Anne Brunet

All Publications

• Lysosome activation clears aggregates and enhances quiescent neural stem cell activation during aging Science Leeman, D. S., Hebestreit, K., Ruetz, T., Webb, A. E., McKay, A., Pollina, E. A., Dulken, B. W., Zhao, X., Yeo, R. W., Ho, T. T., Mahmoudi, S., Devarajan, K., Passegué, E., Rando, T. A., Frydman, J., Brunet, A. 2018: 1277-1283
• Mono-unsaturated fatty acids link H3K4me3 modifiers to C. elegans lifespan NATURE Han, S., Schroeder, E. A., Silva-Garica, C. G., Hebestreit, K., Mair, W. B., Brunet, A. 2017; 544 (7649): 185-?

  Abstract

  Chromatin and metabolic states both influence lifespan, but how they interact in lifespan regulation is largely unknown. The COMPASS chromatin complex, which trimethylates lysine 4 on histone H3 (H3K4me3), regulates lifespan in Caenorhabditis elegans. However, the mechanism by which H3K4me3 modifiers affect longevity, and whether this mechanism involves metabolic changes, remain unclear. Here we show that a deficiency in H3K4me3 methyltransferase, which extends lifespan, promotes fat accumulation in worms with a specific enrichment of mono-unsaturated fatty acids (MUFAs). This fat metabolism switch in H3K4me3 methyltransferase-deficient worms is mediated at least in part by the downregulation of germline targets, including S6 kinase, and by the activation of an intestinal transcriptional network that upregulates delta-9 fatty acid desaturases. Notably, the accumulation of MUFAs is necessary for the lifespan extension of H3K4me3 methyltransferase-deficient worms, and dietary MUFAs are sufficient to extend lifespan. Given the conservation of lipid metabolism, dietary or endogenous MUFAs could extend lifespan and healthspan in other species, including mammals.

  View details for DOI 10.1038/nature21686

  View details for Web of Science ID 000398897900028

  View details for PubMedID 28379943

  View details for PubMedCentralID PMC5391274

• Single-Cell Transcriptomic Analysis Defines Heterogeneity and Transcriptional Dynamics in the Adult Neural Stem Cell Lineage. Cell reports Dulken, B. W., Leeman, D. S., Boutet, S. C., Hebestreit, K., Brunet, A. 2017; 18 (3): 777-790

  Abstract

  Neural stem cells (NSCs) in the adult mammalian brain serve as a reservoir for the generation of new neurons, oligodendrocytes, and astrocytes. Here, we use single-cell RNA sequencing to characterize adult NSC populations and examine the molecular identities and heterogeneity of in vivo NSC populations. We find that cells in the NSC lineage exist on a continuum through the processes of activation and differentiation. Interestingly, rare intermediate states with distinct molecular profiles can be identified and experimentally validated, and our analysis identifies putative surface markers and key intracellular regulators for these subpopulations of NSCs. Finally, using the power of single-cell profiling, we conduct a meta-analysis to compare in vivo NSCs and in vitro cultures, distinct fluorescence-activated cell sorting strategies, and different neurogenic niches. These data provide a resource for the field and contribute to an integrative understanding of the adult NSC lineage.

  View details for DOI 10.1016/j.celrep.2016.12.060

  View details for PubMedID 28099854

  View details for PubMedCentralID PMC5269583

• Efficient genome engineering approaches for the short-lived African turquoise killifish. Nature protocols Harel, I., Valenzano, D. R., Brunet, A. 2016; 11 (10): 2010-2028

  Abstract

  A central challenge in experimental aging research is the lack of short-lived vertebrate models for genetic studies. Here we present a comprehensive protocol for efficient genome engineering in the African turquoise killifish (Nothobranchius furzeri), which is the shortest-lived vertebrate in captivity with a median life span of 4-6 months. By taking advantage of the clustered regularly interspaced short palindromic repeats/CRISPR-associated protein-9 nuclease (CRISPR/Cas9) system and the turquoise killifish genome, this platform enables the generation of knockout alleles via nonhomologous end joining (NHEJ) and knock-in alleles via homology-directed repair (HDR). We include guidelines for guide RNA (gRNA) target design, embryo injection and hatching, germ-line transmission and for minimizing off-target effects. We also provide strategies for Tol2-based transgenesis and large-scale husbandry conditions that are critical for success. Because of the fast life cycle of the turquoise killifish, stable lines can be generated as rapidly as 2-3 months, which is much faster than other fish models. This protocol provides powerful genetic tools for studying vertebrate aging and aging-related diseases.

  View details for DOI 10.1038/nprot.2016.103

  View details for PubMedID 27658015

• The African Turquoise Killifish Genome Provides Insights into Evolution and Genetic Architecture of Lifespan CELL Valenzano, D. R., Benayoun, B. A., Singh, P. P., Zhang, E., Etter, P. D., Hu, C., Clement-Ziza, M., Willemsen, D., Cui, R., Harel, I., Machado, B. E., Yee, M., Sharp, S. C., Bustamante, C. D., Beyer, A., Johnson, E. A., Brunet, A. 2015; 163 (6): 1539-1554

  Abstract

  Lifespan is a remarkably diverse trait ranging from a few days to several hundred years in nature, but the mechanisms underlying the evolution of lifespan differences remain elusive. Here we de novo assemble a reference genome for the naturally short-lived African turquoise killifish, providing a unique resource for comparative and experimental genomics. The identification of genes under positive selection in this fish reveals potential candidates to explain its compressed lifespan. Several aging genes are under positive selection in this short-lived fish and long-lived species, raising the intriguing possibility that the same gene could underlie evolution of both compressed and extended lifespans. Comparative genomics and linkage analysis identify candidate genes associated with lifespan differences between various turquoise killifish strains. Remarkably, these genes are clustered on the sex chromosome, suggesting that short lifespan might have co-evolved with sex determination. Our study provides insights into the evolutionary forces that shape lifespan in nature.

  View details for DOI 10.1016/j.cell.2015.11.008

  View details for Web of Science ID 000366044800024

  View details for PubMedID 26638078

  View details for PubMedCentralID PMC4684691

• A platform for rapid exploration of aging and diseases in a naturally short-lived vertebrate. Cell Harel, I., Benayoun, B. A., Machado, B., Singh, P. P., Hu, C., Pech, M. F., Valenzano, D. R., Zhang, E., Sharp, S. C., Artandi, S. E., Brunet, A. 2015; 160 (5): 1013-1026

  View details for DOI 10.1016/j.cell.2015.01.038

  View details for PubMedID 25684364

• H3K4me3 Breadth Is Linked to Cell Identity and Transcriptional Consistency. Cell Benayoun, B. A., Pollina, E. A., Ucar, D., Mahmoudi, S., Karra, K., Wong, E. D., Devarajan, K., Daugherty, A. C., Kundaje, A. B., Mancini, E., Hitz, B. C., Gupta, R., Rando, T. A., Baker, J. C., Snyder, M. P., Cherry, J. M., Brunet, A. 2014; 158 (3): 673-688

  Abstract

  Trimethylation of histone H3 at lysine 4 (H3K4me3) is a chromatin modification known to mark the transcription start sites of active genes. Here, we show that H3K4me3 domains that spread more broadly over genes in a given cell type preferentially mark genes that are essential for the identity and function of that cell type. Using the broadest H3K4me3 domains as a discovery tool in neural progenitor cells, we identify novel regulators of these cells. Machine learning models reveal that the broadest H3K4me3 domains represent a distinct entity, characterized by increased marks of elongation. The broadest H3K4me3 domains also have more paused polymerase at their promoters, suggesting a unique transcriptional output. Indeed, genes marked by the broadest H3K4me3 domains exhibit enhanced transcriptional consistency and [corrected] increased transcriptional levels, and perturbation of H3K4me3 breadth leads to changes in transcriptional consistency. Thus, H3K4me3 breadth contains information that could ensure transcriptional precision at key cell identity/function genes.

  View details for DOI 10.1016/j.cell.2014.06.027

  View details for PubMedID 25083876

  View details for PubMedCentralID PMC4137894

• Males Shorten the Life Span of C. elegans Hermaphrodites via Secreted Compounds SCIENCE Maures, T. J., Booth, L. N., Benayoun, B. A., Izrayelit, Y., Schroeder, F. C., Brunet, A. 2014; 343 (6170): 541-544

  Abstract

  How an individual's longevity is affected by the opposite sex is still largely unclear. In the nematode Caenorhabditis elegans, the presence of males accelerated aging and shortened the life span of individuals of the opposite sex (hermaphrodites), including long-lived or sterile hermaphrodites. The male-induced demise could occur without mating and required only exposure of hermaphrodites to medium in which males were once present. Such communication through pheromones or other diffusible substances points to a nonindividual autonomous mode of aging regulation. The male-induced demise also occurred in other species of nematodes, suggesting an evolutionary conserved process whereby males may induce the disposal of the opposite sex to save resources for the next generation or to prevent competition from other males.

  View details for DOI 10.1126/science.1244160

  View details for Web of Science ID 000330343700047

  View details for PubMedID 24292626

• Progranulin, lysosomal regulation and neurodegenerative disease NATURE REVIEWS NEUROSCIENCE Kao, A. W., McKay, A., Singh, P. P., Brunet, A., Huang, E. J. 2017; 18 (6): 325-333

  Abstract

  The discovery that heterozygous and homozygous mutations in the gene encoding progranulin are causally linked to frontotemporal dementia and lysosomal storage disease, respectively, reveals previously unrecognized roles of the progranulin protein in regulating lysosome biogenesis and function. Given the importance of lysosomes in cellular homeostasis, it is not surprising that progranulin deficiency has pleiotropic effects on neural circuit development and maintenance, stress response, innate immunity and ageing. This Progress article reviews recent advances in progranulin biology emphasizing its roles in lysosomal function and brain innate immunity, and outlines future avenues of investigation that may lead to new therapeutic approaches for neurodegeneration.

  View details for DOI 10.1038/nrn.2017.36

  View details for Web of Science ID 000401517300006

  View details for PubMedID 28435163

• AMPKa1-LDH pathway regulates muscle stem cell self-renewal by controlling metabolic homeostasis. EMBO journal Theret, M., Gsaier, L., Schaffer, B., Juban, G., Ben Larbi, S., Weiss-Gayet, M., Bultot, L., Caterina, C., Foretz, M., Desplanches, D., Sanz, P., Zang, Z., Yang, L., Vial, G., Viollet, B., Sakamoto, K., Brunet, A., Chazaud, B., Mounier, R. 2017

  Abstract

  Control of stem cell fate to either enter terminal differentiation versus returning to quiescence (self-renewal) is crucial for tissue repair. Here, we showed that AMP-activated protein kinase (AMPK), the master metabolic regulator of the cell, controls muscle stem cell (MuSC) self-renewal. AMPKα1(-/-) MuSCs displayed a high self-renewal rate, which impairs muscle regeneration. AMPKα1(-/-) MuSCs showed a Warburg-like switch of their metabolism to higher glycolysis. We identified lactate dehydrogenase (LDH) as a new functional target of AMPKα1. LDH, which is a non-limiting enzyme of glycolysis in differentiated cells, was tightly regulated in stem cells. In functional experiments, LDH overexpression phenocopied AMPKα1(-/-) phenotype, that is shifted MuSC metabolism toward glycolysis triggering their return to quiescence, while inhibition of LDH activity rescued AMPKα1(-/-) MuSC self-renewal. Finally, providing specific nutrients (galactose/glucose) to MuSCs directly controlled their fate through the AMPKα1/LDH pathway, emphasizing the importance of metabolism in stem cell fate.

  View details for DOI 10.15252/embj.201695273

  View details for PubMedID 28515121

• Interaction between epigenetic and metabolism in aging stem cells. Current opinion in cell biology Brunet, A., Rando, T. A. 2017; 45: 1-7

  Abstract

  Aging is accompanied by a decline in tissue function, regeneration, and repair. A large part of this decline is caused by the deterioration of tissue stem cell function. Understanding the mechanisms that drive stem cell aging and how to counteract them is a critical step for enhancing tissue repair and maintenance during aging. Emerging evidence indicates that epigenetic modifiers and metabolism regulators interact to impact lifespan, suggesting that this mechanism may also affect stem cell function with age. This review focuses on the interaction between chromatin and metabolism in the regulation of tissue stem cells during aging. We also discuss how these mechanisms integrate environmental stimuli such as nutrient stress to regulate stem cell function. Finally, this review examines new perspectives for regeneration, rejuvenation, and treatment of age-related decline of stem cell function.

  View details for DOI 10.1016/j.ceb.2016.12.009

  View details for PubMedID 28129586

• Bursts of Reprogramming: A Path to Extend Lifespan? CELL Mahmoudi, S., Brunet, A. 2016; 167 (7): 1672-1674

  Abstract

  In a thought-provoking study, Ocampo et al. show that the cyclic expression of stem cell reprogramming factors in vivo increases the lifespan of a mouse model of premature aging and provides health benefits to chronologically old, normal mice.

  View details for DOI 10.1016/j.cell.2016.11.050

  View details for Web of Science ID 000393114700005

  View details for PubMedID 27984716

• Characterization of the direct targets of FOXO transcription factors throughout evolution. Aging cell Webb, A. E., Kundaje, A., Brunet, A. 2016; 15 (4): 673-685

  Abstract

  FOXO transcription factors (FOXOs) are central regulators of lifespan across species, yet they also have cell-specific functions, including adult stem cell homeostasis and immune function. Direct targets of FOXOs have been identified genome-wide in several species and cell types. However, whether FOXO targets are specific to cell types and species or conserved across cell types and throughout evolution remains uncharacterized. Here, we perform a meta-analysis of direct FOXO targets across tissues and organisms, using data from mammals as well as Caenorhabditis elegans and Drosophila. We show that FOXOs bind cell type-specific targets, which have functions related to that particular cell. Interestingly, FOXOs also share targets across different tissues in mammals, and the function and even the identity of these shared mammalian targets are conserved in invertebrates. Evolutionarily conserved targets show enrichment for growth factor signaling, metabolism, stress resistance, and proteostasis, suggesting an ancestral, conserved role in the regulation of these processes. We also identify candidate cofactors at conserved FOXO targets that change in expression with age, including CREB and ETS family factors. This meta-analysis provides insight into the evolution of the FOXO network and highlights downstream genes and cofactors that may be particularly important for FOXO's conserved function in adult homeostasis and longevity.

  View details for DOI 10.1111/acel.12479

  View details for PubMedID 27061590

  View details for PubMedCentralID PMC4933671

• FoxO3 regulates neuronal reprogramming of cells from postnatal and aging mice PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Ahlenius, H., Chanda, S., Webb, A. E., Yousif, I., Karmazin, J., Prusiner, S. B., Brunet, A., Suedhof, T. C., Wernig, M. 2016; 113 (30): 8514-8519

  Abstract

  We and others have shown that embryonic and neonatal fibroblasts can be directly converted into induced neuronal (iN) cells with mature functional properties. Reprogramming of fibroblasts from adult and aged mice, however, has not yet been explored in detail. The ability to generate fully functional iN cells from aged organisms will be particularly important for in vitro modeling of diseases of old age. Here, we demonstrate production of functional iN cells from fibroblasts that were derived from mice close to the end of their lifespan. iN cells from aged mice had apparently normal active and passive neuronal membrane properties and formed abundant synaptic connections. The reprogramming efficiency gradually decreased with fibroblasts derived from embryonic and neonatal mice, but remained similar for fibroblasts from postnatal mice of all ages. Strikingly, overexpression of a transcription factor, forkhead box O3 (FoxO3), which is implicated in aging, blocked iN cell conversion of embryonic fibroblasts, whereas knockout or knockdown of FoxO3 increased the reprogramming efficiency of adult-derived but not of embryonic fibroblasts and also enhanced functional maturation of resulting iN cells. Hence, FoxO3 has a central role in the neuronal reprogramming susceptibility of cells, and the importance of FoxO3 appears to change during development.

