RESUMEN
The mammalian SWI/SNF complex, or BAF complex, has a conserved and direct role in antagonizing Polycomb-mediated repression. Yet, BAF also promotes repression by Polycomb in stem cells and cancer. How BAF both antagonizes and promotes Polycomb-mediated repression remains unknown. Here, we utilize targeted protein degradation to dissect the BAF-Polycomb axis in mouse embryonic stem cells on short timescales. We report that rapid BAF depletion redistributes Polycomb repressive complexes PRC1 and PRC2 from highly occupied domains, like Hox clusters, to weakly occupied sites normally opposed by BAF. Polycomb redistribution from highly repressed domains results in their decompaction, gain of active epigenomic features and transcriptional derepression. Surprisingly, through dose-dependent degradation of PRC1 and PRC2, we identify a conventional role for BAF in Polycomb-mediated repression, in addition to global Polycomb redistribution. These findings provide new mechanistic insight into the highly dynamic state of the Polycomb-Trithorax axis.
Asunto(s)
Ensamble y Desensamble de Cromatina/fisiología , Represión Epigenética/fisiología , Regulación de la Expresión Génica/fisiología , Complejos Multiproteicos/fisiología , Proteínas del Grupo Polycomb/fisiología , Animales , Sistemas CRISPR-Cas , Células Cultivadas , Ensamble y Desensamble de Cromatina/genética , ADN Helicasas/genética , ADN Helicasas/metabolismo , Proteínas de Unión al ADN/fisiología , Células Madre Embrionarias/metabolismo , Epigénesis Genética , Represión Epigenética/genética , Edición Génica , Regulación de la Expresión Génica/genética , Genes Homeobox , Genoma , Células HEK293 , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/metabolismo , Humanos , Mutación con Pérdida de Función , Ratones , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Proteolisis , Proteínas Recombinantes de Fusión/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Factores de Transcripción/fisiologíaRESUMEN
Lysine methylation on histone tails impacts genome regulation and cell fate determination in many developmental processes. Apicomplexa intracellular parasites cause major diseases and they have developed complex life cycles with fine-tuned differentiation events. Yet, apicomplexa genomes have few transcription factors and little is known about their epigenetic control systems. Tick-borne Theileria apicomplexa species have relatively small, compact genomes and a remarkable ability to transform leucocytes in their bovine hosts. Here we report enriched H3 lysine 18 monomethylation (H3K18me1) on the gene bodies of repressed genes in Theileria macroschizonts. Differentiation to merozoites (merogony) leads to decreased H3K18me1 in parasite nuclei. Pharmacological manipulation of H3K18 acetylation or methylation impacted parasite differentiation and expression of stage-specific genes. Finally, we identify a parasite SET-domain methyltransferase (TaSETup1) that can methylate H3K18 and represses gene expression. Thus, H3K18me1 emerges as an important epigenetic mark which controls gene expression and stage differentiation in Theileria parasites.
Asunto(s)
Represión Epigenética/fisiología , Regulación del Desarrollo de la Expresión Génica/fisiología , Histonas/metabolismo , Estadios del Ciclo de Vida/genética , Theileria/crecimiento & desarrollo , Acetilación/efectos de los fármacos , Animales , Bovinos , Línea Celular , Pollos , Secuenciación de Inmunoprecipitación de Cromatina , Represión Epigenética/efectos de los fármacos , Regulación del Desarrollo de la Expresión Génica/efectos de los fármacos , Células HEK293 , Humanos , Proteínas de Insectos/metabolismo , Estadios del Ciclo de Vida/efectos de los fármacos , Lisina/metabolismo , Metilación/efectos de los fármacos , Metiltransferasas/genética , Metiltransferasas/aislamiento & purificación , Metiltransferasas/metabolismo , Mutagénesis Sitio-Dirigida , Péptidos Cíclicos/farmacología , Péptidos Cíclicos/uso terapéutico , RNA-Seq , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Theileria/genética , Theileriosis/tratamiento farmacológico , Theileriosis/parasitología , Tranilcipromina/farmacología , Tranilcipromina/uso terapéuticoRESUMEN
Enhancer of zeste homolog 2 (EZH2) is enzymatic catalytic subunit of polycomb repressive complex 2 (PRC2) that can alter downstream target genes expression by trimethylation of Lys-27 in histone 3 (H3K27me3). EZH2 could also regulate gene expression in ways besides H3K27me3. Functions of EZH2 in cells proliferation, apoptosis, and senescence have been identified. Its important roles in the pathophysiology of cancer are now widely concerned. Therefore, targeting EZH2 for cancer therapy is a hot research topic now and different types of EZH2 inhibitors have been developed. In this review, we summarize the structure and action modes of EZH2, focusing on up-to-date findings regarding the role of EZH2 in cancer initiation, progression, metastasis, metabolism, drug resistance, and immunity regulation. Furtherly, we highlight the advance of targeting EZH2 therapies in experiments and clinical studies.
