RESUMEN
Protein arginine methyltransferase 5 (PRMT5) activity is dysregulated in many aggressive cancers and its enhanced levels are associated with increased tumour growth and survival. However, the role of PRMT5 in breast cancer remains underexplored. In this study, we show that PRMT5 is overexpressed in breast cancer cell lines, and that it promotes WNT/ß-CATENIN proliferative signalling through epigenetic silencing of pathway antagonists, DKK1 and DKK3, leading to enhanced expression of c-MYC, CYCLIN D1 and SURVIVIN. Through chromatin immunoprecipitation (ChIP) studies, we found that PRMT5 binds to the promoter region of WNT antagonists, DKK1 and DKK3, and induces symmetric methylation of H3R8 and H4R3 histones. Our findings also show that PRMT5 inhibition using a specific small molecule inhibitor, compound 5 (CMP5), reduces PRMT5 recruitment as well as methylation of H3R8 and H4R3 histones in the promoter regions of DKK1 and DKK3, which consequently results in reduced expression CYCLIN D1 and SURVIVIN. Furthermore, CMP5 treatment either alone or in combination with 5-Azacytidine and Trichostatin A restored expression of DKK1 and DKK3 in TNBCs. PRMT5 inhibition also altered the growth characteristics of breast cancer cells and induced their death. Collectively, these results show that PRMT5 controls breast cancer cell growth through epigenetic silencing of WNT/ß-CATENIN pathway antagonists, DKK1 and DKK3, resulting in up-regulation of WNT/ß-CATENIN proliferative signalling.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/genética , Epigénesis Genética , Silenciador del Gen , Péptidos y Proteínas de Señalización Intercelular/genética , Proteína-Arginina N-Metiltransferasas/metabolismo , Vía de Señalización Wnt , Apoptosis/efectos de los fármacos , Neoplasias de la Mama/genética , Línea Celular Tumoral , Metilación de ADN , Decitabina/farmacología , Femenino , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Histonas/metabolismo , Humanos , Unión Proteica , Proteína-Arginina N-Metiltransferasas/antagonistas & inhibidoresRESUMEN
Protein arginine methyltransferase-5 (PRMT5) is overexpressed in aggressive B-cell non-Hodgkin's lymphomas, including mantle cell lymphoma and diffuse large B-cell lymphoma, and supports constitutive expression of CYCLIN D1 and c-MYC. Here, we combined ChIP analysis with next-generation sequencing to identify microRNA (miRNA) genes that are targeted by PRMT5 in aggressive lymphoma cell lines. We identified enrichment of histone 3 dimethylation at Arg-8 (H3(Me2)R8) in the promoter regions of miR33b, miR96, and miR503. PRMT5 knockdown de-repressed transcription of all three miRNAs, accompanied by loss of recruitment of epigenetic repressor complexes containing PRMT5 and either histone deacetylase 2 (HDAC2) or HDAC3, enhanced binding of co-activator complexes containing p300 or CREB-binding protein (CBP), and increased acetylation of specific histones, including H2BK12, H3K9, H3K14, and H4K8 at the miRNA promoters. Re-expression of individual miRNAs in B-cell lymphoma cells down-regulated expression of PRMT5, CYCLIN D1, and c-MYC, which are all predicted targets of these miRNAs, and reduced lymphoma cell survival. Luciferase reporter assays with WT and mutant 3'UTRs of CYCLIN D1 and c-MYC mRNAs revealed that binding sites for miR33b, miR96, and miR503 are critical for translational regulation of the transcripts of these two genes. Our findings link altered PRMT5 expression to transcriptional silencing of tumor-suppressing miRNAs in lymphoma cells and reinforce PRMT5's relevance for promoting lymphoma cell growth and survival.
Asunto(s)
Ciclina D1/genética , Linfoma de Células B/enzimología , MicroARNs/metabolismo , Proteína-Arginina N-Metiltransferasas/metabolismo , Proteínas Proto-Oncogénicas c-myc/genética , Regiones no Traducidas 3' , Acetilación , Proteína de Unión a CREB/metabolismo , Secuenciación de Inmunoprecipitación de Cromatina , Ciclina D1/metabolismo , Regulación hacia Abajo , Proteína p300 Asociada a E1A/metabolismo , Silenciador del Gen , Genes Supresores de Tumor , Histona Desacetilasas/genética , Histona Desacetilasas/metabolismo , Histonas/metabolismo , Humanos , Linfoma de Células B/genética , Linfoma de Células B/metabolismo , Metilación , MicroARNs/genética , Regiones Promotoras Genéticas , Proteína-Arginina N-Metiltransferasas/genética , Proteínas Proto-Oncogénicas c-myc/metabolismoRESUMEN
Breast cancer (BC) is the most common malignancy and the leading cause of death in women worldwide. Only 5%-10% of mutations in BRCA genes are associated with familial breast tumours in Eastern countries, suggesting the contribution of other genes. Using a microarray gene expression profiling study of BC, we have recently identified BRIP1 (fivefold up-regulation) as a potential gene associated with BC progression in the Omani population. Although BRIP1 regulates DNA repair and cell proliferation, the precise role of BRIP1 in BC cell invasion/metastasis has not been explored yet; this prompted us to test the hypothesis that BRIP1 promotes BC cell proliferation and invasion. Using a combination of cellular and molecular approaches, our results revealed differential overexpression of BRIP1 in different BC cell lines. Functional assays validated further the physiological relevance of BRIP1 in tumour malignancy, and siRNA-mediated BRIP1 knockdown significantly reduced BC cell motility by targeting key motility-associated genes. Moreover, down-regulation of BRIP1 expression significantly attenuated cell proliferation via cell cycle arrest. Our study is the first to show the novel function of BRIP1 in promoting BC cell invasion by regulating expression of various downstream target genes. Furthermore, these findings provide us with a unique opportunity to identify BRIP1-induced pro-invasive genes that could serve as biomarkers and/or targets to guide the design of appropriate BC targeted therapies.
