Integrated genomic analysis of AgRP neurons reveals that IRF3 regulates leptin’s hunger-suppressing effects

AgRP neurons in the arcuate nucleus of the hypothalamus (ARC) coordinate homeostatic changes in appetite associated with fluctuations in food availability and leptin signaling. Identifying the relevant transcriptional regulatory pathways in these neurons has been a priority, yet such attempts have been stymied due to their low abundance and the rich cellular diversity of the ARC. Here we generated AgRP neuron-specific transcriptomic and chromatin accessibility profiles during opposing states of fasting-induced hunger and leptin-induced hunger suppression. Cis-regulatory analysis of these integrated datasets enabled the identification of 28 putative hunger-promoting and 29 putative hunger-suppressing transcriptional regulators in AgRP neurons, 16 of which were predicted to be transcriptional effectors of leptin. Within our dataset, Interferon regulatory factor 3 (IRF3) emerged as a leading candidate mediator of leptin-induced hunger-suppression. Gain- and loss-of-function experiments in vivo confirm the role of IRF3 in mediating the acute satiety-evoking effects of leptin in AgRP neurons, while live-cell imaging in vitro indicate that leptin can activate neuronal IRF3 in a cell autonomous manner. Finally, we employ CUT&RUN to uncover direct transcriptional targets of IRF3 in AgRP neurons in vivo. Thus, our findings identify AgRP neuron-expressed IRF3 as a key transcriptional effector of the hunger-suppressing effects of leptin.

[1]  R. Harris.  Phosphorylation of STAT3 in hypothalamic nuclei is stimulated by lower doses of leptin than are needed to inhibit food intake. , 2021, American journal of physiology. Endocrinology and metabolism.

[2]  M. Kumari,et al.  IRF3 reduces adipose thermogenesis via ISG15-mediated reprogramming of glycolysis. , 2021, The Journal of clinical investigation.

[3]  J. Rabinowitz,et al.  The hepatocyte clock and feeding control chronophysiology of multiple liver cell types , 2020, Science.

[4]  Christopher P. Davis,et al.  An Activity-Mediated Transition in Transcription in Early Postnatal Neurons , 2020, Neuron.

[5]  M. Low,et al.  The transcriptional regulator PRDM12 is critical for Pomc expression in the mouse hypothalamus and controlling food intake, adiposity, and body weight , 2020, Molecular metabolism.

[6]  M. Myers,et al.  Identification of the leptin receptor sequences crucial for the STAT3-Independent control of metabolism , 2020, Molecular metabolism.

[7]  Steven Henikoff,et al.  Improved CUT&RUN chromatin profiling tools , 2019, eLife.

[8]  H. Bluyssen,et al.  Direct Inhibition of IRF-Dependent Transcriptional Regulatory Mechanisms Associated With Disease , 2019, Front. Immunol..

[9]  J. Takahashi,et al.  Transcriptional Basis for Rhythmic Control of Hunger and Metabolism within the AgRP Neuron. , 2019, Cell metabolism.

[10]  Karl K. Murphy,et al.  Genomic and epigenomic mapping of leptin-responsive neuronal populations involved in body weight regulation , 2019, Nature Metabolism.

[11]  Michael E. Greenberg,et al.  Activity-Regulated Transcription: Bridging the Gap between Neural Activity and Behavior , 2018, Neuron.

[12]  C. Saper,et al.  A Glutamatergic Hypothalamomedullary Circuit Mediates Thermogenesis, but Not Heat Conservation, during Stress-Induced Hyperthermia , 2018, Current Biology.

[13]  M. Kumari,et al.  Warming Induces Significant Reprogramming of Beige, but Not Brown, Adipocyte Cellular Identity. , 2018, Cell metabolism.

[14]  Martha L. Bulyk,et al.  Direct Promoter Repression by BCL11A Controls the Fetal to Adult Hemoglobin Switch , 2018, Cell.

[15]  D. Kong,et al.  Genetic identification of leptin neural circuits in energy and glucose homeostases , 2018, Nature.

[16]  Alan C. Rupp,et al.  Defining the Transcriptional Targets of Leptin Reveals a Role for Atf3 in Leptin Action , 2018, Diabetes.

[17]  Yiming Chen,et al.  Dynamics of Gut-Brain Communication Underlying Hunger , 2017, Neuron.

[18]  S. Henikoff,et al.  Targeted in situ genome-wide profiling with high efficiency for low cell numbers , 2018, Nature Protocols.

[19]  M. Andermann,et al.  Toward a Wiring Diagram Understanding of Appetite Control , 2017, Neuron.

[20]  H. Aburatani,et al.  NFIA co-localizes with PPARγ and transcriptionally controls the brown fat gene program , 2017, Nature Cell Biology.

[21]  Jing Wang,et al.  WebGestalt 2017: a more comprehensive, powerful, flexible and interactive gene set enrichment analysis toolkit , 2017, Nucleic Acids Res..

[22]  G. Ming,et al.  Neuronal activity modifies the chromatin accessibility landscape in the adult brain , 2017, Nature Neuroscience.

[23]  Evan Z. Macosko,et al.  A Molecular Census of Arcuate Hypothalamus and Median Eminence Cell Types , 2017, Nature Neuroscience.