Asunto(s)
Antineoplásicos/uso terapéutico , Proteína Potenciadora del Homólogo Zeste 2/antagonistas & inhibidores , Represión Epigenética/fisiología , Código de Histonas/fisiología , Proteínas de Neoplasias/antagonistas & inhibidores , Neoplasias/tratamiento farmacológico , Adenosina/análogos & derivados , Adenosina/farmacología , Adenosina/uso terapéutico , Antineoplásicos/farmacología , Benzamidas/farmacología , Benzamidas/uso terapéutico , Compuestos de Bifenilo/farmacología , Compuestos de Bifenilo/uso terapéutico , Ciclo Celular/fisiología , Transformación Celular Neoplásica , Ensayos Clínicos como Asunto , Terapia Combinada , Resistencia a Antineoplásicos/fisiología , Proteína Potenciadora del Homólogo Zeste 2/química , Proteína Potenciadora del Homólogo Zeste 2/fisiología , Histonas/metabolismo , Humanos , Metilación , Morfolinas/farmacología , Morfolinas/uso terapéutico , Estudios Multicéntricos como Asunto , Metástasis de la Neoplasia/fisiopatología , Proteínas de Neoplasias/química , Proteínas de Neoplasias/fisiología , Neoplasias/metabolismo , Complejo Represivo Polycomb 2/antagonistas & inhibidores , Piridonas/farmacología , Piridonas/uso terapéutico , Relación Estructura-Actividad , Activación Transcripcional/fisiología , Microambiente Tumoral/inmunologíaRESUMEN
Diabetic retinopathy (DR) is a microvascular complication of diabetes and one of the most common causes of blindness in active stage. This study is performed to explore the effects of microRNA-21 (miR-21) on retinal vascular endothelial cell (RVEC) viability and angiogenesis in rats with DR via the phosphatidylinositiol 3-kinase/protein kinase B (PI3K/Akt)/vascular endothelial growth factor (VEGF) signaling pathway by binding to phosphatase and tensin homolog (PTEN). Sprague Dawley (SD) rats were used for establishment of DR models. Target relationship between miR-21 and PTEN was assessed by bioinformatics prediction in combination with dual-luciferase reporter gene assay. Identification of expression of miR-21, PTEN and PI3K/Akt/VEGF signaling pathway-related genes in the retinal tissues was then conducted. In order to assess the contributory role of miR-21 in DR, the RVECs were transfected with mimic or inhibitor of miR-21, or siRNA-PTEN, followed by the detection of expression of PTEN and PI3K/Akt/VEGF-related genes, as well as the measurement of cell viability, cell cycle and apoptosis. Increased expression of miR-21 and PI3K/Akt/VEGF related genes, along with a reduced expression of PTEN was observed in the retinal tissues of DR rats. PTEN was targeted and negatively regulated by miR-21, while the PI3K/Akt/VEGF signaling pathway was activated by miR-21. RVECs transfected with miR-21 inhibitor exhibited promoted viability and angiogenesis, and inhibited apoptosis. To conclude, our results indicated that miR-21 overexpression could potentially stimulate RVEC viability and angiogenesis in rats with DR through activation of the PI3K/Akt/VEGF signaling pathway via repressing PTEN expression, highlighting the potential of miR-21 as a target for DR treatment.
Asunto(s)
Retinopatía Diabética/metabolismo , Células Endoteliales/patología , MicroARNs/genética , Neovascularización Patológica/prevención & control , Fosfohidrolasa PTEN/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Factor A de Crecimiento Endotelial Vascular/metabolismo , Animales , Apoptosis , Western Blotting , Proliferación Celular , Diabetes Mellitus Experimental/genética , Diabetes Mellitus Experimental/metabolismo , Retinopatía Diabética/genética , Células Endoteliales/metabolismo , Represión Epigenética/fisiología , Citometría de Flujo , Inmunohistoquímica , Masculino , Neovascularización Patológica/metabolismo , Fosfatidilinositol 3-Quinasas/genética , Proteínas Proto-Oncogénicas c-akt/genética , Ratas , Ratas Sprague-Dawley , Vasos Retinianos/patología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transducción de Señal/fisiología , Transfección , Factor A de Crecimiento Endotelial Vascular/genéticaRESUMEN
Transposable elements dominate the mammalian genome, but their contribution to genetic and epigenetic regulation has been largely overlooked. This was in part due to technical limitations, which made the study of repetitive sequences at single copy resolution difficult. The advancement of next-generation sequencing assays in the last decade has greatly enhanced our understanding of transposable element function. In some instances, specific transposable elements are thought to have been co-opted into regulatory roles during both mouse and human development, while in disease such regulatory potential can contribute to malignancy. DNA methylation is arguably the best characterised regulator of transposable element activity. DNA methylation is associated with transposable element repression, and acts to limit their genotoxic potential. In specific developmental contexts, erasure of DNA methylation is associated with a burst of transposable element expression. Developmental regulation of DNA methylation enables transposon activation, ensuring their survival and propagation throughout the host genome, and also allows the host access to regulatory sequences encoded within the elements. Here I discuss DNA methylation at transposable elements, describing its function and dynamic regulation throughout murine and human development.
