RESUMEN
Epigenetic marks including histone modifications and DNA methylation are associated with the regulation of gene expression and activity. In addition, an increasing number of non-coding RNAs with regulatory activity on gene expression have been identified. Alongside, technological advancements allow for the analysis of these mechanisms with high resolution up to the single-cell level. For instance, the assay for transposase-accessible chromatin using sequencing (ATAC-seq) simultaneously probes for chromatin accessibility and nucleosome positioning. Thus, it provides information on two levels of epigenetic regulation. Development and differentiation of T cells into functional subset cells including memory T cells are dynamic processes driven by environmental signals. Here, we briefly review the current knowledge of how epigenetic regulation contributes to subset specification, differentiation and memory development in T cells. Specifically, we focus on epigenetic mechanisms differentially active in the two distinct T cell populations expressing αß or γδ T cell receptors. We also discuss examples of epigenetic alterations of T cells in autoimmune diseases. DNA methylation and histone acetylation are subject to modification by several classes of 'epigenetic modifiers', some of which are in clinical use or in preclinical development. Therefore, we address the impact of some epigenetic modifiers on T-cell activation and differentiation, and discuss possible synergies with T cell-based immunotherapeutic strategies.
Asunto(s)
Diferenciación Celular , Plasticidad de la Célula , Epigénesis Genética , Epigenómica , Linfocitos T/fisiología , Animales , Metilación de ADN , Humanos , Activación de Linfocitos , Procesamiento Proteico-PostraduccionalRESUMEN
CONTEXT: Only approximately 85% of patients with a clinical diagnosis complete androgen insensitivity syndrome and less than 30% with partial androgen insensitivity syndrome can be explained by inactivating mutations in the androgen receptor (AR) gene. OBJECTIVE: The objective of the study was to clarify this discrepancy by in vitro determination of AR transcriptional activity in individuals with disorders of sex development (DSD) and male controls. DESIGN: Quantification of DHT-dependent transcriptional induction of the AR target gene apolipoprotein D (APOD) in cultured genital fibroblasts (GFs) (APOD assay) and next-generation sequencing of the complete coding and noncoding AR locus. SETTING: The study was conducted at a university hospital endocrine research laboratory. PATIENTS: GFs from 169 individuals were studied encompassing control males (n = 68), molecular defined DSD other than androgen insensitivity syndrome (AIS; n = 18), AR mutation-positive AIS (n = 37), and previously undiagnosed DSD including patients with a clinical suspicion of AIS (n = 46). INTERVENTION(S): There were no interventions. MAIN OUTCOME MEASURE(S): DHT-dependent APOD expression in cultured GF and AR mutation status in 169 individuals was measured. RESULTS: The APOD assay clearly separated control individuals (healthy males and molecular defined DSD patients other than AIS) from genetically proven AIS (cutoff < 2.3-fold APOD-induction; 100% sensitivity, 93.3% specificity, P < .0001). Of 46 DSD individuals with no AR mutation, 17 (37%) fell below the cutoff, indicating disrupted androgen signaling. CONCLUSIONS: AR mutation-positive AIS can be reliably identified by the APOD assay. Its combination with next-generation sequencing of the AR locus uncovered an AR mutation-negative, new class of androgen resistance, which we propose to name AIS type II. Our data support the existence of cellular components outside the AR affecting androgen signaling during sexual differentiation with high clinical relevance.
