RESUMEN
Although most acute skin wounds heal rapidly, non-healing skin ulcers represent an increasing and substantial unmet medical need that urgently requires effective therapeutics. Keratinocytes resurface wounds to re-establish the epidermal barrier by transitioning to an activated, migratory state, but this ability is lost in dysfunctional chronic wounds. Small-molecule regulators of keratinocyte plasticity with the potential to reverse keratinocyte malfunction in situ could offer a novel therapeutic approach in skin wound healing. Utilizing high-throughput phenotypic screening of primary keratinocytes, we identify such small molecules, including bromodomain and extra-terminal domain (BET) protein family inhibitors (BETi). BETi induce a sustained activated, migratory state in keratinocytes in vitro, increase activation markers in human epidermis ex vivo and enhance skin wound healing in vivo. Our findings suggest potential clinical utility of BETi in promoting keratinocyte re-epithelialization of skin wounds. Importantly, this novel property of BETi is exclusively observed after transient low-dose exposure, revealing new potential for this compound class.
Asunto(s)
Proteínas de Ciclo Celular/genética , Epidermis/efectos de los fármacos , Repitelización/efectos de los fármacos , Úlcera Cutánea/tratamiento farmacológico , Bibliotecas de Moléculas Pequeñas/farmacología , Factores de Transcripción/genética , Heridas no Penetrantes/tratamiento farmacológico , Animales , Proteínas de Ciclo Celular/antagonistas & inhibidores , Proteínas de Ciclo Celular/metabolismo , Modelos Animales de Enfermedad , Epidermis/metabolismo , Epidermis/patología , Transferencia Resonante de Energía de Fluorescencia , Regulación de la Expresión Génica , Ensayos Analíticos de Alto Rendimiento , Humanos , Queratinocitos/efectos de los fármacos , Queratinocitos/metabolismo , Queratinocitos/patología , Masculino , Ratones , Ratones Endogámicos C57BL , Cultivo Primario de Células , Isoformas de Proteínas/antagonistas & inhibidores , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Precursores de Proteínas/antagonistas & inhibidores , Precursores de Proteínas/genética , Precursores de Proteínas/metabolismo , Repitelización/genética , Úlcera Cutánea/genética , Úlcera Cutánea/metabolismo , Úlcera Cutánea/patología , Bibliotecas de Moléculas Pequeñas/química , Relación Estructura-Actividad , Factores de Transcripción/antagonistas & inhibidores , Factores de Transcripción/metabolismo , Transcripción Genética , Heridas no Penetrantes/genética , Heridas no Penetrantes/metabolismo , Heridas no Penetrantes/patologíaRESUMEN
The constitutive androstane receptor (CAR) is a xenobiotic sensor expressed in hepatocytes that activates genes involved in drug metabolism, lipid homeostasis, and cell proliferation. Much progress has been made in understanding the mechanism of activation of human CAR by drugs and xenobiotics. However, many aspects of the activation pathway remain to be elucidated. In this report, we have used viral constructs to express human CAR, its splice variants, and mutant CAR forms in hepatocytes from Car-/- mice in vitro and in vivo. We demonstrate CAR expression rescued the ability of Car-/- hepatocytes to respond to a wide range of CAR activators including phenobarbital. Additionally, two major splice isoforms of human CAR, CAR2 and CAR3, were inactive with almost all the agents tested. In contrast to the current model of CAR activation, ectopic CAR1 is constitutively localized in the nucleus and is loaded onto Cyp2b10 gene in the absence of an inducing agent. In studies to elucidate the role of threonine T38 in CAR regulation, we found that the T38D mutant was inactive even in the presence of CAR activators. However, the T38A mutant was activated by CAR inducers, showing that T38 is not essential for CAR activation. Also, using the inhibitor erlotinib, we could not confirm a role for the epidermal growth factor receptor in CAR regulation. Our data suggest that CAR is constitutively bound to gene regulatory regions and is regulated by exogenous agents through a mechanism which involves protein phosphorylation in the nucleus.