Asunto(s)
Metilación de ADN/fisiología , Elementos Transponibles de ADN/fisiología , Animales , Represión Epigenética/fisiología , HumanosRESUMEN
The formation of new memories requires transcription. However, the mechanisms that limit signaling of relevant gene programs in space and time for precision of information coding remain poorly understood. We found that, during learning, the cellular patterns of expression of early response genes (ERGs) are regulated by class IIa HDACs 4 and 5, transcriptional repressors that transiently enter neuronal nuclei from cytoplasm after sensory input. Mice lacking these repressors in the forebrain have abnormally broad experience-dependent expression of ERGs, altered synaptic architecture and function, elevated anxiety, and severely impaired memory. By acutely manipulating the nuclear activity of class IIa HDACs in behaving animals using a chemical-genetic technique, we further demonstrate that rapid induction of transcriptional programs is critical for memory acquisition but these programs may become dispensable when a stable memory is formed. These results provide new insights into the molecular basis of memory storage.
Asunto(s)
Represión Epigenética/fisiología , Histona Desacetilasas/metabolismo , Memoria/fisiología , Transcripción Genética/fisiología , Animales , Conducta Animal/fisiología , Núcleo Celular/metabolismo , Células Cultivadas , Corteza Cerebral/citología , Corteza Cerebral/metabolismo , Femenino , Células HEK293 , Humanos , Masculino , Ratones , Ratones Noqueados , Neuronas , Cultivo Primario de Células , Transducción de Señal/genética , Análisis Espacio-TemporalRESUMEN
Expressional changes of pain-associated genes in primary sensory neurons of DRG are critical for neuropathic pain genesis. DNA methyltransferase (DNMT)-triggered DNA methylation silences gene expression. We show here that DNMT1, a canonical maintenance methyltransferase, acts as the de novo DNMT and is required for neuropathic pain genesis likely through repressing at least DRG Kcna2 gene expression in male mice. Peripheral nerve injury upregulated DNMT1 expression in the injured DRG through the transcription factor cAMP response element binding protein-triggered transcriptional activation of Dnmt1 gene. Blocking this upregulation prevented nerve injury-induced DNA methylation within the promoter and 5'-untranslated region of Kcna2 gene, rescued Kcna2 expression and total Kv current, attenuated hyperexcitability in the injured DRG neurons, and alleviated nerve injury-induced pain hypersensitivities. Given that Kcna2 is a key player in neuropathic pain, our findings suggest that DRG DNMT1 may be a potential target for neuropathic pain management.SIGNIFICANCE STATEMENT In the present study, we reported that DNMT1, a canonical DNA maintenance methyltransferase, is upregulated via the activation of the transcription factor CREB in the injured DRG after peripheral nerve injury. This upregulation was responsible for nerve injury-induced de novo DNA methylation within the promoter and 5'-untranslated region of the Kcna2 gene, reductions in Kcna2 expression and Kv current and increases in neuronal excitability in the injured DRG. Since pharmacological inhibition or genetic knockdown of DRG DNMT1 alleviated nerve injury-induced pain hypersensitivities, DRG DNMT1 contributes to neuropathic pain genesis partially through repression of DRG Kcna2 gene expression.
Asunto(s)
ADN (Citosina-5-)-Metiltransferasa 1/metabolismo , Represión Epigenética/fisiología , Canal de Potasio Kv.1.2/metabolismo , Neuralgia/metabolismo , Neuronas Aferentes/metabolismo , Animales , Ganglios Espinales/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Traumatismos de los Nervios Periféricos/metabolismoRESUMEN
Folate deficiency has been known to contribute to neural tube and neural crest defects, but why these tissues are particularly affected, and which are the molecular mechanisms involved in those abnormalities are important human health questions that remain unanswered. Here we study the function of two of the main folate transporters, FolR1 and Rfc1, which are robustly expressed in these tissues. Folate is the precursor of S-adenosylmethionine, which is the main donor for DNA, protein and RNA methylation. Our results show that knockdown of FolR1 and/or Rfc1 reduced the abundance of histone H3 lysine and DNA methylation, two epigenetic modifications that play an important role during neural and neural crest development. Additionally, by knocking down folate transporter or pharmacologically inhibiting folate transport and metabolism, we observed ectopic Sox2 expression at the expense of neural crest markers in the dorsal neural tube. This is correlated with neural crest associated defects, with particular impact on orofacial formation. By using bisulfite sequencing, we show that this phenotype is consequence of reduced DNA methylation on the Sox2 locus at the dorsal neural tube, which can be rescued by the addition of folinic acid. Taken together, our in vivo results reveal the importance of folate as a source of the methyl groups necessary for the establishment of the correct epigenetic marks during neural and neural crest fate-restriction.