Asunto(s)
Neoplasias de la Mama/genética , Neoplasias de la Mama/patología , Proteínas del Grupo de Complementación de la Anemia de Fanconi/metabolismo , ARN Helicasas/metabolismo , Puntos de Control del Ciclo Celular/genética , Línea Celular Tumoral , Movimiento Celular/genética , Proliferación Celular/genética , Regulación hacia Abajo/genética , Proteínas del Grupo de Complementación de la Anemia de Fanconi/genética , Femenino , Regulación Neoplásica de la Expresión Génica , Humanos , Invasividad Neoplásica , ARN Helicasas/genética , ARN Mensajero/genética , ARN Mensajero/metabolismo , ARN Interferente Pequeño/metabolismo , Cicatrización de HeridasRESUMEN
Epigenetic regulation by the type II protein arginine methyltransferase, PRMT5, plays an essential role in the control of cancer cell proliferation and tumorigenesis. In this report, we investigate the relationship between PRMT5 and WNT/ß-CATENIN as well as AKT/GSK3ß proliferative signaling in three different types of non-Hodgkin's lymphoma cell lines, clinical samples, and mouse primary lymphoma cells. We show that PRMT5 stimulates WNT/ß-CATENIN signaling through direct epigenetic silencing of pathway antagonists, AXIN2 and WIF1, and indirect activation of AKT/GSK3ß signaling. PRMT5 inhibition with either shRNA-mediated knockdown or a specific small molecule PRMT5 inhibitor, CMP-5, not only leads to derepression of WNT antagonists and decreased levels of active phospho-AKT (Thr-450 and Ser-473) and inactive phospho-GSK3ß (Ser-9) but also results in decreased transcription of WNT/ß-CATENIN target genes, CYCLIN D1, c-MYC, and SURVIVIN, and enhanced lymphoma cell death. Furthermore, PRMT5 inhibition leads to reduced recruitment of co-activators CBP, p300, and MLL1, as well as enhanced recruitment of co-repressors HDAC2 and LSD1 to the WNT/ß-CATENIN target gene promoters. These results indicate that PRMT5 governs expression of prosurvival genes by promoting WNT/ß-CATENIN and AKT/GSK3ß proliferative signaling and that its inhibition induces lymphoma cell death, which warrants further clinical evaluation.
Asunto(s)
Regulación Neoplásica de la Expresión Génica , Glucógeno Sintasa Quinasa 3 beta/metabolismo , Linfoma/metabolismo , Proteína-Arginina N-Metiltransferasas/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Transducción de Señal , Vía de Señalización Wnt , Animales , Línea Celular Tumoral , Supervivencia Celular , Glucógeno Sintasa Quinasa 3 beta/genética , Linfoma/genética , Linfoma/patología , Ratones , Proteína-Arginina N-Metiltransferasas/genética , Proteínas Proto-Oncogénicas c-akt/genética , beta Catenina/genética , beta Catenina/metabolismoRESUMEN
PPARγ2 is a critical lineage-determining transcription factor that is essential for adipogenic differentiation. Here we report characterization of the three-dimensional structure of the PPARγ2 locus after the onset of adipogenic differentiation and the mechanisms by which it forms. We identified a differentiation-dependent loop between the PPARγ2 promoter and an enhancer sequence 10 kb upstream that forms at the onset of PPARγ2 expression. The arginine methyltransferase Prmt5 was required for loop formation, and overexpression of Prmt5 resulted in premature loop formation and earlier onset of PPARγ2 expression. Kinetic studies of regulatory factor interactions at the PPARγ2 promoter and enhancer revealed enhanced interaction of Prmt5 with the promoter that preceded stable association of Prmt5 with enhancer sequences. Prmt5 knockdown prevented binding of both MED1, a subunit of Mediator complex that facilitates enhancer-promoter interactions, and Brg1, the ATPase of the mammalian SWI/SNF chromatin remodeling enzyme required for PPARγ2 activation and adipogenic differentiation. The data indicate a dynamic association of Prmt5 with the regulatory sequences of the PPARγ2 gene that facilitates differentiation-dependent, three-dimensional organization of the locus. In addition, other differentiation-specific, long-range chromatin interactions showed Prmt5-dependence, indicating a more general role for Prmt5 in mediating higher-order chromatin connections in differentiating adipocytes.