Asunto(s)
Cromatina/genética , Hepatocitos/fisiología , Receptores Citoplasmáticos y Nucleares/genética , Activación Transcripcional/genética , Animales , Núcleo Celular/efectos de los fármacos , Núcleo Celular/genética , Células Cultivadas , Receptor de Androstano Constitutivo , Receptores ErbB/genética , Clorhidrato de Erlotinib/farmacología , Femenino , Hepatocitos/efectos de los fármacos , Humanos , Ratones , Ratones Endogámicos C57BL , Fenobarbital/farmacología , Isoformas de Proteínas/genética , Activación Transcripcional/efectos de los fármacosRESUMEN
Gene regulatory interactions underlying the early stages of non-genotoxic carcinogenesis are poorly understood. Here, we have identified key candidate regulators of phenobarbital (PB)-mediated mouse liver tumorigenesis, a well-characterized model of non-genotoxic carcinogenesis, by applying a new computational modeling approach to a comprehensive collection of in vivo gene expression studies. We have combined our previously developed motif activity response analysis (MARA), which models gene expression patterns in terms of computationally predicted transcription factor binding sites with singular value decomposition (SVD) of the inferred motif activities, to disentangle the roles that different transcriptional regulators play in specific biological pathways of tumor promotion. Furthermore, transgenic mouse models enabled us to identify which of these regulatory activities was downstream of constitutive androstane receptor and ß-catenin signaling, both crucial components of PB-mediated liver tumorigenesis. We propose novel roles for E2F and ZFP161 in PB-mediated hepatocyte proliferation and suggest that PB-mediated suppression of ESR1 activity contributes to the development of a tumor-prone environment. Our study shows that combining MARA with SVD allows for automated identification of independent transcription regulatory programs within a complex in vivo tissue environment and provides novel mechanistic insights into PB-mediated hepatocarcinogenesis.
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Carcinogénesis/genética , Regulación Neoplásica de la Expresión Génica , Neoplasias Hepáticas/genética , Fenobarbital/toxicidad , Transcripción Genética/efectos de los fármacos , Animales , Sitios de Unión , Proliferación Celular/efectos de los fármacos , Biología Computacional/métodos , Simulación por Computador , Receptor de Androstano Constitutivo , Redes Reguladoras de Genes , Hígado/efectos de los fármacos , Hígado/metabolismo , Neoplasias Hepáticas/inducido químicamente , Neoplasias Hepáticas/metabolismo , Masculino , Ratones , Motivos de Nucleótidos , Receptores Citoplasmáticos y Nucleares/metabolismo , Transducción de Señal , Factores de Transcripción/metabolismo , beta Catenina/metabolismoRESUMEN
Aberrant DNA methylation is a common feature of neoplastic lesions, and early detection of such changes may provide powerful mechanistic insights and biomarkers for carcinogenesis. Here, we investigate dynamic changes in the mouse liver DNA methylome associated with short (1 day) and prolonged (7, 28 and 91 days) exposure to the rodent liver non-genotoxic carcinogen, phenobarbital (PB). We find that the distribution of 5mC/5hmC is highly consistent between untreated individuals of a similar age; yet, changes during liver maturation in a transcriptionally dependent manner. Following drug treatment, we identify and validate a series of differentially methylated or hydroxymethylated regions: exposure results in staged transcriptional responses with distinct kinetic profiles that strongly correlate with promoter proximal region 5hmC levels. Furthermore, reciprocal changes for both 5mC and 5hmC in response to PB suggest that active demethylation may be taking place at each set of these loci via a 5hmC intermediate. Finally, we identify potential early biomarkers for non-genotoxic carcinogenesis, including several genes aberrantly expressed in liver cancer. Our work suggests that 5hmC profiling can be used as an indicator of cell states during organ maturation and drug-induced responses and provides novel epigenetic signatures for non-genotoxic carcinogen exposure.