Asunto(s)
Deficiencia de Ácido Fólico/fisiopatología , Cresta Neural/metabolismo , Factores de Transcripción SOXB1/fisiología , Animales , Embrión de Pollo , Metilación de ADN/efectos de los fármacos , Epigénesis Genética/genética , Represión Epigenética/genética , Represión Epigenética/fisiología , Epigenómica , Receptor 1 de Folato , Ácido Fólico/metabolismo , Deficiencia de Ácido Fólico/metabolismo , Regulación del Desarrollo de la Expresión Génica/efectos de los fármacos , Histonas/metabolismo , Humanos , Tubo Neural/metabolismo , Defectos del Tubo Neural/genética , Defectos del Tubo Neural/fisiopatologíaRESUMEN
Subsets of endogenous retroviruses (ERVs) are derepressed in mouse embryonic stem cells (mESCs) deficient for Setdb1, which catalyzes histone H3 lysine 9 trimethylation (H3K9me3). Most of those ERVs, including IAPs, remain silent if Setdb1 is deleted in differentiated embryonic cells; however they are derepressed when deficient for Dnmt1, suggesting that Setdb1 is dispensable for ERV silencing in somatic cells. However, H3K9me3 enrichment on ERVs is maintained in differentiated cells and is mostly diminished in mouse embryonic fibroblasts (MEFs) lacking Setdb1. Here we find that distinctive sets of ERVs are reactivated in different types of Setdb1-deficient somatic cells, including the VL30-class of ERVs in MEFs, whose derepression is dependent on cell-type-specific transcription factors (TFs). These data suggest a more general role for Setdb1 in ERV silencing, which provides an additional layer of epigenetic silencing through the H3K9me3 modification.
Asunto(s)
Metilación de ADN/fisiología , Retrovirus Endógenos/fisiología , Represión Epigenética/fisiología , N-Metiltransferasa de Histona-Lisina/metabolismo , Activación Viral/genética , Animales , Diferenciación Celular/fisiología , ADN (Citosina-5-)-Metiltransferasa 1/genética , ADN (Citosina-5-)-Metiltransferasa 1/metabolismo , Fibroblastos , Genes de Partícula A Intracisternal/genética , N-Metiltransferasa de Histona-Lisina/genética , Histonas/genética , Interacciones Huésped-Patógeno/genética , Ratones , Ratones Noqueados , Células Madre Embrionarias de RatonesRESUMEN
The achievement of animal cloning and subsequent development of cell reprogramming technology are having a profound impact on our view of the mechanisms of aging in complex organisms. The experimental evidence showing that an adult somatic nucleus implanted into an enucleated oocyte can give rise to a whole new individual strongly suggests that the integrity of the genome of an adult nucleus is fully preserved. Here, we will review recent experimental evidence showing that pluripotency gene-based cell reprogramming can erase the epigenetic marks of aging and rejuvenate cells from old individuals reversing most signs of aging and that when induced pluripotent stem cells are differentiated back to the cell type of origin, the rejuvenated cells share many of the features of wild-type counterparts from young donors. This evidence supports the idea that progressive epigenetic dysregulation may be the key driver of organismal aging and challenges the conventional view of aging as an irreversible process. The model of aging as an epigenetic process provides an elegant explanation of a number of age-related processes difficult to explain by conventional theories of aging.
