolism, function and dynamics, and has been implicated in autophagy and immune regulation. ERRα is abundantly expressed in the intestine and in cells of the immune system. However, its role in inflammatory bowel disease (IBD) remains unknown. Here, we report a protective role of ERRα in the intestine. We found that mice deficient in ERRα were susceptible to experimental colitis, exhibiting increased colon inflammation and tissue damage. This phenotype was mediated by impaired compensatory proliferation of intestinal epithelial cells (IEC) following injury, enhanced IEC apoptosis and necrosis and reduced mucus-producing goblet cell counts. Longitudinal analysis of the microbiota demonstrated that loss of ERRα lead to a reduction in microbiome α-diversity and depletion of healthy gut bacterial constituents. Mechanistically, ERRα mediated its protective effects by acting within the radio-resistant compartment of the intestine. It promoted disease tolerance through transcriptional control of key genes involved in intestinal tissue homeostasis and repair. These findings provide new insights on the role of ERRα in the gut and extends our current knowledge of nuclear receptors implicated in IBD., (© 2021. The Author(s).)

DNA methylation is implicated in the acquisition of malignant phenotypes, and the use of epigenetic modulating drugs is a promising anti-cancer therapeutic strategy. 5-aza-2'deoxycytidine (decitabine, 5-azadC) is an FDA-approved DNA methyltransferase (DNMT) inhibitor with proven effectiveness against hematological malignancies and more recently triple-negative breast cancer (BC). Herein, genetic or pharmacological studies uncovered a hitherto unknown feedforward molecular link between DNMT1 and the estrogen related receptor α (ERRα), a key transcriptional regulator of cellular metabolism. Mechanistically, DNMT1 promotes ERRα stability which in turn couples DNMT1 transcription with that of the methionine cycle and S-adenosylmethionine synthesis to drive DNA methylation. In vitro and in vivo investigation using a pre-clinical mouse model of BC demonstrated a clear therapeutic advantage for combined administration of the ERRα inhibitor C29 with 5-azadC. A large-scale bisulfite genomic sequencing analysis revealed specific methylation perturbations fostering the discovery that reversal of promoter hypermethylation and consequently derepression of the tumor suppressor gene, IRF4, is a factor underlying the observed BC suppressive effects. This work thus uncovers a critical role of ERRα in the crosstalk between transcriptional control of metabolism and epigenetics and illustrates the potential for targeting ERRα in combination with DNMT inhibitors for BC treatment and other epigenetics-driven malignancies.

Excessive reactive oxygen species (ROS) can cause oxidative stress and consequently cell injury contributing to a wide range of diseases. Addressing the critical gaps in our understanding of the adaptive molecular events downstream ROS provocation holds promise for the identification of druggable metabolic vulnerabilities. Here, we unveil a direct molecular link between the activity of two estrogen-related receptor (ERR) isoforms and the control of glutamine utilization and glutathione antioxidant production. ERRα down-regulation restricts glutamine entry into the TCA cycle, while ERRγ up-regulation promotes glutamine-driven glutathione production. Notably, we identify increased ERRγ expression/activation as a hallmark of oxidative stress triggered by mitochondrial disruption or chemotherapy. Enhanced tumor antioxidant capacity is an underlying feature of human breast cancer (BCa) patients that respond poorly to treatment. We demonstrate that pharmacological inhibition of ERRγ with the selective inverse agonist GSK5182 increases antitumor efficacy of the chemotherapeutic paclitaxel on poor outcome BCa tumor organoids. Our findings thus underscore the ERRs as novel redox sensors and effectors of a ROS defense program and highlight the potential therapeutic advantage of exploiting ERRγ inhibitors for the treatment of BCa and other diseases where oxidative stress plays a central role., (© 2020 Vernier et al.; Published by Cold Spring Harbor Laboratory Press.)

