RESUMEN
G9a is a histone methyltransferase responsible for the dimethylation of histone H3 at lysine 9 (H3K9me2). G9a plays key roles in transcriptional silencing of developmentally regulated genes, but its role in X-chromosome inactivation (XCI) has been under debate. Here, we uncover a female-specific function of G9a and demonstrate that deleting G9a has a disproportionate impact on the X chromosome relative to the rest of the genome. G9a deficiency causes a failure of XCI and female-specific hypersensitivity to drug inhibition of H3K9me2. We show that G9a interacts with Tsix and Xist RNAs, and that competitive inhibition of the G9a-RNA interaction recapitulates the XCI defect. During XCI, Xist recruits G9a to silence X-linked genes on the future inactive X. In parallel on the future Xa, Tsix recruits G9a to silence Xist in cis Thus, RNA tethers G9a for allele-specific targeting of the H3K9me2 modification and the G9a-RNA interaction is essential for XCI.
Asunto(s)
Cromosomas Humanos X , Antígenos de Histocompatibilidad/metabolismo , N-Metiltransferasa de Histona-Lisina/metabolismo , Metiltransferasas , ARN Largo no Codificante , Femenino , Histonas/metabolismo , Humanos , Metiltransferasas/genética , ARN Largo no Codificante/genética , Inactivación del Cromosoma X/genéticaRESUMEN
In mammals, dosage compensation between XX and XY individuals occurs through X chromosome inactivation (XCI). The noncoding Xist RNA is expressed and initiates XCI only when more than one X chromosome is present. Current models invoke a dependency on the X-to-autosome ratio (X:A), but molecular factors remain poorly defined. Here, we demonstrate that molecular titration between an X-encoded RNA and an autosomally encoded protein dictates Xist induction. In pre-XCI cells, CTCF protein represses Xist transcription. At the onset of XCI, Jpx RNA is upregulated, binds CTCF, and extricates CTCF from one Xist allele. We demonstrate that CTCF is an RNA-binding protein and is titrated away from the Xist promoter by Jpx RNA. Thus, Jpx activates Xist by evicting CTCF. The functional antagonism via molecular titration reveals a role for long noncoding RNA in epigenetic regulation.
Asunto(s)
ARN Largo no Codificante/metabolismo , Proteínas Represoras/metabolismo , Regulación hacia Arriba , Inactivación del Cromosoma X , Animales , Factor de Unión a CCCTC , Cromosomas de los Mamíferos/metabolismo , Células Madre Embrionarias/metabolismo , Femenino , Masculino , Ratones , Regiones Promotoras Genéticas , ARN Largo no Codificante/genética , Cromosoma X/metabolismoRESUMEN
CTCF is a master regulator that plays important roles in genome architecture and gene expression. How CTCF is recruited in a locus-specific manner is not fully understood. Evidence from epigenetic processes, such as X chromosome inactivation (XCI), indicates that CTCF associates functionally with RNA. Using genome-wide approaches to investigate the relationship between its RNA interactome and epigenomic landscape, here we report that CTCF binds thousands of transcripts in mouse embryonic stem cells, many in close proximity to CTCF's genomic binding sites. CTCF is a specific and high-affinity RNA-binding protein (Kd < 1 nM). During XCI, CTCF differentially binds the active and inactive X chromosomes and interacts directly with Tsix, Xite, and Xist RNAs. Tsix and Xite RNAs target CTCF to the X inactivation center, thereby inducing homologous X chromosome pairing. Our work elucidates one mechanism by which CTCF is recruited in a locus-specific manner and implicates CTCF-RNA interactions in long-range chromosomal interactions.