Asunto(s)
Citosina/análogos & derivados , Metilación de ADN , Epigénesis Genética , Hígado/metabolismo , 5-Metilcitosina/metabolismo , Animales , Carcinógenos/toxicidad , Transformación Celular Neoplásica , Sistema Enzimático del Citocromo P-450/genética , Citosina/metabolismo , Epigénesis Genética/efectos de los fármacos , Marcadores Genéticos , Hígado/efectos de los fármacos , Hígado/crecimiento & desarrollo , Masculino , Ratones , Análisis de Secuencia por Matrices de Oligonucleótidos , Fenobarbital/toxicidad , Regiones Promotoras Genéticas , Transcriptoma/efectos de los fármacosRESUMEN
The epigenetic modification of 5-hydroxymethylcytosine (5hmC) is receiving great attention due to its potential role in DNA methylation reprogramming and as a cell state identifier. Given this interest, it is important to identify reliable and cost-effective methods for the enrichment of 5hmC marked DNA for downstream analysis. We tested three commonly used affinity-based enrichment techniques; (i) antibody, (ii) chemical capture and (iii) protein affinity enrichment and assessed their ability to accurately and reproducibly report 5hmC profiles in mouse tissues containing high (brain) and lower (liver) levels of 5hmC. The protein-affinity technique is a poor reporter of 5hmC profiles, delivering 5hmC patterns that are incompatible with other methods. Both antibody and chemical capture-based techniques generate highly similar genome-wide patterns for 5hmC, which are independently validated by standard quantitative PCR (qPCR) and glucosyl-sensitive restriction enzyme digestion (gRES-qPCR). Both antibody and chemical capture generated profiles reproducibly link to unique chromatin modification profiles associated with 5hmC. However, there appears to be a slight bias of the antibody to bind to regions of DNA rich in simple repeats. Ultimately, the increased specificity observed with chemical capture-based approaches makes this an attractive method for the analysis of locus-specific or genome-wide patterns of 5hmC.
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Citosina/análogos & derivados , ADN/química , 5-Metilcitosina/análogos & derivados , Animales , Anticuerpos , Cromatina/metabolismo , Islas de CpG , Citosina/análisis , Citosina/inmunología , Proteínas de Unión al ADN/análisis , Sitios Genéticos , Impresión Genómica , Inmunoensayo/métodos , Hígado/química , Masculino , Ratones , Ratones Endogámicos C57BL , Análisis de Secuencia por Matrices de Oligonucleótidos , Secuencias Repetidas en TándemRESUMEN
Topoisomerases are essential for DNA replication in dividing cells, but their genomic targets and function in postmitotic cells remain poorly understood. Here we show that a switch in the expression from Topoisomerases IIα (Top2α) to IIß (Top2ß) occurs during neuronal differentiation in vitro and in vivo. Genome-scale location analysis in stem cell-derived postmitotic neurons reveals Top2ß binding to chromosomal sites that are methylated at lysine 4 of histone H3, a feature of regulatory regions. Indeed Top2ß-bound sites are preferentially promoters and become targets during the transition from neuronal progenitors to neurons, at a time when cells exit the cell cycle. Absence of Top2ß protein or its activity leads to changes in transcription and chromatin accessibility at many target genes. Top2ß deficiency does not impair stem cell properties and early steps of neuronal differentiation but causes premature death of postmitotic neurons. This neuronal degeneration is caused by up-regulation of Ngfr p75, a gene bound and repressed by Top2ß. These findings suggest a chromatin-based targeting of Top2ß to regulatory regions in the genome to govern the transcriptional program associated with neuronal differentiation and longevity.