  View details for DOI 10.1073/pnas.1607079113

  View details for Web of Science ID 000380346200057

  View details for PubMedID 27402759

• The Aging Epigenome MOLECULAR CELL Booth, L. N., Brunet, A. 2016; 62 (5): 728-744

  Abstract

  During aging, the mechanisms that normally maintain health and stress resistance strikingly decline, resulting in decrepitude, frailty, and ultimately death. Exactly when and how this decline occurs is unknown. Changes in transcriptional networks and chromatin state lie at the heart of age-dependent decline. These epigenomic changes are not only observed during aging but also profoundly affect cellular function and stress resistance, thereby contributing to the progression of aging. We propose that the dysregulation of transcriptional and chromatin networks is a crucial component of aging. Understanding age-dependent epigenomic changes will yield key insights into how aging begins and progresses and should lead to the development of new therapeutics that delay or even reverse aging and age-related diseases.

  View details for DOI 10.1016/j.molcel.2016.05.013

  View details for Web of Science ID 000377049500010

  View details for PubMedID 27259204

• AMPK: An Energy-Sensing Pathway with Multiple Inputs and Outputs TRENDS IN CELL BIOLOGY Hardie, D. G., Schaffer, B. E., Brunet, A. 2016; 26 (3): 190-201

  Abstract

  AMP-activated protein kinase (AMPK) is a key regulator of energy balance expressed ubiquitously in eukaryotic cells. Here we review the canonical adenine nucleotide-dependent mechanism that activates AMPK when cellular energy status is compromised, as well as other, noncanonical activation mechanisms. Once activated, AMPK acts to restore energy homeostasis by promoting catabolic pathways, resulting in ATP generation, and inhibiting anabolic pathways that consume ATP. We also review the various hypothesis-driven and unbiased approaches that have been used to identify AMPK substrates and have revealed substrates involved in both metabolic and non-metabolic processes. We particularly focus on methods for identifying the AMPK target recognition motif and how it can be used to predict new substrates.

  View details for DOI 10.1016/j.tcb.2015.10.013

  View details for Web of Science ID 000370900100004

  View details for PubMedID 26616193

• High telomerase is a hallmark of undifferentiated spermatogonia and is required for maintenance of male germline stem cells. Genes & development Pech, M. F., Garbuzov, A., Hasegawa, K., Sukhwani, M., Zhang, R. J., Benayoun, B. A., Brockman, S. A., Lin, S., Brunet, A., Orwig, K. E., Artandi, S. E. 2015; 29 (23): 2420-2434

  Abstract

  Telomerase inactivation causes loss of the male germline in worms, fish, and mice, indicating a conserved dependence on telomere maintenance in this cell lineage. Here, using telomerase reverse transcriptase (Tert) reporter mice, we found that very high telomerase expression is a hallmark of undifferentiated spermatogonia, the mitotic population where germline stem cells reside. We exploited these high telomerase levels as a basis for purifying undifferentiated spermatogonia using fluorescence-activated cell sorting. Telomerase levels in undifferentiated spermatogonia and embryonic stem cells are comparable and much greater than in somatic progenitor compartments. Within the germline, we uncovered an unanticipated gradient of telomerase activity that also enables isolation of more mature populations. Transcriptomic comparisons of Tert(High) undifferentiated spermatogonia and Tert(Low) differentiated spermatogonia by RNA sequencing reveals marked differences in cell cycle and key molecular features of each compartment. Transplantation studies show that germline stem cell activity is confined to the Tert(High) cKit(-) population. Telomere shortening in telomerase knockout strains causes depletion of undifferentiated spermatogonia and eventual loss of all germ cells after undifferentiated spermatogonia drop below a critical threshold. These data reveal that high telomerase expression is a fundamental characteristic of germline stem cells, thus explaining the broad dependence on telomerase for germline immortality in metazoans.

  View details for DOI 10.1101/gad.271783.115

  View details for PubMedID 26584619

  View details for PubMedCentralID PMC4691947

• Identification of AMPK Phosphorylation Sites Reveals a Network of Proteins Involved in Cell Invasion and Facilitates Large-Scale Substrate Prediction. Cell metabolism Schaffer, B. E., Levin, R. S., Hertz, N. T., Maures, T. J., Schoof, M. L., Hollstein, P. E., Benayoun, B. A., Banko, M. R., Shaw, R. J., Shokat, K. M., Brunet, A. 2015; 22 (5): 907-921

  View details for DOI 10.1016/j.cmet.2015.09.009

  View details for PubMedID 26456332

• Epigenetic regulation of ageing: linking environmental inputs to genomic stability. Nature reviews. Molecular cell biology Benayoun, B. A., Pollina, E. A., Brunet, A. 2015; 16 (10): 593-610

  View details for DOI 10.1038/nrm4048

  View details for PubMedID 26373265

• Shockingly Early: Chromatin-Mediated Loss of the Heat Shock Response. Molecular cell Booth, L. N., Brunet, A. 2015; 59 (4): 515-516

  Abstract

  In this issue of Molecular Cell, Labbadia and Morimoto (2015) show that there is a precipitous decline in stress resistance at the onset of reproduction in C. elegans and that this transition is regulated by changes in repressive chromatin marks.

  View details for DOI 10.1016/j.molcel.2015.08.004

  View details for PubMedID 26295957

• AMP-Activated Protein Kinase Directly Phosphorylates and Destabilizes Hedgehog Pathway Transcription Factor GLI1 in Medulloblastoma. Cell reports Li, Y., Luo, J., Mosley, Y. C., Hedrick, V. E., Paul, L. N., Chang, J., Zhang, G., Wang, Y., Banko, M. R., Brunet, A., Kuang, S., Wu, J., Chang, C., Scott, M. P., Yang, J. 2015; 12 (4): 599-609

  Abstract

  The Hedgehog (Hh) pathway regulates cell differentiation and proliferation during development by controlling the Gli transcription factors. Cell fate decisions and progression toward organ and tissue maturity must be coordinated, and how an energy sensor regulates the Hh pathway is not clear. AMP-activated protein kinase (AMPK) is an important sensor of energy stores and controls protein synthesis and other energy-intensive processes. AMPK is directly responsive to intracellular AMP levels, inhibiting a wide range of cell activities if ATP is low and AMP is high. Thus, AMPK can affect development by influencing protein synthesis and other processes needed for growth and differentiation. Activation of AMPK reduces GLI1 protein levels and stability, thus blocking Sonic-hedgehog-induced transcriptional activity. AMPK phosphorylates GLI1 at serines 102 and 408 and threonine 1074. Mutation of these three sites into alanine prevents phosphorylation by AMPK. This leads to increased GLI1 protein stability, transcriptional activity, and oncogenic potency.

  View details for DOI 10.1016/j.celrep.2015.06.054

  View details for PubMedID 26190112

  View details for PubMedCentralID PMC4521589

• Inhibition of pluripotency networks by the rb tumor suppressor restricts reprogramming and tumorigenesis. Cell stem cell Kareta, M. S., Gorges, L. L., Hafeez, S., Benayoun, B. A., Marro, S., Zmoos, A., Cecchini, M. J., Spacek, D., Batista, L. F., O'Brien, M., Ng, Y., Ang, C. E., Vaka, D., Artandi, S. E., Dick, F. A., Brunet, A., Sage, J., Wernig, M. 2015; 16 (1): 39-50

  Abstract

  Mutations in the retinoblastoma tumor suppressor gene Rb are involved in many forms of human cancer. In this study, we investigated the early consequences of inactivating Rb in the context of cellular reprogramming. We found that Rb inactivation promotes the reprogramming of differentiated cells to a pluripotent state. Unexpectedly, this effect is cell cycle independent, and instead reflects direct binding of Rb to pluripotency genes, including Sox2 and Oct4, which leads to a repressed chromatin state. More broadly, this regulation of pluripotency networks and Sox2 in particular is critical for the initiation of tumors upon loss of Rb in mice. These studies therefore identify Rb as a global transcriptional repressor of pluripotency networks, providing a molecular basis for previous reports about its involvement in cell fate pliability, and implicate misregulation of pluripotency factors such as Sox2 in tumorigenesis related to loss of Rb function.

  View details for DOI 10.1016/j.stem.2014.10.019

  View details for PubMedID 25467916

  View details for PubMedCentralID PMC4389904

• Cell biology. Lysosomal lipid lengthens life span. Science Han, S., Brunet, A. 2015; 347 (6217): 32-33

  View details for DOI 10.1126/science.aaa4565

  View details for PubMedID 25554778

• The African Turquoise Killifish: A Model for Exploring Vertebrate Aging and Diseases in the Fast Lane. Cold Spring Harbor symposia on quantitative biology Harel, I., Brunet, A. 2015; 80: 275-279

  Abstract

  Why and how organisms age remains a mystery, and it defines one of the biggest challenges in biology. Aging is also the primary risk factor for many human pathologies, such as cancer, diabetes, cardiovascular diseases, and neurodegenerative diseases. Thus, manipulating the aging rate and potentially postponing the onset of these devastating diseases could have a tremendous impact on human health. Recent studies, relying primarily on nonvertebrate short-lived model systems, have shown the importance of both genetic and environmental factors in modulating the aging rate. However, relatively little is known about aging in vertebrates or what processes may be unique and specific to these complex organisms. Here we discuss how advances in genomics and genome editing have significantly expanded our ability to probe the aging process in a vertebrate system. We highlight recent findings from a naturally short-lived vertebrate, the African turquoise killifish, which provides an attractive platform for exploring mechanisms underlying vertebrate aging and age-related diseases.

  View details for DOI 10.1101/sqb.2015.80.027524

  View details for PubMedID 26642856

• Epigenetics of Aging and Aging-related Disease JOURNALS OF GERONTOLOGY SERIES A-BIOLOGICAL SCIENCES AND MEDICAL SCIENCES Brunet, A., Berger, S. L. 2014; 69: S17-S20

  Abstract

  Aging is associated with a wide range of human disorders, including cancer, diabetes, cardiovascular, and neurodegenerative diseases. Long thought to be an inexorable road toward decline and diseases, aging is in fact remarkably plastic. Such plasticity could be harnessed to approach age-related diseases from a novel perspective. Although many studies have focused on the genes that impact aging, the nongenetic regulation of aging is gaining increasing attention. Specifically, aging is associated with profound epigenetic changes, resulting in alterations of gene expression and disturbances in broad genome architecture and the epigenomic landscape. The potential reversibility of these epigenetic changes that occur as a hallmark of aging offers exciting opportunities to alter the trajectory of age-related diseases. This short review highlights key epigenetic players in the regulation of aging, as well as both future goals and challenges to the utilization of epigenetic strategies to delay and reverse the main diseases of aging.

  View details for DOI 10.1093/gerona/glu042

  View details for Web of Science ID 000337130200004

  View details for PubMedID 24833581

• FOXO3 Promotes Quiescence in Adult Muscle Stem Cells during the Process of Self-Renewal. Stem cell reports Gopinath, S. D., Webb, A. E., Brunet, A., Rando, T. A. 2014; 2 (4): 414-426

  Abstract

  Skeletal muscle stem cells, or "satellite cells" (SCs), are required for the regeneration of damaged muscle tissue. Although SCs self-renew during regeneration, the mechanisms that govern SC re-entry into quiescence remain elusive. We show that FOXO3, a member of the forkhead family of transcription factors, is expressed in quiescent SCs (QSCs). Conditional deletion of Foxo3 in QSCs impairs self-renewal and increases the propensity of SCs to adopt a differentiated fate. Transcriptional analysis of SCs lacking FOXO3 revealed a downregulation of Notch signaling, a key regulator of SC quiescence. Conversely, overexpression of Notch intracellular domain (NICD) rescued the self-renewal deficit of FOXO3-deficient SCs. We show that FOXO3 regulates NOTCH1 and NOTCH3 receptor expression and that decreasing expression of NOTCH1 and NOTCH3 receptors phenocopies the effect of FOXO3 deficiency in SCs. We demonstrate that FOXO3, perhaps by activating Notch signaling, promotes the quiescent state during SC self-renewal in adult muscle regeneration.

  View details for DOI 10.1016/j.stemcr.2014.02.002

  View details for PubMedID 24749067

  View details for PubMedCentralID PMC3986584

• FOXO transcription factors: key regulators of cellular quality control TRENDS IN BIOCHEMICAL SCIENCES Webb, A. E., Brunet, A. 2014; 39 (4): 159-169

  Abstract

  FOXO transcription factors are conserved regulators of longevity downstream of insulin signaling. These transcription factors integrate signals emanating from nutrient deprivation and stress stimuli to coordinate programs of genes involved in cellular metabolism and resistance to oxidative stress. Here, we discuss emerging evidence for a pivotal role of FOXO factors in promoting the expression of genes involved in autophagy and the ubiquitin-proteasome system--two cell clearance processes that are essential for maintaining organelle and protein homeostasis (proteostasis). The ability of FOXO to maintain cellular quality control appears to be critical in processes and pathologies where damaged proteins and organelles accumulate, including aging and neurodegenerative diseases.

  View details for DOI 10.1016/j.tibs.2014.02.003

  View details for Web of Science ID 000335426200004

  View details for PubMedID 24630600

• Stem cells: Sex specificity in the blood. Nature Leeman, D. S., Brunet, A. 2014; 505 (7484): 488-490

  View details for DOI 10.1038/505488a

  View details for PubMedID 24451537

• Hierarchical Mechanisms for Direct Reprogramming of Fibroblasts to Neurons CELL Wapinski, O. L., Vierbuchen, T., Qu, K., Lee, Q. Y., Chanda, S., Fuentes, D. R., Giresi, P. G., Ng, Y. H., Marro, S., Neff, N. F., Drechsel, D., Martynoga, B., Castro, D. S., Webb, A. E., Suedhof, T. C., Brunet, A., Guillemot, F., Chang, H. Y., Wernig, M. 2013; 155 (3): 621-635

  Abstract

  Direct lineage reprogramming is a promising approach for human disease modeling and regenerative medicine, with poorly understood mechanisms. Here, we reveal a hierarchical mechanism in the direct conversion of fibroblasts into induced neuronal (iN) cells mediated by the transcription factors Ascl1, Brn2, and Myt1l. Ascl1 acts as an "on-target" pioneer factor by immediately occupying most cognate genomic sites in fibroblasts. In contrast, Brn2 and Myt1l do not access fibroblast chromatin productively on their own; instead, Ascl1 recruits Brn2 to Ascl1 sites genome wide. A unique trivalent chromatin signature in the host cells predicts the permissiveness for Ascl1 pioneering activity among different cell types. Finally, we identified Zfp238 as a key Ascl1 target gene that can partially substitute for Ascl1 during iN cell reprogramming. Thus, a precise match between pioneer factors and the chromatin context at key target genes is determinative for transdifferentiation to neurons and likely other cell types.