Asunto(s)
Síndrome de Resistencia Androgénica/diagnóstico , Apolipoproteínas D , Bioensayo/normas , Trastornos del Desarrollo Sexual/diagnóstico , Receptores Androgénicos/metabolismo , Testosterona/análogos & derivados , Adulto , Síndrome de Resistencia Androgénica/genética , Síndrome de Resistencia Androgénica/metabolismo , Células Cultivadas , Trastornos del Desarrollo Sexual/genética , Trastornos del Desarrollo Sexual/metabolismo , Fibroblastos , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Masculino , Mutación , Receptores Androgénicos/genética , Sensibilidad y Especificidad , Testosterona/metabolismo , Transcripción GenéticaRESUMEN
Follicular lymphoma (FL) with a t(14;18) is a B-cell neoplasm clinically characterized by multiple recurrencies. In order to investigate the clonal evolution of this lymphoma, we studied paired primary and relapse tumor samples from 33 patients with recurrent non-transformed t(14;18)-positive FL. We reconstructed phylogenetic trees of the evolution by taking advantage of the activation-induced cytidine deaminase (AID)-mediated somatic hypermutation (SHM) active in the germinal center reaction using sequences of the clonal VHDHJH rearrangements of the immunoglobulin heavy chain (IGH) locus. Mutational analysis of the IGH locus showed evidence for ongoing somatic mutation and for counter-selection of mutations affecting the BCR conformation during tumor evolution. We further followed evolutionary divergence by targeted sequencing of gene loci affected by aberrant SHM as well as of known driver genes of lymphomagenesis, and by array-based genome-wide chromosomal imbalance and DNA methylation analysis. We observed a wide spectrum of evolutionary patterns ranging from almost no evolution to divergent evolution within recurrent non-transformed t(14;18) FL. Remarkably, we observed a correlation of the magnitude of evolutionary divergence across all genetic and epigenetic levels suggesting co-evolution. The distribution of coding mutations in driver genes and the correlation with SHM suggest CREBBP and AID to be potential modifiers of genetic and epigenetic co-evolution in FL.
Asunto(s)
Epigénesis Genética , Linfoma Folicular/genética , Linfoma Folicular/inmunología , Adulto , Anciano , Anciano de 80 o más Años , Cromosomas/ultraestructura , Biología Computacional , Citidina Desaminasa/genética , Metilación de ADN , Análisis Mutacional de ADN , Epigenómica , Evolución Molecular , Eliminación de Gen , Genómica , Humanos , Inmunoglobulinas/inmunología , Persona de Mediana Edad , Mutación , Análisis de Secuencia por Matrices de Oligonucleótidos , Filogenia , Polimorfismo de Nucleótido Simple , Receptores de Antígenos de Linfocitos B/genética , Recurrencia , Translocación GenéticaAsunto(s)
Linfoma de Burkitt/genética , Metilación de ADN , Herpesvirus Humano 4 , Secuenciación de Nucleótidos de Alto Rendimiento , Linfoma de Burkitt/virología , Línea Celular Tumoral , Exoma , Regulación Neoplásica de la Expresión Génica , Herpesvirus Humano 4/genética , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Humanos , Cariotipificación , Polimorfismo de Nucleótido SimpleRESUMEN
Male external genital differentiation is accompanied by implementation of a long-term, male-specific gene expression pattern indicating androgen programming in cultured genital fibroblasts. We hypothesized the existence of an epigenetic background contributing to this phenomenon. DNA methylation levels in 2 normal scrotal fibroblast strains from 46,XY males compared to 2 labia majora fibroblast strains from 46,XY females with complete androgen insensitivity syndrome (AIS) due to androgen receptor (AR) mutations were analyzed by Illumina GoldenGate methylation arrays®. Results were validated with pyrosequencing in labia majora fibroblast strains from fifteen 46,XY patients and compared to nine normal male scrotal fibroblast strains. HOXA5 showed a significantly higher methylation level in complete AIS. This finding was confirmed by bisulfite pyrosequencing of 14 CpG positions within the HOXA5 promoter in the same strains. Extension of the 2 groups revealed a constant low HOXA5 methylation pattern in the controls in contrast to a highly variable methylation pattern in the AIS patients. HOXA5 represents a candidate gene of androgen-mediated promoter methylation. The constantly low HOXA5 DNA methylation level of normal male scrotal fibroblast strains and the frequently high methylation levels in labia majora fibroblast strains in AIS indicate for the first time that androgen programming in sexual differentiation is not restricted to global gene transcription but also occurs at the epigenetic level.