Asunto(s)
Envejecimiento/fisiología , Clonación de Organismos , Represión Epigenética/fisiología , Animales , Reprogramación Celular , Rejuvenecimiento/fisiologíaRESUMEN
BACKGROUND: Repression of the KAI1 metastasis suppressor gene is closely associated with malignancy and poor prognosis in many human cancer types including prostate cancer. Since gene repression in human cancers frequently results from epigenetic alterations by DNA methylation and histone modifications, we examined whether the KAI1 gene becomes silenced through these epigenetic mechanisms in prostate cancer. METHODS: KAI1 mRNA and protein levels were determined by RT-PCR and immunoblotting analyses, respectively. Methylation status of the KAI1 promoter DNA in prostate cancer cell lines and tissues was evaluated by methylation-specific PCR analysis of bisulfite-modified genomic DNAs. Methylated CpG sites in the KAI1 promoter were identified by sequencing the PCR clones of the bisulfite-modified KAI1 promoter DNA. KAI1 protein levels in human prostate cancer tissue samples were examined by immunofluorescence staining of the tissues with an anti-KAI1 antibody. RESULTS: Among the three human prostate cancer cell lines examined, PC3 and DU145 cells exhibited markedly decreased levels of KAI1 mRNA and protein as compared to LNCaP cells, even though the exogenous KAI1 promoter not being methylated was normally functional in all these cell lines. Treatment of the low KAI1-expressing cell lines with a demethylating agent, 5'-aza-2'-deoxycytidine, significantly elevated KAI1 expression levels, implicating the involvement of DNA methylation in KAI1 downregulation. Methylation of CpG islands within the KAI1 promoter region was observed in the low KAI1-expressing cells, but not in the high KAI1-expressing cells. Also, methyl CpG-binding proteins such as MBD2 and MeCP2 were complexed to the KAI1 promoter in the low KAI1-expressing cells. Bisulfite sequencing analysis identified the intensively methylated CpG residues in the KAI1 promoter clones derived from prostate cancer cells and tissues with no or low KAI1 expression. As in prostate cancer cell lines, prostate cancer tissues from patients also displayed a negative association between KAI1 expression levels and methylation status of the KAI1 promoter. CONCLUSIONS: The present data suggest that the KAI1 gene might be repressed by epigenetic alterations through the promoter CpG-site methylation during prostate cancer progression. This epigenetic mechanism could provide a clue for understanding how the KAI1 gene was silenced in metastatic prostate cancers. Prostate 77: 350-360, 2017. © 2016 Wiley Periodicals, Inc.
Asunto(s)
Islas de CpG/fisiología , Represión Epigenética/fisiología , Genes Supresores de Tumor/fisiología , Proteína Kangai-1/metabolismo , Regiones Promotoras Genéticas/fisiología , Neoplasias de la Próstata/metabolismo , Secuencia de Bases , Línea Celular Tumoral , Humanos , Proteína Kangai-1/genética , Masculino , Neoplasias de la Próstata/genéticaRESUMEN
The structural maintenance of chromosomes (SMC) proteins are fundamental to chromosome organization. They share a characteristic domain structure, featuring a central SMC hinge domain that is critical for forming SMC dimers and interacting with nucleic acids. The structural maintenance of chromosomes flexible hinge domain containing 1 (Smchd1) is a non-canonical member of the SMC family. Although it has been well established that Smchd1 serves crucial roles in epigenetic silencing events implicated in development and disease, much less is known about the structure and function of the Smchd1 protein. Recently, we demonstrated that the C-terminal hinge domain of Smchd1 forms a nucleic acid-binding homodimer; however, it is unclear how the protomers are assembled within the hinge homodimer and how the full-length Smchd1 protein is organized with respect to the hinge region. In the present study, by employing SAXS we demonstrate that the hinge domain of Smchd1 probably adopts an unconventional homodimeric arrangement augmented by an intermolecular coiled coil formed between the two monomers. Such a dimeric structure differs markedly from that of archetypical SMC proteins, raising the possibility that Smchd1 binds chromatin in an unconventional manner.
Asunto(s)
Proteínas Cromosómicas no Histona/metabolismo , Represión Epigenética/fisiología , Regulación de la Expresión Génica/fisiología , Animales , Proteínas Cromosómicas no Histona/genética , Células HEK293 , Humanos , Inmunoprecipitación , Ratones , Modelos Moleculares , Conformación Proteica , Estructura Terciaria de Proteína , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMEN
Maintenance of vascular integrity is required for embryogenesis and organ homeostasis. However, the gene expression programs that stabilize blood vessels are poorly understood. Here, we show that the histone methyltransferase Ezh2 maintains integrity of the developing vasculature by repressing a transcriptional program that activates expression of Mmp9. Inactivation of Ezh2 in developing mouse endothelium caused embryonic lethality with compromised vascular integrity and increased extracellular matrix degradation. Genome-wide approaches showed that Ezh2 targets Mmp9 and its activators Fosl1 and Klf5. In addition, we uncovered Creb3l1 as an Ezh2 target that directly activates Mmp9 gene expression in the endothelium. Furthermore, genetic inactivation of Mmp9 rescued vascular integrity defects in Ezh2-deficient embryos. Thus, epigenetic repression of Creb3l1, Fosl1, Klf5 and Mmp9 by Ezh2 in endothelial cells maintains the integrity of the developing vasculature, potentially linking this transcriptional network to diseases with compromised vascular integrity.
