RESUMEN
Chromatin immunoprecipitation followed by high-throughput sequencing (ChIP-seq) is currently the method of choice to determine binding sites of chromatin-associated factors in a genome-wide manner. Here, we describe a method to investigate the binding preferences of mammalian DNA methyltransferases (DNMT) based on ChIP-seq using biotin-tagging. Stringent ChIP of DNMT proteins based on the strong interaction between biotin and avidin circumvents limitations arising from low antibody specificity and ensures reproducible enrichment. DNMT-bound DNA fragments are ligated to sequencing adaptors, amplified and sequenced on a high-throughput sequencing instrument. Bioinformatic analysis gives valuable information about the binding preferences of DNMTs genome-wide and around promoter regions. This method is unconventional due to the use of genetically engineered cells; however, it allows specific and reliable determination of DNMT binding.
Asunto(s)
Metilación de ADN , Metilasas de Modificación del ADN/genética , ADN/genética , Estudio de Asociación del Genoma Completo , Análisis por Matrices de Proteínas , Animales , Avidina/química , Sitios de Unión , Biotina/química , Cromatina/química , Cromatina/genética , ADN/química , Metilasas de Modificación del ADN/química , Humanos , Regiones Promotoras Genéticas , Programas InformáticosRESUMEN
The indisputable role of epigenetics in cancer and the fact that epigenetic alterations can be reversed have favoured development of epigenetic drugs. In this study, we design and synthesize potent novel, selective and reversible chemical probes that simultaneously inhibit the G9a and DNMTs methyltransferase activity. In vitro treatment of haematological neoplasia (acute myeloid leukaemia-AML, acute lymphoblastic leukaemia-ALL and diffuse large B-cell lymphoma-DLBCL) with the lead compound CM-272, inhibits cell proliferation and promotes apoptosis, inducing interferon-stimulated genes and immunogenic cell death. CM-272 significantly prolongs survival of AML, ALL and DLBCL xenogeneic models. Our results represent the discovery of first-in-class dual inhibitors of G9a/DNMTs and establish this chemical series as a promising therapeutic tool for unmet needs in haematological tumours.
Asunto(s)
Antineoplásicos/farmacología , Metilasas de Modificación del ADN/antagonistas & inhibidores , Diseño de Fármacos , Inhibidores Enzimáticos/farmacología , Neoplasias Hematológicas/tratamiento farmacológico , N-Metiltransferasa de Histona-Lisina/antagonistas & inhibidores , Animales , Antineoplásicos/química , Antineoplásicos/uso terapéutico , Apoptosis/efectos de los fármacos , Apoptosis/genética , Apoptosis/inmunología , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Proliferación Celular/genética , Cristalografía por Rayos X , Metilasas de Modificación del ADN/química , Metilasas de Modificación del ADN/genética , Metilasas de Modificación del ADN/metabolismo , Relación Dosis-Respuesta a Droga , Evaluación Preclínica de Medicamentos , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/uso terapéutico , Epigénesis Genética/efectos de los fármacos , Femenino , Neoplasias Hematológicas/genética , Neoplasias Hematológicas/inmunología , Neoplasias Hematológicas/mortalidad , Antígenos de Histocompatibilidad/química , Antígenos de Histocompatibilidad/genética , Antígenos de Histocompatibilidad/metabolismo , N-Metiltransferasa de Histona-Lisina/química , N-Metiltransferasa de Histona-Lisina/genética , N-Metiltransferasa de Histona-Lisina/metabolismo , Humanos , Interferones/inmunología , Interferones/metabolismo , Ratones , Ratones Endogámicos BALB C , Microsomas Hepáticos , Simulación del Acoplamiento Molecular , Análisis de Supervivencia , Resultado del Tratamiento , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Inhibition of DNA methyltransferases (DNMTs) is a promising approach for the therapeutic treatment of cancer and other diseases. In this work, we review the recent progress on the molecular modeling and virtual screening toward the identification of key structural features associated with the enzyme inhibitory action of active compounds and to identify DNMT inhibitors with novel molecular scaffolds. We discuss the molecular modeling with the co-factor binding site using a recent crystallographic structure of the methyltransferase domain of human DNMT1. We also review the emerging synergy of molecular modeling and chemoinformatic approaches applied to epigenetic therapies targeting DNMTs.