  View details for DOI 10.1016/j.cell.2013.09.028

  View details for Web of Science ID 000326571800016

  View details for PubMedID 24243019

  View details for PubMedCentralID PMC3871197

• FOXO3 Shares Common Targets with ASCL1 Genome-wide and Inhibits ASCL1-Dependent Neurogenesis. Cell reports Webb, A. E., Pollina, E. A., Vierbuchen, T., Urbán, N., Ucar, D., Leeman, D. S., Martynoga, B., Sewak, M., Rando, T. A., Guillemot, F., Wernig, M., Brunet, A. 2013; 4 (3): 477-491

  Abstract

  FOXO transcription factors are central regulators of longevity from worms to humans. FOXO3, the FOXO isoform associated with exceptional human longevity, preserves adult neural stem cell pools. Here, we identify FOXO3 direct targets genome-wide in primary cultures of adult neural progenitor cells (NPCs). Interestingly, FOXO3-bound sites are enriched for motifs for bHLH transcription factors, and FOXO3 shares common targets with the proneuronal bHLH transcription factor ASCL1/MASH1 in NPCs. Analysis of the chromatin landscape reveals that FOXO3 and ASCL1 are particularly enriched at the enhancers of genes involved in neurogenic pathways. Intriguingly, FOXO3 inhibits ASCL1-dependent neurogenesis in NPCs and direct neuronal conversion in fibroblasts. FOXO3 also restrains neurogenesis in vivo. Our study identifies a genome-wide interaction between the prolongevity transcription factor FOXO3 and the cell-fate determinant ASCL1 and raises the possibility that FOXO3's ability to restrain ASCL1-dependent neurogenesis may help preserve the neural stem cell pool.

  View details for DOI 10.1016/j.celrep.2013.06.035

  View details for PubMedID 23891001

• Chromatin Modifications as Determinants of Muscle Stem Cell Quiescence and Chronological Aging CELL REPORTS Liu, L., Cheung, T. H., Charville, G. W., Hurgo, B. M., Leavitt, T., Shih, J., Brunet, A., Rando, T. A. 2013; 4 (1): 189-204

  Abstract

  The ability to maintain quiescence is critical for the long-term maintenance of a functional stem cell pool. To date, the epigenetic and transcriptional characteristics of quiescent stem cells and how they change with age remain largely unknown. In this study, we explore the chromatin features of adult skeletal muscle stem cells, or satellite cells (SCs), which reside predominantly in a quiescent state in fully developed limb muscles of both young and aged mice. Using a ChIP-seq approach to obtain global epigenetic profiles of quiescent SCs (QSCs), we show that QSCs possess a permissive chromatin state in which few genes are epigenetically repressed by Polycomb group (PcG)-mediated histone 3 lysine 27 trimethylation (H3K27me3), and a large number of genes encoding regulators that specify nonmyogenic lineages are demarcated by bivalent domains at their transcription start sites (TSSs). By comparing epigenetic profiles of QSCs from young and old mice, we also provide direct evidence that, with age, epigenetic changes accumulate and may lead to a functional decline in quiescent stem cells. These findings highlight the importance of chromatin mapping in understanding unique features of stem cell identity and stem cell aging.

  View details for DOI 10.1016/j.celrep.2013.05.043

  View details for Web of Science ID 000321901900018

  View details for PubMedID 23810552

• Expansion of oligodendrocyte progenitor cells following SIRT1 inactivation in the adult brain. Nature cell biology Rafalski, V. A., Ho, P. P., Brett, J. O., Ucar, D., Dugas, J. C., Pollina, E. A., Chow, L. M., Ibrahim, A., Baker, S. J., Barres, B. A., Steinman, L., Brunet, A. 2013; 15 (6): 614-624

  View details for DOI 10.1038/ncb2735

  View details for PubMedID 23644469

• FOXO flips the longevity SWItch. Nature cell biology Webb, A. E., Brunet, A. 2013; 15 (5): 444-446

  Abstract

  FOXO transcription factors promote longevity from worms to mammals, but the mechanisms by which FOXO extends lifespan have remained elusive. In the nematode Caenorhabditis elegans, FOXO is now shown to recruit the nucleosome remodelling complex SWI/SNF to its target genes, which is essential for FOXO to elicit stress resistance and longevity.

  View details for DOI 10.1038/ncb2749

  View details for PubMedID 23636422

• Bridging the transgenerational gap with epigenetic memory TRENDS IN GENETICS Lim, J. P., Brunet, A. 2013; 29 (3): 176-186

  Abstract

  It is textbook knowledge that inheritance of traits is governed by genetics, and that the epigenetic modifications an organism acquires are largely reset between generations. Recently, however, transgenerational epigenetic inheritance has emerged as a rapidly growing field, providing evidence suggesting that some epigenetic changes result in persistent phenotypes across generations. Here, we survey some of the most recent examples of transgenerational epigenetic inheritance in animals, ranging from Caenorhabditis elegans to humans, and describe approaches and limitations to studying this phenomenon. We also review the current body of evidence implicating chromatin modifications and RNA molecules in mechanisms underlying this unconventional mode of inheritance and discuss its evolutionary implications.

  View details for DOI 10.1016/j.tig.2012.12.008

  View details for Web of Science ID 000316243500008

  View details for PubMedID 23410786

  View details for PubMedCentralID PMC3595609

• FoxO6 regulates memory consolidation and synaptic function GENES & DEVELOPMENT Salih, D. A., Rashid, A. J., Colas, D., de la Torre-Ubieta, L., Zhu, R. P., Morgan, A. A., Santo, E. E., Ucar, D., Devarajan, K., Cole, C. J., Madison, D. V., Shamloo, M., Butte, A. J., Bonni, A., Josselyn, S. A., Brunet, A. 2012; 26 (24): 2780-2801

  Abstract

  The FoxO family of transcription factors is known to slow aging downstream from the insulin/IGF (insulin-like growth factor) signaling pathway. The most recently discovered FoxO isoform in mammals, FoxO6, is highly enriched in the adult hippocampus. However, the importance of FoxO factors in cognition is largely unknown. Here we generated mice lacking FoxO6 and found that these mice display normal learning but impaired memory consolidation in contextual fear conditioning and novel object recognition. Using stereotactic injection of viruses into the hippocampus of adult wild-type mice, we found that FoxO6 activity in the adult hippocampus is required for memory consolidation. Genome-wide approaches revealed that FoxO6 regulates a program of genes involved in synaptic function upon learning in the hippocampus. Consistently, FoxO6 deficiency results in decreased dendritic spine density in hippocampal neurons in vitro and in vivo. Thus, FoxO6 may promote memory consolidation by regulating a program coordinating neuronal connectivity in the hippocampus, which could have important implications for physiological and pathological age-dependent decline in memory.

  View details for DOI 10.1101/gad.208926.112

  View details for Web of Science ID 000312775700011

  View details for PubMedID 23222102

  View details for PubMedCentralID PMC3533081

• Energy metabolism and energy-sensing pathways in mammalian embryonic and adult stem cell fate JOURNAL OF CELL SCIENCE Rafalski, V. A., Mancini, E., Brunet, A. 2012; 125 (23): 5597-5608

  Abstract

  Metabolism is influenced by age, food intake, and conditions such as diabetes and obesity. How do physiological or pathological metabolic changes influence stem cells, which are crucial for tissue homeostasis? This Commentary reviews recent evidence that stem cells have different metabolic demands than differentiated cells, and that the molecular mechanisms that control stem cell self-renewal and differentiation are functionally connected to the metabolic state of the cell and the surrounding stem cell niche. Furthermore, we present how energy-sensing signaling molecules and metabolism regulators are implicated in the regulation of stem cell self-renewal and differentiation. Finally, we discuss the emerging literature on the metabolism of induced pluripotent stem cells and how manipulating metabolic pathways might aid cellular reprogramming. Determining how energy metabolism regulates stem cell fate should shed light on the decline in tissue regeneration that occurs during aging and facilitate the development of therapies for degenerative or metabolic diseases.

  View details for DOI 10.1242/jcs.114827

  View details for Web of Science ID 000315164200002

  View details for PubMedID 23420198

• Aging and reprogramming: a two-way street CURRENT OPINION IN CELL BIOLOGY Mahmoudi, S., Brunet, A. 2012; 24 (6): 744-756

  Abstract

  Aging is accompanied by the functional decline of cells, tissues, and organs, as well as a striking increase in a wide range of diseases. The reprogramming of somatic cells to induced pluripotent stem cells (iPSCs) opens new avenues for the aging field and has important applications for therapeutic treatments of age-related diseases. Here we review emerging studies on how aging and age-related pathways influence iPSC generation and property. We discuss the exciting possibility that reverting to a pluripotent stem cell stage erases several deficits associated with aging and offers new strategies for rejuvenation. Finally, we argue that reprogramming provides a unique opportunity to model aging and perhaps exceptional longevity.

  View details for DOI 10.1016/j.ceb.2012.10.004

  View details for Web of Science ID 000314743100006

  View details for PubMedID 23146768

• Methylation by Set9 modulates FoxO3 stability and transcriptional activity AGING-US Calnan, D. R., Webb, A. E., White, J. L., Stowe, T. R., Goswami, T., Shi, X., Espejo, A., Bedford, M. T., Gozani, O., Gygi, S. P., Brunet, A. 2012; 4 (7): 462-479

  Abstract

  The FoxO family of transcription factors plays an important role in longevity and tumor suppression by regulating the expression of a wide range of target genes. FoxO3 has recently been found to be associated with extreme longevity in humans and to regulate the homeostasis of adult stem cell pools in mammals, which may contribute to longevity. The activity of FoxO3 is controlled by a variety of post-translational modifications that have been proposed to form a 'code' affecting FoxO3 subcellular localization, DNA binding ability, protein-protein interactions and protein stability. Lysine methylation is a crucial post-translational modification on histones that regulates chromatin accessibility and is a key part of the 'histone code'. However, whether lysine methylation plays a role in modulating FoxO3 activity has never been examined. Here we show that the methyltransferase Set9 directly methylates FoxO3 in vitro and in cells. Using a combination of tandem mass spectrometry and methyl-specific antibodies, we find that Set9 methylates FoxO3 at a single residue, lysine 271, a site previously known to be deacetylated by Sirt1. Methylation of FoxO3 by Set9 decreases FoxO3 protein stability, while moderately increasing FoxO3 transcriptional activity. The modulation of FoxO3 stability and activity by methylation may be critical for fine-tuning cellular responses to stress stimuli, which may in turn affect FoxO3's ability to promote tumor suppression and longevity.

  View details for Web of Science ID 000307474000004

  View details for PubMedID 22820736

• Aging and the control of the insulin-FOXO signaling pathway M S-MEDECINE SCIENCES Brunet, A. 2012; 28 (3): 316-320

  Abstract

  Aging is a complex process that is accompanied by the onset of a series of age-related diseases, including Alzheimer's disease. Aging is controlled by a combination of genetic and environmental factors. Among the genes that regulate aging, the insulin-FOXO signaling pathway plays a central role, as this pathway regulates lifespan in multiple species, such as worms, flies, and mice. In humans, exceptional longevity - being a centenarian - is also associated with genetic variation in this insulin-FOXO pathway. Recent evidence indicates that the FOXO family of transcription factors plays a key role in the self-renewal of adult and embryonic stem cells, which could contribute to tissue regeneration. Understanding the mechanisms underlying aging should help better prevent and treat age-dependent diseases.

  View details for DOI 10.1051/medsci/2012283021

  View details for Web of Science ID 000303365100021

  View details for PubMedID 22480657

• Histone methylation makes its mark on longevity TRENDS IN CELL BIOLOGY Han, S., Brunet, A. 2012; 22 (1): 42-49

  Abstract

  How long organisms live is not entirely written in their genes. Recent findings reveal that epigenetic factors that regulate histone methylation, a type of chromatin modification, can affect lifespan. The reversible nature of chromatin modifications suggests that therapeutic targeting of chromatin regulators could be used to extend lifespan and healthspan. This review describes the epigenetic regulation of lifespan in diverse model organisms, focusing on the role and mode of action of chromatin regulators that affect two epigenetic marks, trimethylated lysine 4 of histone H3 (H3K4me3) and trimethylated lysine 27 of histone H3 (H3K27me3), in longevity.

  View details for DOI 10.1016/j.tcb.2011.11.001

  View details for Web of Science ID 000299450400005

  View details for PubMedID 22177962

  View details for PubMedCentralID PMC3253950

• Epigenetic memory of longevity in Caenorhabditis elegans. Worm Benayoun, B. A., Brunet, A. 2012; 1 (1): 77-81

  Abstract

  A recent study by Greer et al. in the nematode C. elegans has shown transgenerational epigenetic inheritance of longevity in the descendants of worms deficient for subunits of a complex responsible for histone H3 lysine 4 trimethylation (H3K4me3). In this commentary, we discuss the implications of this epigenetic memory of longevity and the potential mechanisms underlying this phenomenon. The transgenerational inheritance of longevity could result from heritable depletion of H3K4me3 at particular aging-regulating gene loci that would only be progressively replenished. The epigenetic memory of longevity could also be explained by the transgenerational transmission of other molecules, for example other proteins or non-coding RNAs. The discovery of an epigenetic memory of longevity in worms raises the intriguing possibility that environmental cues modulating longevity in ancestors might affect subsequent generations in a non-Mendelian manner. Another remaining intriguing question is whether transgenerational inheritance of longevity also exists in other species, including mammals.

  View details for DOI 10.4161/worm.19157

  View details for PubMedID 24058828

• Chemical Genetic Screen for AMPK alpha 2 Substrates Uncovers a Network of Proteins Involved in Mitosis MOLECULAR CELL Banko, M. R., Allen, J. J., Schaffer, B. E., Wilker, E. W., Tsou, P., White, J. L., Villen, J., Wang, B., Kim, S. R., Sakamoto, K., Gygi, S. P., Cantley, L. C., Yaffe, M. B., Shokat, K. M., Brunet, A. 2011; 44 (6): 878-892

  Abstract

  The energy-sensing AMP-activated protein kinase (AMPK) is activated by low nutrient levels. Functions of AMPK, other than its role in cellular metabolism, are just beginning to emerge. Here we use a chemical genetics screen to identify direct substrates of AMPK in human cells. We find that AMPK phosphorylates 28 previously unidentified substrates, several of which are involved in mitosis and cytokinesis. We identify the residues phosphorylated by AMPK in vivo in several substrates, including protein phosphatase 1 regulatory subunit 12C (PPP1R12C) and p21-activated protein kinase (PAK2). AMPK-induced phosphorylation is necessary for PPP1R12C interaction with 14-3-3 and phosphorylation of myosin regulatory light chain. Both AMPK activity and PPP1R12C phosphorylation are increased in mitotic cells and are important for mitosis completion. These findings suggest that AMPK coordinates nutrient status with mitosis completion, which may be critical for the organism's response to low nutrients during development, or in adult stem and cancer cells.

  View details for DOI 10.1016/j.molcel.2011.11.005

  View details for Web of Science ID 000298827200007

  View details for PubMedID 22137581

• Transposon-Mediated Transgenesis in the Short-Lived African Killifish Nothobranchius furzeri, a Vertebrate Model for Aging. G3 (Bethesda, Md.) Valenzano, D. R., Sharp, S., Brunet, A. 2011; 1 (7): 531-538

  Abstract

  The African killifish Nothobranchius furzeri is the shortest-lived vertebrate that can be bred in captivity. N. furzeri comprises several wild-derived strains with striking differences in longevity ranging from 3 to 9 months, which makes it a powerful vertebrate model for aging research. The short life cycle of N. furzeri should also facilitate studies on adult traits that are specific to vertebrates. Although progress has been made to generate a genetic linkage map and to start sequencing the genome of N. furzeri, tools to genetically manipulate this species of fish have not yet been developed. Here, we report the first establishment of transgenesis in N. furzeri. We use the Tol2 transposase system to generate transgenic N. furzeri that express green fluorescent protein driven by the Xenopus cytoskeletal actin promoter or the zebrafish heat-shock protein 70 promoter. We successfully generate stable transgenic lines of N. furzeri with germline transmission of integrated transgene. The development of transgenesis in N. furzeri provides a powerful tool to investigate the mechanisms underlying aging and longevity in a short-lived vertebrate model. Transgenesis in this fish will also facilitate the study of other phenotypes, including adult tissue regeneration and cognitive behavior.