Surface free energy remains a fundamental material property to characterize the interfacial interactions between liquid and solid. Here, we developed a precise approach to determine surface energy by using contact angles of binary mixtures of water-dimethyl sulfoxide (DMSO), water-formamide, water-ethylene glycol, and water-glycerol and analyzed using the Owens-Wendt method. A mixing equation was developed to estimate liquid-dispersive surface tension (γ L,mix d ) and polar surface tension (γ L,mix p ) parameters for binary mixtures. To test the approach, two hydrophobic surfaces, flat polydimethylsiloxane (PDMS), and silane-derivatized glass were prepared and the contact angle of mixtures on the surfaces were obtained. Surface energy of PDMS determined by three binary mixtures agrees with that from pure solvents, but the uncertainty decreases to less than 13%; remarkably, the uncertainty drops to around 5% once we combined measured contact angles from all the mixtures, namely, water-DMSO, water-formamide, and water-ethylene glycol. Surface energies of silane-derivatized glass bearing ethyl (C 2 ), hexyl (C 6 ), and octadecyl (C 18 ) alkyl chains were determined with water-formamide and water-glycerol mixtures. Measured contact angles fit the Owens-Wendt model, and surface energy value determined from different binary mixtures agree with each other within error. Contact angle measurement of liquid mixtures is a simple method for determination of surface energy that improves the precision of surface energy determined by measurements of multiple pure solvents.

Dysregulation of histone modifications promotes carcinogenesis by altering transcription. Breast cancers frequently overexpress the histone methyltransferase EZH2, the catalytic subunit of Polycomb Repressor Complex 2 (PRC2). However, the role of EZH2 in this setting is unclear due to the context-dependent functions of PRC2 and the heterogeneity of breast cancer. Moreover, the mechanisms underlying PRC2 overexpression in cancer are obscure. Here, using multiple models of breast cancer driven by the oncogene ErbB2, we show that the tyrosine kinase c-Src links energy sufficiency with PRC2 overexpression via control of mRNA translation. By stimulating mitochondrial ATP production, c-Src suppresses energy stress, permitting sustained activation of the mammalian/mechanistic target of rapamycin complex 1 (mTORC1), which increases the translation of mRNAs encoding the PRC2 subunits Ezh2 and Suz12. We show that Ezh2 overexpression and activity are pivotal in ErbB2-mediated mammary tumourigenesis. These results reveal the hitherto unknown c-Src/mTORC1/PRC2 axis, which is essential for ErbB2-driven carcinogenesis.

As transcriptional factors, nuclear receptors (NRs) function as major regulators of gene expression. In particular, dysregulation of NR activity has been shown to significantly alter metabolic homeostasis in various contexts leading to metabolic disorders and cancers. The orphan estrogen-related receptor (ERR) subfamily of NRs, comprised of ERRα, ERRβ, and ERRγ, for which a natural ligand has yet to be identified, are known as central regulators of energy metabolism. If AMP-activated protein kinase (AMPK) and mechanistic target of rapamycin (mTOR) can be viewed as sensors of the metabolic needs of a cell and responding acutely via post-translational control of proteins, then the ERRs can be regarded as downstream effectors of metabolism via transcriptional regulation of genes for a long-term and sustained adaptive response. In this review, we will focus on recent findings centered on the transcriptional roles played by ERRα in hepatocytes. Modulation of ERRα activity in both in vitro and in vivo models via genetic or pharmacological manipulation coupled with chromatin-immunoprecipitation (ChIP)-on-chip and ChIP-sequencing (ChIP-seq) studies have been fundamental in delineating the direct roles of ERRα in the control of hepatic gene expression. These studies have identified crucial roles for ERRα in lipid and carbohydrate metabolism as well as in mitochondrial function under both physiological and pathological conditions. The regulation of ERRα expression and activity via ligand-independent modes of action including coregulator binding, post-translational modifications (PTMs) and control of protein stability will be discussed in the context that may serve as valuable tools to modulate ERRα function as new therapeutic avenues for the treatment of hepatic metabolic dysfunction and related diseases.

