RESUMEN
In accordance with the World Health Organization, cancer is the second leading cause of death in patients. In recent years, the number of cancer patients has been growing, and the occurrence of cancer in people is becoming more common, primarily due to lifestyle factors. Yin Yang 1 (YY1) is a transcription factor that is widespread throughout. It is a zinc finger protein, falling under the GLI-Kruppel class. YY1 is known to regulate transcriptional activation and repression of various genes associated with different cellular processes such as DNA repair, autophagy, cell survival and apoptosis, and cell division. Meanwhile, EZH2 is a histone-lysine N-methyltransferase enzyme encoded by gene 7 in humans. Its main function involves catalyzing the addition of methyl groups to histone H3 at lysine 27 (H3K27me3), and it is involved in regulating CD8 + T cell fate and function. It is a subunit of a Polycomb repressor complex 2 (PRC2). The EZH2 gene encodes for an enzyme that is involved in histone methylation and transcriptional repression. It adds methyl groups to lysine 27 on histone H3 (H3K27me3) with the help of the cofactor S-adenosyl-L-methionine. In addition to its role in epigenetic regulation, EZH2 also acts as a regulator of CD8+ T cell fate and function. EZH2 has been implicated in T Cell Receptor (TCR) signaling via the regulation of actin polymerization. In fact, EZH2 is involved in numerous signaling pathways that lead to tumorigenesis. EZH2 is mutated in cancer and shows overexpression. Due to its mutation and overexpression, the cells that help combat cancer are suppressed and carcinogenicity is promoted. The association of EZH2 and YY1 poses an intriguing mechanism in relation to cancer.
Asunto(s)
Proteína Potenciadora del Homólogo Zeste 2 , Neoplasias , Humanos , Proteína Potenciadora del Homólogo Zeste 2/genética , Proteína Potenciadora del Homólogo Zeste 2/metabolismo , Histonas/genética , Complejo Represivo Polycomb 2/genética , Lisina , Epigénesis Genética , Yin-Yang , Neoplasias/genética , Factor de Transcripción YY1/genética , Factor de Transcripción YY1/metabolismoRESUMEN
The first-generation enhancer of zeste homologue 2 (EZH2) inhibitors suffer from several limitations, such as high dosage, cofactor S-adenosylmethionine (SAM) competition, and acquired drug resistance. Development of covalent EZH2 inhibitors that are noncompetitive with cofactor SAM offers an opportunity to overcome these disadvantages. The structure-based design of compound 16 (BBDDL2059) as a highly potent and selective covalent inhibitor of EZH2 is presented in this context. 16 inhibits EZH2 enzymatic activity at sub-nanomolar concentrations and achieves low nanomolar potencies in cell growth inhibition. The kinetic assay revealed that 16 is noncompetitive with the cofactor SAM, providing the basis for its superior activity over noncovalent and positive controls by reducing competition with cofactor SAM and offering a preliminary proof for its covalent inhibition nature. Mass spectrometric analysis and washout experiments firmly establish its covalent inhibition mechanism. This study demonstrates that covalent inhibition of EZH2 can offer a new opportunity for the development of promising new-generation drug candidates.
Asunto(s)
Lisina , S-Adenosilmetionina , S-Adenosilmetionina/farmacología , S-Adenosilmetionina/química , Proteína Potenciadora del Homólogo Zeste 2 , Complejo Represivo Polycomb 2 , Proliferación Celular , Línea Celular TumoralRESUMEN
The hypothalamus displays staggering cellular diversity, chiefly established during embryogenesis by the interplay of several signalling pathways and a battery of transcription factors. However, the contribution of epigenetic cues to hypothalamus development remains unclear. We mutated the polycomb repressor complex 2 gene Eed in the developing mouse hypothalamus, which resulted in the loss of H3K27me3, a fundamental epigenetic repressor mark. This triggered ectopic expression of posteriorly expressed regulators (e.g. Hox homeotic genes), upregulation of cell cycle inhibitors and reduced proliferation. Surprisingly, despite these effects, single cell transcriptomic analysis revealed that most neuronal subtypes were still generated in Eed mutants. However, we observed an increase in glutamatergic/GABAergic double-positive cells, as well as loss/reduction of dopamine, hypocretin and Tac2-Pax6 neurons. These findings indicate that many aspects of the hypothalamic gene regulatory flow can proceed without the key H3K27me3 epigenetic repressor mark, but points to a unique sensitivity of particular neuronal subtypes to a disrupted epigenomic landscape.