  View details for DOI 10.1534/g3.111.001271

  View details for PubMedID 22384364

• Transposon-Mediated Transgenesis in the Short-Lived African Killifish Nothobranchius furzeri, a Vertebrate Model for Aging G3-GENES GENOMES GENETICS Valenzano, D. R., Sharp, S., Brunet, A. 2011; 1 (7): 531-538

  View details for DOI 10.1534/g3.111.001271

  View details for Web of Science ID 000312410300002

• The H3K27 demethylase UTX-1 regulates C. elegans lifespan in a germline-independent, insulin-dependent manner AGING CELL Maures, T. J., Greer, E. L., Hauswirth, A. G., Brunet, A. 2011; 10 (6): 980-990

  Abstract

  Aging is accompanied by alterations in epigenetic marks that control chromatin states, including histone acetylation and methylation. Enzymes that reversibly affect histone marks associated with active chromatin have recently been found to regulate aging in Caenorhabditis elegans. However, relatively little is known about the importance for aging of histone marks associated with repressed chromatin. Here, we use a targeted RNAi screen in C. elegans to identify four histone demethylases that significantly regulate worm lifespan, UTX-1, RBR-2, LSD-1, and T26A5.5. Interestingly, UTX-1 belongs to a conserved family of histone demethylases specific for lysine 27 of histone H3 (H3K27me3), a mark associated with repressed chromatin. Both utx-1 knockdown and heterozygous mutation of utx-1 extend lifespan and increase the global levels of the H3K27me3 mark in worms. The H3K27me3 mark significantly drops in somatic cells during the normal aging process. UTX-1 regulates lifespan independently of the presence of the germline, but in a manner that depends on the insulin-FoxO signaling pathway. These findings identify the H3K27me3 histone demethylase UTX-1 as a novel regulator of worm lifespan in somatic cells.

  View details for DOI 10.1111/j.1474-9726.2011.00738.x

  View details for Web of Science ID 000297003800007

  View details for PubMedID 21834846

• Transgenerational epigenetic inheritance of longevity in Caenorhabditis elegans NATURE Greer, E. L., Maures, T. J., Ucar, D., Hauswirth, A. G., Mancini, E., Lim, J. P., Benayoun, B. A., Shi, Y., Brunet, A. 2011; 479 (7373): 365-U204

  Abstract

  Chromatin modifiers regulate lifespan in several organisms, raising the question of whether changes in chromatin states in the parental generation could be incompletely reprogrammed in the next generation and thereby affect the lifespan of descendants. The histone H3 lysine 4 trimethylation (H3K4me3) complex, composed of ASH-2, WDR-5 and the histone methyltransferase SET-2, regulates Caenorhabditis elegans lifespan. Here we show that deficiencies in the H3K4me3 chromatin modifiers ASH-2, WDR-5 or SET-2 in the parental generation extend the lifespan of descendants up until the third generation. The transgenerational inheritance of lifespan extension by members of the ASH-2 complex is dependent on the H3K4me3 demethylase RBR-2, and requires the presence of a functioning germline in the descendants. Transgenerational inheritance of lifespan is specific for the H3K4me3 methylation complex and is associated with epigenetic changes in gene expression. Thus, manipulation of specific chromatin modifiers only in parents can induce an epigenetic memory of longevity in descendants.

  View details for DOI 10.1038/nature10572

  View details for Web of Science ID 000297059700038

  View details for PubMedID 22012258

  View details for PubMedCentralID PMC3368121

• The pro-longevity gene FoxO3 is a direct target of the p53 tumor suppressor ONCOGENE Renault, V. M., Thekkat, P. U., Hoang, K. L., WHITE, J. L., Brady, C. A., Broz, D. K., Venturelli, O. S., Johnson, T. M., Oskoui, P. R., Xuan, Z., Santo, E. E., Zhang, M. Q., Vogel, H., Attardi, L. D., Brunet, A. 2011; 30 (29): 3207-3221

  Abstract

  FoxO transcription factors have a conserved role in longevity, and act as tissue-specific tumor suppressors in mammals. Several nodes of interaction have been identified between FoxO transcription factors and p53, a major tumor suppressor in humans and mice. However, the extent and importance of the functional interaction between FoxO and p53 have not been fully explored. Here, we show that p53 regulates the expression of FoxO3, one of the four mammalian FoxO genes, in response to DNA damaging agents in both mouse embryonic fibroblasts and thymocytes. We find that p53 transactivates FoxO3 in cells by binding to a site in the second intron of the FoxO3 gene, a genomic region recently found to be associated with extreme longevity in humans. While FoxO3 is not necessary for p53-dependent cell cycle arrest, FoxO3 appears to modulate p53-dependent apoptosis. We also find that FoxO3 loss does not interact with p53 loss for tumor development in vivo, although the tumor spectrum of p53-deficient mice appears to be affected by FoxO3 loss. Our findings indicate that FoxO3 is a p53 target gene, and suggest that FoxO3 and p53 are part of a regulatory transcriptional network that may have an important role during aging and cancer.

  View details for DOI 10.1038/onc.2011.35

  View details for Web of Science ID 000293006800001

  View details for PubMedID 21423206

  View details for PubMedCentralID PMC3136551

• Epigenetic regulation of aging stem cells ONCOGENE Pollina, E. A., Brunet, A. 2011; 30 (28): 3105-3126

  Abstract

  The function of adult tissue-specific stem cells declines with age, which may contribute to the physiological decline in tissue homeostasis and the increased risk of neoplasm during aging. Old stem cells can be 'rejuvenated' by environmental stimuli in some cases, raising the possibility that a subset of age-dependent stem cell changes is regulated by reversible mechanisms. Epigenetic regulators are good candidates for such mechanisms, as they provide a versatile checkpoint to mediate plastic changes in gene expression and have recently been found to control organismal longevity. Here, we review the importance of chromatin regulation in adult stem cell compartments. We particularly focus on the roles of chromatin-modifying complexes and transcription factors that directly impact chromatin in aging stem cells. Understanding the regulation of chromatin states in adult stem cells is likely to have important implications for identifying avenues to maintain the homeostatic balance between sustained function and neoplastic transformation of aging stem cells.

  View details for DOI 10.1038/onc.2011.45

  View details for Web of Science ID 000292726300001

  View details for PubMedID 21441951

• MicroRNA programs in normal and aberrant stem and progenitor cells GENOME RESEARCH Arnold, C. P., Tan, R., Zhou, B., Yue, S., Schaffert, S., Biggs, J. R., Doyonnas, R., Lo, M., Perry, J. M., Renault, V. M., Sacco, A., Somervaille, T., Viatour, P., Brunet, A., Cleary, M. L., Li, L., Sage, J., Zhang, D., Blau, H. M., Chen, C., Chen, C. 2011; 21 (5): 798-810

  Abstract

  Emerging evidence suggests that microRNAs (miRNAs), an abundant class of ∼22-nucleotide small regulatory RNAs, play key roles in controlling the post-transcriptional genetic programs in stem and progenitor cells. Here we systematically examined miRNA expression profiles in various adult tissue-specific stem cells and their differentiated counterparts. These analyses revealed miRNA programs that are common or unique to blood, muscle, and neural stem cell populations and miRNA signatures that mark the transitions from self-renewing and quiescent stem cells to proliferative and differentiating progenitor cells. Moreover, we identified a stem/progenitor transition miRNA (SPT-miRNA) signature that predicts the effects of genetic perturbations, such as loss of PTEN and the Rb family, AML1-ETO9a expression, and MLL-AF10 transformation, on self-renewal and proliferation potentials of mutant stem/progenitor cells. We showed that some of the SPT-miRNAs control the self-renewal of embryonic stem cells and the reconstitution potential of hematopoietic stem cells (HSCs). Finally, we demonstrated that SPT-miRNAs coordinately regulate genes that are known to play roles in controlling HSC self-renewal, such as Hoxb6 and Hoxa4. Together, these analyses reveal the miRNA programs that may control key processes in normal and aberrant stem and progenitor cells, setting the foundations for dissecting post-transcriptional regulatory networks in stem cells.

  View details for DOI 10.1101/gr.111385.110

  View details for Web of Science ID 000290088000018

  View details for PubMedID 21451113

• A CRTCal Link between Energy and Life Span CELL METABOLISM Brunet, A. 2011; 13 (4): 358-360

  Abstract

  Cutting down calories prolongs life, but how this works remains largely unknown. A recent study in Nature (Mair et al., 2011) shows that life span extension triggered by the energy-sensing protein kinase AMPK is mediated by an evolutionarily conserved transcriptional circuit involving CRTC-1 and CREB.

  View details for DOI 10.1016/j.cmet.2011.03.012

  View details for Web of Science ID 000289381300006

  View details for PubMedID 21459320

• Energy metabolism in adult neural stem cell fate PROGRESS IN NEUROBIOLOGY Rafalski, V. A., Brunet, A. 2011; 93 (2): 182-203

  Abstract

  The adult mammalian brain contains a population of neural stem cells that can give rise to neurons, astrocytes, and oligodendrocytes and are thought to be involved in certain forms of memory, behavior, and brain injury repair. Neural stem cell properties, such as self-renewal and multipotency, are modulated by both cell-intrinsic and cell-extrinsic factors. Emerging evidence suggests that energy metabolism is an important regulator of neural stem cell function. Molecules and signaling pathways that sense and influence energy metabolism, including insulin/insulin-like growth factor I (IGF-1)-FoxO and insulin/IGF-1-mTOR signaling, AMP-activated protein kinase (AMPK), SIRT1, and hypoxia-inducible factors, are now implicated in neural stem cell biology. Furthermore, these signaling modules are likely to cooperate with other pathways involved in stem cell maintenance and differentiation. This review summarizes the current understanding of how cellular and systemic energy metabolism regulate neural stem cell fate. The known consequences of dietary restriction, exercise, aging, and pathologies with deregulated energy metabolism for neural stem cells and their differentiated progeny will also be discussed. A better understanding of how neural stem cells are influenced by changes in energy availability will help unravel the complex nature of neural stem cell biology in both the normal and diseased state.

  View details for DOI 10.1016/j.pneurobio.2010.10.007

  View details for Web of Science ID 000287950400003

  View details for PubMedID 21056618

• The MicroRNA Cluster miR-106b similar to 25 Regulates Adult Neural Stem/Progenitor Cell Proliferation and Neuronal Differentiation AGING-US Brett, J. O., Renault, V. M., Rafalski, V. A., Webb, A. E., Brunet, A. 2011; 3 (2): 108-124

  Abstract

  In adult mammals, neural stem cells (NSCs) generate new neurons that are important for specific types of learning and memory. Controlling adult NSC number and function is fundamental for preserving the stem cell pool and ensuring proper levels of neurogenesis throughout life. Here we study the importance of the microRNA gene cluster miR-106b~25 (miR-106b, miR-93, and miR-25) in primary cultures of neural stem/progenitor cells (NSPCs) isolated from adult mice. We find that knocking down miR-25 decreases NSPC proliferation, whereas ectopically expressing miR-25 promotes NSPC proliferation. Expressing the entire miR-106b~25 cluster in NSPCs also increases their ability to generate new neurons. Interestingly, miR-25 has a number of potential target mRNAs involved in insulin/insulin-like growth factor-1 (IGF) signaling, a pathway implicated in aging. Furthermore, the regulatory region of miR-106b~25 is bound by FoxO3, a member of the FoxO family of transcription factors that maintains adult stem cells and extends lifespan downstream of insulin/IGF signaling. These results suggest that miR-106b~25 regulates NSPC function and is part of a network involving the insulin/IGF-FoxO pathway, which may have important implications for the homeostasis of the NSC pool during aging.

  View details for Web of Science ID 000288170400008

  View details for PubMedID 21386132

  View details for PubMedCentralID PMC3082007

• Members of the H3K4 trimethylation complex regulate lifespan in a germline-dependent manner in C. elegans NATURE Greer, E. L., Maures, T. J., Hauswirth, A. G., Green, E. M., Leeman, D. S., Maro, G. S., Han, S., Banko, M. R., Gozani, O., Brunet, A. 2010; 466 (7304): 383-U137

  Abstract

  The plasticity of ageing suggests that longevity may be controlled epigenetically by specific alterations in chromatin state. The link between chromatin and ageing has mostly focused on histone deacetylation by the Sir2 family, but less is known about the role of other histone modifications in longevity. Histone methylation has a crucial role in development and in maintaining stem cell pluripotency in mammals. Regulators of histone methylation have been associated with ageing in worms and flies, but characterization of their role and mechanism of action has been limited. Here we identify the ASH-2 trithorax complex, which trimethylates histone H3 at lysine 4 (H3K4), as a regulator of lifespan in Caenorhabditis elegans in a directed RNA interference (RNAi) screen in fertile worms. Deficiencies in members of the ASH-2 complex-ASH-2 itself, WDR-5 and the H3K4 methyltransferase SET-2-extend worm lifespan. Conversely, the H3K4 demethylase RBR-2 is required for normal lifespan, consistent with the idea that an excess of H3K4 trimethylation-a mark associated with active chromatin-is detrimental for longevity. Lifespan extension induced by ASH-2 complex deficiency requires the presence of an intact adult germline and the continuous production of mature eggs. ASH-2 and RBR-2 act in the germline, at least in part, to regulate lifespan and to control a set of genes involved in lifespan determination. These results indicate that the longevity of the soma is regulated by an H3K4 methyltransferase/demethylase complex acting in the C. elegans germline.

  View details for DOI 10.1038/nature09195

  View details for Web of Science ID 000279867100052

  View details for PubMedID 20555324

  View details for PubMedCentralID PMC3075006

• A FOXO-Pak1 transcriptional pathway controls neuronal polarity GENES & DEVELOPMENT de la Torre-Ubieta, L., Gaudilliere, B., Yang, Y., Ikeuchi, Y., Yamada, T., DiBacco, S., Stegmueller, J., Schueller, U., Salih, D. A., Rowitch, D., Brunet, A., Bonni, A. 2010; 24 (8): 799-813

  Abstract

  Neuronal polarity is essential for normal brain development and function. However, cell-intrinsic mechanisms that govern the establishment of neuronal polarity remain to be identified. Here, we report that knockdown of endogenous FOXO proteins in hippocampal and cerebellar granule neurons, including in the rat cerebellar cortex in vivo, reveals a requirement for the FOXO transcription factors in the establishment of neuronal polarity. The FOXO transcription factors, including the brain-enriched protein FOXO6, play a critical role in axo-dendritic polarization of undifferentiated neurites, and hence in a switch from unpolarized to polarized neuronal morphology. We also identify the gene encoding the protein kinase Pak1, which acts locally in neuronal processes to induce polarity, as a critical direct target gene of the FOXO transcription factors. Knockdown of endogenous Pak1 phenocopies the effect of FOXO knockdown on neuronal polarity. Importantly, exogenous expression of Pak1 in the background of FOXO knockdown in both primary neurons and postnatal rat pups in vivo restores the polarized morphology of neurons. These findings define the FOXO proteins and Pak1 as components of a cell-intrinsic transcriptional pathway that orchestrates neuronal polarity, thus identifying a novel function for the FOXO transcription factors in a unique aspect of neural development.

