RESUMEN
Tuft cells in mucosal tissues are key regulators of type 2 immunity. Here, we examined the impact of the microbiota on tuft cell biology in the intestine. Succinate induction of tuft cells and type 2 innate lymphoid cells was elevated with loss of gut microbiota. Colonization with butyrate-producing bacteria or treatment with butyrate suppressed this effect and reduced intestinal histone deacetylase activity. Epithelial-intrinsic deletion of the epigenetic-modifying enzyme histone deacetylase 3 (HDAC3) inhibited tuft cell expansion in vivo and impaired type 2 immune responses during helminth infection. Butyrate restricted stem cell differentiation into tuft cells, and inhibition of HDAC3 in adult mice and human intestinal organoids blocked tuft cell expansion. Collectively, these data define a HDAC3 mechanism in stem cells for tuft cell differentiation that is dampened by a commensal metabolite, revealing a pathway whereby the microbiota calibrate intestinal type 2 immunity.
Asunto(s)
Mucosa Intestinal , Microbiota , Adulto , Ratones , Humanos , Animales , Células en Penacho , Butiratos/farmacología , Butiratos/metabolismo , Inmunidad Innata , Linfocitos/metabolismo , Intestinos , Histona Desacetilasas/metabolismo , Diferenciación CelularRESUMEN
Pioneer transcription factors (TFs) regulate cell fate by establishing transcriptionally primed and active states. However, cell fate control requires the coordination of both lineage-specific gene activation and repression of alternative-lineage programs, a process that is poorly understood. Here, we demonstrate that the pioneer TF FOXA coordinates with PRDM1 TF to recruit nucleosome remodeling and deacetylation (NuRD) complexes and Polycomb repressive complexes (PRCs), which establish highly occupied, accessible nucleosome conformation with bivalent epigenetic states, thereby preventing precocious and alternative-lineage gene expression during human endoderm differentiation. Similarly, the pioneer TF OCT4 coordinates with PRDM14 to form bivalent enhancers and repress cell differentiation programs in human pluripotent stem cells, suggesting that this may be a common and critical function of pioneer TFs. We propose that pioneer and PRDM TFs coordinate to safeguard cell fate through epigenetic repression mechanisms.
Asunto(s)
Nucleosomas , Factores de Transcripción , Humanos , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Nucleosomas/genética , Diferenciación Celular/genética , Proteínas del Grupo Polycomb/metabolismo , Epigénesis GenéticaRESUMEN
SNPs affecting disease risk often reside in non-coding genomic regions. Here, we show that SNPs are highly enriched at mouse strain-selective adipose tissue binding sites for PPARγ, a nuclear receptor for anti-diabetic drugs. Many such SNPs alter binding motifs for PPARγ or cooperating factors and functionally regulate nearby genes whose expression is strain selective and imbalanced in heterozygous F1 mice. Moreover, genetically determined binding of PPARγ accounts for mouse strain-specific transcriptional effects of TZD drugs, providing proof of concept for personalized medicine related to nuclear receptor genomic occupancy. In human fat, motif-altering SNPs cause differential PPARγ binding, provide a molecular mechanism for some expression quantitative trait loci, and are risk factors for dysmetabolic traits in genome-wide association studies. One PPARγ motif-altering SNP is associated with HDL levels and other metabolic syndrome parameters. Thus, natural genetic variation in PPARγ genomic occupancy determines individual disease risk and drug response.
Asunto(s)
Hipoglucemiantes/metabolismo , PPAR gamma/genética , PPAR gamma/metabolismo , Polimorfismo de Nucleótido Simple , Tejido Adiposo , Animales , Expresión Génica , Humanos , Ratones , Ratones de la Cepa 129 , Ratones Endogámicos C57BL , Factores de Transcripción/metabolismoRESUMEN
How homeodomain proteins gain sufficient specificity to control different cell fates has been a long-standing problem in developmental biology. The conserved Gsx homeodomain proteins regulate specific aspects of neural development in animals from flies to mammals, and yet they belong to a large transcription factor family that bind nearly identical DNA sequences in vitro. Here, we show that the mouse and fly Gsx factors unexpectedly gain DNA binding specificity by forming cooperative homodimers on precisely spaced and oriented DNA sites. High-resolution genomic binding assays revealed that Gsx2 binds both monomer and homodimer sites in the developing mouse ventral telencephalon. Importantly, reporter assays showed that Gsx2 mediates opposing outcomes in a DNA binding site-dependent manner: Monomer Gsx2 binding represses transcription, whereas homodimer binding stimulates gene expression. In Drosophila, the Gsx homolog, Ind, similarly represses or stimulates transcription in a site-dependent manner via an autoregulatory enhancer containing a combination of monomer and homodimer sites. Integrating these findings, we test a model showing how the homodimer to monomer site ratio and the Gsx protein levels defines gene up-regulation versus down-regulation. Altogether, these data serve as a new paradigm for how cooperative homeodomain transcription factor binding can increase target specificity and alter regulatory outcomes.