Emerging evidence has illustrated the importance of epigenomic reprogramming in cancer, with altered post-translational modifications of histones contributing to pathogenesis. However, the contributions of histone modifiers to breast cancer progression are unclear, and how these processes vary between molecular subtypes has yet to be adequately addressed. Here we report that genetic or pharmacological targeting of the epigenetic modifier Ezh2 dramatically hinders metastatic behaviour in both a mouse model of breast cancer and patient-derived xenografts reflective of the Luminal B subtype. We further define a subtype-specific molecular mechanism whereby EZH2 maintains H3K27me3-mediated repression of the FOXC1 gene, thereby inactivating a FOXC1-driven, anti-invasive transcriptional program. We demonstrate that higher FOXC1 is predictive of favourable outcome specifically in Luminal B breast cancer patients and establish the use of EZH2 methyltransferase inhibitors as a viable strategy to block metastasis in Luminal B breast cancer, where options for targeted therapy are limited.

Given the increasing prevalence of obesity and the metabolic syndrome, identification of intrinsic molecular programs responsible for ensuring fuel homeostasis and preventing metabolic disease is needed. We investigated whether the orphan nuclear receptor estrogen-related receptor α (ERRα), a major regulator of energy metabolism, plays a role in lipid homeostasis and the development of nonalcoholic fatty liver disease (NAFLD) in response to chronic high-fat diet (HFD) consumption and long-term fasting. Systemic ablation of ERRα in mice demonstrated clear beneficial effects for loss of ERRα function in protection from HFD-provoked body weight gain manifested not only from a reduction in white adipose tissue stores but also from an impediment in intrahepatic lipid accumulation. The prevention of HFD-induced NAFLD in ERRα-null mice was underscored by transcriptional repression of de novo lipogenesis, which was upregulated in wild-type mice, a known contributing factor to lipid-stimulated hepatic steatosis. Surprisingly, given these findings, ERRα deficiency had no significant impact on the degree of fasting-induced NAFLD, involving the mobilization of adipocyte triglyceride (TG) stores into the liver. However, the presence of ERRα was essential for acute refeeding-mediated reversal of fasting-induced hepatic TG accretion, underpinned by impaired downregulation of adipose TG lipolysis and reduced hepatic mitochondrial oxidative activity. Taken together, the regulation of lipid handling by ERRα depended on the nutritional state, suggesting that negative modulation of ERRα activity could be envisaged to prevent lipid-induced NAFLD, whereas inducing its activity would be useful to treat and reverse the instilled disease.

The orphan nuclear receptor ESRRA (estrogen-related receptor α) is a key regulator of energy homeostasis and mitochondrial function. Macroautophagy/autophagy, an intracellular degradation process, is a critical innate effector against intracellular microbes. Here, we demonstrate that ESRRA is required for the activation of autophagy to promote innate antimicrobial defense against mycobacterial infection. AMP-activated protein kinase pathway and SIRT1 (sirtuin 1) activation led to induction of ESRRA, which is essential for autophagosome formation, in bone marrow-derived macrophages. ESRRA enhanced the transcriptional activation of numerous autophagy-related (Atg) genes containing ERR response elements in their promoter regions. Furthermore, ESRRA, operating in a feed-forward loop with SIRT1, was required for autophagy activation through deacetylation of ATG5, BECN1, and ATG7. Importantly, ESRRA deficiency resulted in a decrease of phagosomal maturation and antimicrobial responses against mycobacterial infection. Thus, we identify ESRRA as a critical activator of autophagy via both transcriptional and post-translational control to promote antimicrobial host responses.

