RESUMEN
Behavioral plasticity is key to animal survival. Harpegnathos saltator ants can switch between worker and queen-like status (gamergate) depending on the outcome of social conflicts, providing an opportunity to study how distinct behavioral states are achieved in adult brains. Using social and molecular manipulations in live ants and ant neuronal cultures, we show that ecdysone and juvenile hormone drive molecular and functional differences in the brains of workers and gamergates and direct the transcriptional repressor Kr-h1 to different target genes. Depletion of Kr-h1 in the brain caused de-repression of "socially inappropriate" genes: gamergate genes were upregulated in workers, whereas worker genes were upregulated in gamergates. At the phenotypic level, loss of Kr-h1 resulted in the emergence of worker-specific behaviors in gamergates and gamergate-specific traits in workers. We conclude that Kr-h1 is a transcription factor that maintains distinct brain states established in response to socially regulated hormones.
Asunto(s)
Hormigas/genética , Ecdisterona/farmacología , Jerarquia Social , Proteínas de Insectos/metabolismo , Neuronas/metabolismo , Sesquiterpenos/farmacología , Conducta Social , Transcriptoma/genética , Animales , Hormigas/efectos de los fármacos , Hormigas/fisiología , Conducta Animal/efectos de los fármacos , Encéfalo/metabolismo , Regulación de la Expresión Génica/efectos de los fármacos , Genoma , Neuronas/efectos de los fármacos , Fenotipo , Proteínas Represoras/metabolismo , Transducción de Señal/efectos de los fármacos , Transcriptoma/efectos de los fármacosRESUMEN
Interferon (IFN)-Is are crucial mediators of antiviral immunity and homeostatic immune system regulation. However, the source of IFN-I signaling under homeostatic conditions is unclear. We discovered that commensal microbes regulate the IFN-I response through induction of IFN-ß by colonic DCs. Moreover, the mechanism by which a specific commensal microbe induces IFN-ß was identified. Outer membrane (OM)-associated glycolipids of gut commensal microbes belonging to the Bacteroidetes phylum induce expression of IFN-ß. Using Bacteroides fragilis and its OM-associated polysaccharide A, we determined that IFN-ß expression was induced via TLR4-TRIF signaling. Antiviral activity of this purified microbial molecule against infection with either vesicular stomatitis virus (VSV) or influenza was demonstrated to be dependent on the induction of IFN-ß. In a murine VSV infection model, commensal-induced IFN-ß regulated natural resistance to virus infection. Due to the physiological importance of IFN-Is, discovery of an IFN-ß-inducing microbial molecule represents a potential approach for the treatment of some human diseases.
Asunto(s)
Inmunidad Innata , Microbiota , Virosis/microbiología , Animales , Bacteroides fragilis/fisiología , Células de la Médula Ósea/efectos de los fármacos , Células de la Médula Ósea/metabolismo , Membrana Celular/efectos de los fármacos , Membrana Celular/metabolismo , Colon/patología , Colon/virología , Células Dendríticas/efectos de los fármacos , Células Dendríticas/metabolismo , Femenino , Regulación de la Expresión Génica/efectos de los fármacos , Glucolípidos/metabolismo , Inmunidad Innata/efectos de los fármacos , Interferón beta/sangre , Interferón beta/metabolismo , Masculino , Ratones Endogámicos C57BL , Microbiota/efectos de los fármacos , Polisacáridos Bacterianos/farmacología , Receptor Toll-Like 4/metabolismo , Vesiculovirus/fisiología , Virosis/genéticaRESUMEN
Insulin receptor (IR) signaling is central to normal metabolic control and dysregulated in prevalent chronic diseases. IR binds insulin at the cell surface and transduces rapid signaling via cytoplasmic kinases. However, mechanisms mediating long-term effects of insulin remain unclear. Here, we show that IR associates with RNA polymerase II in the nucleus, with striking enrichment at promoters genome-wide. The target genes were highly enriched for insulin-related functions including lipid metabolism and protein synthesis and diseases including diabetes, neurodegeneration, and cancer. IR chromatin binding was increased by insulin and impaired in an insulin-resistant disease model. Promoter binding by IR was mediated by coregulator host cell factor-1 (HCF-1) and transcription factors, revealing an HCF-1-dependent pathway for gene regulation by insulin. These results show that IR interacts with transcriptional machinery at promoters and identify a pathway regulating genes linked to insulin's effects in physiology and disease.