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Proteínas de Drosophila/metabolismo , Drosophila/embriología , Drosophila/genética , Regulación del Desarrollo de la Expresión Génica/genética , Proteínas de Homeodominio/metabolismo , Animales , Proteínas de Drosophila/genética , Genoma/genética , Estudio de Asociación del Genoma Completo , Proteínas de Homeodominio/genética , Ratones , Unión Proteica , Telencéfalo/embriologíaRESUMEN
Exercise can alter the skeletal muscle DNA methylome, yet little is known about the role of the DNA methylation machinery in exercise capacity. Here, we show that DNMT3A expression in oxidative red muscle increases greatly following a bout of endurance exercise. Muscle-specific Dnmt3a knockout mice have reduced tolerance to endurance exercise, accompanied by reduction in oxidative capacity and mitochondrial respiration. Moreover, Dnmt3a-deficient muscle overproduces reactive oxygen species (ROS), the major contributors to muscle dysfunction. Mechanistically, we show that DNMT3A suppresses the Aldh1l1 transcription by binding to its promoter region, altering its epigenetic profile. Forced expression of ALDH1L1 elevates NADPH levels, which results in overproduction of ROS by the action of NADPH oxidase complex, ultimately resulting in mitochondrial defects in myotubes. Thus, inhibition of ALDH1L1 pathway can rescue oxidative stress and mitochondrial dysfunction from Dnmt3a deficiency in myotubes. Finally, we show that in vivo knockdown of Aldh1l1 largely rescues exercise intolerance in Dnmt3a-deficient mice. Together, we establish that DNMT3A in skeletal muscle plays a pivotal role in endurance exercise by controlling intracellular oxidative stress.
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ADN (Citosina-5-)-Metiltransferasas/genética , Músculo Esquelético/metabolismo , Oxidorreductasas actuantes sobre Donantes de Grupo CH-NH/genética , Resistencia Física/genética , Animales , Línea Celular , ADN (Citosina-5-)-Metiltransferasas/metabolismo , ADN Metiltransferasa 3A , Perfilación de la Expresión Génica , Técnicas de Inactivación de Genes , Ratones , Mitocondrias Musculares/metabolismo , Estrés Oxidativo , Oxidorreductasas actuantes sobre Donantes de Grupo CH-NH/metabolismo , Ratas , Especies Reactivas de Oxígeno/metabolismo , Análisis de Secuencia de ARNRESUMEN
Homeodomain proteins constitute one of the largest families of metazoan transcription factors. Genetic studies have demonstrated that homeodomain proteins regulate many developmental processes. Yet, biochemical data reveal that most bind highly similar DNA sequences. Defining how homeodomain proteins achieve DNA binding specificity has therefore been a long-standing goal. Here, we developed a novel computational approach to predict cooperative dimeric binding of homeodomain proteins using High-Throughput (HT) SELEX data. Importantly, we found that 15 of 88 homeodomain factors form cooperative homodimer complexes on DNA sites with precise spacing requirements. Approximately one third of the paired-like homeodomain proteins cooperatively bind palindromic sequences spaced 3 bp apart, whereas other homeodomain proteins cooperatively bind sites with distinct orientation and spacing requirements. Combining structural models of a paired-like factor with our cooperativity predictions identified key amino acid differences that help differentiate between cooperative and non-cooperative factors. Finally, we confirmed predicted cooperative dimer sites in vivo using available genomic data for a subset of factors. These findings demonstrate how HT-SELEX data can be computationally mined to predict cooperativity. In addition, the binding site spacing requirements of select homeodomain proteins provide a mechanism by which seemingly similar AT-rich DNA sequences can preferentially recruit specific homeodomain factors.