Asunto(s)
Adipogénesis/genética , Diferenciación Celular , Elementos de Facilitación Genéticos , Sitios Genéticos , PPAR gamma/genética , Regiones Promotoras Genéticas , Proteína-Arginina N-Metiltransferasas/metabolismo , Células 3T3-L1 , Animales , Proteínas Potenciadoras de Unión a CCAAT/metabolismo , Línea Celular , Ensamble y Desensamble de Cromatina , ADN Helicasas/metabolismo , Regulación del Desarrollo de la Expresión Génica , Técnicas de Silenciamiento del Gen , Subunidad 1 del Complejo Mediador/metabolismo , Ratones , Proteínas Nucleares/metabolismo , Unión Proteica , Transporte de Proteínas , Factores de Transcripción/metabolismo , Activación TranscripcionalRESUMEN
Epigenetic events that are essential drivers of lymphocyte transformation remain incompletely characterized. We used models of Epstein-Barr virus (EBV)-induced B-cell transformation to document the relevance of protein arginine methyltransferase 5 (PRMT5) to regulation of epigenetic-repressive marks during lymphomagenesis. EBV(+) lymphomas and transformed cell lines exhibited abundant expression of PRMT5, a type II PRMT enzyme that promotes transcriptional silencing of target genes by methylating arginine residues on histone tails. PRMT5 expression was limited to EBV-transformed cells, not resting or activated B lymphocytes, validating it as an ideal therapeutic target. We developed a first-in-class, small-molecule PRMT5 inhibitor that blocked EBV-driven B-lymphocyte transformation and survival while leaving normal B cells unaffected. Inhibition of PRMT5 led to lost recruitment of a PRMT5/p65/HDAC3-repressive complex on the miR96 promoter, restored miR96 expression, and PRMT5 downregulation. RNA-sequencing and chromatin immunoprecipitation experiments identified several tumor suppressor genes, including the protein tyrosine phosphatase gene PTPROt, which became silenced during EBV-driven B-cell transformation. Enhanced PTPROt expression following PRMT5 inhibition led to dephosphorylation of kinases that regulate B-cell receptor signaling. We conclude that PRMT5 is critical to EBV-driven B-cell transformation and maintenance of the malignant phenotype, and that PRMT5 inhibition shows promise as a novel therapeutic approach for B-cell lymphomas.
Asunto(s)
Linfocitos B/efectos de los fármacos , Transformación Celular Viral/efectos de los fármacos , Inhibidores Enzimáticos/farmacología , Proteína-Arginina N-Metiltransferasas/antagonistas & inhibidores , Animales , Linfocitos B/metabolismo , Linfocitos B/virología , Western Blotting , Línea Celular Transformada , Transformación Celular Viral/genética , Células Cultivadas , Herpesvirus Humano 4/fisiología , Histona Desacetilasas/genética , Histona Desacetilasas/metabolismo , Interacciones Huésped-Patógeno/efectos de los fármacos , Humanos , Linfoma/genética , Linfoma/metabolismo , Linfoma/virología , Ratones SCID , MicroARNs/genética , MicroARNs/metabolismo , Microscopía Confocal , Proteína-Arginina N-Metiltransferasas/genética , Proteína-Arginina N-Metiltransferasas/metabolismo , Interferencia de ARN , Proteínas Tirosina Fosfatasas Clase 3 Similares a Receptores/genética , Proteínas Tirosina Fosfatasas Clase 3 Similares a Receptores/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Bibliotecas de Moléculas Pequeñas/farmacología , Factor de Transcripción ReIA/genética , Factor de Transcripción ReIA/metabolismo , Transcriptoma/efectos de los fármacos , Transcriptoma/genética , Proteínas Supresoras de Tumor/genética , Proteínas Supresoras de Tumor/metabolismoRESUMEN
Jumonji domain-containing protein 6 (JMJD6) is a nuclear protein involved in histone modification, transcription and RNA processing. Although JMJD6 is crucial for tissue development, the link between its molecular functions and its roles in any given differentiation process is unknown. We report that JMJD6 is required for adipogenic gene expression and differentiation in a manner independent of Jumonji C domain catalytic activity. JMJD6 knockdown led to a reduction of C/EBPß and C/EBPδ protein expression without affecting mRNA levels in the early phase of differentiation. However, ectopic expression of C/EBPß and C/EBPδ did not rescue differentiation. Further analysis demonstrated that JMJD6 was associated with the Pparγ2 and Cebpα loci and putative enhancers. JMJD6 was previously found associated with bromodomain and extra-terminal domain (BET) proteins, which can be targeted by the bromodomain inhibitor JQ1. JQ1 treatment prevented chromatin binding of JMJD6, Pparγ2 and Cebpα expression, and adipogenic differentiation, yet had no effect on C/EBPß and C/EBPδ expression or chromatin binding. These results indicate dual roles for JMJD6 in promoting adipogenic gene expression program by post-transcriptional regulation of C/EBPß and C/EBPδ and direct transcriptional activation of Pparγ2 and Cebpα during adipocyte differentiation.