Asunto(s)
ARN Mensajero/metabolismo , Proteínas de Unión al ARN/metabolismo , Proteínas Represoras/metabolismo , Cromosoma X/genética , Animales , Factor de Unión a CCCTC , Células Cultivadas , Emparejamiento Cromosómico , Células Madre Embrionarias/metabolismo , Epigénesis Genética , Sitios Genéticos , Ratones , Unión ProteicaRESUMEN
BACKGROUND: Treatment options for malignant pleural mesothelioma are scarce. Tazemetostat, a selective oral enhancer of zeste homolog 2 (EZH2) inhibitor, has shown antitumour activity in several haematological cancers and solid tumours. We aimed to evaluate the anti-tumour activity and safety of tazemetostat in patients with measurable relapsed or refractory malignant pleural mesothelioma. METHODS: We conducted an open-label, single-arm phase 2 study at 16 hospitals in France, the UK, and the USA. Eligible patients were aged 18 years or older with malignant pleural mesothelioma of any histology that was relapsed or refractory after treatment with at least one pemetrexed-containing regimen, an Eastern Cooperative Oncology Group performance status of 0 or 1, and a life expectancy of greater than 3 months. In part 1 of the study, participants received oral tazemetostat 800 mg once on day 1 and then twice daily from day 2 onwards. In part 2, participants received oral tazemetostat 800 mg twice daily starting on day 1 of cycle 1, using a two-stage Green-Dahlberg design. Tazemetostat was administered in 21-day cycles for approximately 17 cycles. The primary endpoint of part 1 was the pharmacokinetics of tazemetostat and its metabolite at day 15 after administration of 800 mg tazemetostat, as measured by maximum serum concentration (Cmax), time to Cmax (Tmax), area under the concentration-time curve (AUC) to day 15 (AUC0-t), area under the curve from time 0 extrapolated to infinity (AUC0-∞), and the half-life (t1/2) of tazemetostat, assessed in all patients enrolled in part 1. The primary endpoint of part 2 was the disease control rate (the proportion of patients with a complete response, partial response, or stable disease) at week 12 in patients with malignant pleural mesothelioma per protocol with BAP1 inactivation determined by immunohistochemistry. The safety population included all the patients who had at least one post-dose safety assessment. This trial is now complete and is registered with ClinicalTrials.gov, NCT02860286. FINDINGS: Between July 29, 2016, and June 2, 2017, 74 patients were enrolled (13 in part 1 and 61 in part 2) and received tazemetostat, 73 (99%) of whom had BAP1-inactivated tumours. In part 1, following repeat dosing of tazemetostat at steady state, on day 15 of cycle 1, the mean Cmax was 829 ng/mL (coefficient of variation 56·3%), median Tmax was 2 h (range 1-4), mean AUC0-twas 3310 h·ng/mL (coefficient of variation 50·4%), mean AUC0-∞ was 3180 h·ng/mL (46·6%), and the geometric mean t1/2 was 3·1 h (13·9%). After a median follow-up of 35·9 weeks (IQR 20·6-85·9), the disease control rate in part 2 in patients with BAP1-inactivated malignant pleural mesothelioma was 54% (95% CI 42-67; 33 of 61 patients) at week 12. No patients had a confirmed complete response. Two patients had a confirmed partial response: one had an ongoing partial response with a duration of 18 weeks and the other had a duration of 42 weeks. The most common grade 3-4 treatment-emergent adverse events were hyperglycaemia (five [7%] patients), hyponatraemia (five [7%]), and anaemia (four [5%]); serious adverse events were reported in 25 (34%) of 74 patients. Five (7%) of 74 patients died while on study; no treatment-related deaths occurred. INTERPRETATION: Further refinement of biomarkers for tazemetostat activity in malignant pleural mesothelioma beyond BAP1 inactivation could help identify a subset of tumours that are most likely to derive prolonged benefit or shrinkage from this therapy. FUNDING: Epizyme.
Asunto(s)
Mesotelioma Maligno , Mesotelioma , Neoplasias , Benzamidas/efectos adversos , Compuestos de Bifenilo , Proteína Potenciadora del Homólogo Zeste 2/genética , Inhibidores Enzimáticos/uso terapéutico , Humanos , Mesotelioma/tratamiento farmacológico , Mesotelioma/patología , Morfolinas/uso terapéutico , Neoplasias/inducido químicamente , Piridonas , Proteínas Supresoras de Tumor , Ubiquitina TiolesterasaRESUMEN
A key issue in the immune system is to generate specific cell types, often with opposing activities. The mechanisms of differentiation and subtype specification of immune cells such as macrophages and dendritic cells are critical to understand the regulatory principles and logic of the immune system. In addition to cytokines and pathogens, it is increasingly appreciated that lipid signaling also has a key role in differentiation and subtype specification. In this review we explore how intracellular lipid signaling via a set of transcription factors regulates cellular differentiation, subtype specification, and immune as well as metabolic homeostasis. We introduce macrophages and dendritic cells and then we focus on a group of transcription factors, nuclear receptors, which regulate gene expression upon receiving lipid signals. The receptors we cover are the ones with a recognized physiological function in these cell types and ones which heterodimerize with the retinoid X receptor. These are as follows: the receptor for a metabolite of vitamin A, retinoic acid: retinoic acid receptor (RAR), the vitamin D receptor (VDR), the fatty acid receptor: peroxisome proliferator-activated receptor γ (PPARγ), the oxysterol receptor liver X receptor (LXR), and their obligate heterodimeric partner, the retinoid X receptor (RXR). We discuss how they can get activated and how ligand is generated and eliminated in these cell types. We also explore how activation of a particular target gene contributes to biological functions and how the regulation of individual target genes adds up to the coordination of gene networks. It appears that RXR heterodimeric nuclear receptors provide these cells with a coordinated and interrelated network of transcriptional regulators for interpreting the lipid milieu and the metabolic changes to bring about gene expression changes leading to subtype and functional specification. We also show that these networks are implicated in various immune diseases and are amenable to therapeutic exploitation.