Asunto(s)
Proteínas de Homeodominio , Animales , Sitios de Unión , ADN/química , Proteínas de Homeodominio/metabolismo , Secuenciación de Nucleótidos de Alto RendimientoRESUMEN
The transcription factor early B-cell factor 2 (EBF2) is an essential mediator of brown adipocyte commitment and terminal differentiation. However, the mechanisms by which EBF2 regulates chromatin to activate brown fat-specific genes in adipocytes were unknown. ChIP-seq (chromatin immunoprecipitation [ChIP] followed by deep sequencing) analyses in brown adipose tissue showed that EBF2 binds and regulates the activity of lineage-specific enhancers. Mechanistically, EBF2 physically interacts with the chromatin remodeler BRG1 and the BAF chromatin remodeling complex in brown adipocytes. We identified the histone reader protein DPF3 as a brown fat-selective component of the BAF complex that was required for brown fat gene programming and mitochondrial function. Loss of DPF3 in brown adipocytes reduced chromatin accessibility at EBF2-bound enhancers and led to a decrease in basal and catecholamine-stimulated expression of brown fat-selective genes. Notably, Dpf3 is a direct transcriptional target of EBF2 in brown adipocytes, thereby establishing a regulatory module through which EBF2 activates and also recruits DPF3-anchored BAF complexes to chromatin. Together, these results reveal a novel mechanism by which EBF2 cooperates with a tissue-specific chromatin remodeling complex to activate brown fat identity genes.
Asunto(s)
Adipogénesis/genética , Tejido Adiposo Pardo/citología , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/fisiología , Ensamble y Desensamble de Cromatina , Cromatina/metabolismo , Proteínas Cromosómicas no Histona/metabolismo , Proteínas de Unión al ADN/genética , Histonas/metabolismo , Factores de Transcripción/genética , Tejido Adiposo Pardo/metabolismo , Animales , Linaje de la Célula/genética , Células Cultivadas , Regulación de la Expresión Génica , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Transcripción GenéticaRESUMEN
The projection neurons of the striatum, the principal nucleus of the basal ganglia, belong to one of the following two major pathways: the striatopallidal (indirect) pathway or the striatonigral (direct) pathway. Striatonigral axons project long distances and encounter ascending tracts (thalamocortical) while coursing alongside descending tracts (corticofugal) as they extend through the internal capsule and cerebral peduncle. These observations suggest that striatal circuitry may help to guide their trajectories. To investigate the developmental contributions of striatonigral axons to internal capsule formation, we have made use of Sox8-EGFP (striatal direct pathway) and Fezf2-TdTomato (corticofugal pathway) BAC transgenic reporter mice in combination with immunohistochemical markers to trace these axonal pathways throughout development. We show that striatonigral axons pioneer the internal capsule and cerebral peduncle and are temporally and spatially well positioned to provide guidance for corticofugal and thalamocortical axons. Using Isl1 conditional knock-out (cKO) mice, which exhibit disrupted striatonigral axon outgrowth, we observe both corticofugal and thalamocortical axon defects with either ventral forebrain- or telencephalon-specific Isl1 inactivation, despite Isl1 not being expressed in either cortical or thalamic projection neurons. Striatonigral axon defects can thus disrupt internal capsule formation. Our genome-wide transcriptomic analysis in Isl1 cKOs reveals changes in gene expression relevant to cell adhesion, growth cone dynamics, and extracellular matrix composition, suggesting potential mechanisms by which the striatonigral pathway exerts this guidance role. Together, our data support a novel pioneering role for the striatal direct pathway in the correct assembly of the ascending and descending axon tracts within the internal capsule and cerebral peduncle.SIGNIFICANCE STATEMENT The basal ganglia are a group of subcortical nuclei with established roles in the coordination of voluntary motor programs, aspects of cognition, and the selection of appropriate social behaviors. Hence, disruptions in basal ganglia connectivity have been implicated in the motor, cognitive, and social dysfunction characterizing common neurodevelopmental disorders such as attention-deficit/hyperactivity disorder, autism spectrum disorder, obsessive-compulsive disorder, and tic disorder. Here, we identified a novel role for the striatonigral (direct) pathway in pioneering the internal capsule and cerebral peduncle, and in guiding axons extending to and from the cortex. Our findings suggest that the abnormal development of basal ganglia circuits can drive secondary internal capsule defects and thereby may contribute to the pathology of these disorders.