Asunto(s)
Adipocitos/metabolismo , Adipogénesis/genética , Regulación de la Expresión Génica , Receptores de Superficie Celular/metabolismo , Tejido Adiposo/metabolismo , Animales , Proteína alfa Potenciadora de Unión a CCAAT/genética , Proteína alfa Potenciadora de Unión a CCAAT/metabolismo , Proteína beta Potenciadora de Unión a CCAAT/metabolismo , Línea Celular , Cromatina/metabolismo , Femenino , Masculino , Ratones , PPAR gamma/genética , Estructura Terciaria de Proteína , ARN Polimerasa II/metabolismo , Receptores de Superficie Celular/antagonistas & inhibidores , Receptores de Superficie Celular/química , Receptores de Superficie Celular/fisiología , Transcripción GenéticaRESUMEN
Arginine methylation governs important cellular processes that impact growth and proliferation, as well as differentiation and development. Through their ability to catalyze symmetric or asymmetric methylation of histone and non-histone proteins, members of the protein arginine methyltransferase (PRMT) family regulate chromatin structure and expression of a wide spectrum of target genes. Unlike other PRMTs, PRMT5 works in concert with a variety of cellular proteins including ATP-dependent chromatin remodelers and co-repressors to induce epigenetic silencing. Recent work also implicates PRMT5 in the control of growth-promoting and pro-survival pathways, which demonstrates its versatility as an enzyme involved in both epigenetic regulation of anti-cancer target genes and organelle biogenesis. These studies not only provide insight into the molecular mechanisms by which PRMT5 contributes to growth control, but also justify therapeutic targeting of PRMT5.
Asunto(s)
Desarrollo Embrionario , Proteína Metiltransferasas/metabolismo , Animales , Arginina/metabolismo , Diferenciación Celular , Proliferación Celular , Humanos , Metilación , Proteína Metiltransferasas/genética , Proteína-Arginina N-MetiltransferasasRESUMEN
The SWI/SNF chromatin remodeling complex facilitates gene transcription by remodeling chromatin using the energy of ATP hydrolysis. Recent studies have indicated an interplay between the SWI/SNF complex and protein-arginine methyltransferases (PRMTs). Little is known, however, about the role of SWI/SNF and PRMTs in vitamin D receptor (VDR)-mediated transcription. Using SWI/SNF-defective cells, we demonstrated that Brahma-related gene 1 (BRG1), an ATPase that is a component of the SWI/SNF complex, plays a fundamental role in induction by 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) of the transcription of Cyp24a1 encoding the enzyme 25-hydroxyvitamin D3 24-hydroxylase involved in the catabolism of 1,25(OH)2D3. BRG1 was found to associate with CCAAT-enhancer-binding protein (C/EBP) ß and cooperate with VDR and C/EBPß in regulating Cyp24a1 transcription. PRMT5, a type II PRMT that interacts with BRG1, repressed Cyp24a1 transcription and mRNA expression. Our findings indicate the requirement of the C/EBP site for the inhibitory effect of PRMT5 via its methylation of H3R8 and H4R3. These findings indicate that the SWI/SNF complex and PRMT5 may be key factors involved in regulation of 1,25(OH)2D3 catabolism and therefore in the maintenance of calcium homeostasis by vitamin D. These studies also define epigenetic events linked to a novel mechanism of negative regulation of VDR-mediated transcription.
Asunto(s)
Calcitriol/metabolismo , Calcio/metabolismo , Proteínas Cromosómicas no Histona/genética , Epigénesis Genética , Proteína-Arginina N-Metiltransferasas/genética , Factores de Transcripción/genética , Vitamina D3 24-Hidroxilasa/genética , Animales , Células COS , Línea Celular Tumoral , Chlorocebus aethiops , Proteínas Cromosómicas no Histona/metabolismo , Células Epiteliales/citología , Células Epiteliales/metabolismo , Retroalimentación Fisiológica , Humanos , Osteoblastos/citología , Osteoblastos/metabolismo , Proteína-Arginina N-Metiltransferasas/metabolismo , Transducción de Señal , Factores de Transcripción/metabolismo , Transcripción Genética , Vitamina D3 24-Hidroxilasa/metabolismoRESUMEN
The microphthalmia-associated transcription factor (MITF) is required for terminal osteoclast differentiation and is a signaling effector engaged by macrophage colony-stimulating factor 1 (CSF-1) and receptor activator of nuclear factor-κB ligand (RANKL). MITF exerts its regulatory functions through its association with cofactors. Discovering the identity of its various partners will provide insights into the mechanisms governing gene expression during osteoclastogenesis. Here, we demonstrate that the proto-oncogene fused in sarcoma (FUS), the chromatin remodeling ATPase BRG1, and MITF form a trimeric complex that is regulated by phosphorylation of MITF at Ser-307 by p38 MAPK during osteoclast differentiation. FUS was recruited to MITF target gene promoters Acp5 and Ctsk during osteoclast differentiation, and FUS knockdown abolished efficient transcription of Acp5 and Ctsk. Furthermore, sumoylation of MITF at Lys-316, known to negatively regulate MITF transcriptional activity, inhibited MITF interactions with FUS and BRG1 in a p38 MAPK phosphorylation-dependent manner. These results demonstrate that FUS is a coregulator of MITF activity and provide new insights into how the RANKL/p38 MAPK signaling nexus controls gene expression in osteoclasts.