Androgen receptor (AR) signaling reprograms cellular metabolism to support prostate cancer (PCa) growth and survival. Another key regulator of cellular metabolism is mTOR, a kinase found in diverse protein complexes and cellular localizations, including the nucleus. However, whether nuclear mTOR plays a role in PCa progression and participates in direct transcriptional cross-talk with the AR is unknown. Here, via the intersection of gene expression, genomic, and metabolic studies, we reveal the existence of a nuclear mTOR-AR transcriptional axis integral to the metabolic rewiring of PCa cells. Androgens reprogram mTOR-chromatin associations in an AR-dependent manner in which activation of mTOR-dependent metabolic gene networks is essential for androgen-induced aerobic glycolysis and mitochondrial respiration. In models of castration-resistant PCa cells, mTOR was capable of transcriptionally regulating metabolic gene programs in the absence of androgens, highlighting a potential novel castration resistance mechanism to sustain cell metabolism even without a functional AR. Remarkably, we demonstrate that increased mTOR nuclear localization is indicative of poor prognosis in patients, with the highest levels detected in castration-resistant PCa tumors and metastases. Identification of a functional mTOR targeted multigene signature robustly discriminates between normal prostate tissues, primary tumors, and hormone refractory metastatic samples but is also predictive of cancer recurrence. This study thus underscores a paradigm shift from AR to nuclear mTOR as being the master transcriptional regulator of metabolism in PCa., (© 2017 Audet-Walsh et al.; Published by Cold Spring Harbor Laboratory Press.)

The tumor suppressor folliculin (FLCN) forms a repressor complex with AMP-activated protein kinase (AMPK). Given that AMPK is a master regulator of cellular energy homeostasis, we generated an adipose-specific Flcn (Adipoq-FLCN) knockout mouse model to investigate the role of FLCN in energy metabolism. We show that loss of FLCN results in a complete metabolic reprogramming of adipose tissues, resulting in enhanced oxidative metabolism. Adipoq-FLCN knockout mice exhibit increased energy expenditure and are protected from high-fat diet (HFD)-induced obesity. Importantly, FLCN ablation leads to chronic hyperactivation of AMPK, which in turns induces and activates two key transcriptional regulators of cellular metabolism, proliferator-activated receptor γ (PPARγ) coactivator-1α (PGC-1α) and estrogen-related receptor α (ERRα). Together, the AMPK/PGC-1α/ERRα molecular axis positively modulates the expression of metabolic genes to promote mitochondrial biogenesis and activity. In addition, mitochondrial uncoupling proteins as well as other markers of brown fat are up-regulated in both white and brown FLCN-null adipose tissues, underlying the increased resistance of Adipoq-FLCN knockout mice to cold exposure. These findings identify a key role of FLCN as a negative regulator of mitochondrial function and identify a novel molecular pathway involved in the browning of white adipocytes and the activity of brown fat., (© 2016 Yan et al.; Published by Cold Spring Harbor Laboratory Press.)

The orphan nuclear receptor estrogen-related receptor α (ERRα; NR3B1) is a key metabolic regulator, but its function in regulating inflammation remains largely unknown. Here, we demonstrate that ERRα negatively regulates Toll-like receptor (TLR)-induced inflammation by promoting Tnfaip3 transcription and fine-tuning of metabolic reprogramming in macrophages. ERRα-deficient (Esrra(-/-)) mice showed increased susceptibility to endotoxin-induced septic shock, leading to more severe pro-inflammatory responses than control mice. ERRα regulated macrophage inflammatory responses by directly binding the promoter region of Tnfaip3, a deubiquitinating enzyme in TLR signaling. In addition, Esrra(-/-) macrophages showed an increased glycolysis, but impaired mitochondrial respiratory function and biogenesis. Further, ERRα was required for the regulation of NF-κB signaling by controlling p65 acetylation via maintenance of NAD(+) levels and sirtuin 1 activation. These findings unravel a previously unappreciated role for ERRα as a negative regulator of TLR-induced inflammatory responses through inducing Tnfaip3 transcription and controlling the metabolic reprogramming., (Copyright © 2015 Elsevier Inc. All rights reserved.)