  View details for DOI 10.1101/gad.1880510

  View details for Web of Science ID 000276730300008

  View details for PubMedID 20395366

• Mapping Loci Associated With Tail Color and Sex Determination in the Short-Lived Fish Nothobranchius furzeri GENETICS Valenzano, D. R., Kirschner, J., Kamber, R. A., Zhang, E., Weber, D., Cellerino, A., Englert, C., Platzer, M., Reichwald, K., Brunet, A. 2009; 183 (4): 1385-1395

  Abstract

  The African fish Nothobranchius furzeri is the shortest-lived vertebrate species that can reproduce in captivity, with a median life span of 9-11 weeks for the shortest-lived strain. Natural populations of N. furzeri display differences in life span, aging biomarkers, behavior, and color, which make N. furzeri a unique vertebrate system for studying the genetic basis of these traits. We mapped regions of the genome involved in sex determination and tail color by genotyping microsatellite markers in the F(2) progeny of a cross between a short-lived, yellow-tailed strain and a long-lived, red-tailed strain of N. furzeri. We identified one region linked with the yellow/red tail color that maps close to melanocortin 1 receptor (mc1r), a gene involved in pigmentation in several vertebrate species. Analysis of the segregation of sex-linked markers revealed that N. furzeri has a genetic sex determination system with males as the heterogametic sex and markedly reduced recombination in the male sex-determining region. Our results demonstrate that both naturally-evolved pigmentation differences and sex determination in N. furzeri are controlled by simple genetic mechanisms and set the stage for the molecular genetic dissection of factors underlying such traits. The microsatellite-based linkage map we developed for N. furzeri will also facilitate analysis of the genetic architecture of traits that characterize this group of vertebrates, including short life span and adaptation to extreme environmental conditions.

  View details for DOI 10.1534/genetics.109.108670

  View details for Web of Science ID 000272435000016

  View details for PubMedID 19786620

  View details for PubMedCentralID PMC2787427

• FoxO3 Regulates Neural Stem Cell Homeostasis CELL STEM CELL Renault, V. M., Rafalski, V. A., Morgan, A. A., Salih, D. A., Brett, J. O., Webb, A. E., Villeda, S. A., Thekkat, P. U., Guillerey, C., Denko, N. C., Palmer, T. D., Bufte, A. J., Brunet, A. 2009; 5 (5): 527-539

  Abstract

  In the nervous system, neural stem cells (NSCs) are necessary for the generation of new neurons and for cognitive function. Here we show that FoxO3, a member of a transcription factor family known to extend lifespan in invertebrates, regulates the NSC pool. We find that adult FoxO3(-/-) mice have fewer NSCs in vivo than wild-type counterparts. NSCs isolated from adult FoxO3(-/-) mice have decreased self-renewal and an impaired ability to generate different neural lineages. Identification of the FoxO3-dependent gene expression profile in NSCs suggests that FoxO3 regulates the NSC pool by inducing a program of genes that preserves quiescence, prevents premature differentiation, and controls oxygen metabolism. The ability of FoxO3 to prevent the premature depletion of NSCs might have important implications for counteracting brain aging in long-lived species.

  View details for DOI 10.1016/j.stem.2009.09.014

  View details for Web of Science ID 000272019500014

  View details for PubMedID 19896443

  View details for PubMedCentralID PMC2775802

• CANCER When restriction is good NATURE Brunet, A. 2009; 458 (7239): 713-714

  View details for DOI 10.1038/458713a

  View details for Web of Science ID 000265193600026

  View details for PubMedID 19360073

• Different dietary restriction regimens extend lifespan by both independent and overlapping genetic pathways in C-elegans AGING CELL Greer, E. L., Brunet, A. 2009; 8 (2): 113-127

  Abstract

  Dietary restriction (DR) has the remarkable ability to extend lifespan and healthspan. A variety of DR regimens have been described in species ranging from yeast to mammals. However, whether different DR regimens extend lifespan via universal, distinct, or overlapping pathways is still an open question. Here we examine the genetic pathways that mediate longevity by different DR regimens in Caenorhabditis elegans. We have previously shown that the low-energy sensing AMP-activated protein kinase AMPK/aak-2 and the Forkhead transcription factor FoxO/daf-16 are necessary for longevity induced by a DR regimen that we developed (sDR). Here we find that AMPK and FoxO are necessary for longevity induced by another DR regimen, but are dispensable for the lifespan extension induced by two different DR methods. Intriguingly, AMPK is also necessary for the lifespan extension elicited by resveratrol, a natural polyphenol that mimics some aspects of DR. Conversely, we test if genes previously reported to mediate longevity by a variety of DR methods are necessary for sDR-induced longevity. Although clk-1, a gene involved in ubiquinone biosynthesis, is also required for sDR-induced lifespan extension, we find that four other genes (sir-2.1, FoxA/pha-4, skn-1, and hsf-1) are all dispensable for longevity induced by sDR. Consistent with the observation that different DR methods extend lifespan by mostly independent genetic mechanisms, we find that the effects on lifespan of two different DR regimens are additive. Understanding the genetic network by which different DR regimens extend lifespan has important implications for harnessing the full benefits of DR on lifespan and healthspan.

  View details for DOI 10.1111/j.1474-9726.2009.00459.x

  View details for Web of Science ID 000264607200004

  View details for PubMedID 19239417

• AMP-activated Protein Kinase and FoxO Transcription Factors in Dietary Restriction-induced Longevity 15th International Symposium on Olfaction and Taste Greer, E. L., Banko, M. R., Brunet, A. WILEY-BLACKWELL. 2009: 688–692

  Abstract

  Aging is regulated by modifications in single genes and by simple changes in the environment. The signaling pathway connecting insulin to FoxO transcription factors integrates environmental stimuli to regulate lifespan. FoxO transcription factors are directly phosphorylated in response to insulin/growth factor signaling by the protein kinase Akt, thereby causing their sequestration in the cytoplasm. In the absence of insulin/growth factors, FoxO factors translocate to the nucleus where they trigger a range of cellular responses, including resistance to oxidative stress--a phenotype highly coupled with lifespan extension. Our recent results indicate that FoxO transcription factors are also regulated in response to nutrient deprivation by the AMP-activated protein kinase (AMPK) pathway. The energy-sensing AMPK directly phosphorylates FoxO transcription factors at six regulatory sites. AMPK phosphorylation enhances FoxO transcriptional activity, leading to the expression of specific target genes involved in stress resistance and changes in energy metabolism. The AMPK-FoxO pathway plays a crucial role in the ability of a dietary restriction regimen to extend lifespan in Caenorhabditis elegans. Understanding the intricate signaling networks that translate environmental conditions like dietary restriction into changes in gene expression that extend lifespan will be of critical importance to identify ways to delay the onset of aging and age-dependent diseases.

  View details for DOI 10.1111/j.1749-6632.2009.04019.x

  View details for Web of Science ID 000270495700118

  View details for PubMedID 19686213

• Foxo Transcription Factors: Central Sensors of Environmental Stimuli that Regulate Longevity 15th International Symposium on Olfaction and Taste Brunet, A., Greer, E. L., Banko, M. R. OXFORD UNIV PRESS. 2008: S20–S20

  View details for Web of Science ID 000259973600084

• The FoxO code ONCOGENE Calnan, D. R., Brunet, A. 2008; 27 (16): 2276-2288

  Abstract

  The FoxO family of Forkhead transcription factors plays an important role in longevity and tumor suppression by upregulating target genes involved in stress resistance, metabolism, cell cycle arrest and apoptosis. FoxO transcription factors translate a variety of environmental stimuli, including insulin, growth factors, nutrients and oxidative stress, into specific gene-expression programs. These environmental stimuli control FoxO activity primarily by regulating their subcellular localization, but also by affecting their protein levels, DNA-binding properties and transcriptional activity. The precise regulation of FoxO transcription factors is enacted by an intricate combination of post-translational modifications (PTMs), including phosphorylation, acetylation and ubiquitination, and binding protein partners. An intriguing possibility is that FoxO PTMs may act as a 'molecular FoxO code' read by selective protein partners to rapidly regulate gene-expression programs. The effective control of FoxO activity in response to environmental stimuli is likely to be critical to prevent aging and age-dependent diseases, including cancer, neurodegenerative diseases and diabetes.

  View details for DOI 10.1038/onc.2008.21

  View details for Web of Science ID 000254782700003

  View details for PubMedID 18391970

• FoxO transcription factors in the maintenance of cellular homeostasis during aging CURRENT OPINION IN CELL BIOLOGY Salih, D. A., Brunet, A. 2008; 20 (2): 126-136

  Abstract

  The FoxO family of Forkhead transcription factors functions at the interface of tumor suppression, energy metabolism, and organismal longevity. FoxO factors are key downstream targets of insulin, growth factor, nutrient, and oxidative stress stimuli that coordinate a wide range of cellular outputs. FoxO-dependent cellular responses include gluconeogenesis, neuropeptide secretion, atrophy, autophagy, apoptosis, cell cycle arrest, and stress resistance. This review will discuss the roles of the mammalian FoxO family in a variety of cell types, from stem cells to mature cells, in the context of the whole organism. Given the overwhelming evidence that the FoxO factors promote longevity in invertebrates, this review will also discuss the potential role of the FoxO factors in the aging of mammalian organisms.

  View details for DOI 10.1016/j.ceb.2008.02.005

  View details for Web of Science ID 000255546800003

  View details for PubMedID 18394876

• Signaling networks in aging JOURNAL OF CELL SCIENCE Greer, E. L., Brunet, A. 2008; 121 (4): 407-412

  View details for DOI 10.1242/jcs.021519

  View details for Web of Science ID 000253425800001

  View details for PubMedID 18256383

• FOXO transcription factors in ageing and cancer 10th International Symposium on Insulin Receptors and Insulin Action Greer, E. L., Brunet, A. WILEY-BLACKWELL. 2008: 19–28

  Abstract

  Ageing is associated with an increased onset of cancer. Understanding the molecular mechanisms that underlie the age dependency of cancer will have important implications for preventing and treating this pathology. The signalling pathway connecting insulin and FOXO transcription factors provides the most compelling example for a conserved genetic pathway at the interface between ageing and cancer. FOXO transcription factors (FOXO) promote longevity and tumour suppression. FOXO transcription factors are directly phosphorylated in response to insulin/growth factor signalling by the protein kinase Akt, thereby causing their sequestration in the cytoplasm. In the absence of insulin/growth factors, FOXO factors translocate to the nucleus where they trigger a range of cellular responses, including resistance to oxidative stress, a phenotype highly coupled with lifespan extension. FOXO factors integrate stress stimuli via phosphorylation, acetylation and mono-ubiquitination of a series of regulatory sites. Understanding how FOXO proteins integrate environmental conditions to control specific gene expression programmes will be pivotal in identifying ways to slow the onset of cancer in ageing individuals.

  View details for DOI 10.1111/j.1748-1716.2007.01780.x

  View details for Web of Science ID 000251667000004

  View details for PubMedID 18171426

• Aging and cancer: killing two birds with one worm NATURE GENETICS Brunet, A. 2007; 39 (11): 1306-1307

  View details for Web of Science ID 000250575900006

  View details for PubMedID 17968345

• The energy sensor AMP-activated protein kinase directly regulates the mammalian FOXO3 transcription factor JOURNAL OF BIOLOGICAL CHEMISTRY Greer, E. L., Oskoui, P. R., Banko, M. R., Maniar, J. M., Gygi, M. P., Gygi, S. P., Brunet, A. 2007; 282 (41): 30107-30119

  Abstract

  The maintenance of homeostasis throughout an organism's life span requires constant adaptation to changes in energy levels. The AMP-activated protein kinase (AMPK) plays a critical role in the cellular responses to low energy levels by switching off energy-consuming pathways and switching on energy-producing pathways. However, the transcriptional mechanisms by which AMPK acts to adjust cellular energy levels are not entirely characterized. Here, we find that AMPK directly regulates mammalian FOXO3, a member of the FOXO family of Forkhead transcription factors known to promote resistance to oxidative stress, tumor suppression, and longevity. We show that AMPK phosphorylates human FOXO3 at six previously unidentified regulatory sites. Phosphorylation by AMPK leads to the activation of FOXO3 transcriptional activity without affecting FOXO3 subcellular localization. Using a genome-wide microarray analysis, we identify a set of target genes that are regulated by FOXO3 when phosphorylated at these six regulatory sites in mammalian cells. The regulation of FOXO3 by AMPK may play a crucial role in fine tuning gene expression programs that control energy balance and stress resistance in cells throughout life.

  View details for DOI 10.1074/jbc.M705325200

  View details for Web of Science ID 000249981200041

  View details for PubMedID 17711846

• An AMPK-FOXO pathway mediates longevity induced by a novel method of dietary restriction in C-elegans CURRENT BIOLOGY Greer, E. L., Dowlatshahi, D., Banko, M. R., Villen, J., Hoang, K., Blanchard, D., Gygi, S. P., Brunet, A. 2007; 17 (19): 1646-1656

  Abstract

  Dietary restriction (DR) is the most effective environmental intervention to extend lifespan in a wide range of species. However, the molecular mechanisms underlying the benefits of DR on longevity are still poorly characterized. AMP-activated protein kinase (AMPK) is activated by a decrease in energy levels, raising the possibility that AMPK might mediate lifespan extension by DR.By using a novel DR assay that we developed and validated in C. elegans, we find that AMPK is required for this DR method to extend lifespan and delay age-dependent decline. We find that AMPK exerts its effects in part via the FOXO transcription factor DAF-16. FOXO/DAF-16 is necessary for the beneficial effects of this DR method on lifespan. Expression of an active version of AMPK in worms increases stress resistance and extends longevity in a FOXO/DAF-16-dependent manner. Lastly, we find that AMPK activates FOXO/DAF-16-dependent transcription and phosphorylates FOXO/DAF-16 at previously unidentified sites, suggesting a possible direct mechanism of regulation of FOXO/DAF-16 by AMPK.Our study shows that an energy-sensing AMPK-FOXO pathway mediates the lifespan extension induced by a novel method of dietary restriction in C. elegans.

  View details for DOI 10.1016/j.cub.2007.08.047

  View details for Web of Science ID 000250125200023

  View details for PubMedID 17900900

  View details for PubMedCentralID PMC2185793

• Ageing - From stem to stern NATURE Brunet, A., Rando, T. A. 2007; 449 (7160): 288-?

  View details for DOI 10.1038/449288a

  View details for Web of Science ID 000249557300030

  View details for PubMedID 17882206

• FOXO transcription factors CURRENT BIOLOGY Carter, M. E., Brunet, A. 2007; 17 (4): R113-R114

  View details for Web of Science ID 000244463100006

  View details for PubMedID 17307039

• FOXO transcription factors at the interface between longevity and tumor suppression ONCOGENE Greer, E. L., Brunet, A. 2005; 24 (50): 7410-7425

  Abstract

  A wide range of human diseases, including cancer, has a striking age-dependent onset. However, the molecular mechanisms that connect aging and cancer are just beginning to be unraveled. FOXO transcription factors are promising candidates to serve as molecular links between longevity and tumor suppression. These factors are major substrates of the protein kinase Akt. In the presence of insulin and growth factors, FOXO proteins are relocalized from the nucleus to the cytoplasm and degraded via the ubiquitin-proteasome pathway. In the absence of growth factors, FOXO proteins translocate to the nucleus and upregulate a series of target genes, thereby promoting cell cycle arrest, stress resistance, or apoptosis. Stress stimuli also trigger the relocalization of FOXO factors into the nucleus, thus allowing an adaptive response to stress stimuli. Consistent with the notion that stress resistance is highly coupled with lifespan extension, activation of FOXO transcription factors in worms and flies increases longevity. Emerging evidence also suggests that FOXO factors play a tumor suppressor role in a variety of cancers. Thus, FOXO proteins translate environmental stimuli into changes in gene expression programs that may coordinate organismal longevity and tumor suppression.