Asunto(s)
Presentación de Antígeno/inmunología , Células Dendríticas/inmunología , Metabolismo de los Lípidos/inmunología , Macrófagos/inmunología , Receptores Citoplasmáticos y Nucleares/inmunología , Animales , Células Dendríticas/metabolismo , Regulación de la Expresión Génica/inmunología , Humanos , Activación de Linfocitos/inmunología , Macrófagos/metabolismo , Masculino , Ratones , PPAR gamma/inmunología , Ratas , Receptores Citoplasmáticos y Nucleares/genética , Receptores Citoplasmáticos y Nucleares/metabolismo , Factores de Transcripción/metabolismo , Tretinoina/farmacología , Vitamina D/farmacologíaRESUMEN
Peroxisome proliferator-activated receptor γ (PPARγ) is a lipid-activated transcription factor regulating lipid metabolism and inflammatory response in macrophages and dendritic cells (DCs). These immune cells exposed to distinct inflammatory milieu show cell type specification as a result of altered gene expression. We demonstrate here a mechanism how inflammatory molecules modulate PPARγ signaling in distinct subsets of cells. Proinflammatory molecules inhibited whereas interleukin-4 (IL-4) stimulated PPARγ activity in macrophages and DCs. Furthermore, IL-4 signaling augmented PPARγ activity through an interaction between PPARγ and signal transducer and activators of transcription 6 (STAT6) on promoters of PPARγ target genes, including FABP4. Thus, STAT6 acts as a facilitating factor for PPARγ by promoting DNA binding and consequently increasing the number of regulated genes and the magnitude of responses. This interaction, underpinning cell type-specific responses, represents a unique way of controlling nuclear receptor signaling by inflammatory molecules in immune cells.
Asunto(s)
Células Dendríticas/metabolismo , Regulación de la Expresión Génica , Macrófagos/metabolismo , PPAR gamma/metabolismo , Factor de Transcripción STAT6/metabolismo , Animales , Proteínas de Unión a Ácidos Grasos/metabolismo , Humanos , Mediadores de Inflamación/metabolismo , Interleucina-4/metabolismo , Ratones , Regiones Promotoras GenéticasRESUMEN
X-chromosome inactivation is a mechanism of dosage compensation in which one of the two X chromosomes in female mammals is transcriptionally silenced. Once established, silencing of the inactive X (Xi) is robust and difficult to reverse pharmacologically. However, the Xi is a reservoir of >1,000 functional genes that could be potentially tapped to treat X-linked disease. To identify compounds that could reactivate the Xi, here we screened â¼367,000 small molecules in an automated high-content screen using an Xi-linked GFP reporter in mouse fibroblasts. Given the robust nature of silencing, we sensitized the screen by "priming" cells with the DNA methyltransferase inhibitor, 5-aza-2'-deoxycytidine (5azadC). Compounds that elicited GFP activity include VX680, MLN8237, and 5azadC, which are known to target the Aurora kinase and DNA methylation pathways. We demonstrate that the combinations of VX680 and 5azadC, as well as MLN8237 and 5azadC, synergistically up-regulate genes on the Xi. Thus, our work identifies a synergism between the DNA methylation and Aurora kinase pathways as being one of interest for possible pharmacological reactivation of the Xi.