The tyrosine kinase receptor ERBB2 is required for normal development of the heart and is a potent oncogene in breast epithelium. Trastuzumab, a monoclonal antibody targeting ERBB2, improves the survival of breast cancer patients, but cardiac dysfunction is a major side effect of the drug. The molecular mechanisms underlying how ERBB2 regulates cardiac function and why trastuzumab is cardiotoxic remain poorly understood. We show here that ERBB2 hypomorphic mice develop cardiac dysfunction that mimics the side effects observed in patients treated with trastuzumab. We demonstrate that this phenotype is related to the critical role played by ERBB2 in cardiac homeostasis and physiological hypertrophy. Importantly, genetic and therapeutic reduction of ERBB2 activity in mice, as well as ablation of ERBB2 signaling by trastuzumab or siRNAs in human cardiomyocytes, led to the identification of an impaired E2F-1-dependent genetic program critical for the cardiac adaptive stress response. These findings demonstrate the existence of a previously unknown mechanistic link between ERBB2 and E2F-1 transcriptional activity in heart physiology and trastuzumab-induced cardiac dysfunction., (Copyright © 2014, American Society for Microbiology. All Rights Reserved.)

Muscle fitness is an important determinant of health and disease. However, the molecular mechanisms involved in the coordinate regulation of the metabolic and structural determinants of muscle endurance are still poorly characterized. Herein, we demonstrate that estrogen-related receptor α (ERRα, NR3B1) is essential for skeletal muscle fitness. Notably, we show that ERRα-null animals are hypoactive and that genetic or therapeutic disruption of ERRα in mice results in reduced exercise tolerance. Mice lacking ERRα also exhibited lactatemia at exhaustion. Gene expression profiling demonstrates that ERRα plays a key role in various metabolic processes important for muscle function including energy substrate transport and use (Ldhd, Slc16a1, Hk2, and Glul), the tricarboxylic acid cycle (Cycs, and Idh3g), and oxidative metabolism (Pdha1, and Uqcrq). Metabolomics studies revealed impairment in replenishment of several amino acids (eg, glutamine) during recovery to exercise. Moreover, loss of ERRα was found to alter the expression of genes involved in oxidative stress response (Hmox1), maintenance of muscle fiber integrity (Trim63, and Hspa1b), and muscle plasticity and neovascularization (Vegfa). Taken together, our study shows that ERRα plays a key role in directing transcriptional programs required for optimal mitochondrial oxidative potential and muscle fitness, suggesting that modulation of ERRα activity could be used to manage metabolic myopathies and/or promote the adaptive response to physical exercise.

Estrogen-related receptor α (ERRα) is a key regulator of mitochondrial function and metabolism essential for energy-driven cellular processes in both normal and cancer cells. ERRα has also been shown to mediate bone-derived macrophage activation by proinflammatory cytokines. However, the role of ERRα in cancer in which inflammation acts as a tumor promoter has yet to be investigated. Herein we show that global loss of ERRα accelerates the development of diethylnitrosamine (DEN)-induced hepatocellular carcinoma. Biochemical and metabolomics studies revealed that loss of ERRα promotes hepatocyte necrosis over apoptosis in response to DEN due to a deficiency in energy production. We further show that increased hepatocyte death and associated compensatory proliferation observed in DEN-injured ERRα-null livers is concomitant with increased nuclear factor κB (NF-κB)-dependent transcriptional control of cytokine expression in Kupffer cells. In particular, we demonstrate that loss of ERRα-dependent regulation of the NF-κB inhibitor IκBα leads to enhanced NF-κB activity and cytokine gene activation. Our work thus shows that global loss of ERRα activity promotes hepatocellular carcinoma by independent but synergistic mechanisms in hepatocytes and Kupffer cells, implying that pharmacological manipulation of ERRα activity may have a significant clinical impact on carcinogen-induced cancers.