Asunto(s)
Trastorno del Espectro Autista , Pedúnculo Cerebral , Animales , Trastorno del Espectro Autista/metabolismo , Axones/fisiología , Corteza Cerebral/metabolismo , Cápsula Interna , Ratones , Ratones Noqueados , Ratones Transgénicos , Vías Nerviosas/fisiología , TálamoRESUMEN
Brown adipose tissue is a thermogenic organ that dissipates chemical energy as heat to protect animals against hypothermia and to counteract metabolic disease. However, the transcriptional mechanisms that determine the thermogenic capacity of brown adipose tissue before environmental cold are unknown. Here we show that histone deacetylase 3 (HDAC3) is required to activate brown adipose tissue enhancers to ensure thermogenic aptitude. Mice with brown adipose tissue-specific genetic ablation of HDAC3 become severely hypothermic and succumb to acute cold exposure. Uncoupling protein 1 (UCP1) is nearly absent in brown adipose tissue lacking HDAC3, and there is also marked downregulation of mitochondrial oxidative phosphorylation genes resulting in diminished mitochondrial respiration. Remarkably, although HDAC3 acts canonically as a transcriptional corepressor, it functions as a coactivator of oestrogen-related receptor α (ERRα) in brown adipose tissue. HDAC3 coactivation of ERRα is mediated by deacetylation of PGC-1α and is required for the transcription of Ucp1, Ppargc1a (encoding PGC-1α), and oxidative phosphorylation genes. Importantly, HDAC3 promotes the basal transcription of these genes independently of adrenergic stimulation. Thus, HDAC3 uniquely primes Ucp1 and the thermogenic transcriptional program to maintain a critical capacity for thermogenesis in brown adipose tissue that can be rapidly engaged upon exposure to dangerously cold temperature.
Asunto(s)
Tejido Adiposo Pardo/metabolismo , Regulación de la Expresión Génica , Histona Desacetilasas/metabolismo , Termogénesis , Animales , Respiración de la Célula , Frío , Elementos de Facilitación Genéticos/genética , Calor , Humanos , Masculino , Ratones , Mitocondrias/metabolismo , Fosforilación Oxidativa , Coactivador 1-alfa del Receptor Activado por Proliferadores de Peroxisomas gamma/genética , Coactivador 1-alfa del Receptor Activado por Proliferadores de Peroxisomas gamma/metabolismo , Receptores de Estrógenos/metabolismo , Termogénesis/genética , Proteína Desacopladora 1/genética , Proteína Desacopladora 1/metabolismo , Receptor Relacionado con Estrógeno ERRalfaRESUMEN
PR (PRD1-BF1-RIZ1 homologous) domain-containing 16 (PRDM16) drives a brown fat differentiation program, but the mechanisms by which PRDM16 activates brown fat-selective genes have been unclear. Through chromatin immunoprecipitation (ChIP) followed by deep sequencing (ChIP-seq) analyses in brown adipose tissue (BAT), we reveal that PRDM16 binding is highly enriched at a broad set of brown fat-selective genes. Importantly, we found that PRDM16 physically binds to MED1, a component of the Mediator complex, and recruits it to superenhancers at brown fat-selective genes. PRDM16 deficiency in BAT reduces MED1 binding at PRDM16 target sites and causes a fundamental change in chromatin architecture at key brown fat-selective genes. Together, these data indicate that PRDM16 controls chromatin architecture and superenhancer activity in BAT.
Asunto(s)
Tejido Adiposo Pardo/metabolismo , Cromatina/metabolismo , Proteínas de Unión al ADN/metabolismo , Subunidad 1 del Complejo Mediador/metabolismo , Factores de Transcripción/metabolismo , Transcripción Genética/fisiología , Animales , Cromatina/química , Cromatina/genética , Elementos de Facilitación Genéticos , RatonesRESUMEN
Rosiglitazone (rosi) is a powerful insulin sensitizer, but serious toxicities have curtailed its widespread clinical use. Rosi functions as a high-affinity ligand for peroxisome proliferator-activated receptor γ (PPARγ), the adipocyte-predominant nuclear receptor (NR). The classic model, involving binding of ligand to the NR on DNA, explains positive regulation of gene expression, but ligand-dependent repression is not well understood. We addressed this issue by studying the direct effects of rosi on gene transcription using global run-on sequencing (GRO-seq). Rosi-induced changes in gene body transcription were pronounced after 10 min and correlated with steady-state mRNA levels as well as with transcription at nearby enhancers (enhancer RNAs [eRNAs]). Up-regulated eRNAs occurred almost exclusively at PPARγ-binding sites, to which rosi treatment recruited coactivators, including MED1, p300, and CBP. In contrast, transcriptional repression by rosi involved a loss of coactivators from eRNA sites devoid of PPARγ and enriched for other transcription factors, including AP-1 factors and C/EBPs. Thus, rosi activates and represses transcription by fundamentally different mechanisms that could inform the future development of anti-diabetic drugs.