Asunto(s)
Aurora Quinasas/antagonistas & inhibidores , Metilación de ADN/efectos de los fármacos , Inactivación del Cromosoma X/efectos de los fármacos , Animales , Aurora Quinasa A/antagonistas & inhibidores , Aurora Quinasa A/genética , Aurora Quinasa B/antagonistas & inhibidores , Aurora Quinasa B/genética , Aurora Quinasas/genética , Azacitidina/administración & dosificación , Azacitidina/análogos & derivados , Azepinas/administración & dosificación , Línea Celular , Decitabina , Evaluación Preclínica de Medicamentos , Sinergismo Farmacológico , Femenino , Técnicas de Silenciamiento del Gen , Genes Ligados a X , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Ensayos Analíticos de Alto Rendimiento , Ratones , Ratones Transgénicos , Piperazinas/administración & dosificación , Pirimidinas/administración & dosificación , Cromosoma X/efectos de los fármacos , Cromosoma X/genéticaRESUMEN
Cell-type specific gene silencing by histone H3 lysine 27 and lysine 9 methyltransferase complexes PRC2 and G9A-GLP is crucial both during development and to maintain cell identity. Although studying their interaction partners has yielded valuable insight into their functions, how these factors are regulated on a network level remains incompletely understood. Here, we present a new approach that combines quantitative interaction proteomics with global chromatin profiling to functionally characterize repressive chromatin modifying protein complexes in embryonic stem cells. We define binding stoichiometries of 9 new and 12 known interaction partners of PRC2 and 10 known and 29 new interaction partners of G9A-GLP, respectively. We demonstrate that PRC2 and G9A-GLP interact physically and share several interaction partners, including the zinc finger proteins ZNF518A and ZNF518B. Using global chromatin profiling by targeted mass spectrometry, we discover that even sub-stoichiometric binding partners such as ZNF518B can positively regulate global H3K9me2 levels. Biochemical analysis reveals that ZNF518B directly interacts with EZH2 and G9A. Our systematic analysis suggests that ZNF518B may mediate the structural association between PRC2 and G9A-GLP histone methyltransferases and additionally regulates the activity of G9A-GLP.
Asunto(s)
N-Metiltransferasa de Histona-Lisina/metabolismo , Dedos de Zinc/fisiología , Animales , Células Madre Embrionarias/metabolismo , Ratones , ProteómicaRESUMEN
OBJECTIVE: To identify microRNAs (miRNAs) in human T cells that can explain known antiinflammatory properties of steroids. METHODS: Activated human CD4+ T cells from healthy donors were exposed to 1 µM methylprednisolone (MP) in vitro and then subjected to miRNA and messenger RNA microarray analyses. Changes in expression profiles were recorded. Using quantitative polymerase chain reaction (qPCR), flow cytometry, and enzyme-linked immunosorbent assay (ELISA), we confirmed the suppression of predicted targets, and through miRNA transfection experiments, we could suggest mechanistic links. RESULTS: We identified numerous steroid-responsive genes and miRNAs-many known and some novel-including multiple previously unknown proinflammatory genes suppressed by MP. Further studies using qPCR, flow cytometry, and ELISA demonstrated that methylprednisolone increased the expression of miRNA-98 (miR-98) and suppressed the levels of predicted targets, including interleukin-13 and 3 tumor necrosis factor receptors (TNFRs): Fas, FasL, and TNFR superfamily member 1B. Forced expression of miR-98 in T cells resulted in suppression of the same targets. CONCLUSION: The findings of this study demonstrate a link between miR-98 expression and the effects of MP and provide evidence suggesting that MP acts through miR-98 to inhibit specific proinflammatory targets. Identification of this antiinflammatory mechanism of glucocorticoids is important, since it may pave the way toward the elusive goal of dissociating adverse effects from therapeutic effects.
Asunto(s)
Glucocorticoides/farmacología , Metilprednisolona/farmacología , MicroARNs/efectos de los fármacos , Linfocitos T/efectos de los fármacos , Perfilación de la Expresión Génica , Humanos , MicroARNs/metabolismo , Linfocitos T/metabolismo , Regulación hacia ArribaRESUMEN
The nuclear receptor peroxisome proliferator-activated receptor-gamma (PPARgamma) has important roles in adipogenesis and immune response as well as roles in both lipid and carbohydrate metabolism. Although synthetic agonists for PPARgamma are widely used as insulin sensitizers, the identity of the natural ligand(s) for PPARgamma is still not clear. Suggested natural ligands include 15-deoxy-delta12,14-prostaglandin J2 and oxidized fatty acids such as 9-HODE and 13-HODE. Crystal structures of PPARgamma have revealed the mode of recognition for synthetic compounds. Here we report structures of PPARgamma bound to oxidized fatty acids that are likely to be natural ligands for this receptor. These structures reveal that the receptor can (i) simultaneously bind two fatty acids and (ii) couple covalently with conjugated oxo fatty acids. Thermal stability and gene expression analyses suggest that such covalent ligands are particularly effective activators of PPARgamma and thus may serve as potent and biologically relevant ligands.