mTOR and ERRα are key regulators of common metabolic processes, including lipid homeostasis. However, it is currently unknown whether these factors cooperate in the control of metabolism. ChIP-sequencing analyses of mouse liver reveal that mTOR occupies regulatory regions of genes on a genome-wide scale including enrichment at genes shared with ERRα that are involved in the TCA cycle and lipid biosynthesis. Genetic ablation of ERRα and rapamycin treatment, alone or in combination, alter the expression of these genes and induce the accumulation of TCA metabolites. As a consequence, both genetic and pharmacological inhibition of ERRα activity exacerbates hepatic hyperlipidemia observed in rapamycin-treated mice. We further show that mTOR regulates ERRα activity through ubiquitin-mediated degradation via transcriptional control of the ubiquitin-proteasome pathway. Our work expands the role of mTOR action in metabolism and highlights the existence of a potent mTOR/ERRα regulatory axis with significant clinical impact., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Metabolic homeostasis and circadian rhythms are closely intertwined biological processes. Nuclear receptors, as sensors of hormonal and nutrient status, are actively implicated in maintaining this physiological relationship. Although the orphan nuclear receptor estrogen-related receptor α (ERRα, NR3B1) plays a central role in the control of energy metabolism and its expression is known to be cyclic in the liver, its role in temporal control of metabolic networks is unknown. Here we report that ERRα directly regulates all major components of the molecular clock. ERRα-null mice also display deregulated locomotor activity rhythms and circadian period lengths under free-running conditions, as well as altered circulating diurnal bile acid and lipid profiles. In addition, the ERRα-null mice exhibit time-dependent hypoglycemia and hypoinsulinemia, suggesting a role for ERRα in modulating insulin sensitivity and glucose handling during the 24-hour light/dark cycle. We also provide evidence that the newly identified ERRα corepressor PROX1 is implicated in rhythmic control of metabolic outputs. To help uncover the molecular basis of these phenotypes, we performed genome-wide location analyses of binding events by ERRα, PROX1, and BMAL1, an integral component of the molecular clock. These studies revealed the existence of transcriptional regulatory loops among ERRα, PROX1, and BMAL1, as well as extensive overlaps in their target genes, implicating these three factors in the control of clock and metabolic gene networks in the liver. Genomic convergence of ERRα, PROX1, and BMAL1 transcriptional activity thus identified a novel node in the molecular circuitry controlling the daily timing of metabolic processes., Competing Interests: The authors have declared that no competing interests exist.

IRF8 (Interferon Regulatory Factor 8) plays an important role in defenses against intracellular pathogens, including several aspects of myeloid cells function. It is required for ontogeny and maturation of macrophages and dendritic cells, for activation of anti-microbial defenses, and for production of the Th1-polarizing cytokine interleukin-12 (IL-12) in response to interferon gamma (IFNγ) and protection against infection with Mycobacterium tuberculosis. The transcriptional programs and cellular pathways that are regulated by IRF8 in response to IFNγ and that are important for defenses against M. tuberculosis are poorly understood. These were investigated by transcript profiling and chromatin immunoprecipitation on microarrays (ChIP-chip). Studies in primary macrophages identified 368 genes that are regulated by IRF8 in response to IFNγ/CpG and that behave as stably segregating expression signatures (eQTLs) in F2 mice fixed for a wild-type or mutant allele at IRF8. A total of 319 IRF8 binding sites were identified on promoters genome-wide (ChIP-chip) in macrophages treated with IFNγ/CpG, defining a functional G/AGAAnTGAAA motif. An analysis of the genes bearing a functional IRF8 binding site, and showing regulation by IFNγ/CpG in macrophages and/or in M. tuberculosis-infected lungs, revealed a striking enrichment for the pathways of antigen processing and presentation, including multiple structural and enzymatic components of the Class I and Class II MHC (major histocompatibility complex) antigen presentation machinery. Also significantly enriched as IRF8 targets are the group of endomembrane- and phagosome-associated small GTPases of the IRG (immunity-related GTPases) and GBP (guanylate binding proteins) families. These results identify IRF8 as a key regulator of early response pathways in myeloid cells, including phagosome maturation, antigen processing, and antigen presentation by myeloid cells., Competing Interests: The authors have declared that no competing interests exist.