  View details for DOI 10.1038/sj.onc.1209086

  View details for Web of Science ID 000233201900004

  View details for PubMedID 16288288

• Stress-dependent regulation of FOXO transcription factors by the SIRT1 deacetylase SCIENCE Brunet, A., Sweeney, L. B., Sturgill, J. F., Chua, K. F., Greer, P. L., Lin, Y. X., Tran, H., Ross, S. E., Mostoslavsky, R., Cohen, H. Y., Hu, L. S., Cheng, H. L., Jedrychowski, M. P., Gygi, S. P., Sinclair, D. A., Alt, F. W., Greenberg, M. E. 2004; 303 (5666): 2011-2015

  Abstract

  The Sir2 deacetylase modulates organismal life-span in various species. However, the molecular mechanisms by which Sir2 increases longevity are largely unknown. We show that in mammalian cells, the Sir2 homolog SIRT1 appears to control the cellular response to stress by regulating the FOXO family of Forkhead transcription factors, a family of proteins that function as sensors of the insulin signaling pathway and as regulators of organismal longevity. SIRT1 and the FOXO transcription factor FOXO3 formed a complex in cells in response to oxidative stress, and SIRT1 deacetylated FOXO3 in vitro and within cells. SIRT1 had a dual effect on FOXO3 function: SIRT1 increased FOXO3's ability to induce cell cycle arrest and resistance to oxidative stress but inhibited FOXO3's ability to induce cell death. Thus, one way in which members of the Sir2 family of proteins may increase organismal longevity is by tipping FOXO-dependent responses away from apoptosis and toward stress resistance.

  View details for DOI 10.1126/science.1094637

  View details for Web of Science ID 000220429800040

  View details for PubMedID 14976264

• PEA-15 binding to ERK1/2 MAPKs is required for its modulation of integrin activation JOURNAL OF BIOLOGICAL CHEMISTRY Chou, F. L., Hill, J. M., Hsieh, J. C., Pouyssegur, J., Brunet, A., Glading, A., Uberall, F., Ramos, J. W., Werner, M. H., Ginsberg, M. H. 2003; 278 (52): 52587-52597

  Abstract

  Activation of Raf-1 suppresses integrin activation, potentially through the activation of extracellular signal-regulated kinases 1 and 2 (ERK1/2). However, bulk ERK1/2 activation does not correlate with suppression. PEA-15 reverses suppression of integrin activation and binds ERK1/2. Here we report that PEA-15 reversal of integrin suppression depends on its capacity to bind ERK1/2, indicating that ERK1/2 function is indeed required for suppression. Mutations in either the death effector domain or C-terminal tail of PEA-15 that block ERK1/2 binding abrogated the reversal of integrin suppression. Furthermore, we used ERK/p38 chimeras and site-directed mutagenesis to identify ERK1/2 residues required for binding PEA-15. Mutations of residues that precede the alphaG helix and within the mitogen-activated protein kinase insert blocked ERK2 binding to PEA-15, but not activation of ERK2. These ERK2 mutants blocked the ability of PEA-15 to reverse suppression of integrin activation. Thus, PEA-15 regulation of integrin activation depends on its binding to ERK1/2. To directly test the role of ERK1/2 localization in suppression, we enforced membrane association of ERK1 and 2 by joining a membrane-targeting CAAX box sequence to them. Both ERK1-CAAX and ERK2-CAAX were membrane-localized and suppressed integrin activation. In contrast to suppression by membrane-targeted Raf-CAAX, suppression by ERK1/2-CAAX was not reversed by PEA-15. Thus, ERK1/2 are the Raf effectors for suppression of integrin activation, and PEA-15 reverses suppression by binding ERK1/2.

  View details for DOI 10.1074/jbc.M309322200

  View details for Web of Science ID 000187480700077

  View details for PubMedID 14506247

• The many forks in FOXO's road. Science's STKE : signal transduction knowledge environment Tran, H., Brunet, A., Griffith, E. C., Greenberg, M. E. 2003; 2003 (172): RE5-?

  Abstract

  The FOXO family of transcription factors constitute an evolutionarily conserved subgroup within the larger family known as winged helix or Forkhead transcriptional regulators. Building upon work in the nematode, researchers have uncovered a role for these proteins in a diverse set of cellular responses that include glucose metabolism, stress response, cell cycle regulation, and apoptosis. At the organismal level, FOXO transcription factors are believed to function in various pathological processes ranging from cancer and diabetes to organismal aging. A number of studies have also shed light on the signaling pathways that regulate FOXO activity in response to external stimuli and have identified multiple FOXO target genes that mediate this varied set of biological responses.

  View details for PubMedID 12621150

• DNA repair pathway stimulated by the forkhead transcription factor FOXO3a through the Gadd45 protein SCIENCE Tran, H., Brunet, A., Grenier, J. M., Datta, S. R., Fornace, A. J., DiStefano, P. S., Chiang, L. W., Greenberg, M. E. 2002; 296 (5567): 530-534

  Abstract

  The signaling pathway from phosphoinositide 3-kinase to the protein kinase Akt controls organismal life-span in invertebrates and cell survival and proliferation in mammals by inhibiting the activity of members of the FOXO family of transcription factors. We show that mammalian FOXO3a also functions at the G2 to M checkpoint in the cell cycle and triggers the repair of damaged DNA. By gene array analysis, FOXO3a was found to modulate the expression of several genes that regulate the cellular response to stress at the G2-M checkpoint. The growth arrest and DNA damage response gene Gadd45a appeared to be a direct target of FOXO3a that mediates part of FOXO3a's effects on DNA repair. These findings indicate that in mammals FOXO3a regulates the resistance of cells to stress by inducing DNA repair and thereby may also affect organismal life-span.

  View details for Web of Science ID 000175179400046

  View details for PubMedID 11964479

• 14-3-3 transits to the nucleus and participates in dynamic nucleocytoplasmic transport JOURNAL OF CELL BIOLOGY Brunet, A., Kanai, F., Stehn, J., Xu, J., Sarbassova, D., Frangioni, J. V., Dalal, S. N., DeCaprio, J. A., Greenberg, M. E., Yaffe, M. B. 2002; 156 (5): 817-828

  Abstract

  14-3-3 proteins regulate the cell cycle and prevent apoptosis by controlling the nuclear and cytoplasmic distribution of signaling molecules with which they interact. Although the majority of 14-3-3 molecules are present in the cytoplasm, we show here that in the absence of bound ligands 14-3-3 homes to the nucleus. We demonstrate that phosphorylation of one important 14-3-3 binding molecule, the transcription factor FKHRL1, at the 14-3-3 binding site occurs within the nucleus immediately before FKHRL1 relocalization to the cytoplasm. We show that the leucine-rich region within the COOH-terminal alpha-helix of 14-3-3, which had been proposed to function as a nuclear export signal (NES), instead functions globally in ligand binding and does not directly mediate nuclear transport. Efficient nuclear export of FKHRL1 requires both intrinsic NES sequences within FKHRL1 and phosphorylation/14-3-3 binding. Finally, we present evidence that phosphorylation/14-3-3 binding may also prevent FKHRL1 nuclear reimport. These results indicate that 14-3-3 can mediate the relocalization of nuclear ligands by several mechanisms that ensure complete sequestration of the bound 14-3-3 complex in the cytoplasm.

  View details for DOI 10.1083/jcb.200112059

  View details for Web of Science ID 000176426300007

  View details for PubMedID 11864996

• Transforming growth factor beta enhances epithelial cell survival via Akt-dependent regulation of FKHRL1 MOLECULAR BIOLOGY OF THE CELL Shin, I., Bakin, A. V., Rodeck, U., Brunet, A., ARTEAGA, C. L. 2001; 12 (11): 3328-3339

  Abstract

  The Forkhead family of transcription factors participates in the induction of death-related genes. In NMuMG and 4T1 mammary epithelial cells, transforming growth factor beta (TGF beta) induced phosphorylation and cytoplasmic retention of the Forkhead factor FKHRL1, while reducing FHKRL1-dependent transcriptional activity. TGF beta-induced FKHRL1 phosphorylation and nuclear exclusion were inhibited by LY294002, an inhibitor of phosphatidylinositol-3 kinase. A triple mutant of FKHRL1, in which all three Akt phosphorylation sites have been mutated (TM-FKHRL1), did not translocate to the cytoplasm in response to TGF beta. In HaCaT keratinocytes, expression of dominant-negative Akt prevented TGF beta-induced 1) reduction of Forkhead-dependent transcription, 2) FKHRL1 phosphorylation, and 3) nuclear exclusion of FKRHL1. Forced expression of either wild-type (WT) or TM-FKHRL1, but not a FKHRL1 mutant with deletion of the transactivation domain, resulted in NMuMG mammary cell apoptosis. Evidence of nuclear fragmentation colocalized to cells with expression of WT- or TM-FKHRL1. The apoptotic effect of WT-FKHRL1 but not TM-FKHRL1 was prevented by exogenous TGF beta. Serum starvation-induced apoptosis was also inhibited by TGF beta in NMuMG and HaCaT cells. Finally, dominant-negative Akt abrogated the antiapoptotic effect of TGF beta. Taken together, these data suggest that TGF beta may play a role in epithelial cell survival via Akt-dependent regulation of FKHRL1.

  View details for Web of Science ID 000172357200003

  View details for PubMedID 11694570

• Transcription-dependent and -independent control of neuronal survival by the PI3K-Akt signaling pathway CURRENT OPINION IN NEUROBIOLOGY Brunet, A., Datta, S. R., Greenberg, M. E. 2001; 11 (3): 297-305

  Abstract

  The PI3K-Akt signaling pathway plays a critical role in mediating survival signals in a wide range of neuronal cell types. The recent identification of a number of substrates for the serine/threonine kinase Akt suggests that it blocks cell death by both impinging on the cytoplasmic cell death machinery and by regulating the expression of genes involved in cell death and survival. In addition, recent experiments suggest that Akt may also use metabolic pathways to regulate cell survival.

  View details for Web of Science ID 000169286200005

  View details for PubMedID 11399427

• Protein kinase SGK mediates survival signals by phosphorylating the forkhead transcription factor FKHRL1 (FOXO3a) MOLECULAR AND CELLULAR BIOLOGY Brunet, A., Park, J., Tran, H., Hu, L. S., HEMMINGS, B. A., Greenberg, M. E. 2001; 21 (3): 952-965

  Abstract

  Serum- and glucocorticoid-inducible kinases (SGKs) form a novel family of serine/threonine kinases that are activated in response to a variety of extracellular stimuli. SGKs are related to Akt (also called PKB), a serine/threonine kinase that plays a crucial role in promoting cell survival. Like Akt, SGKs are activated by the phosphoinositide-3 kinase (PI3K) and translocate to the nucleus upon growth factor stimulation. However the physiological substrates and cellular functions of SGKs remained to be identified. We hypothesized that SGKs regulate cellular functions in concert with Akt by phosphorylating common targets within the nucleus. The best-characterized nuclear substrates of Akt are transcription factors of the Forkhead family. Akt phosphorylates Forkhead transcription factors such as FKHRL1, leading to FKHRL1's exit from the nucleus and the consequent shutoff of FKHRL1 target genes. We show here that SGK1, like Akt, promotes cell survival and that it does so in part by phosphorylating and inactivating FKHRL1. However, SGK and Akt display differences with respect to the efficacy with which they phosphorylate the three regulatory sites on FKHRL1. While both kinases can phosphorylate Thr-32, SGK displays a marked preference for Ser-315 whereas Akt favors Ser-253. These findings suggest that SGK and Akt may coordinately regulate the function of FKHRL1 by phosphorylating this transcription factor at distinct sites. The efficient phosphorylation of these three sites on FKHRL1 by SGK and Akt appears to be critical to the ability of growth factors to suppress FKHRL1-dependent transcription, thereby preventing FKHRL1 from inducing cell cycle arrest and apoptosis. These findings indicate that SGK acts in concert with Akt to propagate the effects of PI3K activation within the nucleus and to mediate the biological outputs of PI3K signaling, including cell survival and cell cycle progression.

  View details for Web of Science ID 000166353700025

  View details for PubMedID 11154281

• Substrate recognition domains within extracellular signal-regulated kinase mediate binding and catalytic activation of mitogen-activated protein kinase phosphatase-3 JOURNAL OF BIOLOGICAL CHEMISTRY Nichols, A., Camps, M., Gillieron, C., Chabert, C., Brunet, A., Wilsbacher, J., Cobb, M., Pouyssegur, J., Shaw, J. P., Arkinstall, S. 2000; 275 (32): 24613-24621

  Abstract

  Mitogen-activated protein (MAP) kinase phosphatase-3 (MKP-3) is a dual specificity phosphatase that inactivates extracellular signal-regulated kinase (ERK) MAP kinases. This reflects tight and specific binding between ERK and the MKP-3 amino terminus with consequent phosphatase activation and dephosphorylation of the bound MAP kinase. We have used a series of p38/ERK chimeric molecules to identify domains within ERK necessary for binding and catalytic activation of MKP-3. These studies demonstrate that ERK kinase subdomains V-XI are necessary and sufficient for binding and catalytic activation of MKP-3. These domains constitute the major COOH-terminal structural lobe of ERK. p38/ERK chimeras possessing these regions display increased sensitivity to inactivation by MKP-3. These data also reveal an overlap between ERK domains interacting with MKP-3 and those known to confer substrate specificity on the ERK MAP kinase. Consistent with this, we show that peptides representing docking sites within the target substrates Elk-1 and p90(rsk) inhibit ERK-dependent activation of MKP-3. In addition, abolition of ERK-dependent phosphatase activation following mutation of a putative kinase interaction motif (KIM) within the MKP-3 NH(2) terminus suggests that key sites of contact for the ERK COOH-terminal structural lobe include residues localized between the Cdc25 homology domains (CH2) found conserved between members of the DSP gene family.

  View details for Web of Science ID 000088683300053

  View details for PubMedID 10811804

• Cellular survival: a play in three Akts GENES & DEVELOPMENT Datta, S. R., Brunet, A., Greenberg, M. E. 1999; 13 (22): 2905-2927

  View details for Web of Science ID 000084048600001

  View details for PubMedID 10579998

• Cell survival promoted by the Ras-MAPK signaling pathway by transcription-dependent and -independent mechanisms SCIENCE Bonni, A., Brunet, A., West, A. E., Datta, S. R., Takasu, M. A., Greenberg, M. E. 1999; 286 (5443): 1358-1362

  Abstract

  A mechanism by which the Ras-mitogen-activated protein kinase (MAPK) signaling pathway mediates growth factor-dependent cell survival was characterized. The MAPK-activated kinases, the Rsks, catalyzed the phosphorylation of the pro-apoptotic protein BAD at serine 112 both in vitro and in vivo. The Rsk-induced phosphorylation of BAD at serine 112 suppressed BAD-mediated apoptosis in neurons. Rsks also are known to phosphorylate the transcription factor CREB (cAMP response element-binding protein) at serine 133. Activated CREB promoted cell survival, and inhibition of CREB phosphorylation at serine 133 triggered apoptosis. These findings suggest that the MAPK signaling pathway promotes cell survival by a dual mechanism comprising the posttranslational modification and inactivation of a component of the cell death machinery and the increased transcription of pro-survival genes.