Asunto(s)
Cromatina/genética , ADN-Topoisomerasas de Tipo II/genética , Proteínas de Unión al ADN/genética , Neuronas/metabolismo , Animales , Western Blotting , Diferenciación Celular/genética , Supervivencia Celular/genética , Células Cultivadas , ADN-Topoisomerasas de Tipo II/metabolismo , Proteínas de Unión al ADN/antagonistas & inhibidores , Proteínas de Unión al ADN/metabolismo , Dicetopiperazinas , Células Madre Embrionarias/citología , Células Madre Embrionarias/efectos de los fármacos , Células Madre Embrionarias/metabolismo , Femenino , Perfilación de la Expresión Génica , Regulación del Desarrollo de la Expresión Génica , Inmunoprecipitación , Masculino , Ratones , Ratones de la Cepa 129 , Ratones Noqueados , Neuronas/citología , Neuronas/efectos de los fármacos , Análisis de Secuencia por Matrices de Oligonucleótidos , Piperazinas/farmacología , Unión Proteica , Interferencia de ARN , Receptor de Factor de Crecimiento Nervioso/genética , Receptor de Factor de Crecimiento Nervioso/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factores de Tiempo , Inhibidores de Topoisomerasa II/farmacologíaRESUMEN
The process of hepatocarcinogenesis in the diethylnitrosamine (DEN) initiation/phenobarbital (PB) promotion mouse model involves the selective clonal outgrowth of cells harboring oncogene mutations in Ctnnb1, while spontaneous or DEN-only-induced tumors are often Ha-ras- or B-raf-mutated. The molecular mechanisms and pathways underlying these different tumor sub-types are not well characterized. Their identification may help identify markers for xenobiotic promoted versus spontaneously occurring liver tumors. Here, we have characterized mouse liver tumors harboring either Ctnnb1 or Ha-ras mutations via integrated molecular profiling at the transcriptional, translational and post-translational levels. In addition, metabolites of the intermediary metabolism were quantified by high resolution (1)H magic angle nuclear magnetic resonance. We have identified tumor genotype-specific differences in mRNA and miRNA expression, protein levels, post-translational modifications, and metabolite levels that facilitate the molecular and biochemical stratification of tumor phenotypes. Bioinformatic integration of these data at the pathway level led to novel insights into tumor genotype-specific aberrant cell signaling and in particular to a better understanding of alterations in pathways of the cell intermediary metabolism, which are driven by the constitutive activation of the ß-Catenin and Ha-ras oncoproteins in tumors of the two genotypes.
Asunto(s)
Biomarcadores de Tumor/genética , Perfilación de la Expresión Génica , Genes ras/genética , Neoplasias Hepáticas Experimentales/genética , Neoplasias Hepáticas Experimentales/metabolismo , Metabolómica , Mutación/genética , beta Catenina/genética , Animales , Biomarcadores de Tumor/metabolismo , Western Blotting , Redes y Vías Metabólicas , Ratones , MicroARNs/genética , Análisis de Secuencia por Matrices de Oligonucleótidos , Procesamiento Proteico-Postraduccional , ARN Mensajero/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
Sickle cell disease (SCD) is a prevalent, life-threatening condition attributable to a heritable mutation in ß-hemoglobin. Therapeutic induction of fetal hemoglobin (HbF) can ameliorate disease complications and has been intently pursued. However, safe and effective small-molecule inducers of HbF remain elusive. We report the discovery of dWIZ-1 and dWIZ-2, molecular glue degraders of the WIZ transcription factor that robustly induce HbF in erythroblasts. Phenotypic screening of a cereblon (CRBN)-biased chemical library revealed WIZ as a previously unknown repressor of HbF. WIZ degradation is mediated by recruitment of WIZ(ZF7) to CRBN by dWIZ-1, as resolved by crystallography of the ternary complex. Pharmacological degradation of WIZ was well tolerated and induced HbF in humanized mice and cynomolgus monkeys. These findings establish WIZ degradation as a globally accessible therapeutic strategy for SCD.
Asunto(s)
Anemia de Células Falciformes , Antidrepanocíticos , Hemoglobina Fetal , Factores de Transcripción de Tipo Kruppel , Proteínas del Tejido Nervioso , Animales , Humanos , Ratones , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Proteínas Adaptadoras Transductoras de Señales/genética , Anemia de Células Falciformes/tratamiento farmacológico , Anemia de Células Falciformes/metabolismo , Antidrepanocíticos/química , Antidrepanocíticos/farmacología , Antidrepanocíticos/uso terapéutico , Cristalografía por Rayos X , Descubrimiento de Drogas , Hemoglobina Fetal/genética , Hemoglobina Fetal/metabolismo , Factores de Transcripción de Tipo Kruppel/metabolismo , Macaca fascicularis , Proteínas del Tejido Nervioso/metabolismo , Proteolisis/efectos de los fármacos , Bibliotecas de Moléculas Pequeñas/química , Bibliotecas de Moléculas Pequeñas/farmacología , Bibliotecas de Moléculas Pequeñas/uso terapéutico , Ubiquitina-Proteína Ligasas/metabolismo , Ubiquitina-Proteína Ligasas/genéticaRESUMEN
WDR5 is a highly conserved protein that performs multiple scaffolding functions in the context of chromatin. However, efforts to understand the function of WDR5 in normal tissues physiologically are quite limited so far. In our study, we explored the function of Wdr5 in erythropoiesis and hematopoiesis by using a hematopoietic-specific Wdr5 knockout mouse model. We found that loss of Wdr5 mediated by Vav-iCre leads to embryonic lethality with defective erythropoiesis. In addition, Wdr5-deficiency completely impairs the hematopoietic stem and progenitor cells function and might alter the immunophenotype of these stem cells and progenitors by decreasing c-Kit expression. Collectively, we identified the pivotal role of Wdr5 in fetal hematopoiesis and erythropoiesis as the de novo findings.