Asunto(s)
Adipocitos/efectos de los fármacos , Regulación de la Expresión Génica/efectos de los fármacos , Hipoglucemiantes/farmacología , PPAR gamma/metabolismo , Tiazolidinedionas/farmacología , Células 3T3-L1 , Animales , Humanos , Subunidad 1 del Complejo Mediador/metabolismo , Ratones , Unión Proteica , Rosiglitazona , TranscriptomaRESUMEN
Brown adipose has the potential to counteract obesity, and thus, identifying signaling pathways that regulate the activity of this tissue is of great clinical interest. PRDM16 is a transcription factor that activates brown fat-specific genes while repressing white fat and muscle-specific genes in adipocytes. Whether PRDM16 also controls other gene programs to regulate adipocyte function was unclear. Here, we identify a novel role for PRDM16 in suppressing type I interferon (IFN)-stimulated genes (ISGs), including Stat1, in adipocytes in vitro and in vivo Ectopic activation of type I IFN signaling in brown adipocytes induces mitochondrial dysfunction and reduces uncoupling protein 1 (UCP1) expression. Prdm16-deficient adipose displays an exaggerated response to type I IFN, including higher STAT1 levels and reduced mitochondrial gene expression. Mechanistically, PRDM16 represses ISGs through binding to promoter regions of these genes and blocking the activating function of IFN regulatory factor 1 (IRF1). Together, these data indicate that PRDM16 diminishes responsiveness to type I IFN in adipose cells to promote thermogenic and mitochondrial function.
Asunto(s)
Adipocitos/fisiología , Proteínas de Unión al ADN/metabolismo , Regulación de la Expresión Génica , Factor 1 Regulador del Interferón/antagonistas & inhibidores , Interferón Tipo I/metabolismo , Mitocondrias/metabolismo , Termogénesis , Factores de Transcripción/metabolismo , Animales , Ratones , Factor de Transcripción STAT1/antagonistas & inhibidores , Proteína Desacopladora 1/metabolismoRESUMEN
Histone deacetylases (HDACs) are believed to regulate gene transcription by catalyzing deacetylation reactions. HDAC3 depletion in mouse liver upregulates lipogenic genes and results in severe hepatosteatosis. Here we show that pharmacologic HDAC inhibition in primary hepatocytes causes histone hyperacetylation but does not upregulate expression of HDAC3 target genes. Meanwhile, deacetylase-dead HDAC3 mutants can rescue hepatosteatosis and repress lipogenic genes expression in HDAC3-depleted mouse liver, demonstrating that histone acetylation is insufficient to activate gene transcription. Mutations abolishing interactions with the nuclear receptor corepressor (NCOR or SMRT) render HDAC3 nonfunctional in vivo. Additionally, liver-specific knockout of NCOR, but not SMRT, causes metabolic and transcriptomal alterations resembling those of mice without hepatic HDAC3, demonstrating that interaction with NCOR is essential for deacetylase-independent function of HDAC3. These findings highlight nonenzymatic roles of a major HDAC in transcriptional regulation in vivo and warrant reconsideration of the mechanism of action of HDAC inhibitors.