Asunto(s)
Epigénesis Genética/genética , Proteínas de Homeodominio/genética , Receptores Androgénicos/genética , Metilación de ADN/genética , Femenino , Humanos , Masculino , Mutación , Regiones Promotoras Genéticas/genética , Reacción en Cadena de la Polimerasa de Transcriptasa InversaAsunto(s)
Deleción Cromosómica , Cromosomas Humanos Par 14 , Leucemia Linfocítica Crónica de Células B/genética , Linfoma de Células B/genética , Factor 3 Asociado a Receptor de TNF/genética , Animales , Humanos , Leucemia Linfocítica Crónica de Células B/fisiopatología , Linfoma de Células B/fisiopatologíaRESUMEN
To determine the pattern of genetic alterations in primary central nervous system lymphomas (PCNSL), 19 PCNSL were studied by high-density single-nucleotide polymorphism arrays. Recurrent losses involved 6p21.32, 6q21, 8q12-12.2, 9p21.3, 3p14.2, 4q35.2, 10q23.21 and 12p13.2, whereas gains involved 18q21-23, 19q13.31, 19q13.43 and the entire chromosomes X and 12. Partial uniparental disomies (pUPDs) were identified in 6p and 9p21.3. These genomic alterations affected the HLA locus, the CDKN2A/p16, CDKN2B/p15 and MTAP, as well as the PRDM1, FAS, MALT1, and BCL2 genes. Increased methylation values of the CDKN2A/p16 promoter region were detected in 75% (6/8) PCNSL. Gene expression profiling showed 4/21 (20%) minimal common regions of imbalances to be associated with a differential mRNA expression affecting the FAS, STAT6, CD27, ARHGEF6 and SEPT6 genes. Collectively, this study unraveled novel genomic imbalances and pUPD with a high resolution in PCNSL and identified target genes of potential relevance in the pathogenesis of this lymphoma entity.
Asunto(s)
Neoplasias Encefálicas/genética , Aberraciones Cromosómicas , Cromosomas Humanos/genética , Disomía Uniparental , Anciano , Anciano de 80 o más Años , Biomarcadores de Tumor/genética , Biomarcadores de Tumor/metabolismo , Neoplasias Encefálicas/patología , Metilación de ADN , Femenino , Perfilación de la Expresión Génica , Humanos , Hibridación Fluorescente in Situ , Masculino , Persona de Mediana Edad , Análisis de Secuencia por Matrices de Oligonucleótidos , Polimorfismo de Nucleótido Simple/genéticaRESUMEN
BACKGROUND: Pancreatic ductal adenocarcinomas (PDACs) are highly resistant to treatment due to changes in various signalling pathways. CK1 isoforms play important regulatory roles in these pathways. AIMS: We analysed the expression levels of CK1 delta and epsilon (CK1delta/in) in pancreatic tumour cells in order to validate the effects of CK1 inhibition by 3-[2,4,6-(trimethoxyphenyl)methylidenyl]-indolin-2-one (IC261) on their proliferation and sensitivity to anti-CD95 and gemcitabine. METHODS: CK1delta/in expression levels were investigated by using western blotting and immunohistochemistry. Cell death was analysed by FACS analysis. Gene expression was assessed by real-time PCR and western blotting. The putative anti-tumoral effects of IC261 were tested in vivo in a subcutaneous mouse xenotransplantation model for pancreatic cancer. RESULTS: We found that CK1delta/in are highly expressed in pancreatic tumour cell lines and in higher graded PDACs. Inhibition of CK1delta/in by IC261 reduced pancreatic tumour cell growth in vitro and in vivo. Moreover, IC261 decreased the expression levels of several anti-apoptotic proteins and sensitised cells to CD95-mediated apoptosis. However, IC261 did not enhance gemcitabine-mediated cell death either in vitro or in vivo. CONCLUSIONS: Targeting CK1 isoforms by IC261 influences both pancreatic tumour cell growth and apoptosis sensitivity in vitro and the growth of induced tumours in vivo, thus providing a promising new strategy for the treatment of pancreatic tumours.