Asunto(s)
Desarrollo Embrionario/fisiología , Hipotálamo/fisiología , Neuronas/fisiología , Complejo Represivo Polycomb 2/genética , Proteínas del Grupo Polycomb/genética , Animales , Proliferación Celular/genética , Represión Epigenética/genética , Femenino , Masculino , Ratones , Mutación/genética , Transcriptoma/genéticaRESUMEN
Both EZH2 and its homolog EZH1 function as histone H3 Lysine 27 (H3K27) methyltransferases and repress the transcription of target genes. Dysregulation of H3K27 trimethylation (H3K27me3) plays an important role in the development and progression of cancers such as hepatocellular carcinoma (HCC). This study investigated the relationship between the expression of EZH1/2 and the level of H3K27me3 in HCC. Additionally, the role of EZH1/2 in cell growth, tumorigenicity, and resistance to sorafenib were also analyzed. Both the lentiviral knockdown and the pharmacological inhibition of EZH1/2 (UNC1999) diminished the level of H3K27me3 and suppressed cell growth in liver cancer cells, compared with EZH1 or EZH2 single knockdown. Although a significant association was observed between EZH2 expression and H3K27me3 levels in HCC samples, overexpression of EZH1 appeared to contribute to enhanced H3K27me3 levels in some EZH2lowH3K27me3high cases. Akt suppression following sorafenib treatment resulted in an increase of the H3K27me3 levels through a decrease in EZH2 phosphorylation at serine 21. The combined use of sorafenib and UNC1999 exhibited synergistic antitumor effects in vitro and in vivo. Combination treatment canceled the sorafenib-induced enhancement in H3K27me3 levels, indicating that activation of EZH2 function is one of the mechanisms of sorafenib-resistance in HCC. In conclusion, sorafenib plus EZH1/2 inhibitors may comprise a novel therapeutic approach in HCC.
Asunto(s)
Antineoplásicos/uso terapéutico , Benzamidas/uso terapéutico , Carcinoma Hepatocelular/terapia , Proteína Potenciadora del Homólogo Zeste 2/antagonistas & inhibidores , Indazoles/uso terapéutico , Neoplasias Hepáticas/terapia , Piperazinas/uso terapéutico , Complejo Represivo Polycomb 2/antagonistas & inhibidores , Piridonas/uso terapéutico , Sorafenib/uso terapéutico , Anciano , Animales , Carcinoma Hepatocelular/genética , Línea Celular Tumoral , Regulación hacia Abajo/efectos de los fármacos , Proteína Potenciadora del Homólogo Zeste 2/genética , Femenino , Terapia Genética , Humanos , Neoplasias Hepáticas/genética , Masculino , Ratones SCID , Persona de Mediana Edad , Complejo Represivo Polycomb 2/genéticaRESUMEN
Rationale: Tanshinone, a type of diterpenes derived from salvia miltiorrhiza, is a particularly promising herbal medicine compound for the treatment of cancers including acute myeloid leukemia (AML). However, the therapeutic function and the underlying mechanism of Tanshinone in AML are not clear, and the toxic effect of Tanshinone limits its clinical application. Methods: Our work utilizes human leukemia cell lines, zebrafish transgenics and xenograft models to study the cellular and molecular mechanisms of how Tanshinone affects normal and abnormal hematopoiesis. WISH, Sudan Black and O-Dianisidine Staining were used to determine the expression of hematopoietic genes on zebrafish embryos. RNA-seq analysis showed that differential expression genes and enrichment gene signature with Tan I treatment. The surface plasmon resonance (SPR) method was used with a BIAcore T200 (GE Healthcare) to measure the binding affinities of Tan I. In vitro methyltransferase assay was performed to verify Tan I inhibits the histone enzymatic activity of the PRC2 complex. ChIP-qPCR assay was used to determine the H3K27me3 level of EZH2 target genes. Results: We found that Tanshinone I (Tan I), one of the Tanshinones, can inhibit the proliferation of human leukemia cells in vitro and in the xenograft zebrafish model, as well as the normal and malignant definitive hematopoiesis in zebrafish. Mechanistic studies illustrate that Tan I regulates normal and malignant hematopoiesis through direct binding to EZH2, a well-known histone H3K27 methyltransferase, and inhibiting PRC2 enzymatic activity. Furthermore, we identified MMP9 and ABCG2 as two possible downstream genes of Tan I's effects on EZH2. Conclusions: Together, this study confirmed that Tan I is a novel EZH2 inhibitor and suggested MMP9 and ABCG2 as two potential therapeutic targets for myeloid malignant diseases.
Asunto(s)
Transportador de Casetes de Unión a ATP, Subfamilia G, Miembro 2/metabolismo , Abietanos/farmacología , Antineoplásicos Fitogénicos/farmacología , Proteína Potenciadora del Homólogo Zeste 2/antagonistas & inhibidores , Hematopoyesis/efectos de los fármacos , Leucemia/tratamiento farmacológico , Leucemia/metabolismo , Metaloproteinasa 9 de la Matriz/metabolismo , Proteínas de Neoplasias/metabolismo , Transportador de Casetes de Unión a ATP, Subfamilia G, Miembro 2/genética , Animales , Animales Modificados Genéticamente , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Proliferación Celular/genética , Inmunoprecipitación de Cromatina , Proteína Potenciadora del Homólogo Zeste 2/genética , Proteína Potenciadora del Homólogo Zeste 2/metabolismo , Perfilación de la Expresión Génica , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Regulación Neoplásica de la Expresión Génica/genética , Hematopoyesis/genética , Histonas/metabolismo , Humanos , Leucemia/enzimología , Leucemia/genética , Metaloproteinasa 9 de la Matriz/genética , Proteínas de Neoplasias/genética , Complejo Represivo Polycomb 2/metabolismo , Unión Proteica , RNA-Seq , Salvia miltiorrhiza/química , Resonancia por Plasmón de Superficie , Transcriptoma/genética , Ensayos Antitumor por Modelo de Xenoinjerto , Pez CebraRESUMEN
Female puberty is subject to Polycomb Group (PcG)-dependent transcriptional repression. Kiss1, a puberty-activating gene, is a key target of this silencing mechanism. Using a gain-of-function approach and a systems biology strategy we now show that EED, an essential PcG component, acts in the arcuate nucleus of the hypothalamus to alter the functional organization of a gene network involved in the stimulatory control of puberty. A central node of this network is Kdm6b, which encodes an enzyme that erases the PcG-dependent histone modification H3K27me3. Kiss1 is a first neighbor in the network; genes encoding glutamatergic receptors and potassium channels are second neighbors. By repressing Kdm6b expression, EED increases H3K27me3 abundance at these gene promoters, reducing gene expression throughout a gene network controlling puberty activation. These results indicate that Kdm6b repression is a basic mechanism used by PcG to modulate the biological output of puberty-activating gene networks.