  View details for Web of Science ID 000083675500042

  View details for PubMedID 10558990

• Akt promotes cell survival by phosphorylating and inhibiting a forkhead transcription factor CELL Brunet, A., Bonni, A., Zigmond, M. J., Lin, M. Z., Juo, P., Hu, L. S., ANDERSON, M. J., Arden, K. C., Blenis, J., Greenberg, M. E. 1999; 96 (6): 857-868

  Abstract

  Survival factors can suppress apoptosis in a transcription-independent manner by activating the serine/ threonine kinase Akt, which then phosphorylates and inactivates components of the apoptotic machinery, including BAD and Caspase 9. In this study, we demonstrate that Akt also regulates the activity of FKHRL1, a member of the Forkhead family of transcription factors. In the presence of survival factors, Akt phosphorylates FKHRL1, leading to FKHRL1's association with 14-3-3 proteins and FKHRL1's retention in the cytoplasm. Survival factor withdrawal leads to FKHRL1 dephosphorylation, nuclear translocation, and target gene activation. Within the nucleus, FKHRL1 triggers apoptosis most likely by inducing the expression of genes that are critical for cell death, such as the Fas ligand gene.

  View details for Web of Science ID 000079300100012

  View details for PubMedID 10102273

• Nuclear translocation of p42/p44 mitogen-activated protein kinase is required for growth factor-induced gene expression and cell cycle entry EMBO JOURNAL Brunet, A., Roux, D., Lenormand, P., Dowd, S., Keyse, S., Pouyssegur, J. 1999; 18 (3): 664-674

  Abstract

  Mitogen-activated protein kinase (MAPK) modules, composed of three protein kinases activated by successive phosphorylation, are involved in the signal transduction of a wide range of extracellular agents. In mammalian cells, mitogenic stimulation triggers the translocation of p42/p44MAPK from the cytoplasm to the nucleus, whereas the other protein kinases of the module remain cytosolic. Since MAPK has been shown to phosphorylate and activate nuclear targets, such as the transcription factor Elk1, it has been proposed, but not yet demonstrated, that MAPK nuclear translocation could represent a critical step in signal transduction. In this study, we sequestered p42/p44MAPK in the cytoplasm by the expression of a catalytically inactive form of cytoplasmic MAP kinase phosphatase (MKP-3/Pyst-1). Sequestering MAPK in the cytoplasm did not alter its activation or its ability to phosphorylate cytoplasmic substrates of MAPK (p90RSK1 or an engineered cytoplasmic form of Elk1). In contrast, prevention of MAPK nuclear translocation strongly inhibited Elk1-dependent gene transcription and the ability of cells to reinitiate DNA replication in response to growth factors. Thus the relocalization of MAPK to the nucleus appears to be an important regulatory step for mitogen-induced gene expression and cell cycle re-entry.

  View details for Web of Science ID 000078597500017

  View details for PubMedID 9927426

• Growth factor-induced p42/p44 MAPK nuclear translocation and retention requires both MAPK activation and neosynthesis of nuclear anchoring proteins JOURNAL OF CELL BIOLOGY Lenormand, P., Brondello, J. M., Brunet, A., Pouyssegur, J. 1998; 142 (3): 625-633

  Abstract

  Mitogen-activated protein kinases (p42/p44 MAPK, also called Erk2 and Erk1) are key mediators of signal transduction from the cell surface to the nucleus. We have previously shown that the activation of p42/p44 MAPK required for transduction of mitogenic signaling is associated with a rapid nuclear translocation of these kinases. However, the means by which p42 and p44 MAPK translocate into the nucleus after cytoplasmic activation is still not understood and cannot simply be deduced from their protein sequences. In this study, we have demonstrated that activation of the p42/ p44 MAPK pathway was necessary and sufficient for triggering nuclear translocation of p42 and p44 MAPK. First, addition of the MEK inhibitor PD 98059, which blocks activation of the p42/p44 MAPK pathway, impedes the nuclear accumulation, whereas direct activation of the p42/p44 MAPK pathway by the chimera DeltaRaf-1:ER is sufficient to promote nuclear accumulation of p42/p44 MAPK. In addition, we have shown that this nuclear accumulation of p42/p44 MAPK required the neosynthesis of short-lived proteins. Indeed, inhibitors of protein synthesis abrogate nuclear accumulation in response to serum and accelerate p42/p44 MAPK nuclear efflux under conditions of persistent p42/p44 MAPK activation. In contrast, inhibition of targeted proteolysis by the proteasome synergistically potentiated p42/p44 MAPK nuclear localization by nonmitogenic agonists and markedly prolonged nuclear localization of p42/p44 MAPK after mitogenic stimulation. We therefore conclude that the MAPK nuclear translocation requires both activation of the p42/p44 MAPK module and neosynthesis of short-lived proteins that we postulate to be nuclear anchors.

  View details for Web of Science ID 000075415700003

  View details for PubMedID 9700154

• Signal transduction pathways from the membrane to the nucleus: variations on common themes BULLETIN DU CANCER Brunet, A. 1998; 85 (6): 527-537

  Abstract

  During their life, cells are exposed to a wide variety of extracellular stimuli and have to develop appropriate biological responses. Signal transduction from the plasma membrane, which is in contact with the extracellular environment, to the nucleus, where gene expression is achieved, thus represents a fundamental process for the development and maintenance of life in organisms. Signalling pathways are extremely diverse and range from direct strategies, such as the steroid hormone receptor and JAK/STAT (signal transducers and activators of transcription) pathways, to multi-step strategies, such as the NF-kappa B (nuclear factor kappa B), PKA (protein kinase A) and Ras/MAPK (mitogen-activated protein kinase) pathways. In order to modulate gene expression, all these pathways must ultimately achieve nuclear localization. The mechanisms by which these varied signalling components cross the nuclear envelope are equally as diverse. However, despite the variety of the means used, cells have adopted several common themes for signal transduction, particularly interaction between proteins as a mean to transport the signal and phosphorylation as a post-translational modification carrying information. Finally, all signalling pathways have been conserved throughout evolution, inghlighting their advantage for cells. In mammals, proteins that participate in signal transmission represent a frequent target for mutations leading to tumor development. Unraveling signalling pathways thus represents an important step in the fight against cancer.

  View details for Web of Science ID 000074999500006

  View details for PubMedID 9752280

• Inhibition of the mitogen-activated protein kinase pathway triggers B16 melanoma cell differentiation JOURNAL OF BIOLOGICAL CHEMISTRY Englaro, W., Bertolotto, C., Busca, R., Brunet, A., Pages, G., Ortonne, J. P., Ballotti, R. 1998; 273 (16): 9966-9970

  Abstract

  In B16 melanoma cells, mitogen-activated protein (MAP) kinases are activated during cAMP-induced melanogenesis (Englaro, W., Rezzonico, R., Durand-Clément, M., Lallemand, D., Ortonne, J. P., and Ballotti, R. (1995) J. Biol. Chem. 270, 24315-24320). To establish the role of the MAP kinases in melanogenesis, we studied the effects of a specific MAP kinase kinase (MEK) inhibitor PD 98059 on different melanogenic parameters. We showed that PD 98059 inhibits the activation of MAP kinase extracellular signal-regulated kinase 1 by cAMP, but does not impair the effects of cAMP either on the morphological differentiation, characterized by an increase in dendrite outgrowth, or on the up-regulation of tyrosinase that is the key enzyme in melanogenesis. On the contrary, PD 98059 promotes by itself cell dendricity and increases the tyrosinase amount and activity. Moreover, down-regulation of the MAP kinase pathway by PD 98059, or with dominant negative mutants of p21(ras) and MEK, triggers a stimulation of the tyrosinase promoter activity and enhances the effect of cAMP on this parameter. Conversely, activation of the MAP kinase pathway, using constitutive active mutants of p21(ras) and MEK, leads to an inhibition of basal and cAMP-induced tyrosinase gene transcription. These results demonstrate that the MAP kinase pathway activation is not required for cAMP-induced melanogenesis. Furthermore, the inhibition of this pathway induces B16 melanoma cell differentiation, while a sustained activation impairs the melanogenic effect of cAMP-elevating agents.

  View details for Web of Science ID 000073128800087

  View details for PubMedID 9545341

• Involvement of extracellular signal-regulated kinase module in HIV-mediated CD4 signals controlling activation of nuclear factor-kappa B and AP-1 transcription factors JOURNAL OF IMMUNOLOGY Briant, L., Robert-Hebmann, V., Sivan, V., Brunet, A., Pouyssegur, J., Devaux, C. 1998; 160 (4): 1875-1885

  Abstract

  Although the molecular mechanisms by which the HIV-1 triggers either T cell activation, anergy, or apoptosis remain poorly understood, it is well established that the interaction of HIV-1 envelope glycoproteins with cell surface CD4 delivers signals to the target cell, resulting in activation of transcription factors such as NF-kappa B and AP-1. In this study, we report the first evidence indicating that kinases MEK-1 (MAP kinase/Erk kinase) and ERK-1 (extracellular signal-regulated kinase) act as intermediates in the cascade of events that regulate NF-kappa B and AP-1 activation upon HIV-1 binding to cell surface CD4. We found that CEM cells transfected with dominant negative forms of MEK-1 or ERK-1 do not display NF-kappa B activation after HIV-1 binding to CD4. In contrast, NF-kappa B activation was observed in these cells after PMA stimulation. Although the different cell lines studied expressed similar amounts of CD4 and p56(lck), HIV-1 replication and HIV-1-induced apoptosis were slightly delayed in cells expressing dominant negative forms of MEK-1 or ERK-1 compared with parental CEM cells and cells expressing a constitutively active mutant form of MEK-1 or wild-type ERK-1. In light of recently published data, we propose that a positive signal initiated following oligomerization of CD4 by the virus is likely to involve a recruitment of active forms of p56(lck), Raf-1, MEK-1, and ERK-1, before AP-1 and NF-kappa B activation.

  View details for Web of Science ID 000073704800042

  View details for PubMedID 9469449

• The dual specificity mitogen-activated protein kinase phosphatase-1 and -2 are induced by the p42/p44(MAPK) cascade JOURNAL OF BIOLOGICAL CHEMISTRY Brondello, J. M., Brunet, A., Pouyssegur, J., McKenzie, F. R. 1997; 272 (2): 1368-1376

  Abstract

  Mitogen-activated protein (MAP) kinase phosphatase-1 (MKP-1) and MKP-2 are two members of a recently described family of dual specificity phosphatases that are capable of dephosphorylating p42/p44MAPK. Overexpression of MKP-1 or MKP-2 inhibits MAP kinase-dependent intracellular signaling events and fibroblast proliferation. By using specific antibodies that recognize endogenous MKP-1 and MKP-2 in CCL39 cells, we show that MKP-1 and MKP-2 are not expressed in quiescent cells, but are rapidly induced following serum addition, with protein detectable as early as 30 min (MKP-1) or 60 min (MKP-2). Serum induction of MKP-1 and MKP-2 is sustained, with protein detectable up to 14 h after serum addition. Induction of MKP-1 and, to a lesser extent, MKP-2 temporally correlates with p42/p44MAPK inactivation. To analyze the contribution of the MAP kinase cascade to MKP-1 and MKP-2 induction, we examined CCL39 cells transformed with either v-ras or a constitutively active direct upstream activator of MAP kinase, mitogen-activated protein kinase kinase-1 (MKK-1; MKK-1(SD/SD) mutant). In both cell models, MKP-1 and MKP-2 are constitutively expressed, with MKP-2 being prevalent. In addition, in CCL39 cells expressing an estradiol-inducible deltaRaf-1::ER chimera, activation of Raf alone is sufficient to induce MKP-1 and MKP-2. The role of the MAP kinase cascade in MKP induction was highlighted by the MKK-1 inhibitor PD 098059, which blunted both the activation of p42/p44MAPK and the induction of MKP-1 and MKP-2. However, the MAP kinase cascade is not absolutely required for the induction of MKP-1, as this phosphatase, but not MKP-2, was induced to detectable levels by agents that stimulate protein kinases A and C. Thus, activation of the p42/p44MAPK cascade promotes the induction of MKP-1 and MKP-2, which may then attenuate p42/p44MAPK-dependent events in an inhibitory feedback loop.

  View details for Web of Science ID A1997WC04800097

  View details for PubMedID 8995446

• Mammalian MAP kinase modules: how to transduce specific signals ESSAYS IN BIOCHEMISTRY, VOL 32, 1997 Brunet, A., Pouyssegur, J. 1997; 32: 1-16

  Abstract

  MAPK modules are composed of a cascade of three intracellular protein kinases (MKKK, MKK and MAPK) which are activated successively by phosphorylation events. They are used to transduce a variety of information in organisms as diverse as yeasts, worms, flies or mammals. MAPK modules integrate signals coming from membrane receptor activation and, by the ability of MAPK to translocate into the nucleus and phosphorylate nuclear targets such as transcription factors, they relay extracellular signals into a genomic response. Since several MAPK modules transducing different information are expressed in the same cell, in yeast or in mammals, the question arises as to how fidelity is maintained between the distinct MAPK modules of a single cell. Two levels of specificity have been documented: the molecular selectivity of each enzyme for its substrate, which is particularly evident for the MKK-MAPK couple, permits specificity within one particular module; exogenous proteins, such as the yeast Ste5 protein, may serve as 'chaperone' proteins to tether all the members of a module and restrict signal transduction to this module. In mammalian cells, the MAPK modules are not strictly independent and one pathway may interfere with another. It remains to be determined whether this interference is of physiological relevance.

  View details for Web of Science ID 000072180100001

  View details for PubMedID 9493007

• Cyclin D1 expression is regulated positively by the p42/p44(MAPK) and negatively by the p38/HOG(MAPK) pathway JOURNAL OF BIOLOGICAL CHEMISTRY Lavoie, J. N., LALLEMAIN, G., Brunet, A., Muller, R., Pouyssegur, J. 1996; 271 (34): 20608-20616

  Abstract

  We have previously shown that the persistent activation of p42/p44(MAPK) is required to pass the G1 restriction point in fibroblasts (Pagès, G., Lenormand, P., L'Allemain, G., Chambard, J. C., Meloche, S., and Pouysségur, J. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 8319-8323) and postulated that MAPKs control the activation of G1 cyclin-dependent complexes. We examined the mitogen-dependent induction of cyclin D1 expression, one of the earliest cell cycle-related events to occur during the G0/G1 to S-phase transition, as a potential target of MAPK regulation. Effects exerted either by the p42/p44(MAPK) or the p38/HOGMAPK cascade on the regulation of cyclin D1 promoter activity or cyclin D1 expression were compared in CCL39 cells, using a co-transfection procedure. We found that inhibition of the p42/p44(MAPK) signaling by expression of dominant-negative forms of either mitogen-activated protein kinase kinase 1 (MKK1) or p44(MAPK), or by expression of the MAP kinase phosphatase, MKP-1, strongly inhibited expression of a reporter gene driven by the human cyclin D1 promoter as well as the endogenous cyclin D1 protein. Conversely, activation of this signaling pathway by expression of a constitutively active MKK1 mutant dramatically increased cyclin D1 promoter activity and cyclin D1 protein expression, in a growth factor-independent manner. Moreover, the use of a CCL39-derived cell line that stably expresses an inducible chimera of the estrogen receptor fused to a constitutively active Raf-1 mutant (DeltaRaf-1:ER) revealed that in absence of growth factors, activation of the Raf > MKK1 > p42/p44MAPK cascade is sufficient to fully induce cyclin D1. In marked contrast, the p38(MAPK) cascade showed an opposite effect on the regulation of cyclin D1 expression. In cells co-expressing high levels of the p38(MAPK) kinase (MKK3) together with the p38(MAPK), a significant inhibition of mitogen-induced cyclin D1 expression was observed. Furthermore, inhibition of p38(MAPK) activity with the specific inhibitor, SB203580, enhanced cyclin D1 transcription and protein level. Altogether, these results support the notion that MAPK cascades drive specific cell cycle responses to extracellular stimuli, at least in part, through the modulation of cyclin D1 expression and associated cdk activities.

