Molecular and Genetic Crosstalks between mTOR and ERRα Are Key Determinants of Rapamycin-Induced Nonalcoholic Fatty Liver

SummarymTOR 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

Hypothalamic eIF2α signaling regulates food intake.

The reversible phosphorylation of the α subunit of eukaryotic initiation factor 2 (eIF2α) is a highly conserved signal implicated in the cellular adaptation to numerous stresses such as the one caused by amino acid limitation. In response to dietary amino acid deficiency, the brain-specific activation of the eIF2α kinase GCN2 leads to food intake inhibition. We report here that GCN2 is rapidly activated in the mediobasal hypothalamus (MBH) after consumption of a leucine-deficient diet. Furthermore, knockdown of GCN2 in this particular area shows that MBH GCN2 activity controls the onset of the aversive response. Importantly, pharmacological experiments demonstrate that the sole phosphorylation of eIF2α in the MBH is sufficient to regulate food intake. eIF2α signaling being at the crossroad of stress pathways activated in several pathological states, our study indicates that hypothalamic eIF2α phosphorylation could play a critical role in the onset of anorexia associated with certain diseases.This work was supported by grants from “Fondation pour la Recherche Médicale,” “Société Française de Nutrition,” Ajinomoto Amino Acid Research Program (3ARP), and “Agence Nationale pour la Recherche.”This is the final version of the article. It first appeared from Elsevier via http://dx.doi.org/10.1016/j.celrep.2014.01.00

The p300/CBP-associated factor (PCAF) is a cofactor of ATF4 for amino acid-regulated transcription of CHOP

When an essential amino acid is limited, a signaling cascade is triggered that leads to increased translation of the ‘master regulator’, activating transcription factor 4 (ATF4), and resulting in the induction of specific target genes. Binding of ATF4 to the amino acid response element (AARE) is an essential step in the transcriptional activation of CHOP (a CCAAT/enhancer-binding protein-related gene) by amino acid deprivation. We set out to identify proteins that interact with ATF4 and that play a role in the transcriptional activation of CHOP. Using a tandem affinity purification (TAP) tag approach, we identified p300/CBP-associated factor (PCAF) as a novel interaction partner of ATF4 in leucine-starved cells. We show that the N-terminal region of ATF4 is required for a direct interaction with PCAF and demonstrate that PCAF is involved in the full transcriptional response of CHOP by amino acid starvation. Chromatin immunoprecipitation analysis revealed that PCAF is engaged on the CHOP AARE in response to amino acid starvation and that ATF4 is essential for its recruitment. We also show that PCAF stimulates ATF4-driven transcription via its histone acetyltransferase domain. Thus PCAF acts as a coactivator of ATF4 and is involved in the enhancement of CHOP transcription following amino acid starvation

Amino Acid Availability Controls TRB3 Transcription in Liver through the GCN2/eIF2α/ATF4 Pathway

In mammals, plasma amino acid concentrations are markedly affected by dietary or pathological conditions. It has been well established that amino acids are involved in the control of gene expression. Up to now, all the information concerning the molecular mechanisms involved in the regulation of gene transcription by amino acid availability has been obtained in cultured cell lines. The present study aims to investigate the mechanisms involved in transcriptional activation of the TRB3 gene following amino acid limitation in mice liver. The results show that TRB3 is up-regulated in the liver of mice fed a leucine-deficient diet and that this induction is quickly reversible. Using transient transfection and chromatin immunoprecipitation approaches in hepatoma cells, we report the characterization of a functional Amino Acid Response Element (AARE) in the TRB3 promoter and the binding of ATF4, ATF2 and C/EBPβ to this AARE sequence. We also provide evidence that only the binding of ATF4 to the AARE plays a crucial role in the amino acid-regulated transcription of TRB3. In mouse liver, we demonstrate that the GCN2/eIF2α/ATF4 pathway is essential for the induction of the TRB3 gene transcription in response to a leucine-deficient diet. Therefore, this work establishes for the first time that the molecular mechanisms involved in the regulation of gene transcription by amino acid availability are functional in mouse liver

Identification d'une nouvelle voie de signalisation impliquée dans la régulation des gènes par les acides aminés, chez les mammifères

n mammals, environment is a powerful regulator of gene expression. For example, according to nutrition, nutrients -and particularly amino acids- availability is responsible for the induction of the expression of numerous genes. Indeed, specific molecular mechanisms are triggered in order to sense and respond adequately to these variations. Whenever one of the nine essential amino acids is missing, the transcription of specific genes is induced. This activation requires the induction of transcription factor ATF4 and the phosphorylation of transcription factor ATF2. The ATF4 pathway has been identified and relatively well described. However, the elements of the pathway upstream of ATF2 phosphorylation were yet unknown. The aim of my PhD thesis was to identify the different regulatory elements responsible for ATF2 phosphorylation in response to amino acid starvation. I have shown that amino acid starvation induces the activation of a MAPK module composed of MEKK1>MKK7>JNK2 that controls ATF2 phosphorylation on both threonine 69 and 71. I have shown that this module is regulated by two GTPases Cdc42+Rac1 and by Gα12. Finally, I was able to show that this new pathway is involved in the AARE-dependent transcription of ATF3 in response to an amino acid starvation.Chez les mammifères, l'environnement est un puissant régulateur de l'expression des gènes. Par exemple, en fonction de l'alimentation, la disponibilité en nutriments, en particulier en acides aminés, est responsable de l'induction de l'expression d'un certain nombre de gènes. Ainsi, des mécanismes moléculaires sont mis en jeu de façon à permettre la détection de ces variations et d'y répondre de façon appropriée. Lorsque l'un des neuf acides aminés essentiels vient à manquer, on observe une augmentation de la transcription de certains gènes. Cette activation de la transcription requière d'une part l'accumulation du facteur de transcription ATF4 et d'autre part la phosphorylation du facteur de transcription ATF2. La voie ATF4 a été identifiée et relativement bien décrite. En revanche les éléments régulateurs de la voie de signalisation en amonts du facteur ATF2 restent inconnus. Le but de ma thèse était donc de déterminer les différents intermédiaires responsables de la phosphorylation d'ATF2 en réponse à une carence en acides aminés. J'ai ainsi montré qu'une carence en acides aminés provoque la mise en jeu d'un module MAPK composé de MEKK1>MKK7>JNK2 contrôlant la phosphorylation d'ATF2 sur les résidus thréonine 69 et 71. J'ai montré que ce module est régulé en amont par deux GTPases Cdc42+Rac1 et par la protéine Gα12. Enfin, j'ai démontré l'impact de cette nouvelle voie de signalisation sur la transcription AARE-dépendante du gène ATF3 en réponse à une carence en acides aminés