Asunto(s)
Ácidos Grasos/química , Ácidos Grasos/metabolismo , PPAR gamma/química , PPAR gamma/metabolismo , Sustitución de Aminoácidos , Animales , Sitios de Unión/genética , Células COS , Chlorocebus aethiops , Cisteína/química , Humanos , Ligandos , Sustancias Macromoleculares/química , Modelos Moleculares , Estructura Molecular , Mutagénesis Sitio-Dirigida , Oxidación-Reducción , PPAR gamma/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Electricidad EstáticaRESUMEN
Recent studies in cell lines and genetically engineered mice have demonstrated that cytosolic dsDNA could activate dendritic cells (DCs) to become effector APCs. Recognition of DNA might be a major factor in antimicrobial immune responses against cytosolic pathogens and also in human autoimmune diseases such as systemic lupus erythematosus. However, the role of cytosolic dsDNA in human DC activation and its effects on effector T and B cells are still elusive. In this study, we demonstrate that intracellular dsDNA is a potent activator of human monocyte-derived DCs as well as primary DCs. Activation by dsDNA depends on NF-κB activation, partially on the adaptor molecule IFN-promoter stimulator-1 and the novel cytosolic dsDNA receptor IFI16, but not on the previously recognized dsDNA sentinels absent in melanoma 2, DNA-dependent activator of IFN regulatory factor 3, RNA polymerase III, or high-mobility group boxes. More importantly, we report for the first time, to our knowledge, that human dsDNA-activated DCs, rather than LPS- or inflammatory cytokine mixture-activated DCs, represent the most potent inducers of naive CD4(+) T cells to promote Th1-type cytokine production and generate CD4(+) and CD8(+) cytotoxic T cells. dsDNA-DCs, but not LPS- or mixture-activated DCs, induce B cells to produce complement-fixing IgG1 and IgG3 Abs. We propose that cytosolic dsDNA represents a novel, more effective approach to generate DCs to enhance vaccine effectiveness in reprogramming the adaptive immune system to eradicate infectious agents, autoimmunity, allergy, and cancer.
Asunto(s)
Inmunidad Adaptativa , Citosol/inmunología , ADN/inmunología , Células Dendríticas/inmunología , Inmunidad Adaptativa/genética , Animales , Linfocitos B/inmunología , Linfocitos B/metabolismo , Linfocitos T CD4-Positivos/citología , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD8-positivos/citología , Linfocitos T CD8-positivos/inmunología , Muerte Celular/genética , Muerte Celular/inmunología , Diferenciación Celular/genética , Diferenciación Celular/inmunología , Línea Celular Tumoral , Células Cultivadas , Técnicas de Cocultivo , ADN/genética , Células Dendríticas/citología , Células Dendríticas/metabolismo , Humanos , Líquido Intracelular/inmunología , Líquido Intracelular/metabolismo , Ratones , Monocitos/citología , Monocitos/inmunología , Monocitos/metabolismo , Vacunas de ADN/inmunología , Vacunas de ADN/metabolismo , Vacunas de ADN/uso terapéuticoRESUMEN
Expression of CD1a protein defines a human dendritic cell (DC) subset with unique functional activities. We aimed to study the expression of the Nav1.7 sodium channel and the functional consequences of its activity in CD1a(-) and CD1a(+) DC. Single-cell electrophysiology (patch-clamp) and quantitative PCR experiments performed on sorted CD1a(-) and CD1a(+) immature DC (IDC) showed that the frequency of cells expressing Na(+) current, current density, and the relative expression of the SCN9A gene encoding Nav1.7 were significantly higher in CD1a(+) cells than in their CD1a(-) counterparts. The activity of Nav1.7 results in a depolarized resting membrane potential (-8.7 ± 1.5 mV) in CD1a(+) IDC as compared with CD1a(-) cells lacking Nav1.7 (-47 ± 6.2 mV). Stimulation of DC by inflammatory signals or by increased intracellular Ca(2+) levels resulted in reduced Nav1.7 expression. Silencing of the SCN9A gene shifted the membrane potential to a hyperpolarizing direction in CD1a(+) IDC, resulting in decreased cell migration, whereas pharmacological inhibition of Nav1.7 by tetrodotoxin sensitized the cells for activation signals. Fine-tuning of IDC functions by a voltage-gated sodium channel emerges as a new regulatory mechanism modulating the migration and cytokine responses of these DC subsets.