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The possibility that ancestral environmental exposure could result in adaptive inherited effects in mammals has been long debated. Numerous rodent models of transgenerational responses to various environmental factors have been published but due to technical, operational and resource burden, most still await independent confirmation. A previous study reported multigenerational epigenetic adaptation of the hepatic wound healing response upon exposure to the hepatotoxicant carbon tetrachloride (CCl4) in male rats. Here, we comprehensively investigate the transgenerational effects by repeating the original CCl4 multigenerational study with increased power, pedigree tracing, F2 dose-response and suitable randomization schemes. Detailed pathology evaluations do not support adaptive phenotypic suppression of the hepatic wound healing response or a greater fitness of F2 animals with ancestral liver injury exposure. However, transcriptomic analyses identified genes whose expression correlates with ancestral liver injury, although the biological relevance of this apparent transgenerational transmission at the molecular level remains to be determined. This work overall highlights the need for independent evaluation of transgenerational epigenetic inheritance paradigms in mammals.
Asunto(s)
Metilación de ADN , Epigénesis Genética , Hígado , Cicatrización de Heridas , Animales , Masculino , Ratas , Tetracloruro de Carbono/toxicidad , Hígado/lesiones , Cicatrización de Heridas/genéticaRESUMEN
Polycomb Repressive Complex 2 (PRC2) plays an important role in transcriptional regulation during animal development and in cell differentiation, and alteration of PRC2 activity has been associated with cancer. On a molecular level, PRC2 catalyzes methylation of histone H3 lysine 27 (H3K27), resulting in mono-, di-, or trimethylated forms of H3K27, of which the trimethylated form H3K27me3 leads to transcriptional repression of polycomb target genes. Previously, we have shown that binding of the low-molecular-weight compound EED226 to the H3K27me3 binding pocket of the regulatory subunit EED can effectively inhibit PRC2 activity in cells and reduce tumor growth in mouse xenograft models. Here, we report the stepwise optimization of the tool compound EED226 toward the potent and selective EED inhibitor MAK683 (compound 22) and its subsequent preclinical characterization. Based on a balanced PK/PD profile, efficacy, and mitigated risk of forming reactive metabolites, MAK683 has been selected for clinical development.
Asunto(s)
Histonas , Neoplasias , Animales , Inhibidores Enzimáticos , Histonas/metabolismo , Humanos , Metilación , Ratones , Neoplasias/tratamiento farmacológico , Complejo Represivo Polycomb 2RESUMEN
Differentiated cells can be reprogrammed through the formation of heterokaryons and hybrid cells when fused with embryonic stem (ES) cells. Here, we provide evidence that conversion of human B-lymphocytes towards a multipotent state is initiated much more rapidly than previously thought, occurring in transient heterokaryons before nuclear fusion and cell division. Interestingly, reprogramming of human lymphocytes by mouse ES cells elicits the expression of a human ES-specific gene profile, in which markers of human ES cells are expressed (hSSEA4, hFGF receptors and ligands), but markers that are specific to mouse ES cells are not (e.g., Bmp4 and LIF receptor). Using genetically engineered mouse ES cells, we demonstrate that successful reprogramming of human lymphocytes is independent of Sox2, a factor thought to be required for induced pluripotent stem (iPS) cells. In contrast, there is a distinct requirement for Oct4 in the establishment but not the maintenance of the reprogrammed state. Experimental heterokaryons, therefore, offer a powerful approach to trace the contribution of individual factors to the reprogramming of human somatic cells towards a multipotent state.