Asunto(s)
Histona Demetilasas con Dominio de Jumonji/genética , Kisspeptinas/genética , Complejo Represivo Polycomb 2/genética , Pubertad/genética , Animales , Regulación de la Expresión Génica/genética , Redes Reguladoras de Genes/genética , Humanos , Hipotálamo/crecimiento & desarrollo , Hipotálamo/metabolismo , Neuronas/metabolismo , Sistemas Neurosecretores/crecimiento & desarrollo , Sistemas Neurosecretores/metabolismo , Proteínas del Grupo Polycomb/genética , Regiones Promotoras Genéticas/genética , Pubertad/fisiología , Ratas , Biología de SistemasRESUMEN
Firre encodes a lncRNA involved in nuclear organization. Here, we show that Firre RNA expressed from the active X chromosome maintains histone H3K27me3 enrichment on the inactive X chromosome (Xi) in somatic cells. This trans-acting effect involves SUZ12, reflecting interactions between Firre RNA and components of the Polycomb repressive complexes. Without Firre RNA, H3K27me3 decreases on the Xi and the Xi-perinucleolar location is disrupted, possibly due to decreased CTCF binding on the Xi. We also observe widespread gene dysregulation, but not on the Xi. These effects are measurably rescued by ectopic expression of mouse or human Firre/FIRRE transgenes, supporting conserved trans-acting roles. We also find that the compact 3D structure of the Xi partly depends on the Firre locus and its RNA. In common lymphoid progenitors and T-cells Firre exerts a cis-acting effect on maintenance of H3K27me3 in a 26 Mb region around the locus, demonstrating cell type-specific trans- and cis-acting roles of this lncRNA.
Asunto(s)
Epigénesis Genética , ARN Largo no Codificante/genética , Inactivación del Cromosoma X/genética , Alelos , Animales , Secuencia de Bases , Línea Celular , Núcleo Celular/genética , Cromatina/metabolismo , ADN Complementario/genética , Femenino , Eliminación de Gen , Ontología de Genes , Sitios Genéticos , Genoma , Histonas/metabolismo , Lisina/metabolismo , Masculino , Metilación , Ratones Endogámicos C57BL , Complejo Represivo Polycomb 2/metabolismo , ARN Largo no Codificante/metabolismo , Transgenes , Regulación hacia Arriba/genética , Cromosoma X/genéticaRESUMEN
Puberty is regulated by epigenetic mechanisms and is highly sensitive to metabolic and nutritional cues. However, the epigenetic pathways mediating the effects of nutrition and obesity on pubertal timing are unknown. Here, we identify Sirtuin 1 (SIRT1), a fuel-sensing deacetylase, as a molecule that restrains female puberty via epigenetic repression of the puberty-activating gene, Kiss1. SIRT1 is expressed in hypothalamic Kiss1 neurons and suppresses Kiss1 expression. SIRT1 interacts with the Polycomb silencing complex to decrease Kiss1 promoter activity. As puberty approaches, SIRT1 is evicted from the Kiss1 promoter facilitating a repressive-to-permissive switch in chromatin landscape. Early-onset overnutrition accelerates these changes, enhances Kiss1 expression and advances puberty. In contrast, undernutrition raises SIRT1 levels, protracts Kiss1 repression and delays puberty. This delay is mimicked by central pharmacological activation of SIRT1 or SIRT1 overexpression, achieved via transgenesis or virogenetic targeting to the ARC. Our results identify SIRT1-mediated inhibition of Kiss1 as key epigenetic mechanism by which nutritional cues and obesity influence mammalian puberty.