Asunto(s)
Regulación de la Expresión Génica , Estudio de Asociación del Genoma Completo , Receptor de Insulina/metabolismo , Animales , Línea Celular Tumoral , Cromatina/metabolismo , Regulación de la Expresión Génica/efectos de los fármacos , Factor C1 de la Célula Huésped/antagonistas & inhibidores , Factor C1 de la Célula Huésped/genética , Factor C1 de la Célula Huésped/metabolismo , Humanos , Insulina/metabolismo , Insulina/farmacología , Hígado/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Regiones Promotoras Genéticas , Unión Proteica , Subunidades de Proteína/metabolismo , Interferencia de ARN , ARN Polimerasa II/metabolismo , ARN Interferente Pequeño/metabolismo , Receptor de Insulina/química , Transducción de Señal/efectos de los fármacosRESUMEN
The proteasome mediates selective protein degradation and is dynamically regulated in response to proteotoxic challenges. SKN-1A/Nrf1, an endoplasmic reticulum (ER)-associated transcription factor that undergoes N-linked glycosylation, serves as a sensor of proteasome dysfunction and triggers compensatory upregulation of proteasome subunit genes. Here, we show that the PNG-1/NGLY1 peptide:N-glycanase edits the sequence of SKN-1A protein by converting particular N-glycosylated asparagine residues to aspartic acid. Genetically introducing aspartates at these N-glycosylation sites bypasses the requirement for PNG-1/NGLY1, showing that protein sequence editing rather than deglycosylation is key to SKN-1A function. This pathway is required to maintain sufficient proteasome expression and activity, and SKN-1A hyperactivation confers resistance to the proteotoxicity of human amyloid beta peptide. Deglycosylation-dependent protein sequence editing explains how ER-associated and cytosolic isoforms of SKN-1 perform distinct cytoprotective functions corresponding to those of mammalian Nrf1 and Nrf2. Thus, we uncover an unexpected mechanism by which N-linked glycosylation regulates protein function and proteostasis.
Asunto(s)
Proteínas de Caenorhabditis elegans/metabolismo , Proteínas de Unión al ADN/metabolismo , Complejo de la Endopetidasa Proteasomal/metabolismo , Factores de Transcripción/metabolismo , Secuencia de Aminoácidos , Animales , Asparagina/metabolismo , Bortezomib/farmacología , Sistemas CRISPR-Cas/genética , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/química , Proteínas de Caenorhabditis elegans/genética , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/genética , Retículo Endoplásmico/metabolismo , Edición Génica , Regulación de la Expresión Génica/efectos de los fármacos , Estrés Oxidativo , Complejo de la Endopetidasa Proteasomal/genética , Subunidades de Proteína/química , Subunidades de Proteína/genética , Subunidades de Proteína/metabolismo , Alineación de Secuencia , Factores de Transcripción/química , Factores de Transcripción/genéticaRESUMEN
Pediatric-onset colitis and inflammatory bowel disease (IBD) have significant effects on the growth of infants and children, but the etiopathogenesis underlying disease subtypes remains incompletely understood. Here, we report single-cell clustering, immune phenotyping, and risk gene analysis for children with undifferentiated colitis, Crohn's disease, and ulcerative colitis. We demonstrate disease-specific characteristics, as well as common pathogenesis marked by impaired cyclic AMP (cAMP)-response signaling. Specifically, infiltration of PDE4B- and TNF-expressing macrophages, decreased abundance of CD39-expressing intraepithelial T cells, and platelet aggregation and release of 5-hydroxytryptamine at the colonic mucosae were common in colitis and IBD patients. Targeting these pathways by using the phosphodiesterase inhibitor dipyridamole restored immune homeostasis and improved colitis symptoms in a pilot study. In summary, comprehensive analysis of the colonic mucosae has uncovered common pathogenesis and therapeutic targets for children with colitis and IBD.