  View details for Web of Science ID A1996VD33700059

  View details for PubMedID 8702807

• Identification of MAP kinase domains by redirecting stress signals into growth factor responses SCIENCE Brunet, A., Pouyssegur, J. 1996; 272 (5268): 1652-1655

  Abstract

  Mitogen-activated protein kinase (MAPK) cascades, termed MAPK modules, channel extracellular signals into specific cellular responses. Chimeric molecules were constructed between p38 and p44 MAPKs, which transduce stress and growth factor signals, respectively. A discrete region of 40 residues located in the amono-terminal p38MAPK lobe directed the specificity of response to extracellular signals, whereas the p44MAPK chimera, expressed in vivo, redirected stress signals into early mitogenic responses, demonstrating the functional independence of these domains.

  View details for Web of Science ID A1996UR09300047

  View details for PubMedID 8658140

• THE MOUSE P44 MITOGEN-ACTIVATED PROTEIN-KINASE (EXTRACELLULAR SIGNAL-REGULATED KINASE-1) GENE - GENOMIC ORGANIZATION AND STRUCTURE OF THE 5'-FLANKING REGULATORY REGION JOURNAL OF BIOLOGICAL CHEMISTRY Pages, G., Stanley, E. R., Le Gall, M., Brunet, A., Pouyssegur, J. 1995; 270 (45): 26986-26992

  Abstract

  Mitogen-activated protein kinase (MAPK) or extracellular signal-regulated kinase are ubiquitous kinases conserved from fungi to mammals. Their activity is regulated by phosphorylation on both threonine and tyrosine, and they play a crucial role in the regulation of proliferation and differentiation. We report here the cloning of the murine p44 MAP kinase (extracellular signal-regulated kinase 1) gene, the determination of its intron/exon boundaries, and the characterization of its promoter. The gene spans approximately eight kilobases (kb) and can be divided into nine exons and eight introns, each coding region exon containing from one to three of the highly conserved protein kinase domains. Primer extension analysis reveals the existence of two major start sites of transcription located at -183 and -186 base pairs (bp) as well as four discrete start sites for transcription located at -178, -192, -273, and -292 bp of the initiation of translation. However, the start site region lacks TATA-like sequences but does contain initiator-like sequences proximal to the major start sites obtained by primer extension. 1 kb of the promoter region has been sequenced. It contains three putative TATA boxes far upstream of the main start sites region, one AP-1 box, one AP-2 box, one Malt box, one GAGA box, one half serum-responsive element, and putative binding sites for Sp1 (five), GC-rich binding factor (five), CTF-NF1 (one), Myb (one), p53 (two), Ets-1 (one), NF-IL6 (two), MyoD (two), Zeste (one), and hepatocyte nuclear factor-5 (one). To determine the sites critical for the function of the p44 MAPK promoter, we constructed a series of chimeric genes containing variable regions of the 5'-flanking sequence of p44 MAPK gene and the coding region for luciferase. Activity of the promoter, measured by its capacity to direct expression of a luciferase reporter gene, is strong, being comparable with the activity of the Rous sarcoma virus promoter. Progressive deletions of the approximately 1 kb (-1200/-78) promoter region allowed us to define a minimal region of 186 bp (-284/-78) that has maximal promoter activity. Within this context, deletion of the AP-2 binding site reduces by 30-40% the activity of the promoter. Further deletion of this minimal promoter that removes the major start sites (-167/-78) surprisingly preserves promoter activity. This result implicates a major role of this region that contains the Sp1 sites.(ABSTRACT TRUNCATED AT 400 WORDS)

  View details for Web of Science ID A1995TE58300046

  View details for PubMedID 7592946

• B-RAF PROTEIN ISOFORMS INTERACT WITH AND PHOSPHORYLATE MEK-1 ON SERINE RESIDUE-218 AND RESIDUE-222 ONCOGENE Papin, C., Eychene, A., Brunet, A., Pages, G., Pouyssegur, J., Calothy, G., Barnier, J. V. 1995; 10 (8): 1647-1651

  Abstract

  The B-raf/c-Rmil proto-oncogene belongs to the raf/mil family of serine/threonine protein kinases. It encodes multiple protein isoforms resulting from alternative splicing of two exons located upstream of the kinase domain. Recent studies suggested that B-Raf could be the intermediate molecule between Ras and Mek-1 (MAP Kinase Kinase) in signalling pathways specific of neural cells. However, there has been no evidence for a direct interaction between B-Raf and Mek-1. We report here that different B-Raf isoforms can be co-immunoprecipitated with anti-Mek-1 antisera in COS-1 cells and that the kinase activity of B-Raf is not required for its interaction with Mek-1. We also show that all B-Raf isoforms tested phosphorylate Mek-1 in a time-dependent manner, whereas kinase defective mutants fail to do so. Finally, we demonstrate that the constitutively activated S218D, S222D and S218D/S222D mutants of Mek-1 interact similarly with B-Raf. However, only the S218D and S222D mutants, and not the S218D/S222D double mutant, can be phosphorylated by B-Raf isoforms. Therefore, serine residues 218 and 222, previously shown to regulate Mek-1 activity, appear to be the major phosphorylation sites by B-Raf in vitro.

  View details for Web of Science ID A1995QU68100022

  View details for PubMedID 7731720

• [MAP kinase module: role in the control of cell proliferation]. Comptes rendus des séances de la Société de biologie et de ses filiales Brunet, A., Brondello, J. M., L'Allemain, G., Lenormand, P., McKenzie, F., Pagès, G., Pouysségur, J. 1995; 189 (1): 43-57

  Abstract

  A kinase cascade highly conserved throughout evolution, Raf/MAP kinase kinase kinase (MAPKKK)-->MAP kinase kinase (MAPKK)-->MAP kinase (MAPK)-->ribosomal S6 kinase (p90 RSK), is thought to play a crucial role in signal transduction from the membrane to the nucleus. In mammalian cells, this cascade is connected both to tyrosine kinase receptors and G protein-coupled receptors. Although the mode of activation at the receptor level differs, all mitogens activate the ubiquitously expressed isoforms of MAPK, p42 and p44. We have cloned, epitope tagged and expressed in fibroblasts, the Hamster MAPKK and p44 MAPK in order to analyze their time-course of activation, their subcellular localization, their regulatory phosphorylation sites and their role in cell cycle entry. We have demonstrated that MAPK activation was rapid, biphasic and persistent. The sustained phase of activation is only obtained with potent mitogenic agents, correlating with their ability to elicit cell cycle entry. Activation of MAPKK is also rapid and persistent but does not distinguish between mitogenic and non mitogenic factors, indicating that a distinction occurs at the MAPK level, probably by the action of specific phosphatases such as MAPK phosphatase MKP-1. Both isoforms of MAPK are translocated into the nucleus upon growth factor addition whereas the upstream activators (MAPKKK, Raf and MAPKK) remain cytoplasmic. MAPK translocation, together with the ability of MAPK to phosphorylate transcription factors, indicates that MAPK might constitute a relay between cytoplasmic and nuclear events. Finally we show that interfering with the MAP kinase cascade, by expressing either MAPK antisense, a MAPK dominant negative mutant or the MAPK specific phosphatase, MKP-1, suppresses the growth factor induced G0 to G1 transition. In addition, permanently activated versions of MAPKK reduce growth factor requirement, allow autonomous cell growth and induce tumor formation in nude mice. We therefore conclude that MAP kinase activation is both necessary and sufficient to trigger cell cycle entry.

  View details for PubMedID 7648366

• CONSTITUTIVELY ACTIVE MUTANTS OF MAP KINASE KINASE (MEK1) INDUCE GROWTH FACTOR-RELAXATION AND ONCOGENICITY WHEN EXPRESSED IN FIBROBLASTS ONCOGENE Brunet, A., Pages, G., Pouyssegur, J. 1994; 9 (11): 3379-3387

  Abstract

  The Mitogen Activated Protein Kinase (MAPK) module operates downstream of Ras to convey cell surface signals to the nucleus via the nuclear translocation of p42/p44 MAPKs. We have previously established that MAPK activation is obligatory and must persist in the G1 phase to allow resting fibroblasts to exit from G0 (Pagès et al., Proc. Natl. Acad. Sci.1993, 90, 8319-8323). It remained to be established whether MAPK activation was sufficient to trigger cell proliferation. To this aim, we generated and expressed in Chinese hamster lung fibroblasts, constitutively active mutants of hamster MAP kinase kinase (MAPKK). Three mutants: S218D, S222D and S218D/S222D in which we substituted the Raf1/MAPKKK-dependent regulatory phosphorylation sites by aspartic acid residues, displayed increased basal activity when expressed in fibroblasts. Two of them, S218D and S218D/S222D which have a basal activity higher than serum-stimulated wild type-MAPKK (respectively 2- and 5-fold), induced activation of p42 MAPK in growth factor-deprived cells. Interestingly, only these two mutants led to a growth factor-independent state as judged by early gene transcription (activation of the fos promoter), increased sensitivity to growth factors for reinitiation of DNA synthesis, autonomous cell cycling and rapid tumor formation in nude mice. Therefore we conclude that the downstream elements of the growth factor signalling cascade, MAPKK-MAPK, are both necessary and sufficient to promote growth factor signals and autonomous cell cycling in fibroblasts.

  View details for Web of Science ID A1994PM65800035

  View details for PubMedID 7936666

• CONSTITUTIVE MUTANT AND PUTATIVE REGULATORY SERINE PHOSPHORYLATION SITE OF MAMMALIAN MAP KINASE KINASE (MEK1) EMBO JOURNAL Pages, G., Brunet, A., LALLEMAIN, G., Pouyssegur, J. 1994; 13 (13): 3003-3010

  Abstract

  In response to various external stimuli, MAP kinases are activated by phosphorylation on tyrosine and threonine by MAP kinase kinase (MAPKK), a dual specificity kinase. This kinase is in turn activated via Raf-1 and MAPKK kinase (MAPKKK). To determine regulatory phosphorylation sites of MAPKK, we isolated a Chinese hamster cDNA, that we epitope-tagged and expressed in fibroblasts. This hamster MAPKK (MEK1 isoform) can reactivate recombinant p44mapk when immunoprecipitated from growth factor-stimulated cells or when incubated with an active form of MAPKKK. Mutations at either of two residues that are conserved among kinases, D208N or S222A, abolished MAPKK activity. However, only S222A/MAPKK showed a reduction in phosphorylation in response to active MAPKKK and exerted a dominant negative effect on the serum-stimulated endogenous MAPKK. Finally, replacing Ser222 with Asp, a negatively charged residue, restored MAPKK activity independently of the upstream kinase. These results strongly suggest that Ser222 represents one key MAPKKK-dependent phosphorylation site switching on and off the activity of MAPKK, an event crucial for growth control.

  View details for Web of Science ID A1994NW77100007

  View details for PubMedID 8039496

• GROWTH FACTOR-STIMULATED MAP KINASE INDUCES RAPID RETROPHOSPHORYLATION AND INHIBITION OF MAP KINASE KINASE (MEK1) FEBS LETTERS Brunet, A., Pages, G., Pouyssegur, J. 1994; 346 (2-3): 299-303

  Abstract

  The MAP kinase module (Raf/MAPKKK-MAPKK-MAPK) has been shown to be sequentially activated after mitogenic stimulation. Here we demonstrate, by site directed mutagenesis, that MAPK is able to retrophosphorylate its own activator, MAPKK, on two threonine residues Thr-292 and Thr-386 in vitro, and that these sites are also phosphorylated in vivo. A comparison of the kinetics of serum-mediated activation of a wild-type MAPKK and of a mutant unable to undergo phosphorylation by MAPK suggests that this retrophosphorylation may be involved in a negative feedback control of the cascade in vivo.

  View details for Web of Science ID A1994NR56900034

  View details for PubMedID 8013650

• GROWTH-FACTORS INDUCE NUCLEAR TRANSLOCATION OF MAP KINASES (P42(MAPK) AND P44(MAPK)) BUT NOT OF THEIR ACTIVATOR MAP KINASE KINASE (P45(MAPKK)) IN FIBROBLASTS JOURNAL OF CELL BIOLOGY Lenormand, P., Sardet, C., Pages, G., LALLEMAIN, G., Brunet, A., Pouyssegur, J. 1993; 122 (5): 1079-1088

  Abstract

  Mitogen-activated protein kinases (p42mapk and p44mapk) are serine/threonine kinases that are activated rapidly in cells stimulated with various extracellular signals. This activation is mediated via MAP kinase kinase (p45mapkk), a dual specificity kinase which phosphorylates two key regulatory threonine and tyrosine residues of MAP kinases. We reported previously that the persistent phase of MAP kinase activation is essential for mitogenically stimulated cells to pass the "restriction point" of the cell cycle. Here, using specific polyclonal antibodies and transfection of epitope-tagged recombinant MAP kinases we demonstrate that these signaling protein kinases undergo distinct spatio-temporal localization in growth factor-stimulated cells. In G0-arrested hamster fibroblasts the activator p45mapkk and MAP kinases (p42mapk, p44mapk) are mainly cytoplasmic. Subsequent to mitogenic stimulation by serum or alpha-thrombin both MAP kinase isoforms translocate into the nucleus. This translocation is rapid (seen in 15 min), persistent (at least during the entire G1 period up to 6 h), reversible (by removal of the mitogenic stimulus) and apparently 'coupled' to the mitogenic potential; it does not occur in response to nonmitogenic agents such as alpha-thrombin-receptor synthetic peptides and phorbol esters that fail to activate MAP kinases persistently. When p42mapk and p44mapk are expressed stably at high levels, they are found in the nucleus of resting cells; this nuclear localization is also apparent with kinase-deficient mutants (p44mapk T192A or Y194F). In marked contrast the p45mapkk activator remains cytoplasmic even during prolonged growth factor stimulation and even after high expression levels achieved by transfection. We propose that the rapid and persistent nuclear transfer of p42mapk and p44mapk during the entire G0-G1 period is crucial for the function of these kinases in mediating the growth response.

  View details for Web of Science ID A1993LU31000010

  View details for PubMedID 8394845

• MAP KINASE CASCADE - AN ESSENTIAL SIGNALING ROUTE THAT CONTROLS CELL-PROLIFERATION 3rd International Conference on Negative Regulation of Hematopoiesis Pouyssegur, J., Brunet, A., CHAMBARD, J. C., LALLEMAIN, G., Lenormand, P., Pages, G. EDITIONS INSERM. 1993: 55–63

  View details for Web of Science ID A1993BZ85R00009

• MAP KINASE CASCADE AND THE CONTROL OF CELL-PROLIFERATION INTERNATIONAL CONF ON CANCER : BIOLOGICAL MECHANISMS AND CLINICAL APPLICATIONS Pages, G., Brunet, A., CHAMBARD, J. C., LALLEMAIN, G., Lenormand, P., Pouyssegur, J. BIRKHAUSER VERLAG. 1993: 153–164

  View details for Web of Science ID A1993BY70K00018