Asunto(s)
Hepatocitos/enzimología , Histona Desacetilasas/metabolismo , Histonas/metabolismo , Metabolismo de los Lípidos , Hígado/enzimología , Co-Represor 1 de Receptor Nuclear/metabolismo , Transcripción Genética , Acetilación , Animales , Hígado Graso/enzimología , Hígado Graso/genética , Perfilación de la Expresión Génica/métodos , Genotipo , Células HEK293 , Hepatocitos/efectos de los fármacos , Inhibidores de Histona Desacetilasas/farmacología , Histona Desacetilasas/química , Histona Desacetilasas/deficiencia , Histona Desacetilasas/genética , Humanos , Metabolismo de los Lípidos/efectos de los fármacos , Metabolismo de los Lípidos/genética , Hígado/efectos de los fármacos , Masculino , Ratones , Ratones Noqueados , Modelos Moleculares , Mutación , Co-Represor 1 de Receptor Nuclear/genética , Co-Represor 2 de Receptor Nuclear/genética , Co-Represor 2 de Receptor Nuclear/metabolismo , Análisis de Secuencia por Matrices de Oligonucleótidos , Fenotipo , Conformación Proteica , Relación Estructura-Actividad , Transcripción Genética/efectos de los fármacos , TransfecciónRESUMEN
Obesity is characterized by an accumulation of macrophages in adipose, some of which form distinct crown-like structures (CLS) around fat cells. While multiple discrete adipose tissue macrophage (ATM) subsets are thought to exist, their respective effects on adipose tissue, and the transcriptional mechanisms that underlie the functional differences between ATM subsets, are not well understood. We report that obese fat tissue of mice and humans contain multiple distinct populations of ATMs with unique tissue distributions, transcriptomes, chromatin landscapes, and functions. Mouse Ly6c ATMs reside outside of CLS and are adipogenic, while CD9 ATMs reside within CLS, are lipid-laden, and are proinflammatory. Adoptive transfer of Ly6c ATMs into lean mice activates gene programs typical of normal adipocyte physiology. By contrast, adoptive transfer of CD9 ATMs drives gene expression that is characteristic of obesity. Importantly, human adipose tissue contains similar ATM populations, including lipid-laden CD9 ATMs that increase with body mass. These results provide a higher resolution of the cellular and functional heterogeneity within ATMs and provide a framework within which to develop new immune-directed therapies for the treatment of obesity and related sequela.
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Tejido Adiposo/citología , Inflamación/fisiopatología , Macrófagos , Animales , Exosomas/química , Femenino , Humanos , Inflamación/genética , Macrófagos/química , Macrófagos/clasificación , Macrófagos/citología , Macrófagos/metabolismo , Ratones , Ratones Endogámicos C57BL , Obesidad/metabolismo , Obesidad/fisiopatología , Tetraspanina 29/análisis , Tetraspanina 29/metabolismo , Transcriptoma/genéticaRESUMEN
Sequence-specific DNA binding recruits transcription factors (TFs) to the genome to regulate gene expression. Here, we perform high resolution mapping of CEBP proteins to determine how sequence dictates genomic occupancy. We demonstrate a fundamental difference between the sequence repertoire utilized by CEBPs in vivo versus the palindromic sequence preference reported by classical in vitro models, by identifying a palindromic motif at <1% of the genomic binding sites. On the native genome, CEBPs bind a diversity of related 10 bp sequences resulting from the fusion of degenerate and canonical half-sites. Altered DNA specificity of CEBPs in cells occurs through heterodimerization with other bZip TFs, and approximately 40% of CEBP-binding sites in primary human cells harbor motifs characteristic of CEBP heterodimers. In addition, we uncover an important role for sequence bias at core-motif-flanking bases for CEBPs and demonstrate that flanking bases regulate motif function across mammalian bZip TFs. Favorable flanking bases confer efficient TF occupancy and transcriptional activity, and DNA shape may explain how the flanks alter TF binding. Importantly, motif optimization within the 10-mer is strongly correlated with cell-type-independent recruitment of CEBPß, providing key insight into how sequence sub-optimization affects genomic occupancy of widely expressed CEBPs across cell types.
Asunto(s)
Proteínas Potenciadoras de Unión a CCAAT/química , Motivos de Nucleótidos , Transcripción Genética , Animales , Secuencia de Bases , Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/química , Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/metabolismo , Sitios de Unión , Proteínas Potenciadoras de Unión a CCAAT/metabolismo , Inmunoprecipitación de Cromatina , Dimerización , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Ratones , Polimorfismo de Nucleótido Simple , Unión Proteica , Organismos Libres de Patógenos Específicos , Relación Estructura-Actividad , Especificidad por SustratoRESUMEN
Glucocorticoids (GCs) are commonly prescribed drugs, but their anti-inflammatory benefits are mitigated by metabolic side effects. Their transcriptional effects, including tissue-specific gene activation and repression, are mediated by the glucocorticoid receptor (GR), which is known to bind as a homodimer to a palindromic DNA sequence. Using ChIP-exo in mouse liver under endogenous corticosterone exposure, we report here that monomeric GR interaction with a half-site motif is more prevalent than homodimer binding. Monomers colocalize with lineage-determining transcription factors in both liver and primary macrophages, and the GR half-site motif drives transcription, suggesting that monomeric binding is fundamental to GR's tissue-specific functions. In response to exogenous GC in vivo, GR dimers assemble on chromatin near ligand-activated genes, concomitant with monomer evacuation of sites near repressed genes. Thus, pharmacological GCs mediate gene expression by favoring GR homodimer occupancy at classic palindromic sites at the expense of monomeric binding. The findings have important implications for improving therapies that target GR.