Asunto(s)
Antineoplásicos/farmacología , Metilasas de Modificación del ADN/antagonistas & inhibidores , Diseño de Fármacos , Inhibidores Enzimáticos/farmacología , Modelos Moleculares , Animales , Antineoplásicos/química , Antineoplásicos/metabolismo , Biología Computacional , ADN (Citosina-5-)-Metiltransferasas/antagonistas & inhibidores , ADN (Citosina-5-)-Metiltransferasas/química , ADN (Citosina-5-)-Metiltransferasas/metabolismo , Metilasas de Modificación del ADN/química , Metilasas de Modificación del ADN/metabolismo , Evaluación Preclínica de Medicamentos/métodos , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/metabolismo , Humanos , Ligandos , Relación Estructura-ActividadRESUMEN
Dnmt3a1 and Dnmt3a2 are two de novo DNA methyltransferases expressed in mouse embryonic stem cells (mESCs). They differ in that a 219-amino-acid (aa) amino (N)-terminal noncatalytic domain is present only in Dnmt3a1. Here, we examined the unique functions of Dnmt3a1 in mESCs by targeting the coding sequence of the Dnmt3a1 N-terminal domain tagged with enhanced green fluorescent protein (GFP) for insertion into the mouse Rosa26 locus. Using these targeted cells (GFP-3a1Nter), we showed that Dnmt3a1 was efficiently recruited to the silenced Oct3/4 and activated Vtn (vitronectin) gene promoters via its unique N-terminal domain. This recruitment affected the two genes in contrasting ways, compromising Oct3/4 gene promoter DNA methylation to prevent consolidation of the silent state while significantly reducing Vtn transcription. We used this negative effect of the Dnmt3a1 N-terminal domain to investigate the extent of transcriptional regulation by Dnmt3a1 in mESCs by using microarrays. A small group of all-trans retinoic acid (tRA)-inducible genes had lower transcript levels in GFP-3a1Nter cells than in wild-type mESCs. Intriguingly, this group included genes that are important for fetal nutrition, placenta development, and metabolic functions and is enriched for a distinct set of imprinted genes. We also identified a larger group of genes that showed higher transcript levels in the GFP-3a1Nter-expressing cells than in wild-type mESCs, including pluripotency factors and key regulators of primordial germ cell differentiation. Thus, Dnmt3a1 in mESCs functions primarily as a negative and to a lesser extent as a positive regulator of transcription. Our findings suggest that Dnmt3a1 positively affects transcription of specific genes at the promoter level and targets chromosomal domains to epigenetically silence gene clusters in mESCs.
Asunto(s)
Cromosomas de los Mamíferos/genética , ADN (Citosina-5-)-Metiltransferasas/metabolismo , Metilasas de Modificación del ADN/metabolismo , Células Madre Embrionarias/metabolismo , Silenciador del Gen , Familia de Multigenes/genética , Transcripción Genética , Regulación hacia Arriba/genética , Animales , ADN (Citosina-5-)-Metiltransferasas/química , ADN Metiltransferasa 3A , Metilasas de Modificación del ADN/química , ADN Complementario/genética , Células Madre Embrionarias/efectos de los fármacos , Silenciador del Gen/efectos de los fármacos , Sitios Genéticos/genética , Proteínas Fluorescentes Verdes/metabolismo , Proteínas de Homeodominio/metabolismo , Ratones , Proteína Homeótica Nanog , Factor 3 de Transcripción de Unión a Octámeros/metabolismo , Análisis de Secuencia por Matrices de Oligonucleótidos , Regiones Promotoras Genéticas/genética , Unión Proteica/efectos de los fármacos , Estructura Terciaria de Proteína , Proteínas/genética , Proteínas/metabolismo , ARN no Traducido , Transcripción Genética/efectos de los fármacos , Activación Transcripcional/efectos de los fármacos , Activación Transcripcional/genética , Tretinoina/farmacología , Regulación hacia Arriba/efectos de los fármacos , Vitronectina/genéticaRESUMEN