Metabolism and circadian rhythms must be closely integrated to support the energetic needs of the organism linked to the daily timing of physiological and behavioral processes. Although components of the molecular clock can directly target some metabolic genes, the control of metabolic clock output is believed to be mediated mostly through the action of transcription factors whose patterns of expression are rhythmic in metabolic tissues. Our recent work has identified the orphan nuclear receptor estrogen-related receptor α (ERRα), a potent effector of metabolic gene networks, as a direct regulator of the molecular clock. Thus, by acting both upstream of and downstream from the molecular clock, ERRα serves as a key transcription factor linking the clock with metabolic control.

Cancer cell metabolism is often characterized by a shift from an oxidative to a glycolytic bioenergetics pathway, a phenomenon known as the Warburg effect. miR-378(∗) is embedded within PPARGC1b which encodes PGC-1β, a transcriptional regulator of oxidative energy metabolism. Here we show that miR-378(∗) expression is regulated by ERBB2 and induces a metabolic shift in breast cancer cells. miR-378(∗) performs this function by inhibiting the expression of two PGC-1β partners, ERRγ and GABPA, leading to a reduction in tricarboxylic acid cycle gene expression and oxygen consumption as well as an increase in lactate production and in cell proliferation. In situ hybridization experiments show that miR-378(∗) expression correlates with progression of human breast cancer. These results identify miR-378(∗) as a molecular switch involved in the orchestration of the Warburg effect in breast cancer cells via interference with a well-integrated bioenergetics transcriptional pathway., (Copyright © 2010 Elsevier Inc. All rights reserved.)

Estrogen-related receptor alpha (ERRalpha) and proliferator-activated receptor gamma coactivator-1alpha (PGC-1alpha) play central roles in the transcriptional control of energy homeostasis, but little is known about factors regulating their activity. Here we identified the homeobox protein prospero-related homeobox 1 (Prox1) as one such factor. Prox1 interacts with ERRalpha and PGC-1alpha, occupies promoters of metabolic genes on a genome-wide scale, and inhibits the activity of the ERRalpha/PGC-1alpha complex. DNA motif analysis suggests that Prox1 interacts with the genome through tethering to ERRalpha and other factors. Importantly, ablation of Prox1 and ERRalpha have opposite effects on the respiratory capacity of liver cells, revealing an unexpected role for Prox1 in the control of energy homeostasis.

Estrogen-related receptor alpha (ERRalpha) is an orphan nuclear receptor highly expressed in the kidney, an organ playing a central role in blood pressure regulation through electrolyte homeostasis and the renin-angiotensin system. Physiological analysis revealed that, relative to wild-type mice, ERRalpha null mice are hypotensive despite significant hypernatremia, hypokalemia, and slight hyperreninemia. Using a combination of genome-wide location analysis and expression profiling, we demonstrate that ERRalpha regulates the expression of channels involved in renal Na(+) and K(+) handling (Scnn1a, Atp1a1, Atp1b1) and altered in Bartter syndrome (Bsnd, Kcnq1). In addition, ERRalpha regulates the expression of receptors implicated in the systemic regulation of blood pressure (Ghr, Gcgr, Lepr, Npy1r) and of genes within the renin-angiotensin pathway (Ren1, Agt, Ace2). Our study thus identifies ERRalpha as a pleiotropic regulator of renal control of blood pressure, renal Na(+)/K(+) homeostasis, and renin-angiotensin pathway and suggests that modulation of ERRalpha activity could represent a potential avenue for the management of hypertension.

Downregulation and functional deactivation of the transcriptional coactivator PGC-1alpha has been implicated in heart failure pathogenesis. We hypothesized that the estrogen-related receptor alpha (ERRalpha), which recruits PGC-1alpha to metabolic target genes in heart, exerts protective effects in the context of stressors known to cause heart failure. ERRalpha(-/-) mice subjected to left ventricular (LV) pressure overload developed signatures of heart failure including chamber dilatation and reduced LV fractional shortening. (31)P-NMR studies revealed abnormal phosphocreatine depletion in ERRalpha(-/-) hearts subjected to hemodynamic stress, indicative of a defect in ATP reserve. Mitochondrial respiration studies demonstrated reduced maximal ATP synthesis rates in ERRalpha(-/-) hearts. Cardiac ERRalpha target genes involved in energy substrate oxidation, ATP synthesis, and phosphate transfer were downregulated in ERRalpha(-/-) mice at baseline or with pressure overload. These results demonstrate that the nuclear receptor ERRalpha is required for the adaptive bioenergetic response to hemodynamic stressors known to cause heart failure.