Asunto(s)
Enfermedades Inflamatorias del Intestino/patología , Enfermedades Inflamatorias del Intestino/terapia , Mucosa Intestinal/patología , Antígenos CD/metabolismo , Apirasa/metabolismo , Linfocitos B/efectos de los fármacos , Linfocitos B/inmunología , Muerte Celular/efectos de los fármacos , Microambiente Celular/efectos de los fármacos , Niño , Estudios de Cohortes , Colon/patología , Células Dendríticas/efectos de los fármacos , Células Dendríticas/metabolismo , Dipiridamol/farmacología , Células Endoteliales/efectos de los fármacos , Células Endoteliales/metabolismo , Células Epiteliales/efectos de los fármacos , Células Epiteliales/metabolismo , Células Epiteliales/patología , Fibroblastos/efectos de los fármacos , Fibroblastos/metabolismo , Regulación de la Expresión Génica/efectos de los fármacos , Predisposición Genética a la Enfermedad , Homeostasis/efectos de los fármacos , Humanos , Inmunoglobulina G/sangre , Memoria Inmunológica , Inflamación/patología , Enfermedades Inflamatorias del Intestino/sangre , Enfermedades Inflamatorias del Intestino/genética , Interferón Tipo I/metabolismo , Macrófagos/efectos de los fármacos , Macrófagos/metabolismo , Metilprednisolona/farmacología , Células Mieloides/efectos de los fármacos , Células Mieloides/metabolismoRESUMEN
The central dogma of molecular biology, that DNA is transcribed into RNA and RNA translated into protein, was coined in the early days of modern biology. Back in the 1950s and 1960s, bacterial genetics first opened the way toward understanding life as the genetically encoded interaction of macromolecules. As molecular biology progressed and our knowledge of gene control deepened, it became increasingly clear that expression relied on many more levels of regulation. In the process of dissecting mechanisms of gene expression, specific small-molecule inhibitors played an important role and became valuable tools of investigation. Small molecules offer significant advantages over genetic tools, as they allow inhibiting a process at any desired time point, whereas mutating or altering the gene of an important regulator would likely result in a dead organism. With the advent of modern sequencing technology, it has become possible to monitor global cellular effects of small-molecule treatment and thereby overcome the limitations of classical biochemistry, which usually looks at a biological system in isolation. This review focuses on several molecules, especially natural products, that have played an important role in dissecting gene expression and have opened up new fields of investigation as well as clinical venues for disease treatment.
Asunto(s)
Regulación de la Expresión Génica/efectos de los fármacos , Transporte Activo de Núcleo Celular/efectos de los fármacos , Animales , Metilación de ADN/efectos de los fármacos , Epigénesis Genética/efectos de los fármacos , Código de Histonas/efectos de los fármacos , Inhibidores de Histona Desacetilasas/farmacología , Histona Metiltransferasas/antagonistas & inhibidores , Humanos , Modelos Biológicos , Biología Molecular , Biosíntesis de Proteínas/efectos de los fármacos , Empalme del ARN/efectos de los fármacos , Estabilidad del ARN/efectos de los fármacos , Transcripción Genética/efectos de los fármacosRESUMEN
Non-coding genetic variation is a major driver of phenotypic diversity and allows the investigation of mechanisms that control gene expression. Here, we systematically investigated the effects of >50 million variations from five strains of mice on mRNA, nascent transcription, transcription start sites, and transcription factor binding in resting and activated macrophages. We observed substantial differences associated with distinct molecular pathways. Evaluating genetic variation provided evidence for roles of â¼100 TFs in shaping lineage-determining factor binding. Unexpectedly, a substantial fraction of strain-specific factor binding could not be explained by local mutations. Integration of genomic features with chromatin interaction data provided evidence for hundreds of connected cis-regulatory domains associated with differences in transcription factor binding and gene expression. This system and the >250 datasets establish a substantial new resource for investigation of how genetic variation affects cellular phenotypes.
Asunto(s)
Variación Genética , Macrófagos/metabolismo , Factores de Transcripción/metabolismo , Animales , Sitios de Unión , Células de la Médula Ósea/citología , Proteína beta Potenciadora de Unión a CCAAT/genética , Proteína beta Potenciadora de Unión a CCAAT/metabolismo , Análisis por Conglomerados , Elementos de Facilitación Genéticos/genética , Femenino , Regulación de la Expresión Génica/efectos de los fármacos , Lipopolisacáridos/farmacología , Macrófagos/citología , Macrófagos/efectos de los fármacos , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Endogámicos NOD , Regiones Promotoras Genéticas , Unión Proteica , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas/metabolismo , Transactivadores/genética , Transactivadores/metabolismo , Factores de Transcripción/genéticaRESUMEN
Immune checkpoint blockade has provided a paradigm shift in cancer therapy, but the success of this approach is very variable; therefore, biomarkers predictive of clinical efficacy are urgently required. Here, we show that the frequency of PD-1+CD8+ T cells relative to that of PD-1+ regulatory T (Treg) cells in the tumor microenvironment can predict the clinical efficacy of programmed cell death protein 1 (PD-1) blockade therapies and is superior to other predictors, including PD ligand 1 (PD-L1) expression or tumor mutational burden. PD-1 expression by CD8+ T cells and Treg cells negatively impacts effector and immunosuppressive functions, respectively. PD-1 blockade induces both recovery of dysfunctional PD-1+CD8+ T cells and enhanced PD-1+ Treg cell-mediated immunosuppression. A profound reactivation of effector PD-1+CD8+ T cells rather than PD-1+ Treg cells by PD-1 blockade is necessary for tumor regression. These findings provide a promising predictive biomarker for PD-1 blockade therapies.