Asunto(s)
Movimiento Celular/inmunología , Quimiocinas/fisiología , Células Dendríticas/inmunología , Células Dendríticas/metabolismo , Potenciales de la Membrana/inmunología , Monocitos/inmunología , Monocitos/metabolismo , Canales de Sodio/fisiología , Diferenciación Celular/inmunología , Células Cultivadas , Citocinas/metabolismo , Células Dendríticas/citología , Humanos , Monocitos/citología , Canal de Sodio Activado por Voltaje NAV1.7 , Fase de Descanso del Ciclo Celular/inmunologíaRESUMEN
Granulomatous inflammations, characterized by the presence of activated macrophages (MAs) forming epithelioid cell (EPC) clusters, are usually easy to recognize. However, in ambiguous cases the use of a MA marker that expresses selectively in EPCs may be needed. Here, we report that carboxypeptidase-M (CPM), a MA-differentiation marker, is preferentially induced in EPCs of all granuloma types studied, but not in resting MAs. As CPM is not expressed constitutively in MAs, this allows utilization of CPM-immunohistochemistry in diagnostics of minute granuloma detection when dense non-granulomatous MAs are also present. Despite this rule, hardly any detectable CPM was found in advanced/active tubercle caseous disease, albeit in early tuberculosis granuloma, MAs still expressed CPM. Indeed, in vitro both the CPM-protein and -mRNA became downregulated when MAs were infected with live mycobacteria. In vitro, MA-CPM transcript is neither induced remarkably by interferon-γ, known to cause classical MA activation, nor by IL-4, an alternative MA activator. Instead, CPM is selectively expressed in lipid-laden MAs, including the foam cells of atherosclerotic plaques, xanthomatous lesions and lipid pneumonias. By using serum, rich in lipids, and low-density lipoprotein (LDL) or VLDL, CPM upregulation could be reproduced in vitro in monocyte-derived MAs both at transcriptional and protein levels, and the increase is repressed under lipid-depleted conditions. The microarray analyses support the notion that CPM induction correlates with a robust progressive increase in CPM gene expression during monocyte to MA maturation and dendritic cell (DC) differentiation mediated by granulocyte-MA-colony-stimulating factor+IL-4. M-CSF alone also induced CPM. These results collectively indicate that CPM upregulation in MAs is preferentially associated with increased lipid uptake, and exposure to CSF, features of EPCs, also. Therefore, CPM-immunohistochemistry is useful for granuloma and foam MA detections in tissue sections. Furthermore, the present data offer CPM for the first time to be a novel marker and cellular player in lipid uptake and/or metabolism of MAs by promoting foam cell formation.
Asunto(s)
Células Epitelioides/enzimología , Células Espumosas/enzimología , Factor Estimulante de Colonias de Granulocitos y Macrófagos/metabolismo , Granuloma/metabolismo , Metaloendopeptidasas/metabolismo , Biomarcadores/metabolismo , Diferenciación Celular , Células Dendríticas/citología , Células Dendríticas/metabolismo , Proteínas Ligadas a GPI/metabolismo , Humanos , Inmunohistoquímica , Interleucina-4/metabolismo , Metabolismo de los Lípidos , Análisis de Secuencia por Matrices de Oligonucleótidos , ARN Mensajero/metabolismo , Reacción en Cadena en Tiempo Real de la Polimerasa , Activación Transcripcional , Regulación hacia ArribaRESUMEN
BACKGROUND: Chronic obstructive pulmonary disease (COPD) is a chronic inflammatory disease characterized by progressive airflow limitation and significant extrapulmonary (systemic) effects that lead to co-morbid conditions, though the pathomechanism of COPD is largely undetermined. Alveolar macrophages (AM) derived from peripheral monocytes (MO) appear to play a key role in initiating and/or sustaining disease progression. OBJECTIVES: To identify disease- and cell type-specific gene expression profiles and potential overlaps in those in order to diagnose COPD, characterize its progression and determine the effect of drug treatment. METHOD: Global gene expression analysis was used for primary screening in order to obtain expression signatures of AMs and circulating MOs of COPD patients and healthy controls. The results of microarray analyses of AMs (20 controls and 26 COPD patients) and MOs (16 controls and 22 COPD patients) were confirmed and validated by real-time quantitative polymerase chain reaction. RESULTS: We have identified gene sets specifically associated with COPD in AMs and MOs. There were overlapping genes between the two cell types. Our data also show that COPD-specific gene expression signatures in AMs and MOs correlate with percent of predicted FEV(1). CONCLUSION: Disease-specific and overlapping gene expression signatures can be defined in lung-derived macrophages and also in circulating monocytes. Some of the validated expression changes in both cell types correlate with lung function and therefore could serve as biomarkers of disease progression.