[24]  D. Tenen,et al.  IRF3 promotes adipose inflammation and insulin resistance and represses browning. , 2016, The Journal of clinical investigation.

[25]  D. Marks,et al.  A distinct brain pathway links viral RNA exposure to sickness behavior , 2016, Scientific Reports.

[26]  A. Hoffmann,et al.  Limited specificity of IRF3 and ISGF3 in the transcriptional innate‐immune response to double‐stranded RNA , 2015, Journal of leukocyte biology.

[27]  William Stafford Noble,et al.  The MEME Suite , 2015, Nucleic Acids Res..

[28]  Hye-Ri Kang,et al.  Phosphatidylinositol‐3‐kinase and Akt are required for RIG‐I‐mediated anti‐viral signalling through cross‐talk with IPS‐1 , 2015, Immunology.

[29]  Howard Y. Chang,et al.  ATAC‐seq: A Method for Assaying Chromatin Accessibility Genome‐Wide , 2015, Current protocols in molecular biology.

[30]  Pingwei Li,et al.  The mechanism of double-stranded DNA sensing through the cGAS-STING pathway. , 2014, Cytokine & growth factor reviews.

[31]  D. Allison,et al.  Variations in body weight, food intake and body composition after long-term high-fat diet feeding in C57BL/6J Mice , 2014, Obesity.

[32]  J. Betley,et al.  Parallel, Redundant Circuit Organization for Homeostatic Control of Feeding Behavior , 2013, Cell.

[33]  Sam W. Lee,et al.  Rho-kinase Regulates Energy Balance by Targeting Hypothalamic Leptin Receptor Signaling , 2012, Nature Neuroscience.

[34]  U. Klein,et al.  Transcriptional control of adipose lipid handling by IRF4. , 2011, Cell metabolism.

[35]  S. Akira,et al.  Akt Contributes to Activation of the TRIF-Dependent Signaling Pathways of TLRs by Interacting with TANK-Binding Kinase 1 , 2011, The Journal of Immunology.

[36]  Eric S. Lander,et al.  Comparative Epigenomic Analysis of Murine and Human Adipogenesis , 2010, Cell.

[37]  J. Hurst,et al.  Taming anxiety in laboratory mice , 2010, Nature Methods.

[38]  Davis J. McCarthy,et al.  edgeR: a Bioconductor package for differential expression analysis of digital gene expression data , 2009, Bioinform..

[39]  H. Heng,et al.  Signal transducer and activator of transcription-3 is required in hypothalamic agouti-related protein/neuropeptide Y neurons for normal energy homeostasis. , 2008, Endocrinology.

[40]  G. Barsh,et al.  Collective and individual functions of leptin receptor modulated neurons controlling metabolism and ingestion. , 2008, Endocrinology.

[41]  Colm Cunningham,et al.  The sickness behaviour and CNS inflammatory mediator profile induced by systemic challenge of mice with synthetic double-stranded RNA (poly I:C) , 2007, Brain, Behavior, and Immunity.

[42]  R. Palmiter,et al.  NPY/AgRP Neurons Are Essential for Feeding in Adult Mice but Can Be Ablated in Neonates , 2005, Science.

[43]  Kanji A. Takahashi,et al.  Fasting induces a large, leptin-dependent increase in the intrinsic action potential frequency of orexigenic arcuate nucleus neuropeptide Y/Agouti-related protein neurons. , 2005, Endocrinology.

[44]  T. Horvath,et al.  Disruption of neural signal transducer and activator of transcription 3 causes obesity, diabetes, infertility, and thermal dysregulation. , 2004, Proceedings of the National Academy of Sciences of the United States of America.

[45]  J. Konsman.  The mouse brain in stereotaxic coordinates Second Edition (Deluxe) By Paxinos G. and Franklin, K.B.J., Academic Press, New York, 2001, ISBN 0-12-547637-X , 2003, Psychoneuroendocrinology.

[46]  T. Maniatis,et al.  IKKε and TBK1 are essential components of the IRF3 signaling pathway , 2003, Nature Immunology.

[47]  Alexander S. Banks,et al.  STAT3 signalling is required for leptin regulation of energy balance but not reproduction , 2003, Nature.

[48]  Gary K. Beauchamp,et al.  Food Intake, Water Intake, and Drinking Spout Side Preference of 28 Mouse Strains , 2002, Behavior genetics.

[49]  Thomas D. Schmittgen,et al.  Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. , 2001, Methods.

[50]  T. Taniguchi,et al.  Distinct and Essential Roles of Transcription Factors IRF-3 and IRF-7 in Response to Viruses for IFN-α/β Gene Induction , 2000 .

[51]  A. Loudon,et al.  Leptin actions on food intake and body temperature are mediated by IL-1. , 1999, Proceedings of the National Academy of Sciences of the United States of America.

[52]  C. Mantzoros,et al.  Role of leptin in the neuroendocrine response to fasting , 1996, Nature.

[53]  K P Hummel,et al.  Diabetes, a New Mutafton in the Mouse , 1966, Science.

[54]  T. Maniatis,et al.  IKKepsilon and TBK1 are essential components of the IRF3 signaling pathway. , 2003, Nature immunology.

[55]  Thomas D. Schmittgen,et al.  Analysis of Relative Gene Expression Data Using Real-Time Quantitative PCR and the 2 2 DD C T Method , 2022 .