We report the properties of the new AloI restriction and modification enzyme from Acinetobacter lwoffi Ks 4-8 that recognizes the DNA target 5' GGA(N)6GTTC3' (complementary strand 5' GAAC(N)6TCC3'), and the nucleotide sequence of the gene encoding this enzyme. AloI is a bifunctional large polypeptide (deduced M(r) 143 kDa) revealing both DNA endonuclease and methyltransferase activities. Depending on reaction cofactors, AloI cleaves double-stranded DNA on both strands, seven bases on the 5' side, and 12-13 bases on the 3' side of its recognition sequence, and modifies adenine residues in both DNA strands in the target sequence yielding N6-methyladenine. For cleavage activity AloI maintains an absolute requirement for Mg(2+) and does not depend on or is stimulated by either ATP or S-adenosyl-L-methionine. Modification function requires the presence of S-adenosyl-L-methionine and is stimulated by metal ions (Ca(2+)). The C-terminal and central parts of the protein were found to be homologous to certain specificity (HsdS) and modification (HsdM) subunits of type I R-M systems, respectively. The N-terminal part of the protein possesses the putative endonucleolytic motif DXnEXK of restriction endonucleases. The deduced amino acid sequence of AloI shares significant homology with polypeptides encoding HaeIV and CjeI restriction-modification proteins at the N-terminal and central, but not at the C-terminal domains. The organization of AloI implies that its evolution involved fusion of an endonuclease and the two subunits, HsdM and HsdS, of type I restriction enzymes. According to the structure and function properties AloI may be regarded as one more representative of a newly emerging group of HaeIV-like restriction endonucleases. Discovery of these enzymes opens new opportunities for constructing restriction endonucleases with a new specificity.
Asunto(s)
Acinetobacter/enzimología , Metilación de ADN , Metilasas de Modificación del ADN/química , Metilasas de Modificación del ADN/genética , Metilasas de Modificación del ADN/metabolismo , Enzimas de Restricción del ADN/química , Enzimas de Restricción del ADN/metabolismo , Acinetobacter/genética , Adenosina Trifosfato/metabolismo , Secuencia de Aminoácidos , Secuencia de Bases , Calcio/metabolismo , Cationes Bivalentes/metabolismo , Cromatografía en Gel , Clonación Molecular , Coenzimas/metabolismo , Metilasas de Modificación del ADN/aislamiento & purificación , Enzimas de Restricción del ADN/genética , Enzimas de Restricción del ADN/aislamiento & purificación , Concentración de Iones de Hidrógeno , Magnesio/metabolismo , Datos de Secuencia Molecular , Peso Molecular , Complejos Multienzimáticos/química , Complejos Multienzimáticos/genética , Complejos Multienzimáticos/aislamiento & purificación , Complejos Multienzimáticos/metabolismo , Concentración Osmolar , Estructura Terciaria de Proteína , S-Adenosilmetionina/metabolismo , Alineación de Secuencia , Especificidad por SustratoRESUMEN
Methylation of cytosines within the sequence CpG is essential for mouse development and has been linked to transcriptional suppression in vertebrate systems. Methyl-CpG binding proteins (MeCPs) 1 and 2 bind preferentially to methylated DNA and can inhibit transcription. The gene for MeCP2 has been cloned and a methyl-CpG binding domain (MBD) within it has been defined. A search of DNA sequence databases with the MBD sequence identified a human cDNA with potential to encode an MBD-like region. Sequencing of the complete cDNA revealed that the open reading frame also encodes two cysteine-rich domains that are found in animal DNA methyltransferases (DNMTs) and in the mammalian HRX protein (also known as MLL and All-1). HRX is related to Drosophila trithorax. The protein, known as Protein Containing MBD (PCM1), was expressed in bacteria and shown to bind specifically to methylated DNA. PCM1 also repressed transcription in vitro in a methylation-dependent manner. A polyclonal antibody raised against the protein was able to 'supershift' the native MeCP11 complex from HeLa cells, indicating that PCM1 is a component of mammalian MeCP1.