Asunto(s)
Epigénesis Genética , Kisspeptinas/genética , Fenómenos Fisiológicos de la Nutrición , Obesidad/metabolismo , Maduración Sexual , Sirtuina 1/metabolismo , Animales , Núcleo Arqueado del Hipotálamo/metabolismo , Cromatina/metabolismo , Femenino , Histonas/metabolismo , Hipotálamo/metabolismo , Kisspeptinas/metabolismo , Ratones Transgénicos , Modelos Biológicos , Neuronas/metabolismo , Estado Nutricional , Complejo Represivo Polycomb 2/metabolismo , Regiones Promotoras Genéticas , Ratas , Ratas Wistar , Factores de TiempoRESUMEN
Polycomb group (PcG) and Trithorax group (TrxG) proteins are essential for maintaining epigenetic memory in both embryonic stem cells and differentiated cells. To date, how they are localized to hundreds of specific target genes within a vertebrate genome had remained elusive. Here, by focusing on short cis-acting DNA elements of single functions, we discovered three classes of response elements in human genome: Polycomb response elements (PREs), Trithorax response elements (TREs) and Polycomb/Trithorax response elements (P/TREs). In particular, the four PREs (PRE14, 29, 39 and 48) are the first set of, to our knowledge, bona fide vertebrate PREs ever discovered, while many previously reported Drosophila or vertebrate PREs are likely P/TREs. We further demonstrated that YY1 and CpG islands are specifically enriched in the four TREs (PRE30, 41, 44 and 55), but not in the PREs. The three classes of response elements as unraveled in this study should guide further global investigation and open new doors for a deeper understanding of PcG and TrxG mechanisms in vertebrates.
Asunto(s)
Proteínas de Unión al ADN/genética , Represión Epigenética/genética , N-Metiltransferasa de Histona-Lisina/genética , Complejos Multiproteicos/genética , Proteína de la Leucemia Mieloide-Linfoide/genética , Proteínas de Neoplasias/genética , Complejo Represivo Polycomb 2/genética , Elementos de Respuesta/genética , Sistemas CRISPR-Cas , Inmunoprecipitación de Cromatina , Islas de CpG , Técnicas de Inactivación de Genes , Genes Reporteros , Células HEK293 , Células HeLa , Código de Histonas/genética , Humanos , Células K562 , Mutagénesis Insercional , Reacción en Cadena de la Polimerasa , Interferencia de ARN , ARN Interferente Pequeño/genética , Factor de Transcripción YY1/genéticaRESUMEN
Long noncoding RNAs (lncRNAs) have been characterized extensively in animals and are involved in several processes, including homeobox gene expression and X-chromosome inactivation. In comparison, there has been much less detailed characterization of plant lncRNAs, and the number of distinct lncRNAs encoded in plant genomes and their regulation by developmental and epigenetic mechanisms remain largely unknown. Here, we analyzed transcriptome data from Asian rice (Oryza sativa) and identified 6,309 long intergenic noncoding RNAs (lincRNAs), focusing on their expression in reproductive tissues and organs. Most O. sativa lincRNAs were expressed in a highly tissue-specific manner, with an unexpectedly high fraction specifically expressed in male gametes. Mutation of a component of the Polycomb Repressive Complex2 (PRC2) resulted in derepression of another large class of lincRNAs, whose expression is correlated with H3K27 trimethylation in developing panicles. Overlap with the sperm cell-specific lincRNAs suggests that epigenetic repression of lincRNAs in the panicles was partially relieved in the male germline. Expression of a subset of lincRNAs also showed modulation by drought in reproductive tissues. Comparison with other cereal genomes showed that the lincRNAs generally have low levels of conservation at both the sequence and structural levels. Use of a novelty detection support vector machine model enabled the detection of nucleotide sequence and structural homology in â¼10% and â¼4% of the lincRNAs in genomes of purple false brome (Brachypodium distachyon) and maize (Zea mays), respectively. This is the first study to report on a large number of lncRNAs that are targets of repression by PRC2 rather than mediating regulation via PRC2. That the vast majority of the lincRNAs reported here do not overlap with those of other rice studies indicates that these are a significant addition to the known lincRNAs in rice.
Asunto(s)
Oryza/genética , Polen/genética , Complejo Represivo Polycomb 2/genética , ARN Largo no Codificante/genética , Secuencia de Bases , Brachypodium/genética , Cromatina/genética , Secuencia Conservada , Sequías , Represión Epigenética , Regulación de la Expresión Génica de las Plantas , Metilación , Complejo Represivo Polycomb 2/metabolismo , ARN de Planta , Alineación de Secuencia/métodos , Máquina de Vectores de SoporteRESUMEN
UV-B is a high-energy component of the solar radiation perceived by the plant and induces a number of modifications in plant growth and development, including changes in flowering time. However, the molecular mechanisms underlying these changes are largely unknown. In the present work, we demonstrate that Arabidopsis plants grown under white light supplemented with UV-B show a delay in flowering time, and this developmental reprogramming is mediated by the UVR8 photoreceptor. Using a combination of gene expression analyses and UV-B irradiation of different flowering mutants, we gained insight into the pathways involved in the observed flowering time delay in UV-B-exposed Arabidopsis plants. We provide evidence that UV-B light downregulates the expression of MSI1 and CLF, two of the components of the polycomb repressive complex 2, which in consequence drives a decrease in H3K27me3 histone methylation of MIR156 and FLC genes. Modification in the expression of several flowering time genes as a consequence of the decrease in the polycomb repressive complex 2 activity was also determined. UV-B exposure of flowering mutants supports the involvement of this complex in the observed delay in flowering time, mostly through the age pathway.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/genética , Arabidopsis/efectos de la radiación , Flores/fisiología , Flores/efectos de la radiación , MicroARNs/metabolismo , Proteínas Represoras/metabolismo , Rayos Ultravioleta , Arabidopsis/fisiología , Proteínas de Arabidopsis/genética , Regulación hacia Abajo/genética , Flores/genética , Regulación de la Expresión Génica de las Plantas/efectos de la radiación , Genes de Plantas , Histonas/metabolismo , Lisina/metabolismo , Metilación , MicroARNs/genética , Mutación/genética , Complejo Represivo Polycomb 2 , Factores de TiempoRESUMEN
Targeting self-renewal is an important goal in cancer therapy and recent studies have focused on Notch signalling in the maintenance of stemness of glioma stem cells (GSCs). Understanding cancer-specific Notch regulation would improve specificity of targeting this pathway. In this study, we find that Notch1 activation in GSCs specifically induces expression of the lncRNA, TUG1. TUG1 coordinately promotes self-renewal by sponging miR-145 in the cytoplasm and recruiting polycomb to repress differentiation genes by locus-specific methylation of histone H3K27 via YY1-binding activity in the nucleus. Furthermore, intravenous treatment with antisense oligonucleotides targeting TUG1 coupled with a drug delivery system induces GSC differentiation and efficiently represses GSC growth in vivo. Our results highlight the importance of the Notch-lncRNA axis in regulating self-renewal of glioma cells and provide a strong rationale for targeting TUG1 as a specific and potent therapeutic approach to eliminate the GSC population.