Asunto(s)
Regulación de la Expresión Génica/efectos de los fármacos , Inhibidores de Puntos de Control Inmunológico/farmacología , Receptor de Muerte Celular Programada 1/antagonistas & inhibidores , Receptor de Muerte Celular Programada 1/genética , Linfocitos T Reguladores/inmunología , Linfocitos T Reguladores/metabolismo , Antígenos/química , Antígenos/inmunología , Biomarcadores de Tumor , Antígenos CD28/metabolismo , Linfocitos T CD8-positivos/inmunología , Linfocitos T CD8-positivos/metabolismo , Humanos , Inhibidores de Puntos de Control Inmunológico/uso terapéutico , Inmunomodulación , Activación de Linfocitos/inmunología , Linfocitos Infiltrantes de Tumor/inmunología , Linfocitos Infiltrantes de Tumor/metabolismo , Terapia Molecular Dirigida , Metástasis de la Neoplasia , Estadificación de Neoplasias , Neoplasias/tratamiento farmacológico , Neoplasias/etiología , Neoplasias/metabolismo , Neoplasias/mortalidad , Péptidos/química , Péptidos/inmunología , Pronóstico , Receptor de Muerte Celular Programada 1/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Receptores de Antígenos de Linfocitos T/metabolismo , Transducción de Señal , Linfocitos T Reguladores/efectos de los fármacos , Resultado del Tratamiento , Microambiente Tumoral/inmunologíaRESUMEN
Fragile X syndrome (FXS) is a leading genetic cause of intellectual disability and autism. FXS results from the loss of function of fragile X mental retardation protein (FMRP), which represses translation of target transcripts. Most of the well-characterized target transcripts of FMRP are synaptic proteins, yet targeting these proteins has not provided effective treatments. We examined a group of FMRP targets that encode transcriptional regulators, particularly chromatin-associated proteins. Loss of FMRP in mice results in widespread changes in chromatin regulation and aberrant gene expression. To determine if targeting epigenetic factors could reverse phenotypes associated with the disorder, we focused on Brd4, a BET protein and chromatin reader targeted by FMRP. Inhibition of Brd4 function alleviated many of the phenotypes associated with FXS. We conclude that loss of FMRP results in significant epigenetic misregulation and that targeting transcription via epigenetic regulators like Brd4 may provide new treatments for FXS.
Asunto(s)
Azepinas/farmacología , Proteína de la Discapacidad Intelectual del Síndrome del Cromosoma X Frágil/metabolismo , Síndrome del Cromosoma X Frágil/tratamiento farmacológico , Síndrome del Cromosoma X Frágil/metabolismo , Proteínas Nucleares/antagonistas & inhibidores , Proteínas Nucleares/metabolismo , Factores de Transcripción/antagonistas & inhibidores , Factores de Transcripción/metabolismo , Triazoles/farmacología , Animales , Células Cultivadas , Epigénesis Genética , Expresión Génica/efectos de los fármacos , Regulación de la Expresión Génica/efectos de los fármacos , Histonas/metabolismo , Ratones , Ratones Noqueados , Naftiridinas/farmacología , Neuronas/metabolismo , Fenazinas , Transcripción GenéticaRESUMEN
Quantitative mass spectrometry reveals how CD4+ and CD8+ T cells restructure proteomes in response to antigen and mammalian target of rapamycin complex 1 (mTORC1). Analysis of copy numbers per cell of >9,000 proteins provides new understanding of T cell phenotypes, exposing the metabolic and protein synthesis machinery and environmental sensors that shape T cell fate. We reveal that lymphocyte environment sensing is controlled by immune activation, and that CD4+ and CD8+ T cells differ in their intrinsic nutrient transport and biosynthetic capacity. Our data also reveal shared and divergent outcomes of mTORC1 inhibition in naïve versus effector T cells: mTORC1 inhibition impaired cell cycle progression in activated naïve cells, but not effector cells, whereas metabolism was consistently impacted in both populations. This study provides a comprehensive map of naïve and effector T cell proteomes, and a resource for exploring and understanding T cell phenotypes and cell context effects of mTORC1.