Asunto(s)
Carcinoma Ductal Pancreático/patología , Caseína Cinasa 1 épsilon/antagonistas & inhibidores , Quinasa Idelta de la Caseína/antagonistas & inhibidores , Indoles/farmacología , Neoplasias Pancreáticas/patología , Floroglucinol/análogos & derivados , Animales , Antimetabolitos Antineoplásicos/uso terapéutico , Antineoplásicos/farmacología , Antineoplásicos/uso terapéutico , Apoptosis/efectos de los fármacos , Carcinoma Ductal Pancreático/tratamiento farmacológico , Carcinoma Ductal Pancreático/enzimología , Carcinoma Ductal Pancreático/secundario , Caseína Cinasa 1 épsilon/metabolismo , Caseína Cinasa 1 épsilon/fisiología , Quinasa Idelta de la Caseína/metabolismo , Quinasa Idelta de la Caseína/fisiología , Proliferación Celular/efectos de los fármacos , Desoxicitidina/análogos & derivados , Desoxicitidina/uso terapéutico , Evaluación Preclínica de Medicamentos , Humanos , Indoles/uso terapéutico , Metástasis Linfática , Ratones , Ratones SCID , Trasplante de Neoplasias , Neoplasias Pancreáticas/tratamiento farmacológico , Neoplasias Pancreáticas/enzimología , Floroglucinol/farmacología , Floroglucinol/uso terapéutico , Trasplante Heterólogo , Células Tumorales Cultivadas , Receptor fas/fisiología , GemcitabinaRESUMEN
Pancreatic ductal adenocarcinoma (PDAC) is a fatal disease and one of the cancer entities with the lowest life expectancy. Beside surgical therapy, no effective therapeutic options are available yet. Here, we show that 4-phenylbutyrate (4-PB), a known and well-tolerable inhibitor of histone deacetylases (HDAC), induces up to 70% apoptosis in all cell lines tested (Panc 1, T4M-4, COLO 357, BxPc3). In contrast, it leads to cell cycle arrest in only half of the cell lines tested. This drug increases gap junction communication between adjacent T3M-4 cells in a concentration-dependent manner and efficiently inhibits cellular export mechanisms in Panc 1, T4M-4, COLO 357 and BxPc3 cells. Consequently, in combination with gemcitabine 4-PB shows an overadditive effect on induction of apoptosis in BxPc3 and T3M-4 cells (up to 4.5-fold compared to single drug treatment) with accompanied activation of Caspase 8, BH3 interacting domain death agonist (Bid) and poly (ADP-ribose) polymerase family, member 1 (PARP) cleavage. Although the inhibition of the mitogen-activated protein kinase-pathway has no influence on fulminant induction of apoptosis, the inhibition of the JNK-pathway by SP600125 completely abolishes the overadditive effect induced by the combined application of both drugs, firstly reported by this study.
Asunto(s)
Protocolos de Quimioterapia Combinada Antineoplásica/farmacología , Carcinoma Ductal Pancreático/tratamiento farmacológico , Comunicación Celular/efectos de los fármacos , Uniones Comunicantes/efectos de los fármacos , Inhibidores de Histona Desacetilasas , Neoplasias Pancreáticas/tratamiento farmacológico , Fenilbutiratos/farmacología , Antracenos/farmacología , Apoptosis/efectos de los fármacos , Caspasa 8/metabolismo , Ciclo Celular/efectos de los fármacos , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Desoxicitidina/análogos & derivados , Desoxicitidina/farmacología , Relación Dosis-Respuesta a Droga , Resistencia a Antineoplásicos/efectos de los fármacos , Ensayos de Selección de Medicamentos Antitumorales , Sinergismo Farmacológico , Activación Enzimática/efectos de los fármacos , Humanos , Factores de Tiempo , Células Tumorales Cultivadas , GemcitabinaRESUMEN
We report the isolation of the 5' flanking region of GRAF (GTPase regulator associated with the focal adhesion kinase), previously described as a putative tumour suppressor gene of acute myelogenous leukaemia and myelodysplastic syndrome, and demonstrate its promoter activity in reporter gene assays. Two putative protein-binding sites are identified of which one was sensitive to CpG methylation. The suppressed GRAF expression could be restored in leukaemia cell lines by treatment with a demethylating agent and an inhibitor of histone deacetylases. In contrast to normal tissues, which tested negative for GRAF promoter methylation, 11 of 29 (38%) bone marrow samples from patients with acute myeloid leukaemia or myelodysplastic syndrome were positive.