Asunto(s)
Vasos Sanguíneos/embriología , Represión Epigenética/fisiología , Regulación del Desarrollo de la Expresión Génica/fisiología , Complejo Represivo Polycomb 2/metabolismo , Transducción de Señal/fisiología , Animales , Benzotiazoles , Western Blotting , Inmunoprecipitación de Cromatina , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/metabolismo , Cartilla de ADN/genética , Diaminas , Proteína Potenciadora del Homólogo Zeste 2 , Represión Epigenética/genética , Matriz Extracelular/genética , Matriz Extracelular/metabolismo , Hibridación in Situ , Factores de Transcripción de Tipo Kruppel , Luciferasas , Metaloproteinasa 9 de la Matriz/genética , Metaloproteinasa 9 de la Matriz/metabolismo , Ratones , Microscopía Electrónica de Transmisión , Proteínas del Tejido Nervioso/metabolismo , Compuestos Orgánicos , Complejo Represivo Polycomb 2/genética , Proteínas Proto-Oncogénicas c-fos/metabolismo , Quinolinas , Reacción en Cadena en Tiempo Real de la Polimerasa , Análisis de Secuencia de ARNRESUMEN
OBJECTIVE: To investigate whether the changes in collagen gene expression in osteoarthritic (OA) human chondrocytes are associated with changes in the DNA methylation status in the COL2A1 enhancer and COL9A1 promoter. METHODS: Expression levels were determined using quantitative reverse transcription-polymerase chain reaction, and the percentage of DNA methylation was quantified by pyrosequencing. The effect of CpG methylation on COL9A1 promoter activity was determined using a CpG-free vector; cotransfections with expression vectors encoding SOX9, hypoxia-inducible factor 1α (HIF-1α), and HIF-2α were carried out to analyze COL9A1 promoter activities in response to changes in the methylation status. Chromatin immunoprecipitation assays were carried out to validate SOX9 binding to the COL9A1 promoter and the influence of DNA methylation. RESULTS: Although COL2A1 messenger RNA (mRNA) levels in OA chondrocytes were 19-fold higher than those in the controls, all of the CpG sites in the COL2A1 enhancer were totally demethylated in both samples. The levels of COL9A1 mRNA in OA chondrocytes were 6,000-fold lower than those in controls; 6 CpG sites of the COL9A1 promoter were significantly hypermethylated in OA patients as compared with controls. Treatment with 5-azadeoxycitidine enhanced COL9A1 gene expression and prevented culture-induced hypermethylation. In vitro methylation decreased COL9A1 promoter activity. Mutations in the 5 CpG sites proximal to the transcription start site decreased COL9A1 promoter activity. Cotransfection with SOX9 enhanced COL9A1 promoter activity; CpG methylation attenuated SOX9 binding to the COL9A1 promoter. CONCLUSION: This first demonstration that hypermethylation is associated with down-regulation of COL9A1 expression in OA cartilage highlights the pivotal role of epigenetics in OA, involving not only hypomethylation, but also hypermethylation, with important therapeutic implications for OA treatment.
Asunto(s)
Condrocitos/metabolismo , Colágeno Tipo IX/metabolismo , Metilación de ADN/fisiología , Regulación hacia Abajo/fisiología , Represión Epigenética/fisiología , Osteoartritis/metabolismo , Osteoartritis/fisiopatología , Anciano , Anciano de 80 o más Años , Secuencia de Bases , Estudios de Casos y Controles , Células Cultivadas , Condrocitos/patología , Colágeno Tipo II/sangre , Colágeno Tipo II/metabolismo , Colágeno Tipo IX/genética , Islas de CpG/fisiología , Femenino , Regulación de la Expresión Génica/fisiología , Humanos , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Técnicas In Vitro , Masculino , Datos de Secuencia Molecular , Osteoartritis/patología , Regiones Promotoras Genéticas/fisiología , Factor de Transcripción SOX9/metabolismoRESUMEN
DNA methylation is a fundamental epigenetic modification in vertebrate genomes and a small fraction of genomic regions is hypomethylated. Previous studies have implicated hypomethylated regions in gene regulation, but their functions in vertebrate development remain elusive. To address this issue, we generated epigenomic profiles that include base-resolution DNA methylomes and histone modification maps from both pluripotent cells and mature organs of medaka fish and compared the profiles with those of human ES cells. We found that a subset of hypomethylated domains harbor H3K27me3 (K27HMDs) and their size positively correlates with the accumulation of H3K27me3. Large K27HMDs are conserved between medaka and human pluripotent cells and predominantly contain promoters of developmental transcription factor genes. These key genes were found to be under strong transcriptional repression, when compared with other developmental genes with smaller K27HMDs. Furthermore, human-specific K27HMDs show an enrichment of neuronal activity-related genes, which suggests a distinct regulation of these genes in medaka and human. In mature organs, some of the large HMDs become shortened by elevated DNA methylation and associate with sustained gene expression. This study highlights the significance of domain size in epigenetic gene regulation. We propose that large K27HMDs play a crucial role in pluripotent cells by strictly repressing key developmental genes, whereas their shortening consolidates long-term gene expression in adult differentiated cells.