Asunto(s)
Neoplasias Encefálicas/genética , Neoplasias Encefálicas/terapia , Glioma/genética , Glioma/terapia , ARN Largo no Codificante/metabolismo , Receptor Notch1/metabolismo , Animales , Carcinogénesis/genética , Carcinogénesis/patología , Diferenciación Celular/genética , Proteína Potenciadora del Homólogo Zeste 2/metabolismo , Exones/genética , Femenino , Regulación Neoplásica de la Expresión Génica , Humanos , Ratones Endogámicos NOD , Ratones SCID , MicroARNs/genética , MicroARNs/metabolismo , Células Madre Neoplásicas/metabolismo , Células Madre Neoplásicas/patología , Neuronas/metabolismo , Complejo Represivo Polycomb 2/metabolismo , Unión Proteica , Proteínas Proto-Oncogénicas c-myc/metabolismo , ARN Largo no Codificante/genética , ARN Mensajero/genética , ARN Mensajero/metabolismo , Factores de Transcripción SOXB1/metabolismo , Ensayos Antitumor por Modelo de Xenoinjerto , Factor de Transcripción YY1/metabolismoRESUMEN
Understanding inhibitor binding modes is a key aspect of drug development. Early in a drug discovery effort these considerations often impact hit finding strategies and hit prioritization. Multiple inhibitor experiments, where enzyme inhibition is measured in the presence of two simultaneously varied inhibitors, can provide valuable information about inhibitor binding. These experiments utilize the inhibitor concentration dependence of the observed combined inhibition to determine the relationship between two compounds. In this way, it can be determined whether two inhibitors bind exclusively, independently, synergistically, or antagonistically. Novel inhibitors can be tested against each other or reference compounds to assist hit classification and characterization of inhibitor binding. In this chapter, we discuss the utility and design of multiple inhibitor experiments and present a new local curve fitting method for analyzing these data utilizing IC50 replots. The IC50 replot method is analogous to that used for determining mechanisms of inhibition with respect to substrate, as originally proposed by Cheng and Prusoff (Cheng and Prusoff Biochem Pharmacol 22: 3099-3108, 1973). The IC50 replot generated by this method reveals distinct patterns that are diagnostic of the nature of the interaction between two inhibitors. Multiple inhibition of the histone methyltransferase EZH2 by EPZ-5687 and the reaction product S-adenosylhomocysteine is presented as an example of the method.
Asunto(s)
Benzamidas/farmacología , Evaluación Preclínica de Medicamentos/métodos , Proteína Potenciadora del Homólogo Zeste 2/antagonistas & inhibidores , Pruebas de Enzimas/métodos , Inhibidores Enzimáticos/farmacología , Complejo Represivo Polycomb 2/antagonistas & inhibidores , Piridonas/farmacología , S-Adenosilhomocisteína/farmacología , Animales , Sitios de Unión , Unión Competitiva , Compuestos de Bifenilo , Proteína Potenciadora del Homólogo Zeste 2/metabolismo , Humanos , Concentración 50 Inhibidora , Cinética , Morfolinas , Complejo Represivo Polycomb 2/metabolismoRESUMEN
Enhancer of zeste homolog 2 (EZH2) catalyses histone H3 lysine 27 trimethylation (H3K27me3) to silence tumour-suppressor genes in hepatocellular carcinoma (HCC) but the process of locus-specific recruitment remains elusive. Here we investigated the transcription factors involved and the molecular consequences in HCC development. The genome-wide distribution of H3K27me3 was determined by chromatin immunoprecipitation coupled with high-throughput sequencing or promoter array analyses in HCC cells from hepatitis B virus (HBV) X protein transgenic mouse and human cell models. Transcription factor binding site analysis was performed to identify EZH2-interacting transcription factors followed by functional characterization. Our cross-species integrative analysis revealed a crucial link between Yin Yang 1 (YY1) and EZH2-mediated H3K27me3 in HCC. Gene expression analysis of human HBV-associated HCC specimens demonstrated concordant overexpression of YY1 and EZH2, which correlated with poor survival of patients in advanced stages. The YY1 binding motif was significantly enriched in both in vivo and in vitro H3K27me3-occupied genes, including genes for 15 tumour-suppressive microRNAs. Knockdown of YY1 reduced not only global H3K27me3 levels, but also EZH2 and H3K27me3 promoter occupancy and DNA methylation, leading to the transcriptional up-regulation of microRNA-9 isoforms in HCC cells. Concurrent EZH2 knockdown and 5-aza-2'-deoxycytidine treatment synergistically increased the levels of microRNA-9, which reduced the expression and transcriptional activity of nuclear factor-κB (NF-κB). Functionally, YY1 promoted HCC tumourigenicity and inhibited apoptosis of HCC cells, at least partially through NF-κB activation. In conclusion, YY1 overexpression contributes to EZH2 recruitment for H3K27me3-mediated silencing of tumour-suppressive microRNAs, thereby activating NF-κB signalling in hepatocarcinogenesis.