Asunto(s)
Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD8-positivos/inmunología , Diferenciación Celular/inmunología , Proteoma/metabolismo , Animales , Linfocitos T CD4-Positivos/metabolismo , Linfocitos T CD8-positivos/metabolismo , Puntos de Control del Ciclo Celular/inmunología , Diferenciación Celular/efectos de los fármacos , Diferenciación Celular/genética , Células Cultivadas , Femenino , Dosificación de Gen , Perfilación de la Expresión Génica , Regulación de la Expresión Génica/efectos de los fármacos , Regulación de la Expresión Génica/inmunología , Masculino , Espectrometría de Masas , Diana Mecanicista del Complejo 1 de la Rapamicina/antagonistas & inhibidores , Diana Mecanicista del Complejo 1 de la Rapamicina/metabolismo , Ratones , Ratones Transgénicos , Proteoma/inmunología , Proteómica , Sirolimus/farmacologíaRESUMEN
Birth presents a metabolic challenge to cardiomyocytes as they reshape fuel preference from glucose to fatty acids for postnatal energy production1,2. This adaptation is triggered in part by post-partum environmental changes3, but the molecules orchestrating cardiomyocyte maturation remain unknown. Here we show that this transition is coordinated by maternally supplied γ-linolenic acid (GLA), an 18:3 omega-6 fatty acid enriched in the maternal milk. GLA binds and activates retinoid X receptors4 (RXRs), ligand-regulated transcription factors that are expressed in cardiomyocytes from embryonic stages. Multifaceted genome-wide analysis revealed that the lack of RXR in embryonic cardiomyocytes caused an aberrant chromatin landscape that prevented the induction of an RXR-dependent gene expression signature controlling mitochondrial fatty acid homeostasis. The ensuing defective metabolic transition featured blunted mitochondrial lipid-derived energy production and enhanced glucose consumption, leading to perinatal cardiac dysfunction and death. Finally, GLA supplementation induced RXR-dependent expression of the mitochondrial fatty acid homeostasis signature in cardiomyocytes, both in vitro and in vivo. Thus, our study identifies the GLA-RXR axis as a key transcriptional regulatory mechanism underlying the maternal control of perinatal cardiac metabolism.
Asunto(s)
Ácidos Grasos , Glucosa , Corazón , Leche Humana , Ácido gammalinolénico , Femenino , Humanos , Recién Nacido , Embarazo , Cromatina/genética , Ácidos Grasos/metabolismo , Ácido gammalinolénico/metabolismo , Ácido gammalinolénico/farmacología , Regulación de la Expresión Génica/efectos de los fármacos , Glucosa/metabolismo , Corazón/efectos de los fármacos , Corazón/embriología , Corazón/crecimiento & desarrollo , Homeostasis , Técnicas In Vitro , Leche Humana/química , Mitocondrias/efectos de los fármacos , Mitocondrias/metabolismo , Miocitos Cardíacos/efectos de los fármacos , Miocitos Cardíacos/metabolismo , Receptores X Retinoide/metabolismo , Factores de Transcripción/metabolismoRESUMEN
Steroid hormone receptors are ligand-binding transcription factors essential for mammalian physiology. The androgen receptor (AR) binds androgens mediating gene expression for sexual, somatic and behavioural functions, and is involved in various conditions including androgen insensitivity syndrome and prostate cancer1. Here we identified functional mutations in the formin and actin nucleator DAAM2 in patients with androgen insensitivity syndrome. DAAM2 was enriched in the nucleus, where its localization correlated with that of the AR to form actin-dependent transcriptional droplets in response to dihydrotestosterone. DAAM2 AR droplets ranged from 0.02 to 0.06 µm3 in size and associated with active RNA polymerase II. DAAM2 polymerized actin directly at the AR to promote droplet coalescence in a highly dynamic manner, and nuclear actin polymerization is required for prostate-specific antigen expression in cancer cells. Our data uncover signal-regulated nuclear actin assembly at a steroid hormone receptor necessary for transcription.
Asunto(s)
Actinas , Forminas , Proteínas Nucleares , Receptores Androgénicos , Transcripción Genética , Humanos , Actinas/metabolismo , Síndrome de Resistencia Androgénica/genética , Síndrome de Resistencia Androgénica/metabolismo , Andrógenos/farmacología , Andrógenos/metabolismo , Forminas/metabolismo , Regulación de la Expresión Génica/efectos de los fármacos , Proteínas Nucleares/metabolismo , Polimerizacion/efectos de los fármacos , Antígeno Prostático Específico/metabolismo , Neoplasias de la Próstata/genética , Neoplasias de la Próstata/metabolismo , Neoplasias de la Próstata/patología , Receptores Androgénicos/metabolismo , ARN Polimerasa II/metabolismo , Transducción de Señal/efectos de los fármacos , Esteroides/metabolismo , Esteroides/farmacología , Testosterona/análogos & derivados , Transcripción Genética/efectos de los fármacosRESUMEN
While the role of transcription factors and coactivators in controlling enhancer activity and chromatin structure linked to gene expression is well established, the involvement of corepressors is not. Using inflammatory macrophage activation as a model, we investigate here a corepressor complex containing GPS2 and SMRT both genome-wide and at the Ccl2 locus, encoding the chemokine CCL2 (MCP-1). We report that corepressors co-occupy candidate enhancers along with the coactivators CBP (H3K27 acetylase) and MED1 (mediator) but act antagonistically by repressing eRNA transcription-coupled H3K27 acetylation. Genome editing, transcriptional interference, and cistrome analysis reveals that apparently related enhancer and silencer elements control Ccl2 transcription in opposite ways. 4C-seq indicates that corepressor depletion or inflammatory signaling functions mechanistically similarly to trigger enhancer activation. In ob/ob mice, adipose tissue macrophage-selective depletion of the Ccl2 enhancer-transcribed eRNA reduces metaflammation. Thus, the identified corepressor-eRNA-chemokine pathway operates in vivo and suggests therapeutic opportunities by targeting eRNAs in immuno-metabolic diseases.