Asunto(s)
ADN Helicasas/metabolismo , Factor de Transcripción Asociado a Microftalmía/metabolismo , Complejos Multiproteicos/metabolismo , Proteínas Nucleares/metabolismo , Osteoclastos/metabolismo , Regiones Promotoras Genéticas/fisiología , Factores de Transcripción/metabolismo , Transcripción Genética/fisiología , Fosfatasa Ácida/biosíntesis , Fosfatasa Ácida/genética , Animales , Células COS , Catepsina K/biosíntesis , Catepsina K/genética , Chlorocebus aethiops , ADN Helicasas/genética , Regulación de la Expresión Génica/fisiología , Humanos , Isoenzimas/biosíntesis , Isoenzimas/genética , Sistema de Señalización de MAP Quinasas/fisiología , Ratones , Factor de Transcripción Asociado a Microftalmía/genética , Complejos Multiproteicos/genética , Proteínas Nucleares/genética , Osteoclastos/citología , Fosforilación/fisiología , Proto-Oncogenes Mas , Ligando RANK/genética , Ligando RANK/metabolismo , Proteína FUS de Unión a ARN , Fosfatasa Ácida Tartratorresistente , Factores de Transcripción/genética , Proteínas Quinasas p38 Activadas por Mitógenos/genética , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismoRESUMEN
Epigenetic regulation mediated by lysine- and arginine-specific enzymes plays an essential role in tumorigenesis, and enhanced expression of the type II protein arginine methyltransferase PRMT5 as well as the polycomb repressor complex PRC2 has been associated with increased cell proliferation and survival. Here, we show that PRMT5 is overexpressed in three different types of non-Hodgkin lymphoma cell lines and clinical samples as well as in mouse primary lymphoma cells and that it up-regulates PRC2 expression through inactivation of the retinoblastoma proteins RB1 and RBL2. Although PRMT5 epigenetically controls RBL2 expression, it indirectly promotes RB1 phosphorylation through enhanced cyclin D1 expression. Furthermore, we demonstrate that PRMT5 knockdown in non-Hodgkin lymphoma cell lines and mouse primary lymphoma cells leads to RBL2 derepression and RB1 reactivation, which in turn inhibit PRC2 expression and trigger derepression of its CASP10, DAP1, HOXA5, and HRK pro-apoptotic target genes. We also show that reduced PRMT5 expression leads to cyclin D1 transcriptional repression via loss of TP53K372 methylation, which results in decreased BCL3 expression and enhanced recruitment of NF-κB p52-HDAC1 repressor complexes to the cyclin D1 promoter. These findings indicate that PRMT5 is a master epigenetic regulator that governs expression of its own target genes and those regulated by PRC2 and that its inhibition could offer a promising therapeutic strategy for lymphoma patients.
Asunto(s)
Linfoma/genética , Linfoma/metabolismo , Complejo Represivo Polycomb 2/genética , Proteína Metiltransferasas/antagonistas & inhibidores , Proteína Metiltransferasas/genética , Proteína-Arginina N-Metiltransferasas/antagonistas & inhibidores , Proteína-Arginina N-Metiltransferasas/genética , Proteína de Retinoblastoma/metabolismo , Animales , Muerte Celular , Línea Celular Tumoral , Ciclina D1/metabolismo , Epigénesis Genética , Técnicas de Silenciamiento del Gen , Genes de Retinoblastoma , Histona Desacetilasa 2/genética , Histona Desacetilasa 2/metabolismo , Humanos , Linfoma/patología , Linfoma no Hodgkin/genética , Linfoma no Hodgkin/metabolismo , Linfoma no Hodgkin/patología , Ratones , Complejo Represivo Polycomb 2/antagonistas & inhibidores , Regiones Promotoras Genéticas , Proteína p130 Similar a la del Retinoblastoma/genética , Proteína p130 Similar a la del Retinoblastoma/metabolismo , Transducción de Señal , Células Tumorales CultivadasRESUMEN
Transcriptional repression of methylated genes can be mediated by the methyl-CpG binding protein MeCP2. Here we show that human Brahma (Brm), a catalytic component of the SWI/SNF-related chromatin-remodeling complex, associates with MeCP2 in vivo and is functionally linked with repression. We used a number of different molecular approaches and chromatin immunoprecipitation strategies to show a unique cooperation between Brm, BAF57 and MeCP2. We show that Brm and MeCP2 assembly on chromatin occurs on methylated genes in cancer and the gene FMR1 in fragile X syndrome. These experimental findings identify a new role for SWI/SNF in gene repression by MeCP2.
Asunto(s)
Proteínas de Ciclo Celular/fisiología , Proteínas Cromosómicas no Histona/fisiología , Proteínas de Unión al ADN/fisiología , Silenciador del Gen/fisiología , Proteínas Represoras/fisiología , Transactivadores/fisiología , Factores de Transcripción/fisiología , Animales , Proteínas de Drosophila , Proteína de la Discapacidad Intelectual del Síndrome del Cromosoma X Frágil , Histonas/fisiología , Humanos , Proteína 2 de Unión a Metil-CpG , Ratones , Microscopía Fluorescente , Datos de Secuencia Molecular , Células 3T3 NIH , Proteínas del Tejido Nervioso/genética , Proteínas de Unión al ARN/genéticaRESUMEN
Telomeres, the safeguarding caps at the tips of chromosomes, are pivotal in the aging process of cells and have been linked to skin ailments and inflammatory conditions. Telomeres undergo a gradual reduction in length and factors such as oxidative stress hasten this diminishing process. Skin diseases including inflammatory conditions can be correlated with the shortening of telomeres and the persistent activation of DNA damage response in skin tissues. Telomere dysfunction could disrupt the balance of the skin, impairs wound healing, and may contribute to abnormal cytokine production. Skin aging and processes related to telomeres may function as one of the triggers for skin diseases. The presence of proinflammatory cytokines and dysfunctional telomeres in conditions such as Dyskeratosis Congenita implies a possible connection between the shortening of telomeres and the onset of chronic inflammatory skin disorders. In autoinflammatory skin diseases, chronic inflammation hinders wound healing thus aggravating the progression of the disease. The NF-ĸB pathway might contribute to the initiation or progression of chronic disorders by influencing mechanisms associated with telomere biology. The intricate connections between telomeres, telomerase, telomere-associated proteins, and skin diseases are still a complex puzzle to be solved. Here, we provide an overview of the impact of telomeres on both health and disease with a specific emphasis on their role in skin, inflammation and autoinflammatory skin disorders.