At birth, the heart undergoes a critical metabolic switch from a predominant dependence on carbohydrates during fetal life to a greater dependence on postnatal oxidative metabolism. This remains the principle metabolic state throughout life, although pathologic conditions such as heart failure and cardiac hypertrophy reactivate components of the fetal genetic program to increase carbohydrate utilization. Disruption of the ERRgamma gene (Esrrg), which is expressed at high levels in the fetal and postnatal mouse heart, blocks this switch, resulting in lactatemia, electrocardiographic abnormalities, and death during the first week of life. Genomic ChIP-on-chip and expression analysis identifies ERRgamma as both a direct and an indirect regulator of a nuclear-encoded mitochondrial genetic network that coordinates the postnatal metabolic transition. These findings reveal an unexpected and essential molecular genetic component of the oxidative metabolic gene program in the heart and highlight ERRgamma in the study of cardiac hypertrophy and failure.

Orphan nuclear receptor ERRalpha (NR3B1) is recognized as a key regulator of mitochondrial biogenesis, but it is not known whether ERRalpha and other ERR isoforms play a broader role in cardiac energetics and function. We used genome-wide location analysis and expression profiling to appraise the role of ERRalpha and gamma (NR3B3) in the adult heart. Our data indicate that the two receptors, acting as nonobligatory heterodimers, target a common set of promoters involved in the uptake of energy substrates, production and transport of ATP across the mitochondrial membranes, and intracellular fuel sensing, as well as Ca(2+) handling and contractile work. Motif-finding algorithms assisted by functional studies indicated that ERR target promoters are enriched for NRF-1, CREB, and STAT3 binding sites. Our study thus reveals that the ERRs orchestrate a comprehensive cardiac transcriptional program and further suggests that modulation of ERR activities could be used to manage cardiomyopathies.

The orphan nuclear estrogen-related receptor alpha (ERRalpha) and transcriptional cofactor peroxisome proliferator-activated receptor gamma coactivator-1alpha (PGC-1alpha) are involved in the regulation of energy metabolism. Recently, extensive cross-talk between PGC-1alpha and ERRalpha has been demonstrated. The presence of PGC-1alpha is associated with an elevated expression of ERRalpha, and the two proteins can influence the transcriptional activities of one another. Using a candidate gene approach to detect regulatory variants within genes encoding nuclear receptors, we have identified a 23-bp sequence (ESRRA23) containing two nuclear receptor recognition half-site motifs that is present in 1-4 copies within the promoter of the human ESRRA gene encoding ERRalpha. The ESRRA23 sequence contains a functional ERR response element that is specifically bound by ERRalpha, and chromatin immunoprecipitation shows that endogenous ERRalpha occupies its own promoter in vivo. Strikingly, introduction of PGC-1alpha in HeLa cells by transient transfection induces the activity of the ESRRA promoter in a manner that is dependent on the presence of the ESRRA23 element and on its dosage. Coexpression of ERRalpha and PGC-1alpha results in a synergistic activation of the ESRRA promoter. In experiments using ERRalpha null fibroblasts, the ability of PGC-1alpha to stimulate the ESRRA promoter is considerably reduced but can be restored by addition of ERRalpha. Taken together, these results demonstrate that an interdependent ERRalpha/PGC-1alpha-based transcriptional pathway targets the ESRRA23 element to dictate the level of ERRalpha expression. This study further suggests that this regulatory polymorphism may provide differential responses to ERRalpha/PGC-1alpha-mediated metabolic cues in the human population.