Asunto(s)
Linfocitos B/citología , Reprogramación Celular/fisiología , Proteínas de Unión al ADN/metabolismo , Células Madre Embrionarias/metabolismo , Proteínas HMGB/metabolismo , Factor 3 de Transcripción de Unión a Octámeros/metabolismo , Células Madre Pluripotentes/metabolismo , Factores de Transcripción/metabolismo , Animales , Diferenciación Celular , Fusión Celular , Núcleo Celular/metabolismo , Células Madre Embrionarias/citología , Proteínas de Homeodominio/metabolismo , Humanos , Células Híbridas/metabolismo , Ratones , Proteína Homeótica Nanog , Células Madre Pluripotentes/citología , Factores de Transcripción SOXB1RESUMEN
Liver cancer susceptibility varies amongst humans and between experimental animal models because of multiple genetic and epigenetic factors. The molecular characterization of such susceptibilities has the potential to enhance cancer risk assessment of xenobiotic exposures and disease prevention strategies. Here, using DNase I hypersensitivity mapping coupled with transcriptomic profiling, we investigate perturbations in cis-acting gene regulatory elements associated with the early stages of phenobarbital (PB)-mediated liver tumor promotion in susceptible versus resistant mouse strains (B6C3F1 versus C57BL/6J). Integrated computational analyses of strain-selective changes in liver chromatin accessibility underlying PB response reveal differential epigenetic regulation of molecular pathways associated with PB-mediated tumor promotion, including Wnt/ß-catenin signaling. Complementary transcription factor motif analyses reveal mouse strain-selective gene regulatory networks and a novel role for Stat, Smad, and Fox transcription factors in the early stages of PB-mediated tumor promotion. Mapping perturbations in cis-acting gene regulatory elements provides novel insights into the molecular basis for susceptibility to xenobiotic-induced rodent liver tumor promotion and has the potential to enhance mechanism-based cancer risk assessments of xenobiotic exposures.
Asunto(s)
Cromatina/genética , Perfilación de la Expresión Génica/métodos , Neoplasias Hepáticas/patología , Fenobarbital/efectos adversos , Animales , Cromatina/efectos de los fármacos , Ensamble y Desensamble de Cromatina/efectos de los fármacos , Biología Computacional , Epigénesis Genética/efectos de los fármacos , Redes Reguladoras de Genes , Humanos , Neoplasias Hepáticas/inducido químicamente , Neoplasias Hepáticas/genética , Masculino , Ratones , Vía de Señalización Wnt/efectos de los fármacosRESUMEN
Resident adult epithelial stem cells maintain tissue homeostasis by balancing self-renewal and differentiation. The stem cell potential of human epidermal keratinocytes is retained in vitro but lost over time suggesting extrinsic and intrinsic regulation. Transcription factor-controlled regulatory circuitries govern cell identity, are sufficient to induce pluripotency and transdifferentiate cells. We investigate whether transcriptional circuitry also governs phenotypic changes within a given cell type by comparing human primary keratinocytes with intrinsically high versus low stem cell potential. Using integrated chromatin and transcriptional profiling, we implicate IRF2 as antagonistic to stemness and show that it binds and regulates active cis-regulatory elements at interferon response and antigen presentation genes. CRISPR-KD of IRF2 in keratinocytes with low stem cell potential increases self-renewal, migration and epidermis formation. These data demonstrate that transcription factor regulatory circuitries, in addition to maintaining cell identity, control plasticity within cell types and offer potential for therapeutic modulation of cell function.
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Factor 2 Regulador del Interferón/metabolismo , Queratinocitos/citología , Queratinocitos/metabolismo , Células Madre/citología , Células Madre/metabolismo , Diferenciación Celular/genética , Diferenciación Celular/fisiología , Humanos , Factor 2 Regulador del Interferón/genética , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Activación Transcripcional/genética , Activación Transcripcional/fisiologíaRESUMEN
Constitutive heterochromatin, key for the structuration of the centromeres, was long thought as a transcriptionally inert compartment, relatively invariant in composition and structure. Numerous studies have now challenged this view by showing the versatility of these epigenetically defined regions. This review highlights some important aspects of (peri)-centromeric heterochromatin structural and functional plasticity in development and differentiation. The integrity of centromeric regions is crucial for the stability of the genome and the maintenance of ploidy. I outline recent evidence suggesting that epigenetic alterations of heterochromatin can cause defects in chromosome segregation leading to aneuploidy and tumorigenesis.