Asunto(s)
Telómero , Humanos , Enfermedades de la Piel/genética , Inflamación/genética , Acortamiento del Telómero/fisiología , Telomerasa/metabolismo , Telomerasa/genética , Disqueratosis Congénita/genética , Envejecimiento de la Piel/genética , Envejecimiento de la Piel/fisiologíaRESUMEN
Covalent modification of histones by protein arginine methyltransferases (PRMTs) impacts genome organization and gene expression. In this report, we show that PRMT7 interacts with the BRG1-based hSWI/SNF chromatin remodeling complex and specifically methylates histone H2A Arg-3 (H2AR3) and histone H4 Arg-3 (H4R3). To elucidate the biological function of PRMT7, we knocked down its expression in NIH 3T3 cells and analyzed global gene expression. Our findings show that PRMT7 negatively regulates expression of genes involved in DNA repair, including ALKBH5, APEX2, POLD1, and POLD2. Chromatin immunoprecipitation (ChIP) revealed that PRMT7 and dimethylated H2AR3 and H4R3 are enriched at target DNA repair genes in parental cells, whereas PRMT7 knockdown caused a significant decrease in PRMT7 recruitment and H2AR3/H4R3 methylation. Decreased PRMT7 expression also resulted in derepression of target DNA repair genes and enhanced cell resistance to DNA-damaging agents. Furthermore, we show that BRG1 co-localizes with PRMT7 on target promoters and that expression of a catalytically inactive form of BRG1 results in derepression of PRMT7 target DNA repair genes. Remarkably, reducing expression of individual PRMT7 target DNA repair genes showed that only the catalytic subunit of DNA polymerase, POLD1, was able to resensitize PRMT7 knock-down cells to DNA-damaging agents. These results provide evidence for the important role played by PRMT7 in epigenetic regulation of DNA repair genes and cellular response to DNA damage.
Asunto(s)
Daño del ADN , ADN Polimerasa III/biosíntesis , Regulación Enzimológica de la Expresión Génica , Histonas/metabolismo , Regiones Promotoras Genéticas , Proteína-Arginina N-Metiltransferasas/biosíntesis , Desmetilasa de ARN, Homólogo 5 de AlkB , Animales , Núcleo Celular/genética , Núcleo Celular/metabolismo , ADN Helicasas/biosíntesis , ADN Helicasas/genética , ADN Polimerasa III/genética , ADN-(Sitio Apurínico o Apirimidínico) Liasa/biosíntesis , ADN-(Sitio Apurínico o Apirimidínico) Liasa/genética , Dioxigenasas/biosíntesis , Dioxigenasas/genética , Endonucleasas/biosíntesis , Endonucleasas/genética , Epigénesis Genética/genética , Técnicas de Silenciamiento del Gen , Células HeLa , Histonas/genética , Humanos , Proteínas de la Membrana/biosíntesis , Proteínas de la Membrana/genética , Metilación , Ratones , Enzimas Multifuncionales , Células 3T3 NIH , Proteínas Nucleares/biosíntesis , Proteínas Nucleares/genética , Proteína-Arginina N-Metiltransferasas/genética , Factores de Transcripción/biosíntesis , Factores de Transcripción/genéticaRESUMEN
Histone modification regulates gene expression, and one major regulatory step in this process is the ability of proteins that recognize epigenetic marks to recruit enzymes required to specify transcriptional outcome. Here we show that BRD7 is a component of hSWI-SNF complexes that interacts with PRMT5 and PRC2. Recruitment studies revealed that BRD7 co-localizes with PRMT5 and PRC2 on 'suppressor of tumorigenecity 7' (ST7) and retinoblastoma-like protein 2 (RBL2) promoters in patient-derived B cell lines, and that its association with these target genes correlates with hypermethylation of H3R8, H4R3 and H3K27. Furthermore, inhibition of BRD7 expression reduces PRMT5 and PRC2 recruitment to ST7 and RBL2 promoters; however, only ST7 becomes transcriptionally derepressed. Evaluation of the PRMT5- and PRC2-induced epigenetic marks revealed that while H3(Me(2))R8, H4(Me(2))R3 and H3(Me(3))K27 marks are erased from the ST7 promoter, demethylation of RBL2 promoter histones is incomplete. We also show that the arginine demethylase (RDM) JMJD6, which can erase PRMT5-induced H4R3 methylation, and the H3K27-lysine-specific demethylases, KDM6A/UTX and KDM6B/JMJD3, are differentially recruited to ST7 and RBL2. These findings highlight the role played by BRD7 in PRMT5- and PRC2-induced transcriptional silencing, and indicate that recruitment of specific RDMs and KDMs is required for efficient transcriptional derepression.