Asunto(s)
Carcinoma Hepatocelular/metabolismo , Silenciador del Gen , Neoplasias Hepáticas/metabolismo , MicroARNs/metabolismo , FN-kappa B/metabolismo , Factor de Transcripción YY1/metabolismo , Animales , Apoptosis , Sitios de Unión , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/patología , Carcinoma Hepatocelular/virología , Línea Celular Tumoral , Proliferación Celular , Metilación de ADN , Proteína Potenciadora del Homólogo Zeste 2 , Regulación Neoplásica de la Expresión Génica , Histonas/metabolismo , Humanos , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/patología , Neoplasias Hepáticas/virología , Lisina , Metilación , Ratones Desnudos , Ratones Transgénicos , MicroARNs/genética , Complejo Represivo Polycomb 2/genética , Complejo Represivo Polycomb 2/metabolismo , Regiones Promotoras Genéticas , Interferencia de ARN , Transducción de Señal , Factores de Tiempo , Transactivadores/genética , Transactivadores/metabolismo , Transfección , Carga Tumoral , Regulación hacia Arriba , Proteínas Reguladoras y Accesorias Virales , Factor de Transcripción YY1/genéticaRESUMEN
NUT midline carcinoma is a rare entity arising primarily in the midline of teenagers and young adults. Genomically, it is associated with a translocation involving a nuclear protein in testis (NUT) gene with other genes, most commonly, the BRD4 gene. The resultant is a partial or near total block in differentiation of tumor cells into mature squamous elements. Such tumors are resistant to conventional therapy with a reported mean survival at less than 1 year. In this study, we investigated two cases with genomic confirmation as NUT midline carcinoma by morphoproteomic analysis using immunohistochemical antibodies. Our results showed overexpression, largely in the undifferentiated cells of the tumors of: 1) Stemness marker, SRY (sex determining region Y)-box 2 (Sox2); 2) Constitutive activation of the mTORC2 pathway with expression of total insulin-like growth factor-1 receptor (IGF-1R[Tyr1165/1166]), and nuclear p-mTOR (Ser 2448) and p-Akt (Ser 473); and 3) c-Myc, silent mating type information regulation 2 homolog 1 (Sirt1) and histone methyltransferase enhancer of Zeste, Drosophila, homolog 2 (EZH2) as molecular impediments to differentiation. These data were analyzed through the use of QIAGEN's Ingenuity(®) Pathway Analysis (IPA(®), QIAGEN Redwood City, www.qiagen.com/ingenuity). The results established the interconnection of these pathways and molecules, and identified several pharmacogenomic agents--melatonin, metformin, vorinostat, curcumin, and sulforaphane--that have the potential to remove the block in differentiation and lead to the establishment of a more benign form of NUT midline carcinoma.
Asunto(s)
Carcinoma/genética , Carcinoma/patología , Neoplasias del Mediastino/genética , Neoplasias del Mediastino/patología , Proteínas Nucleares/genética , Proteínas Oncogénicas/genética , Adolescente , Adulto , Carcinoma/metabolismo , Carcinoma/terapia , Proteína Potenciadora del Homólogo Zeste 2 , Femenino , Humanos , Inmunohistoquímica , Hibridación Fluorescente in Situ , Isotiocianatos/farmacología , Masculino , Neoplasias del Mediastino/metabolismo , Neoplasias del Mediastino/terapia , Metformina/farmacología , Terapia Molecular Dirigida/métodos , Proteínas de Neoplasias , Proteínas Nucleares/metabolismo , Proteínas Oncogénicas/metabolismo , Complejo Represivo Polycomb 2/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Sirtuina 1/metabolismo , Sulfóxidos , Serina-Treonina Quinasas TOR/metabolismoRESUMEN
Endoplasmic reticulum (ER) and mitochondria dysfunction contribute to insulin resistance generation during obesity and diabetes. ER and mitochondria interact through Mitofusin 2 (MTF2), which anchors in the outer mitochondrial and ER membranes regulating energy metabolism. Ablation of MTF2 leads to ER stress activation and insulin resistance. Here we determine whether lipotoxic insult induced by saturated lipids decreases MTF2 expression leading to ER stress response in hypothalamus and its effects on insulin sensitivity using in vitro and in vivo models. We found that lipotoxic stimulation induced by palmitic acid, but not the monounsaturated palmitoleic acid, decreases MTF2 protein levels in hypothalamic mHypoA-CLU192 cells. Also, palmitic acid incubation activates ER stress response evidenced by increase in the protein levels of GRP78/BIP marker at later stage than MTF2 downregulation. Additionally, we found that MTF2 alterations induced by palmitic, but not palmitoleic, stimulation exacerbate insulin resistance in hypothalamic cells. Insulin resistance induced by palmitic acid is prevented by pre-incubation of the anti-inflammatory and the ER stress release reagents, sodium salicylate and 4 phenylbutirate, respectively. Finally, we demonstrated that lipotoxic insult induced by high fat feeding to mice decreases MTF2 proteins levels in arcuate nucleus of hypothalamus. Our data indicate that saturated lipids modulate MTF2 expression in hypothalamus coordinating the ER stress response and the susceptibility to insulin resistance.