Asunto(s)
Genómica/métodos , Glucocorticoides/farmacología , Receptores de Glucocorticoides/genética , Activación Transcripcional , Animales , Células Cultivadas , Cromatina/genética , Cromatina/metabolismo , Inmunoprecipitación de Cromatina , Clonación Molecular , Expresión Génica , Terapia Genética , Secuenciación de Nucleótidos de Alto Rendimiento , Hígado/efectos de los fármacos , Hígado/metabolismo , Macrófagos/efectos de los fármacos , Macrófagos/metabolismo , Masculino , Ratones , Ratones Endogámicos BALB C , Receptores de Glucocorticoides/metabolismo , Análisis de Secuencia de ARN , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
FGF21 is an atypical member of the FGF family that functions as a hormone to regulate carbohydrate and lipid metabolism. Here we demonstrate that the actions of FGF21 in mouse adipose tissue, but not in liver, are modulated by the nuclear receptor Rev-erbα, a potent transcriptional repressor. Interrogation of genes induced in the absence of Rev-erbα for Rev-erbα-binding sites identified ßKlotho, an essential coreceptor for FGF21, as a direct target gene of Rev-erbα in white adipose tissue but not liver. Rev-erbα ablation led to the robust elevated expression of ßKlotho. Consequently, the effects of FGF21 were markedly enhanced in the white adipose tissue of mice lacking Rev-erbα. A major Rev-erbα-controlled enhancer at the Klb locus was also bound by the adipocytic transcription factor peroxisome proliferator-activated receptor (PPAR) γ, which regulates its activity in the opposite direction. These findings establish Rev-erbα as a specific modulator of FGF21 signaling in adipose tissue.
Asunto(s)
Adipocitos/metabolismo , Tejido Adiposo/metabolismo , Factores de Crecimiento de Fibroblastos/metabolismo , Miembro 1 del Grupo D de la Subfamilia 1 de Receptores Nucleares/metabolismo , Transducción de Señal/fisiología , Adipocitos/citología , Tejido Adiposo/citología , Animales , Femenino , Factores de Crecimiento de Fibroblastos/genética , Proteínas Klotho , Masculino , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Ratones , Ratones Noqueados , Miembro 1 del Grupo D de la Subfamilia 1 de Receptores Nucleares/genética , PPAR gamma/genética , PPAR gamma/metabolismoRESUMEN
Brown adipocytes and muscle and dorsal dermis descend from precursor cells in the dermomyotome, but the factors that regulate commitment to the brown adipose lineage are unknown. Here, we prospectively isolated and determined the molecular profile of embryonic brown preadipose cells. Brown adipogenic precursor activity in embryos was confined to platelet-derived growth factor α(+), myogenic factor 5(Cre)-lineage-marked cells. RNA-sequence analysis identified early B-cell factor 2 (Ebf2) as one of the most selectively expressed genes in this cell fraction. Importantly, Ebf2-expressing cells purified from Ebf2(GFP) embryos or brown fat tissue did not express myoblast or dermal cell markers and uniformly differentiated into brown adipocytes. Interestingly, Ebf2-expressing cells from white fat tissue in adult animals differentiated into brown-like (or beige) adipocytes. Loss of Ebf2 in brown preadipose cells reduced the expression levels of brown preadipose-signature genes, whereas ectopic Ebf2 expression in myoblasts activated brown preadipose-specific genes. Altogether, these results indicate that Ebf2 specifically marks and regulates the molecular profile of brown preadipose cells.