Asunto(s)
Macrófagos Alveolares/metabolismo , Monocitos/metabolismo , Enfermedad Pulmonar Obstructiva Crónica/genética , Adulto , Anciano , Biomarcadores/metabolismo , Líquido del Lavado Bronquioalveolar/inmunología , Femenino , Volumen Espiratorio Forzado , Perfilación de la Expresión Génica , Humanos , Masculino , Persona de Mediana Edad , Análisis de Secuencia por Matrices de Oligonucleótidos , Enfermedad Pulmonar Obstructiva Crónica/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
Although polycomb repressive complex 2 (PRC2) is now recognized as an RNA-binding complex, the full range of binding motifs and why PRC2-RNA complexes often associate with active genes have not been elucidated. Here, we identify high-affinity RNA motifs whose mutations weaken PRC2 binding and attenuate its repressive function in mouse embryonic stem cells. Interactions occur at promoter-proximal regions and frequently coincide with pausing of RNA polymerase II (POL-II). Surprisingly, while PRC2-associated nascent transcripts are highly expressed, ablating PRC2 further upregulates expression via loss of pausing and enhanced transcription elongation. Thus, PRC2-nascent RNA complexes operate as rheostats to fine-tune transcription by regulating transitions between pausing and elongation, explaining why PRC2-RNA complexes frequently occur within active genes. Nascent RNA also targets PRC2 in cis and downregulates neighboring genes. We propose a unifying model in which RNA specifically recruits PRC2 to repress genes through POL-II pausing and, more classically, trimethylation of histone H3 at Lys27.
Asunto(s)
Regulación de la Expresión Génica/genética , Complejo Represivo Polycomb 2/metabolismo , Biosíntesis de Proteínas/fisiología , ARN Polimerasa II/metabolismo , ARN/metabolismo , Animales , Diferenciación Celular/genética , Línea Celular , Células Madre Embrionarias/metabolismo , Histonas/metabolismo , Metilación , Ratones , Motivos de Nucleótidos/genética , Regiones Promotoras Genéticas/genética , Biosíntesis de Proteínas/genética , ARN/genética , Transcripción Genética/genética , Activación Transcripcional/genéticaRESUMEN
BACKGROUND: Whole-genome sequencing (WGS) costs are falling, yet, outside oncology, this information is seldom used in adult clinics. We piloted a rapid WGS (rWGS) workflow, focusing initially on estimating power for a feasibility study of introducing genome information into acute cardiovascular care. METHODS: A prospective implementation study was conducted to test the feasibility and clinical utility of rWGS in acute cardiovascular care. rWGS was performed on 50 adult patients with acute cardiovascular events and cardiac arrest survivors, testing for primary and secondary disease-causing variants, cardiovascular-related pharmacogenomics, and carrier status for recessive diseases. The impact of returning rWGS results on short-term clinical care of participants was investigated. The utility of polygenic risk scores to stratify coronary artery disease was also assessed. RESULTS: Pathogenic variants, typically secondary findings, were identified in 20% (95% CI, 11.7-34.3). About 60% (95% CI, 46.2-72.4) of participants were carriers for one or more recessive traits, most commonly in HFE and SERPINA1 genes. Although 64% (95% CI, 50.1-75.9) of participants carried at least one pharmacogenetic variant of cardiovascular relevance, these were actionable in only 14% (95% CI, 7-26.2). Coronary artery disease prevalence among participants at the 95th percentile of polygenic risk score was 88.2% (95% CI, 71.8-95.7). CONCLUSIONS: We demonstrated the feasibility of rWGS integration into the inpatient management of adults with acute cardiovascular events. Our pilot identified pathogenic variants in one out of 5 acute vascular patients. Integrating rWGS in clinical care will progressively increase actionability.
Asunto(s)
Enfermedades Cardiovasculares/genética , Secuenciación Completa del Genoma , Enfermedad Aguda , Adulto , Anciano , Enfermedades Cardiovasculares/diagnóstico , Femenino , Frecuencia de los Genes , Proteína de la Hemocromatosis/genética , Humanos , Masculino , Persona de Mediana Edad , Farmacogenética , Proyectos Piloto , Estudios Prospectivos , Factores de Riesgo , alfa 1-Antitripsina/química , alfa 1-Antitripsina/genéticaRESUMEN
The noncoding RNA Xist recruits silencing factors to the inactive X chromosome (Xi) and facilitates re-organization of Xi structure. Here, we examine the mouse epigenomic landscape of Xi and assess how Xist alters chromatin accessibility. Xist deletion triggers a gain of accessibility of select chromatin regions that is regulated by BRG1, an ATPase subunit of the SWI/SNF chromatin-remodeling complex. In vitro, RNA binding inhibits nucleosome-remodeling and ATPase activities of BRG1, while in cell culture Xist directly interacts with BRG1 and expels BRG1 from the Xi. Xist ablation leads to a selective return of BRG1 in cis, starting from pre-existing BRG1 sites that are free of Xist. BRG1 re-association correlates with cohesin binding and restoration of topologically associated domains (TADs) and results in the formation of de novo Xi 'superloops'. Thus, Xist binding inhibits BRG1's nucleosome-remodeling activity and results in expulsion of the SWI/SNF complex from the Xi.