Asunto(s)
Proteínas Activadoras de GTPasa/genética , Leucemia Mieloide/genética , Síndromes Mielodisplásicos/genética , Regiones Promotoras Genéticas , Enfermedad Aguda , Secuencia de Bases , Niño , Humanos , Células K562 , Metilación , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
The histone deacetylase (HDAC) inhibitor 4-phenylbutyrate (4-PB) is a non-toxic compound that can induce differentiation and promote maturation of various types of malignant cells. In the present study we show that 4-PB inhibit glioma cell proliferation, induce apoptosis and decrease mRNA expression of glyceraldehyde 3-phosphate dehydrogenase (GAPDH) in a concentration-dependent manner. Proliferation of established rat glioma cell lines (RG2 and C6) in culture was significantly decreased after treatment with 4-PB (2-40 mM). Low concentrations of 4-PB (2-20 mM) induced cell differentiation followed by apoptosis, whereas higher concentrations of 4-PB (40 mM) induced cell necrosis. Also, low concentrations of 4-PB significantly decreased GAPDH mRNA expression in C6 and RG2 rat glioma cells, suggesting a link between decreased cell proliferation, energy consumption, and down-regulation of GAPDH gene expression. We have found that GAPDH mRNA expression is markedly increased in human glioblastoma tissues. Therefore, the novel effect of 4-PB described here may offer means to suppress growth of glioma cells by diminishing the key reaction in glycolysis as a therapeutic approach for cancer.
Asunto(s)
Antineoplásicos/farmacología , Neoplasias Encefálicas/enzimología , Glioblastoma/enzimología , Gliceraldehído-3-Fosfato Deshidrogenasas/genética , Inhibidores de Histona Desacetilasas , Fenilbutiratos/farmacología , ARN Mensajero/metabolismo , Animales , Apoptosis/efectos de los fármacos , Neoplasias Encefálicas/patología , División Celular/efectos de los fármacos , Regulación hacia Abajo , Inhibidores Enzimáticos/farmacología , Glioblastoma/patología , Gliceraldehído-3-Fosfato Deshidrogenasas/metabolismo , Humanos , Necrosis , RatasRESUMEN
Human glioblastoma cell cultures were established and the expression of glial fibrillary acidic protein (GFAP) and the gap-junction protein connexin 43 (Cx43) was confirmed by Western blot. Following treatment with 4-phenylbutyrate (4-PB), increased concentrations of non-phosphorylated GFAP were seen, while phosphorylated isoforms remained intact. Immunocytochemical staining of glioblastoma cells revealed an intracellular redistribution of GFAP. In addition to cytoplasmic immunostaining, GFAP immunoreactivity was also associated with the nucleus and/or the nuclear membrane. Phosphorylated and non-phosphorylated Cx43 proteins were increased 2- to 5-fold following 4-PB treatment, and were redistributed to areas of the cell surface, participating in cell-to-cell contacts. In addition, functional gap-junction coupling was amplified, as indicated by increased fluorescent dye transfer, and elevated levels of Cx43 protein were detected in parallel with enhanced gap-junction communication. Induced cell differentiation, with improved functional coupling of tumour cells, may be of importance for therapeutic strategies involving intercellular transport of low molecular-weight compounds.
Asunto(s)
Antineoplásicos/farmacología , Conexina 43/metabolismo , Uniones Comunicantes/metabolismo , Proteína Ácida Fibrilar de la Glía/metabolismo , Glioblastoma/metabolismo , Inhibidores de Histona Desacetilasas , Fenilbutiratos/farmacología , Western Blotting , Comunicación Celular/fisiología , Histona Desacetilasas/efectos de los fármacos , Humanos , Inmunohistoquímica , Células Tumorales CultivadasRESUMEN
Gene expression profiling revealed ADAM9 to be distinctly overexpressed in pancreatic ductal adenocarcinoma (PDAC). We examined the relevance of ADAM9 expression in PDAC diagnosis and prognosis. A total of 59 infiltrating PDACs, 32 specimens from patients with chronic pancreatitis, 11 endocrine tumours and 24 acinar cell carcinomas were immunohistochemically analysed for ADAM9 expression. Staining for ADAM9 was detected in 58 out of 59 (98.3%) PDACs and in two out of 24 (8.3%) acinar cell carcinomas, but not in endocrine tumours. In the non-neoplastic pancreas, whether normal or chronically inflamed, ADAM9 was expressed in centroacinar and intralobular duct cells, but not in interlobular duct cells and their hyperplastic lesions. Pancreatic ductal adenocarcinomas showing cytoplasmic ADAM9 expression correlated with poor tumour differentiation and also with shorter overall survival than in cases showing only an apical membranous staining pattern (P=0.001). Multivariate analysis identified cytoplasmic ADAM9 expression as an independent marker of shortened survival in a set of 42 curatively (R0) resected PDAC (P<0.05, hazard ratio 2.85, 95% confidence interval: 1.21-6.71). The results show that ADAM9 expression distinguishes PDACs from other solid pancreatic tumours. In addition, cytoplasmic ADAM9 overexpression is associated with poor differentiation and shortened survival. Therefore, ADAM9 overexpression might contribute to the aggressiveness of PDACs.