Asunto(s)
Proteínas Cromosómicas no Histona/metabolismo , Silenciador del Gen , Proteína Metiltransferasas/metabolismo , Proteínas Represoras/metabolismo , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Proteínas Portadoras/metabolismo , Línea Celular Tumoral , Ensamble y Desensamble de Cromatina , Proteínas Cromosómicas no Histona/antagonistas & inhibidores , Células HeLa , Histona Demetilasas/metabolismo , Humanos , Leucemia/genética , Linfoma/genética , Proteínas de Neoplasias , Proteínas Nucleares/metabolismo , Complejo Represivo Polycomb 2 , Proteínas del Grupo Polycomb , Regiones Promotoras Genéticas , Proteína-Arginina N-Metiltransferasas , Proteína p130 Similar a la del Retinoblastoma/genética , Factores de Transcripción , Transcripción Genética , Proteínas Supresoras de Tumor/genéticaRESUMEN
Myogenic microRNAs are important regulators of muscle development and differentiation. To better understand the roles of chromatin-modifying and remodeling enzymes in the activation of myogenic microRNA expression, we have functionally analyzed two different protein arginine methyltransferases, Prmt5 and Prmt4, both of which have previously been implicated in the regulation of myogenic mRNA expression. Both Prmts are required for myogenic microRNA induction during differentiation. Prmt5 is indirectly required due to the necessity of Prmt5 for expression of the transcriptional regulator, myogenin, as ectopic expression of myogenin eliminates Prmt5 dependency. By contrast, Prmt4 binds to the upstream regulatory regions of myogenic microRNAs and is required for dimethylation of the Prmt4 substrate, H3R17, at microRNA regulatory sequences. Deletion of Prmt4 does not alter MyoD binding at myogenic microRNA regulatory sequences but prevents the binding of both myogenin and the Brg1 ATPase that catalyzes SWI/SNF-dependent chromatin remodeling, resulting in an inhibition of microRNA expression.
Asunto(s)
MicroARNs/metabolismo , Desarrollo de Músculos , Proteína Metiltransferasas/fisiología , Proteína-Arginina N-Metiltransferasas/fisiología , Animales , ADN Helicasas/metabolismo , Expresión Génica , Cinética , Ratones , MicroARNs/química , Desarrollo de Músculos/genética , Músculo Esquelético/metabolismo , Miogenina/metabolismo , Células 3T3 NIH , Proteínas Nucleares/metabolismo , Secuencias Reguladoras de Ácido Ribonucleico , Factores de Transcripción/metabolismoRESUMEN
In an effort to understand the epigenetic regulation of ribosomal RNA gene (rDNA) expression we have previously demonstrated the role of DNA methyltransferases and methyl CpG binding proteins in rRNA synthesis. Here, we studied the role of protein arginine methyltransferase PRMT5 and the two methylated histones H3R8Me2 and H4R3Me2, in rDNA expression in Epstein Barr virus- transformed primary B-cells (LCLs) and in HeLa cells responding to serum-regulated growth. Chromatin immunoprecipitation assay showed that histones H3 and H4 associated with rRNA promoters were differentially methylated at arginine residues 8 and 3, respectively, depending on its transcriptional activity. Association of PRMT5 and methylated H3 with the unmethylated promoters in resting B-cells was significantly reduced in rapidly growing LCLs. Unlike PRMT5 and H3R8Me2, histone H4 associated with both methylated and unmethylated rRNA promoters in resting B-cells was methylated at the R3 residue. However, a dramatic decrease in R3 methylation of H4 recruited to the unmethylated rRNA promoters was observed in LCLs while it remained unaltered in the fraction bound to the methylated promoters. Differential interaction of PRMT5 and methylation of H3 and H4 associated with the rRNA promoters was also observed when serum starved HeLa cells were allowed to grow in serum replenished media. Ectopic expression of PRMT5 suppressed activity of both unmethylated and methylated rRNA promoter in transient transfection assay whereas siRNA mediated knockdown of PRMT5 increased rRNA synthesis in HeLa cells. These data suggest a key role of PRMT5 and the two methylated histones in regulating rRNA promoter activity.
Asunto(s)
Histonas/metabolismo , Proteína Metiltransferasas/metabolismo , ARN Ribosómico/genética , Transcripción Genética , Animales , Linfocitos B/metabolismo , Metilación de ADN , Células HeLa , Humanos , Metilación , Ratones , Ratones SCID , Proteína Metiltransferasas/genética , Proteína-Arginina N-Metiltransferasas , ARN Ribosómico/metabolismo , TransfecciónRESUMEN
Skeletal muscle differentiation requires the coordinated activity of transcription factors, histone modifying enzymes, and ATP-dependent chromatin remodeling enzymes. The type II protein arginine methyltransferase Prmt5 symmetrically dimethylates histones H3 and H4 and numerous nonchromatin proteins, and prior work has implicated Prmt5 in transcriptional repression. Here we demonstrate that MyoD-induced muscle differentiation requires Prmt5. One of the first genes activated during differentiation encodes the myogenic regulator myogenin. Prmt5 and dimethylated H3R8 (histone 3 arginine 8) are localized at the myogenin promoter in differentiating cells. Modification of H3R8 required Prmt5, and reduction of Prmt5 resulted in the abrogation of promoter binding by the Brg1 ATPase-associated with the SWI/SNF chromatin remodeling enzymes and all subsequent events associated with gene activation, including increases in chromatin accessibility and stable binding by MyoD. Prmt5 and dimethylated H3R8 were also associated with the myogenin promoter in activated satellite cells isolated from muscle tissue, further demonstrating the physiological relevance of these observations. The data indicate that Prmt5 facilitates myogenesis because it is required for Brg1-dependent chromatin remodeling and gene activation at a locus essential for differentiation. We therefore conclude that a histone modifying enzyme is necessary to permit an ATP-dependent chromatin remodeling enzyme to function.