Asunto(s)
Cromatina/metabolismo , ARN Largo no Codificante/metabolismo , Cromosoma X/metabolismo , Adenosina Trifosfatasas/genética , Adenosina Trifosfatasas/metabolismo , Animales , Línea Celular , Cromatina/genética , Proteínas Cromosómicas no Histona/genética , Proteínas Cromosómicas no Histona/metabolismo , Epigénesis Genética/genética , Epigénesis Genética/fisiología , Femenino , Ratones , Nucleosomas/genética , Nucleosomas/metabolismo , ARN Largo no Codificante/genética , ARN no Traducido/genética , ARN no Traducido/metabolismo , Cromosoma X/genéticaRESUMEN
Subclinical inflammation is a candidate etiological factor in the pathogenesis of metabolic syndrome and in the progression of atherosclerosis. A central role for activated macrophages has been elucidated recently as important regulators of the inflammatory process in atherosclerosis. Macrophage differentiation and function can be modulated by a class of transcription factors termed nuclear receptors. These are activated by intermediary products of basic metabolic processes. In this review the contribution of peroxisome proliferator-activated receptors and liver X receptors to macrophage functions in inflammation and lipid metabolism will be discussed in light of their roles in macrophages during atherosclerosis. In the past decade much effort has been made to understand the mechanisms how lipids are handled by macrophages and how inflammation could promote the atherogenic process. Here, we also provide an overview of these two fields.
Asunto(s)
Proteínas de Unión al ADN/fisiología , Inflamación/metabolismo , Macrófagos/metabolismo , PPAR gamma/fisiología , Receptores Citoplasmáticos y Nucleares/fisiología , Animales , Aterosclerosis/inmunología , Aterosclerosis/metabolismo , Aterosclerosis/patología , Adhesión Celular , Proteínas de Unión al ADN/metabolismo , Humanos , Metabolismo de los Lípidos , Receptores X del Hígado , Receptores Nucleares Huérfanos , PPAR gamma/metabolismo , Receptores Citoplasmáticos y Nucleares/metabolismoRESUMEN
The peroxisome proliferator-activated receptor gamma (PPARgamma) is a member of the nuclear receptor superfamily, a group of transcription factors that regulate expression of their target genes upon ligand binding. As endogenous ligands, oxidized fatty acids and prostanoids can bind to and activate the receptor. Natural and synthetic PPARgamma activators have been studied extensively in many inflammatory settings and in most instances they have been shown to be anti-inflammatory. In this review we give an overview of the different molecular mechanisms how PPARgamma and its agonists exert their anti-inflammatory effects both at the cellular level and the level of the organism. The action of PPARgamma in acute and chronic inflammatory diseases and disease models will be presented.
Asunto(s)
Enfermedades Autoinmunes/inmunología , Inflamación/inmunología , Macrófagos/inmunología , PPAR gamma/inmunología , Animales , Enfermedades Autoinmunes/genética , Humanos , Inflamación/genética , Macrófagos/metabolismo , PPAR gamma/agonistas , PPAR gamma/metabolismoRESUMEN
Cellular differentiation is governed by changes in gene expression, but at the same time, a cell's identity needs to be maintained through multiple cell divisions during maturation. In myeloid cell lines, retinoids induce gene expression and a well-characterized two-step lineage-specific differentiation. To identify mechanisms that contribute to cellular transcriptional memory, we analyzed the epigenetic changes taking place on regulatory regions of tissue transglutaminase, a gene whose expression is tightly linked to retinoid-induced differentiation. Here we report that the induction of an intermediary or "primed" state of myeloid differentiation is associated with increased H4 arginine 3 and decreased H3 lysine 4 methylation. These modifications occur before transcription and appear to prime the chromatin for subsequent hormone-regulated transcription. Moreover, inhibition of methyltransferase activity, pre-acetylation, or activation of the enzyme PAD4 attenuated retinoid-regulated gene expression, while overexpression of PRMT1, a methyltransferase, enhanced retinoid responsiveness. Taken together, our results suggest that H4 arginine 3 methylation is a bona fide positive epigenetic marker and regulator of transcriptional responsiveness as well as a signal integration mechanism during cell differentiation and, as such, may provide epigenetic memory.