Asunto(s)
Carcinoma de Células Acinares/metabolismo , Carcinoma Ductal Pancreático/metabolismo , Desintegrinas/metabolismo , Islotes Pancreáticos/metabolismo , Proteínas de la Membrana/metabolismo , Metaloendopeptidasas/metabolismo , Conductos Pancreáticos/metabolismo , Neoplasias Pancreáticas/metabolismo , Proteínas ADAM , Adenocarcinoma/metabolismo , Adenocarcinoma/patología , Adulto , Anciano , Biomarcadores de Tumor/metabolismo , Carcinoma de Células Acinares/patología , Carcinoma Ductal Pancreático/patología , Diferenciación Celular , Enfermedad Crónica , Citoplasma , Humanos , Técnicas para Inmunoenzimas , Islotes Pancreáticos/patología , Persona de Mediana Edad , Conductos Pancreáticos/patología , Neoplasias Pancreáticas/patología , Pancreatitis/metabolismo , Pancreatitis/patología , Pronóstico , Tasa de SupervivenciaRESUMEN
We describe the screening of lacZ deletions in mammalian cells and the discovery of a novel pair of lacZ deletions that can undergo alpha-complementation only when they are fused to peptides that interact with each other. The two lacZ deletions, delta N 11-75 and delta C 82-1023, were first characterized by fusing to two small interacting peptides and were then further analyzed by fusing to three membrane receptors (G protein-coupled receptors alpha 2cAR and D2DRL and receptor tyrosine kinase insulin receptor) that were known to form homodimers in the membrane. Histochemical and quantitative FACS assays demonstrated that the novel deletions have much lower level of association with each other, thus offering a much lower background in monitoring membrane protein interactions compared to previously published lacZ deletions. Furthermore, our method has the exciting potential to monitor simultaneously membrane receptor dimerization and localization to the cell surface of living cells.
Asunto(s)
Eliminación de Gen , Prueba de Complementación Genética/métodos , Operón Lac/genética , Receptores de Superficie Celular/metabolismo , beta-Galactosidasa/metabolismo , Animales , Células Cultivadas , ADN Complementario/genética , Dimerización , Humanos , Riñón/embriología , Riñón/metabolismo , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Mutación , Plásmidos , Ratas , Receptores Adrenérgicos alfa 2/genética , Receptores Adrenérgicos alfa 2/metabolismo , Receptores de Superficie Celular/genética , Receptores de Dopamina D2/genética , Receptores de Dopamina D2/metabolismo , beta-Galactosidasa/genéticaRESUMEN
Different mechanisms mediating methylation-dependent repression have been demonstrated. Two of these mechanisms play a role in the context of the granulocyte/macrophage-specific lysozyme gene: direct interference with DNA binding of the transcription factor GA-binding protein and deacetylation of histones. Besides enhancement in the unmethylated state, and transcriptional repression upon DNA methylation, the lysozyme downstream enhancer confers tissue-specific demethylation. Because both demethylation activity and repression ability have been attributed to the methyl-CpG-binding domain-containing protein MBD2, we analyzed this protein. The short form MBD2b binds to the methylated lysozyme enhancer and mediates transcriptional repression. MBD2b is capable of binding to the transcriptional repressor Sin3A. The interaction domain of Sin3A required for binding to MBD2b contains the paired amphipathic helix 3. We identified a minimal functional domain that confers both transcriptional repression as well as the interaction to Sin3A. In contrast to the functionally related proteins MeCP2 and MBD1, the repression domain of MBD2b overlaps with the methyl-CpG-binding domain.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Proteínas Represoras/metabolismo , Línea Celular , Islas de CpG , ADN/metabolismo , Metilación de ADN , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/genética , Relación Dosis-Respuesta a Droga , Genes Reporteros , Glutatión Transferasa/metabolismo , Células HeLa , Humanos , Metilación , Muramidasa/metabolismo , Plásmidos/metabolismo , Unión Proteica , Biosíntesis de Proteínas , Estructura Terciaria de Proteína , Proteínas Recombinantes de Fusión/metabolismo , Proteínas Represoras/química , Complejo Correpresor Histona Desacetilasa y Sin3 , Transcripción GenéticaRESUMEN
In many cases, gene repression mediated by CpG methylation has been demonstrated. Two different mechanisms have been postulated to explain the repressive effect of methylated CpG DNA: establishment of a repressive chromatin configuration and inhibition of DNA binding of transactivating factors. Using the M-lysozyme gene, we analyzed gene expression, CpG demethylation and the in vivo formation of enhancer/protein complexes after inducing demethylation or inhibiting histone deacetylases. We show that trans-cription of a methylated and silent mouse M-lysozyme gene can be induced upon the inhibition of histone deacetylases in the absence of demethylation or in vivo transactivating factor binding to the enhancer. In contrast, DNA demethylation induces both gene activity as well as enhancer complex formation. Therefore, both mechanisms play a role in lysozyme gene repression mediated by methylated DNA: (i) the enhancer cannot be loaded with transacting factors; and (ii) histone deacetylation inhibits transcription.