Asunto(s)
Metilación de ADN/fisiología , Células Madre Embrionarias/fisiología , Represión Epigenética/fisiología , Regulación del Desarrollo de la Expresión Génica/fisiología , Histonas/metabolismo , Oryzias/embriología , Animales , Secuencia de Bases , Inmunoprecipitación de Cromatina , Cartilla de ADN/genética , Humanos , Hibridación in Situ , Datos de Secuencia Molecular , Estructura Terciaria de Proteína/fisiología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Análisis de Secuencia de ADNRESUMEN
Molecular targeting of the two receptor interaction domains of the epigenetic repressor silencing mediator of retinoid and thyroid hormone receptors (SMRT(mRID)) produced a transplantable skeletal syndrome that reduced radial bone growth, increased numbers of bone-resorbing periosteal osteoclasts, and increased bone fracture risk. Furthermore, SMRT(mRID) mice develop spontaneous primary myelofibrosis, a chronic, usually idiopathic disorder characterized by progressive bone marrow fibrosis. Frequently linked to polycythemia vera and chronic myeloid leukemia, myelofibrosis displays high patient morbidity and mortality, and current treatment is mostly palliative. To decipher the etiology of this disease, we identified the thrombopoietin (Tpo) gene as a target of the SMRT-retinoic acid receptor signaling pathway in bone marrow stromal cells. Chronic induction of Tpo in SMRT(mRID) mice results in up-regulation of TGF-ß and PDGF in megakaryocytes, uncontrolled proliferation of bone marrow reticular cells, and fibrosis of the marrow compartment. Of therapeutic relevance, we show that this syndrome can be rescued by retinoid antagonists, demonstrating that the physical interface between SMRT and retinoic acid receptor can be a potential therapeutic target to block primary myelofibrosis disease progression.
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Médula Ósea/metabolismo , Citocinas/metabolismo , Represión Epigenética/fisiología , Co-Represor 2 de Receptor Nuclear/antagonistas & inhibidores , Mielofibrosis Primaria/tratamiento farmacológico , Transducción de Señal/fisiología , Trombopoyetina/genética , Fosfatasa Alcalina/sangre , Animales , Benzotiazoles , Calcio/sangre , Proliferación Celular/efectos de los fármacos , Cartilla de ADN/genética , Diaminas , Ensayo de Inmunoadsorción Enzimática , Perfilación de la Expresión Génica , Técnicas de Sustitución del Gen , Luciferasas , Megacariocitos/metabolismo , Ratones , Co-Represor 2 de Receptor Nuclear/genética , Compuestos Orgánicos , Factor de Crecimiento Derivado de Plaquetas/metabolismo , Reacción en Cadena de la Polimerasa , Mielofibrosis Primaria/etiología , Quinolinas , Trombopoyetina/biosíntesis , Factor de Crecimiento Transformador beta/metabolismoRESUMEN
As more and more genomes are being explored and annotated, important features of three-dimensional (3D) genome organization are just being uncovered. In the light of what we know about Polycomb group (PcG) proteins, we will present the latest findings on this topic. The PcG proteins are well-conserved chromatin factors that repress transcription of numerous target genes. They bind the genome at specific sites, forming chromatin domains of associated histone modifications as well as higher-order chromatin structures. These 3D chromatin structures involve the interactions between PcG-bound regulatory regions at short- and long-range distances, and may significantly contribute to PcG function. Recent high throughput "Chromosome Conformation Capture" (3C) analyses have revealed many other higher order structures along the chromatin fiber, partitioning the genomes into well demarcated topological domains. This revealed an unprecedented link between linear epigenetic domains and chromosome architecture, which might be intimately connected to genome function.