Asunto(s)
Adipocitos/metabolismo , Tejido Adiposo Pardo/metabolismo , Tejido Adiposo/metabolismo , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Adipocitos/citología , Adipogénesis/genética , Tejido Adiposo/citología , Tejido Adiposo/embriología , Tejido Adiposo Pardo/citología , Tejido Adiposo Pardo/embriología , Tejido Adiposo Blanco/citología , Tejido Adiposo Blanco/embriología , Tejido Adiposo Blanco/metabolismo , Animales , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Biomarcadores/metabolismo , Linaje de la Célula/genética , Células Cultivadas , Embrión de Mamíferos/citología , Embrión de Mamíferos/embriología , Embrión de Mamíferos/metabolismo , Citometría de Flujo , Perfilación de la Expresión Génica , Regulación del Desarrollo de la Expresión Génica , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Inmunohistoquímica , Ratones de la Cepa 129 , Ratones Endogámicos C57BL , Ratones Transgénicos , Microscopía Confocal , Factor 5 Regulador Miogénico/genética , Factor 5 Regulador Miogénico/metabolismo , Análisis de Secuencia por Matrices de Oligonucleótidos , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
BACKGROUND: Recent mapping of 5-hydroxymethylcytosine (5hmC) provides a genome-wide view of the distribution of this important chromatin mark. However, the role of 5hmC in specific regulatory regions is not clear, especially at enhancers. RESULTS: We found a group of distal transcription factor binding sites highly enriched for 5-hdroxymethylcytosine (5hmC), but lacking any known activating histone marks and being depleted for nascent transcripts, suggesting a repressive role for 5hmC in mouse embryonic stem cells (mESCs). 5-formylcytosine (5fC), which is known to mark poised enhancers where H3K4me1 is enriched, is also observed at these sites. Furthermore, the 5hmC levels were inversely correlated with RNA polymerase II (PolII) occupancy in mESCs as well as in fully differentiated adipocytes. Interestingly, activating H3K4me1/2 histone marks were enriched at these sites when the associated genes become activated following lineage specification. These putative enhancers were shown to be functional in embryonic stem cells when unmethylated. Together, these data suggest that 5hmC suppresses the activity of this group of enhancers, which we termed "silenced enhancers". CONCLUSIONS: Our findings indicate that 5hmC has a repressive role at specific proximal and distal regulatory regions in mESCs, and suggest that 5hmC is a new epigenetic mark for silenced enhancers.
Asunto(s)
Citosina/análogos & derivados , Células Madre Embrionarias/metabolismo , Elementos de Facilitación Genéticos/genética , Epigénesis Genética , 5-Metilcitosina/análogos & derivados , Animales , Sitios de Unión , Linaje de la Célula , Citosina/metabolismo , Células Madre Embrionarias/citología , Histonas/metabolismo , Ratones , ARN Mensajero/genética , ARN Mensajero/metabolismo , Factores de Transcripción/metabolismoRESUMEN
Background: Mesenchymal nephron progenitors (mNPs) give rise to all nephron tubules in the mammalian kidney. Since premature depletion of these cells leads to low nephron numbers, high blood pressure, and various renal diseases, it is critical to understand how mNPs are maintained. While Fgf, Bmp, and Wnt signaling pathways are known to be required for the maintenance of these cells, it is unclear if any other signaling pathways also play roles. Methods: To test the potential role of Hedgehog signaling in mNPs, we conditionally deleted Shh from the collecting duct and Smo from the nephron lineage. To identify the genes regulated by Hedgehog signaling in mNPs, we performed RNA-seq analysis from mNPs with different Smo doses. To test if the upregulation of Notch signaling mimics loss of Hedgehog signaling, we performed Jag1 gain-of-function study in mNPs. Results: We found that loss of either Shh or Smo resulted in premature depletion of mNPs. Our transcriptional profiling data from Smo loss- and gain-of-function mutant mNPs suggested that Hedgehog signaling inhibited the activation of Notch signaling and upregulated the expression of Fox transcription factors such as Foxc1 and Foxp4. Consistent with these observations, we found that ectopic expression of Jag1 caused the premature depletion of mNPs as seen in the Smo mutant kidney. We also found that Foxc1 was capable of binding to mitotic condensed chromatin, a feature of a mitotic bookmarking factor. Conclusions: Our study demonstrates a previously unappreciated role of Hedgehog signaling in preventing premature depletion of mNPs by repressing Notch signaling and likely by activating the expression of Fox factors.