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Heterocromatina/química , Heterocromatina/ultraestructura , Aneuploidia , Animales , Centrómero/fisiología , Centrómero/ultraestructura , Regulación de la Expresión Génica , Genoma , Ratones , Neoplasias/genética , ARN Interferente Pequeño/genéticaRESUMEN
Pleiotropic xenobiotics can trigger dynamic alterations in mammalian chromatin structure and function but many of these are likely non-adverse and simply reflect short-term changes in DNA transactions underlying normal homeostatic, adaptive and protective cellular responses. However, it is plausible that a subset of xenobiotic-induced perturbations of somatic tissue or germline epigenomes result in delayed-onset and long-lasting adverse effects, in particular if they occur during critical stages of growth and development. These could include reprogramming, dedifferentiation, uncontrolled growth, and cumulative toxicity effects through molecular memory of prior xenobiotic exposures or altered susceptibility to subsequent xenobiotic exposures. Here we discuss the current evidence for epigenetic mechanisms underlying latent responses to xenobiotics, and the potential for identifying molecular epigenetic changes that are prodromal to overt morphologic or functional toxicity phenotypes.
RESUMEN
Derisking xenobiotic-induced nongenotoxic carcinogenesis (NGC) represents a significant challenge during the safety assessment of chemicals and therapeutic drugs. The identification of robust mechanism-based NGC biomarkers has the potential to enhance cancer hazard identification. We previously demonstrated Constitutive Androstane Receptor (CAR) and WNT signaling-dependent up-regulation of the pluripotency associated Dlk1-Dio3 imprinted gene cluster noncoding RNAs (ncRNAs) in the liver of mice treated with tumor-promoting doses of phenobarbital (PB). Here, we have compared phenotypic, transcriptional ,and proteomic data from wild-type, CAR/PXR double knock-out and CAR/PXR double humanized mice treated with either PB or chlordane, and show that hepatic Dlk1-Dio3 locus long ncRNAs are upregulated in a CAR/PXR-dependent manner by two structurally distinct CAR activators. We further explored the specificity of Dlk1-Dio3 locus ncRNAs as hepatic NGC biomarkers in mice treated with additional compounds working through distinct NGC modes of action. We propose that up-regulation of Dlk1-Dio3 cluster ncRNAs can serve as an early biomarker for CAR activator-induced nongenotoxic hepatocarcinogenesis and thus may contribute to mechanism-based assessments of carcinogenicity risk for chemicals and novel therapeutics.
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Expresión Génica/efectos de los fármacos , Péptidos y Proteínas de Señalización Intercelular/genética , Yoduro Peroxidasa/genética , Hígado/efectos de los fármacos , ARN Largo no Codificante/genética , Receptores Citoplasmáticos y Nucleares/agonistas , Xenobióticos/toxicidad , Animales , Biomarcadores/metabolismo , Proteínas de Unión al Calcio , Clordano/toxicidad , Receptor de Androstano Constitutivo , Hígado/metabolismo , Hígado/patología , Masculino , Ratones , Ratones Noqueados , Fenobarbital/toxicidad , Regulación hacia Arriba/efectos de los fármacosRESUMEN
High throughput screening and subsequent hit validation identified 4-isopropyl-3-(2-((1-phenylethyl)amino)pyrimidin-4-yl)oxazolidin-2-one as a potent inhibitor of IDH1R132H. Synthesis of the four separate stereoisomers identified the (S,S)-diastereomer (IDH125, 1f) as the most potent isomer. This also showed reasonable cellular activity and excellent selectivity vs IDH1wt. Initial structure-activity relationship exploration identified the key tolerances and potential for optimization. X-ray crystallography identified a functionally relevant allosteric binding site amenable to inhibitors, which can penetrate the blood-brain barrier, and aided rational optimization. Potency improvement and modulation of the physicochemical properties identified (S,S)-oxazolidinone IDH889 (5x) with good exposure and 2-HG inhibitory activity in a mutant IDH1 xenograft mouse model.