Asunto(s)
Elementos de Facilitación Genéticos/fisiología , Histona Desacetilasas/metabolismo , Histonas/metabolismo , Muramidasa/biosíntesis , Factores de Transcripción/metabolismo , Acetilación , Animales , Línea Celular , Metilación de ADN , Represión Enzimática/genética , Inhibidores de Histona Desacetilasas , Ratones , Muramidasa/genética , Factores de Transcripción/antagonistas & inhibidoresRESUMEN
The mouse M-lysozyme downstream enhancer has been previously characterized on several levels of gene regulation. The enhancer was co-localized with a DNase I hypersensitive site in the chromatin of mature macrophages, the in vivo interaction of transcription factor GABP with the enhancer core (MLDE) demonstrated binding being restricted to mature macrophage cells, and analysis of the MLDE methylation state revealed a correlation between demethylation of CpG dinucleotides and the in vivo GABP binding. Here, we analyzed in detail the full-length enhancer in addition to the core element. We identified a total of nine binding sites for nuclear factors. Most of these factors are found ubiquitously in all cell types tested. These factors include several unknown proteins as well as the transcription factor NF-Y. In addition, three binding sites for a new single-stranded DNA binding protein were found. The presence of this factor in mature macrophages correlates with the in vivo DNA melting of one of the binding sites and with the enhancer strength.
Asunto(s)
ADN de Cadena Simple/metabolismo , Proteínas de Unión al ADN/metabolismo , Elementos de Facilitación Genéticos , Muramidasa/genética , Factores de Transcripción/metabolismo , Animales , Secuencia de Bases , Cromatina/fisiología , Huella de ADN , Desoxirribonucleasa I , Fosfatos de Dinucleósidos , Factor de Transcripción de la Proteína de Unión a GA , Regulación Enzimológica de la Expresión Génica , Isoenzimas/biosíntesis , Isoenzimas/genética , Macrófagos/enzimología , Ratones , Datos de Secuencia Molecular , Muramidasa/biosíntesis , Oligodesoxirribonucleótidos/química , Proteínas Recombinantes/biosíntesis , Eliminación de Secuencia , TransfecciónRESUMEN
The mouse lysozyme downstream enhancer was previously colocalized with the DNase I-hypersensitive site in the chromatin of mature macrophages. This hypersensitive site was shown to be macrophage differentiation-dependent. Demethylation of CpG sequences within the enhancer is correlated with lysozyme expression in mature macrophages. Binding of the GABP heterotetrameric transcription factor to the enhancer core element (MLDE), only seen in vivo on the demethylated MLDE element in macrophages, is inhibited by DNA methylation. Here, we analyzed the DNA sequences required for demethylation. In electrophoretic mobility shift experiments we found that in addition to the complete methylated MLDE the hemimethylated form of the lower strand inhibits GABP binding as well. Therefore, GABP is unlikely to be the mediator of demethylation. In addition, we show by stable DNA transfections of methylated mouse lysozyme enhancer sequences that MLDE-flanking sequences are required for demethylation. We narrowed down these DNA elements to two short regions of 163 and 79 base pairs on either side of the MLDE, each of which is sufficient to mediate demethylation of the GABP site.