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Cromatina/química , Genoma/fisiología , Proteínas del Grupo Polycomb/fisiología , Animales , Cromatina/metabolismo , Cromatina/fisiología , Represión Epigenética/genética , Represión Epigenética/fisiología , Humanos , Imagenología Tridimensional , Conformación de Ácido Nucleico , Proteínas del Grupo Polycomb/metabolismoRESUMEN
Large parts of mammalian genomes are transcriptionally inactive and enriched with various classes of interspersed and tandem repeats. Here we show that the tumor suppressor protein p53 cooperates with DNA methylation to maintain silencing of a large portion of the mouse genome. Massive transcription of major classes of short, interspersed nuclear elements (SINEs) B1 and B2, both strands of near-centromeric satellite DNAs consisting of tandem repeats, and multiple species of noncoding RNAs was observed in p53-deficient but not in p53 wild-type mouse fibroblasts treated with the DNA demethylating agent 5-aza-2'-deoxycytidine. The abundance of these transcripts exceeded the level of ß-actin mRNA by more than 150-fold. Accumulation of these transcripts, which are capable of forming double-stranded RNA (dsRNA), was accompanied by a strong, endogenous, apoptosis-inducing type I IFN response. This phenomenon, which we named "TRAIN" (for "transcription of repeats activates interferon"), was observed in spontaneous tumors in two models of cancer-prone mice, presumably reflecting naturally occurring DNA hypomethylation and p53 inactivation in cancer. These observations suggest that p53 and IFN cooperate to prevent accumulation of cells with activated repeats and provide a plausible explanation for the deregulation of IFN function frequently seen in tumors. Overall, this work reveals roles for p53 and IFN that are key for genetic stability and therefore relevant to both tumorigenesis and the evolution of species.
Asunto(s)
Metilación de ADN , Represión Epigenética/fisiología , Interferón Tipo I/metabolismo , ARN no Traducido/genética , Secuencias Repetitivas de Ácidos Nucleicos/genética , Proteína p53 Supresora de Tumor/genética , Actinas/genética , Animales , Azacitidina/análogos & derivados , Biología Computacional , Decitabina , Represión Epigenética/genética , Ratones , Análisis por Micromatrices , Análisis de Secuencia de ARN , Proteína p53 Supresora de Tumor/deficienciaRESUMEN
PURPOSE: Aberrant DNA methylation is common in cancer cells. Epigenetic alterations resulting in the loss of tumor suppression gene functions are frequently involved in tumor development and progression. Recently, methylation of PCDH10 was reported to be associated with multiple hematologic malignancies as well as some solid tumors. Whether the down-regulation of PCDH10 happens in CRC remains unknown. METHODS: Methylation status of PCDH10 was evaluated by methylation-specific PCR analysis. The effects of PCDH10 re-expression were determined in growth, colony formation, cell cycle, and invasion assays. RESULTS: In this study, we found that 100 % (8 of 8) of colorectal cancer cell lines were silenced for PCDH10, but not normal colorectal epithelial cells. Demethylation treatment confirmed that the reduced expression is associated closely with promoter methylation. Hyper-methylation of PCDH10 was also detected in 85 % of primary colorectal tumors, but not in adjacent normal colorectal tissues. CONCLUSIONS: Our results suggest that PCDH10 is an important tumor suppression gene with key roles of suppressing cell proliferation, clonogenicity, and inhibiting cell invasion in the development of colorectal cancer. Thus, PCDH10 methylation may constitute a useful biomarker of colorectal cancer patients.
Asunto(s)
Cadherinas/genética , Carcinoma/genética , Neoplasias Colorrectales/genética , Metilación de ADN/fisiología , Represión Epigenética/fisiología , Células CACO-2 , Cadherinas/metabolismo , Carcinoma/patología , Proliferación Celular , Neoplasias Colorrectales/patología , Represión Epigenética/genética , Regulación Neoplásica de la Expresión Génica , Frecuencia de los Genes , Silenciador del Gen/fisiología , Genes Supresores de Tumor/fisiología , Células HCT116 , Células HT29 , Humanos , Invasividad Neoplásica , Protocadherinas , Células Tumorales CultivadasRESUMEN
During neurogenesis, neural stem/progenitor cells (NPCs) undergo an irreversible fate transition to become neurons. The Notch pathway is important for this process, and repression of Notch-dependent Hes genes is essential for triggering differentiation. However, Notch signaling often remains active throughout neuronal differentiation, implying a change in the transcriptional responsiveness to Notch during the neurogenic transition. We identified Bcl6, an oncogene, as encoding a proneurogenic factor that is required for proper neurogenesis of the mouse cerebral cortex. BCL6 promoted the neurogenic conversion by switching the composition of Notch-dependent transcriptional complexes at the Hes5 promoter. BCL6 triggered exclusion of the co-activator Mastermind-like 1 and recruitment of the NAD(+)-dependent deacetylase Sirt1, which was required for BCL6-dependent neurogenesis. The resulting epigenetic silencing of Hes5 led to neuronal differentiation despite active Notch signaling. Our findings suggest a role for BCL6 in neurogenesis and uncover Notch-BCL6-Sirt1 interactions that may affect other aspects of physiology and disease.