Amino acids as regulators of gene expression in mammals: Molecular mechanisms

Review ArticleInternational audienceIn mammals, the impact of nutrients on gene expression has become an important area of research. Because amino acids have multiple and important functions, their homeostasis has to be finely maintained. However, amino acidemia can be affected in some nutritional conditions and by various forms of stress. Consequently, mammals have to adjust physiological functions involved in the adaptation to amino acid availability. Part of this regulation involves the modulation of numerous gene expression. It has been shown that amino acids by themselves can modify the expression of target genes. This review focuses on the recent advances in the understanding of the mechanisms involved in the control of mammalian gene expression in response to amino acid limitation

Role of the repressor JDP2 in the amino acid-regulated transcription of CHOP

International audienceThe transcriptional activation of CHOP (C/EBP-homologous protein) by amino acid deprivation involves ATF2 and ATF4 binding at the amino acid response element within the promoter. In this report, we investigate the role of JDP2 (Jun Dimerization Protein 2) in the amino acid control of CHOP transcription following amino acid starvation. Our results show that JDP2 binds to the CHOP AARE in unstimulated cells and that its binding decreases following amino acid starvation. We demonstrate that JDP2 acts as a repressor and suggest that it could be functionally associated with HDAC3 to inhibit CHOP transcription

TRB3 inhibits the transcriptional activation of stress-regulated genes by a negative feedback on the ATF4 pathway

International audienceThe integrated stress response (ISR) is defined as a highly conserved response to several stresses that converge to the induction of the activating transcription factor 4 (ATF4). Because an uncontrolled response may have deleterious effects, cells have elaborated several negative feedback loops that attenuate the ISR. In the present study, we describe how induction of the human homolog of Drosophila tribbles (TRB3) attenuates the ISR by a negative feedback mechanism. To investigate the role of TRB3 in the control of the ISR, we used the regulation of gene expression by amino acid limitation as a model. The enhanced production of ATF4 upon amino acid starvation results in the induction of a large number of target genes like CHOP (CAAT/enhancer-binding protein-homologous protein), asparagine synthetase (ASNS), or TRB3. We demonstrate that TRB3 overexpression inhibits the transcriptional induction of CHOP and ASNS whereas TRB3 silencing induces the expression of these genes both under normal and stressed conditions. In addition, transcriptional profiling experiments show that TRB3 affects the expression of many ISR-regulated genes. Our results also suggest that TRB3 and ATF4 belong to the same protein complex bound to the sequence involved in the ATF4-dependent regulation of gene expression by amino acid limitation. Collectively, our data identify TRB3 as a negative feedback regulator of the ATF4-dependent transcription and participates to the fine regulation of the ISR

Nutrient shortage triggers the hexosamine biosynthetic pathway via the GCN2-ATF4 signalling pathway

We thank the Dr. J.D. Minna (University of Texas Southwestern Medical Center, Dallas, USA) for the HBEC 3KT-RL cell line. We are grateful to Dr. D. Ron (Institute of Metabolic Science, Cambridge, UK) for GCN2 deficient Mouse Embryonic Fibroblasts and for the F2vE-PERK construct. The authors thank the Dr. B. Manship for language editing and manuscript corrections. C. Chaveroux. is the recipient of the program for fundamental and clinical research on cancer from the "Fondation de France". S. Manie is supported by the foundation "Association pour la Recherche sur le Cancer" (PJA20131200334), and by the "Programme INCA" (INCA_7981)The hexosamine biosynthetic pathway (HBP) is a nutrient-sensing metabolic pathway that produces the activated amino sugar UDP-N-acetylglucosamine, a critical substrate for protein glycosylation. Despite its biological significance, little is known about the regulation of HBP flux during nutrient limitation. Here, we report that amino acid or glucose shortage increase GFAT1 production, the first and rate-limiting enzyme of the HBP. GFAT1 is a transcriptional target of the activating transcription factor 4 (ATF4) induced by the GCN2-eIF2 alpha signalling pathway. The increased production of GFAT1 stimulates HBP flux and results in an increase in O-linked beta-N-acetylglucosamine protein modifications. Taken together, these findings demonstrate that ATF4 provides a link between nutritional stress and the HBP for the regulation of the O-GlcNAcylation-dependent cellular signallin