Asunto(s)
Adenosina Trifosfato/química , Cromatina/química , Músculos/metabolismo , Proteína Metiltransferasas/fisiología , Animales , Diferenciación Celular , Separación Celular , Cromatina/metabolismo , ADN Helicasas/metabolismo , Metilación de ADN , Enzimas de Restricción del ADN/metabolismo , Citometría de Flujo , Ratones , Músculo Esquelético/metabolismo , Células 3T3 NIH , Proteínas Nucleares/metabolismo , Unión Proteica , Proteína Metiltransferasas/metabolismo , Proteína-Arginina N-Metiltransferasas , ARN Mensajero/metabolismo , Factores de Transcripción/metabolismoRESUMEN
The three basic DNA-binding domain mutations of the microphthalmia-associated transcription factor (Mitf), Mitf(mi/mi), Mitf(or/or), and Mitf(wh/wh) affect osteoclast differentiation with variable penetrance while completely impairing melanocyte development. Mitf(or/or) mice exhibit osteopetrosis that improves with age and their osteoclasts form functional multinuclear osteoclasts, raising the question as to why the Mitf(or/or) mutation results in osteopetrosis. Here we show that Mitf(or/or) osteoclasts express normal levels of acid phosphatase 5 (Acp5) mRNA and significantly lower levels of Cathepsin K (Ctsk) mRNA during receptor activator of nuclear factor kappa B (NFkappaB) ligand (RANKL)-mediated differentiation. Studies using chromatin immunoprecipitation (ChIP) analysis indicate that low levels of Mitf(or/or) protein are recruited to the Ctsk promoter. However, enrichment of Mitf-transcriptional co-activators PU.1 and Brahma-related gene 1 (Brg1) are severely impaired at the Ctsk promoter of Mitf(or/or) osteoclast precursors, indicating that defective recruitment of co-activators by the mutant Mitf(or/or) results in impaired Ctsk expression in osteoclasts. Cathepsin K may thus represent a unique class of Mitf-regulated osteoclast-specific genes that are important for osteoclast function.
Asunto(s)
Diferenciación Celular , ADN Helicasas/metabolismo , Factor de Transcripción Asociado a Microftalmía/metabolismo , Mutación , Proteínas Nucleares/metabolismo , Osteoclastos/metabolismo , Osteogénesis , Osteopetrosis/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Transactivadores/metabolismo , Factores de Transcripción/metabolismo , Fosfatasa Ácida/metabolismo , Factores de Edad , Envejecimiento/metabolismo , Animales , Animales Recién Nacidos , Catepsina K , Catepsinas/metabolismo , Diferenciación Celular/genética , Células Cultivadas , Regulación de la Expresión Génica , Isoenzimas/metabolismo , Ratones , Ratones Mutantes , Factor de Transcripción Asociado a Microftalmía/genética , FN-kappa B/metabolismo , Osteoclastos/enzimología , Osteogénesis/genética , Osteopetrosis/genética , Osteopetrosis/patología , Regiones Promotoras Genéticas , Ligando RANK/metabolismo , ARN Mensajero/metabolismo , Fosfatasa Ácida TartratorresistenteRESUMEN
Mutation of BRG1, hBRM, and their associated factors, INI1 and BAF57, in primary human tumors has suggested that inactivation of human SWI/SNF (hSWI/SNF) complexes may be involved in neoplastic transformation. BT549 is an invasive human breast carcinoma cell line that lacks expression of BAF57, a key hSWI/SNF subunit that mediates interaction with transcriptional activators and corepressors. In this study we investigated the role of BAF57 in suppressing tumorigenesis by establishing BT549 stable cell lines that expresses full-length BAF57 protein. BT549 clones expressing BAF57 demonstrated marked phenotypic changes, slow growth kinetics, and restoration of contact inhibition. Altered growth was found to be due in part to cell cycle arrest and induction of apoptosis. Furthermore, microarray analysis revealed that BAF57-mediated cell death was associated with up-regulation of proapoptotic genes including the tumor suppressor familial cylindromatosis (CYLD), which was found to be a direct target of BAF57 as determined by chromatin immunoprecipitation analysis. Increased expression of CYLD in BT549 cells induced apoptosis, while its suppression by small interfering RNA inhibited cell death in BAF57 expressing BT549 cells. These findings demonstrate the importance of BAF57 in cell growth regulation and provide a novel link between hSWI/SNF chromatin remodelers and apoptosis.