Asunto(s)
Estrés del Retículo Endoplásmico/efectos de los fármacos , Inhibidores Enzimáticos/farmacología , Hipotálamo/citología , Resistencia a la Insulina/fisiología , Neuronas/efectos de los fármacos , Ácido Palmítico/farmacología , Complejo Represivo Polycomb 2/metabolismo , Análisis de Varianza , Animales , Peso Corporal/efectos de los fármacos , Peso Corporal/fisiología , Dieta Alta en Grasa , Relación Dosis-Respuesta a Droga , Chaperón BiP del Retículo Endoplásmico , Ratones , Ratones Endogámicos C57BL , Neuronas/metabolismo , Neuronas/ultraestructura , Proteína Oncogénica v-akt/metabolismo , Complejo Represivo Polycomb 2/genética , Especies Reactivas de Oxígeno/metabolismo , Factores de TiempoRESUMEN
The Class II Transactivator (CIITA) is the master regulator of Major Histocompatibility Class II (MHC II) genes. Transcription of CIITA through the IFN-γ inducible CIITA promoter IV (CIITA pIV) during activation is characterized by a decrease in trimethylation of histone H3 lysine 27 (H3K27me3), catalyzed by the histone methyltransferase Enhancer of Zeste Homolog 2 (EZH2). While EZH2 is the known catalytic subunit of the Polycomb Repressive Complex 2 (PRC2) and is present at the inactive CIITA pIV, the mechanism of PRC2 recruitment to mammalian promoters remains unknown. Here we identify two DNA-binding proteins, which interact with and regulate PRC2 recruitment to CIITA pIV. We demonstrate Yin Yang 1 (YY1) and Jumonji domain containing protein 2 (JARID2) are binding partners along with EZH2 in mammalian cells. Upon IFN-γ stimulation, YY1 dissociates from CIITA pIV while JARID2 binding to CIITA pIV increases, suggesting novel roles for these proteins in regulating expression of CIITA pIV. Knockdown of YY1 and JARID2 yields decreased binding of EZH2 and H3K27me3 at CIITA pIV, suggesting important roles for YY1 and JARID2 at CIITA pIV. JARID2 knockdown also results in significantly elevated levels of CIITA mRNA upon IFN-γ stimulation. This study is the first to identify novel roles of YY1 and JARID2 in the epigenetic regulation of the CIITA pIV by recruitment of PRC2. Our observations indicate the importance of JARID2 in CIITA pIV silencing, and also provide a novel YY1-JARID2-PRC2 regulatory complex as a possible explanation of differential PRC2 recruitment at inducible versus permanently silenced genes.
Asunto(s)
Proteínas Nucleares/genética , Proteínas del Grupo Polycomb/metabolismo , Transactivadores/genética , Proteína Potenciadora del Homólogo Zeste 2 , Regulación de la Expresión Génica/efectos de los fármacos , Técnicas de Silenciamiento del Gen , Células HeLa , Histonas/metabolismo , Humanos , Interferón gamma/farmacología , Lisina/metabolismo , Metilación/efectos de los fármacos , Proteínas Nucleares/metabolismo , Complejo Represivo Polycomb 2/genética , Complejo Represivo Polycomb 2/metabolismo , Regiones Promotoras Genéticas , Unión Proteica/efectos de los fármacos , Unión Proteica/genética , ARN Mensajero/genética , ARN Mensajero/metabolismo , Transactivadores/metabolismo , Transcripción Genética/efectos de los fármacos , Factor de Transcripción YY1/genética , Factor de Transcripción YY1/metabolismoRESUMEN
Long non-protein coding RNAs (lncRNAs) are an important class of molecules that help orchestrate key cellular events. Although their functional roles in cells are not well understood, thousands of lncRNAs and a number of possible mechanisms by which they act have been reported. LncRNAs can exert their regulatory function in cells by interacting with epigenetic enzymes. In this study, we developed a tool to study lncRNA-protein interactions for high-throughput screening of small-molecule modulators using AlphaScreen technology. We tested the interaction of two lncRNAs: brain-derived neurotrophic factor antisense (BDNF-AS) and Hox transcript antisense RNA (HOTAIR), with Enhancer of zeste homolog 2 (EZH2), a histone methyltransferase against a phytochemical library, to look for small-molecule inhibitors that can alter the expression of downstream target genes. We identified ellipticine, a compound that up-regulates BDNF transcription. Our study shows the feasibility of using high-throughput screening to identify modulators of lncRNA-protein interactions and paves the road for targeting lncRNAs that are dysregulated in human disorders using small-molecule therapies.