Asunto(s)
Quimiocina CCL2/genética , Proteínas Co-Represoras/genética , Elementos de Facilitación Genéticos , Péptidos y Proteínas de Señalización Intracelular/genética , Co-Represor 2 de Receptor Nuclear/genética , Obesidad/genética , Elementos Silenciadores Transcripcionales , Tejido Adiposo/inmunología , Tejido Adiposo/patología , Animales , Sistemas CRISPR-Cas , Quimiocina CCL2/inmunología , Proteínas Co-Represoras/inmunología , Edición Génica , Regulación de la Expresión Génica/efectos de los fármacos , Células HEK293 , Histona Acetiltransferasas/genética , Histona Acetiltransferasas/inmunología , Histonas/genética , Histonas/inmunología , Humanos , Péptidos y Proteínas de Señalización Intracelular/inmunología , Lipopolisacáridos/farmacología , Activación de Macrófagos/efectos de los fármacos , Masculino , Subunidad 1 del Complejo Mediador/genética , Subunidad 1 del Complejo Mediador/inmunología , Ratones , Ratones Obesos , Co-Represor 2 de Receptor Nuclear/inmunología , Obesidad/inmunología , Obesidad/patología , Células RAW 264.7 , ARN no Traducido/genética , ARN no Traducido/inmunología , Transducción de SeñalRESUMEN
N6-methyladenosine (m6A) is an abundant internal RNA modification, influencing transcript fate and function in uninfected and virus-infected cells. Installation of m6A by the nuclear RNA methyltransferase METTL3 occurs cotranscriptionally; however, the genomes of some cytoplasmic RNA viruses are also m6A-modified. How the cellular m6A modification machinery impacts coronavirus replication, which occurs exclusively in the cytoplasm, is unknown. Here we show that replication of SARS-CoV-2, the agent responsible for the COVID-19 pandemic, and a seasonal human ß-coronavirus HCoV-OC43, can be suppressed by depletion of METTL3 or cytoplasmic m6A reader proteins YTHDF1 and YTHDF3 and by a highly specific small molecule METTL3 inhibitor. Reduction of infectious titer correlates with decreased synthesis of viral RNAs and the essential nucleocapsid (N) protein. Sites of m6A modification on genomic and subgenomic RNAs of both viruses were mapped by methylated RNA immunoprecipitation sequencing (meRIP-seq). Levels of host factors involved in m6A installation, removal, and recognition were unchanged by HCoV-OC43 infection; however, nuclear localization of METTL3 and cytoplasmic m6A readers YTHDF1 and YTHDF2 increased. This establishes that coronavirus RNAs are m6A-modified and host m6A pathway components control ß-coronavirus replication. Moreover, it illustrates the therapeutic potential of targeting the m6A pathway to restrict coronavirus reproduction.
Asunto(s)
Coronavirus Humano OC43/fisiología , Procesamiento Postranscripcional del ARN/genética , SARS-CoV-2/fisiología , Replicación Viral/genética , Adenosina/análogos & derivados , Adenosina/genética , Adenosina/metabolismo , Línea Celular , Infecciones por Coronavirus/metabolismo , Infecciones por Coronavirus/virología , Regulación de la Expresión Génica/efectos de los fármacos , Interacciones Huésped-Patógeno/efectos de los fármacos , Humanos , Metiltransferasas/antagonistas & inhibidores , Metiltransferasas/metabolismo , Proteínas de la Nucleocápside , ARN Viral/metabolismo , Proteínas de Unión al ARN/metabolismo , Replicación Viral/efectos de los fármacosRESUMEN
Host microbial cross-talk is essential to maintain intestinal homeostasis. However, maladaptation of this response through microbial dysbiosis or defective host defense toward invasive intestinal bacteria can result in chronic inflammation. We have shown that macrophages differentiated in the presence of the bacterial metabolite butyrate display enhanced antimicrobial activity. Butyrate-induced antimicrobial activity was associated with a shift in macrophage metabolism, a reduction in mTOR kinase activity, increased LC3-associated host defense and anti-microbial peptide production in the absence of an increased inflammatory cytokine response. Butyrate drove this monocyte to macrophage differentiation program through histone deacetylase 3 (HDAC3) inhibition. Administration of butyrate induced antimicrobial activity in intestinal macrophages in vivo and increased resistance to enteropathogens. Our data suggest that (1) increased intestinal butyrate might represent a strategy to bolster host defense without tissue damaging inflammation and (2) that pharmacological HDAC3 inhibition might drive selective macrophage functions toward antimicrobial host defense.