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Epigenetic regulation of gene expression plays a pivotal role in the orchestration of immune responses. Chemical allergens form two categories: skin sensitizing chemicals associated with allergic contact dermatitis, and chemicals that cause sensitization of the respiratory tract and occupational asthma. In mice these are characterized by different T helper (Th) cell responses. Changes in DNA methylation in particular have been implicated in the in vivo responses to chemical allergy. As such it was hypothesised that differentially methylated regions (DMR) may provide candidates biomarkers of chemical allergy To examine this, mice were exposed to 2,4-dinitrochlorobenzene (DNCB; a contact allergen) or trimellitic anhydride (TMA; a respiratory allergen). DNA from draining lymph nodes was processed for methylated (5mC) and hydroxymethylated (5hmC) DNA immunoprecipitation (MeDIP/hMeDIP) then selected DMR analysed by qPCR. We describe a number of DMRs which, by combined analysis of 5mC and 5hmC, differentiate between responses induced by DNCB and those by TMA. Furthermore, these changes in methylation are specific to the draining lymph node. The Gmpr DMR is suggested as a possible biomarker for contact allergen-induced immune responses; it is characterised by divergent levels of 5mC and 5hmC DNCB-treated mice only. In contrast, the Nwc DMR was characterised by divergent 5mC and 5hmC specifically in response to TMA, highlighting its possible utility as a biomarker for responses induced by chemical respiratory allergens. These data not only represent novel analysis of 5hmC in response to chemical allergy in vivo, but with further investigation, may also provide a possible basis for differentiation between classes of chemical allergens.
Asunto(s)
5-Metilcitosina/metabolismo , Alérgenos/toxicidad , Citosina/análogos & derivados , Metilación de ADN/efectos de los fármacos , Dinitroclorobenceno/toxicidad , Epigénesis Genética/efectos de los fármacos , Ganglios Linfáticos/efectos de los fármacos , Anhídridos Ftálicos/toxicidad , Animales , Citosina/metabolismo , Fragmentación del ADN , Femenino , Inmunoprecipitación , Ensayo del Nódulo Linfático Local , Ganglios Linfáticos/inmunología , Ganglios Linfáticos/metabolismo , Ganglios Linfáticos/patología , Ratones Endogámicos BALB C , Reacción en Cadena de la Polimerasa , Regiones Promotoras Genéticas/efectos de los fármacosRESUMEN
BACKGROUND: Fragile X syndrome (FXS) is the most common form of inherited intellectual disability, resulting from the loss of function of the fragile X mental retardation 1 (FMR1) gene. The molecular pathways associated with FMR1 epigenetic silencing are still elusive, and their characterization may enhance the discovery of novel therapeutic targets as well as the development of novel clinical biomarkers for disease status. RESULTS: We have deployed customized epigenomic profiling assays to comprehensively map the FMR1 locus chromatin landscape in peripheral mononuclear blood cells (PBMCs) from eight FXS patients and in fibroblast cell lines derived from three FXS patient. Deoxyribonucleic acid (DNA) methylation (5-methylcytosine (5mC)) and hydroxymethylation (5-hydroxymethylcytosine (5hmC)) profiling using methylated DNA immunoprecipitation (MeDIP) combined with a custom FMR1 microarray identifies novel regions of DNA (hydroxy)methylation changes within the FMR1 gene body as well as in proximal flanking regions. At the region surrounding the FMR1 transcriptional start sites, increased levels of 5mC were associated to reciprocal changes in 5hmC, representing a novel molecular feature of FXS disease. Locus-specific validation of FMR1 5mC and 5hmC changes highlighted inter-individual differences that may account for the expected DNA methylation mosaicism observed at the FMR1 locus in FXS patients. Chromatin immunoprecipitation (ChIP) profiling of FMR1 histone modifications, together with 5mC/5hmC and gene expression analyses, support a functional relationship between 5hmC levels and FMR1 transcriptional activation and reveal cell-type specific differences in FMR1 epigenetic regulation. Furthermore, whilst 5mC FMR1 levels positively correlated with FXS disease severity (clinical scores of aberrant behavior), our data reveal for the first time an inverse correlation between 5hmC FMR1 levels and FXS disease severity. CONCLUSIONS: We identify novel, cell-type specific, regions of FMR1 epigenetic changes in FXS patient cells, providing new insights into the molecular mechanisms of FXS. We propose that the combined measurement of 5mC and 5hmC at selected regions of the FMR1 locus may significantly enhance FXS clinical diagnostics and patient stratification.