Asunto(s)
Ensayos Analíticos de Alto Rendimiento , ARN Largo no Codificante/antagonistas & inhibidores , Factor Neurotrófico Derivado del Encéfalo/biosíntesis , Factor Neurotrófico Derivado del Encéfalo/genética , Evaluación Preclínica de Medicamentos , Elipticinas/farmacología , Proteína Potenciadora del Homólogo Zeste 2 , Expresión Génica/efectos de los fármacos , Células HEK293 , Humanos , Complejo Represivo Polycomb 2/biosíntesis , Complejo Represivo Polycomb 2/genética , Unión Proteica/efectos de los fármacos , Interferencia de ARN/efectos de los fármacos , ARN Largo no Codificante/biosíntesis , ARN Largo no Codificante/químicaRESUMEN
Enhancer of Zeste Homolog 2 (EZH2) has emerged as a promising therapeutic target for treatment of a broad spectrum of tumors including gliomas. We explored the interactions of five novel, structurally similar EZH2 inhibitors (EPZ005687, EPZ-6438, UNC1999, GSK343 and GSK126) with P-glycoprotein (P-gp/ABCB1) and breast cancer resistance protein (BCRP/ABCG2). The compounds were screened by in vitro transwell assays and EPZ005687, EPZ-6438 and GSK126 were further tested in vivo using wild-type (WT), Abcb1 and/or Abcg2 knockout mice. All EZH2 inhibitors are transported by P-gp and BCRP, although in vitro the transporter affinity of GSK126 was obscured by very low membrane permeability. Both P-gp and Bcrp1 restrict the brain penetration of EPZ005687 and GSK126, whereas the brain accumulation of EPZ-6438 is limited by P-gp only and efflux of EPZ-6438 was completely abrogated by elacridar. Intriguingly, an unknown factor present in all knockout mouse strains causes EPZ005687 and EPZ-6438 retention in plasma relative to WT mice, a phenomenon not seen with GSK126. In WT mice, the GSK126 tissue-to-plasma ratio for all tissues is lower than for EPZ005687 or EPZ-6438. Moreover, the oral bioavailability of GSK126 is only 0.2% in WT mice, which increases to 14.4% in Abcb1;Abcg2 knockout mice. These results are likely due to poor membrane permeability and question the clinical usefulness of GSK126. Although all tested EZH2 inhibitors are substrates of P-gp and BCRP, restricting the brain penetration and potential utility for treatment of glioma, EPZ-6438 would be the most suitable candidate of this series.
Asunto(s)
Subfamilia B de Transportador de Casetes de Unión a ATP/genética , Subfamilia B de Transportador de Casetes de Unión a ATP/metabolismo , Transportadoras de Casetes de Unión a ATP/genética , Transportadoras de Casetes de Unión a ATP/metabolismo , Barrera Hematoencefálica/efectos de los fármacos , Inhibidores Enzimáticos/farmacocinética , Complejo Represivo Polycomb 2/antagonistas & inhibidores , Administración Oral , Animales , Benzamidas/administración & dosificación , Benzamidas/farmacocinética , Disponibilidad Biológica , Compuestos de Bifenilo , Línea Celular , Perros , Evaluación Preclínica de Medicamentos , Proteína Potenciadora del Homólogo Zeste 2 , Inhibidores Enzimáticos/administración & dosificación , Humanos , Indazoles/administración & dosificación , Indazoles/farmacocinética , Indoles/administración & dosificación , Indoles/farmacocinética , Células de Riñón Canino Madin Darby , Ratones , Ratones Noqueados , Morfolinas , Piridonas/administración & dosificación , Piridonas/farmacocinéticaRESUMEN
Early-life stress (ELS) in mice causes sustained hypomethylation at the downstream Avp enhancer, subsequent overexpression of hypothalamic Avp and increased stress responsivity. The sequence of events leading to Avp enhancer methylation is presently unknown. Here, we used an embryonic stem cell-derived model of hypothalamic-like differentiation together with in vivo experiments to show that binding of polycomb complexes (PcG) preceded the emergence of ELS-responsive DNA methylation and correlated with gene silencing. At the same time, PcG occupancy associated with the presence of Tet proteins preventing DNA methylation. Early hypothalamic-like differentiation triggered PcG eviction, DNA-methyltransferase recruitment and enhancer methylation. Concurrently, binding of the Methyl-CpG-binding and repressor protein MeCP2 increased at the enhancer although Avp expression during later stages of differentiation and the perinatal period continued to increase. Overall, we provide evidence of a new role of PcG proteins in priming ELS-responsive DNA methylation at the Avp enhancer prior to epigenetic programming consistent with the idea that PcG proteins are part of a flexible silencing system during neuronal development.