Asunto(s)
Antiinfecciosos/farmacología , Butiratos/farmacología , Diferenciación Celular/efectos de los fármacos , Macrófagos/efectos de los fármacos , Monocitos/efectos de los fármacos , Animales , Diferenciación Celular/genética , Células Cultivadas , Colon/efectos de los fármacos , Colon/metabolismo , Colon/microbiología , Citocinas/genética , Citocinas/metabolismo , Disbiosis/microbiología , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Intestinos/efectos de los fármacos , Intestinos/microbiología , Macrófagos/metabolismo , Macrófagos/microbiología , Ratones Endogámicos C57BL , Microbiota/efectos de los fármacos , Microbiota/fisiología , Monocitos/metabolismo , Monocitos/microbiologíaRESUMEN
NF-κB (nuclear factor κB) signaling is considered critical for single positive (SP) thymocyte development because loss of upstream activators of NF-κB, such as the IKK complex, arrests their development. We found that the compound ablation of RelA, cRel, and p50, required for canonical NF-κB transcription, had no impact upon thymocyte development. While IKK-deficient thymocytes were acutely sensitive to tumor necrosis factor (TNF)-induced cell death, Rel-deficient cells remained resistant, calling into question the importance of NF-κB as the IKK target required for thymocyte survival. Instead, we found that IKK controlled thymocyte survival by repressing cell-death-inducing activity of the serine/threonine kinase RIPK1. We observed that RIPK1 expression was induced during development of SP thymocytes and that IKK was required to prevent RIPK1-kinase-dependent death of SPs in vivo. Finally, we showed that IKK was required to protect Rel-deficient thymocytes from RIPK1-dependent cell death, underscoring the NF-κB-independent function of IKK during thymic development.
Asunto(s)
Quinasa I-kappa B/metabolismo , FN-kappa B/metabolismo , Proteína Serina-Treonina Quinasas de Interacción con Receptores/metabolismo , Timocitos/metabolismo , Animales , Apoptosis/efectos de los fármacos , Apoptosis/genética , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/genética , Células Cultivadas , Regulación de la Expresión Génica/efectos de los fármacos , Quinasa I-kappa B/genética , Ratones Endogámicos C57BL , Ratones Noqueados , FN-kappa B/genética , Proteína Serina-Treonina Quinasas de Interacción con Receptores/genética , Transducción de Señal/efectos de los fármacos , Transducción de Señal/genética , Timocitos/citología , Timocitos/efectos de los fármacos , Factor de Transcripción ReIA/genética , Factor de Transcripción ReIA/metabolismo , Factor de Necrosis Tumoral alfa/farmacologíaRESUMEN
Young mammals possess a limited regenerative capacity in some tissues, which is lost upon maturation. We investigated whether cellular senescence might play a role in such loss during liver regeneration. We found that following partial hepatectomy, the senescence-associated genes p21, p16Ink4a, and p19Arf become dynamically expressed in different cell types when regenerative capacity decreases, but without a full senescent response. However, we show that treatment with a senescence-inhibiting drug improves regeneration, by disrupting aberrantly prolonged p21 expression. This work suggests that senescence may initially develop from heterogeneous cellular responses, and that senotherapeutic drugs might be useful in promoting organ regeneration.
Asunto(s)
Compuestos de Bifenilo/farmacología , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/genética , Regulación de la Expresión Génica/efectos de los fármacos , Hígado/fisiología , Nitrofenoles/farmacología , Regeneración/efectos de los fármacos , Sulfonamidas/farmacología , Animales , Células Cultivadas , Senescencia Celular/efectos de los fármacos , Inhibidor p16 de la Quinasa Dependiente de Ciclina/genética , Femenino , Masculino , Ratones , Ratones Endogámicos C57BL , Modelos Animales , Piperazinas/farmacologíaRESUMEN
Transcription by RNA polymerase II (RNAPII) is a dynamic process with frequent variations in the elongation rate. However, the physiological relevance of variations in RNAPII elongation kinetics has remained unclear. Here we show in yeast that a RNAPII mutant that reduces the transcription elongation rate causes widespread changes in alternative polyadenylation (APA). We unveil two mechanisms by which APA affects gene expression in the slow mutant: 3' UTR shortening and gene derepression by premature transcription termination of upstream interfering noncoding RNAs. Strikingly, the genes affected by these mechanisms are enriched for functions involved in phosphate uptake and purine synthesis, processes essential for maintenance of the intracellular nucleotide pool. As nucleotide concentration regulates transcription elongation, our findings argue that RNAPII is a sensor of nucleotide availability and that genes important for nucleotide pool maintenance have adopted regulatory mechanisms responsive to reduced rates of transcription elongation.
Asunto(s)
Regulación de la Expresión Génica/efectos de los fármacos , ARN Polimerasa II/genética , Schizosaccharomyces/enzimología , Schizosaccharomyces/genética , Activación Enzimática/efectos de los fármacos , Genes Fúngicos/genética , Mutación , Extensión de la Cadena Peptídica de Translación/efectos de los fármacos , Fosfatos/farmacología , Poliadenilación , Regiones Promotoras Genéticas/genética , ARN Polimerasa II/química , ARN Polimerasa II/metabolismo , Saccharomyces cerevisiae/enzimología , Saccharomyces cerevisiae/genética , Factores de Transcripción/genéticaRESUMEN
Despite being the frontline therapy for type 2 diabetes, the mechanisms of action of the biguanide drug metformin are still being discovered. In particular, the detailed molecular interplays between the AMPK and the mTORC1 pathway in the hepatic benefits of metformin are still ill defined. Metformin-dependent activation of AMPK classically inhibits mTORC1 via TSC/RHEB, but several lines of evidence suggest additional mechanisms at play in metformin inhibition of mTORC1. Here we investigated the role of direct AMPK-mediated serine phosphorylation of RAPTOR in a new RaptorAA mouse model, in which AMPK phospho-serine sites Ser722 and Ser792 of RAPTOR were mutated to alanine. Metformin treatment of primary hepatocytes and intact murine liver requires AMPK regulation of both RAPTOR and TSC2 to fully inhibit mTORC1, and this regulation is critical for both the translational and transcriptional response to metformin. Transcriptionally, AMPK and mTORC1 were both important for regulation of anabolic metabolism and inflammatory programs triggered by metformin treatment. The hepatic transcriptional response in mice on high-fat diet treated with metformin was largely ablated by AMPK deficiency under the conditions examined, indicating the essential role of this kinase and its targets in metformin action in vivo.
Asunto(s)
Proteínas Quinasas Activadas por AMP/metabolismo , Regulación de la Expresión Génica/efectos de los fármacos , Metformina/farmacología , Proteína Reguladora Asociada a mTOR/genética , Transducción de Señal/efectos de los fármacos , Animales , Diabetes Mellitus Tipo 2/tratamiento farmacológico , Modelos Animales de Enfermedad , Técnicas de Sustitución del Gen , Genotipo , Hipoglucemiantes/farmacología , Inflamación , Diana Mecanicista del Complejo 1 de la Rapamicina/metabolismo , Metabolismo/efectos de los fármacos , Metformina/uso terapéutico , Ratones , Fosforilación/efectos de los fármacos , Proteína Reguladora Asociada a mTOR/metabolismo , Serina-Treonina Quinasas TOR/metabolismo , Proteína 2 del Complejo de la Esclerosis Tuberosa/genética , Proteína 2 del Complejo de la Esclerosis Tuberosa/metabolismoRESUMEN
Intercalated cells (ICs) are found in the connecting tubule and the collecting duct. Of the three IC subtypes identified, type B intercalated cells are one of the best characterized and known to mediate Cl- absorption and HCO3- secretion, largely through the anion exchanger pendrin. This exchanger is thought to act in tandem with the Na+-dependent Cl-/HCO3- exchanger, NDCBE, to mediate net NaCl absorption. Pendrin is stimulated by angiotensin II and aldosterone administration via the angiotensin type 1a and the mineralocorticoid receptors, respectively. It is also stimulated in models of metabolic alkalosis, such as with NaHCO3 administration. In some rodent models, pendrin-mediated HCO3- secretion modulates acid-base balance. However, of probably more physiological or clinical significance is the role of these pendrin-positive ICs in blood pressure regulation, which occurs, at least in part, through pendrin-mediated renal Cl- absorption, as well as their effect on the epithelial Na+ channel, ENaC. Aldosterone stimulates ENaC directly through principal cell mineralocorticoid hormone receptor (ligand) binding and also indirectly through its effect on pendrin expression and function. In so doing, pendrin contributes to the aldosterone pressor response. Pendrin may also modulate blood pressure in part through its action in the adrenal medulla, where it modulates the release of catecholamines, or through an indirect effect on vascular contractile force. In addition to its role in Na+ and Cl- balance, pendrin affects the balance of other ions, such as K+ and I-. This review describes how aldosterone and angiotensin II-induced signaling regulate pendrin and the contribution of pendrin-positive ICs in the kidney to distal nephron function and blood pressure.