RESUMEN
The extent to which histone modifying enzymes contribute to DNA methylation in mammals remains unclear. Previous studies suggested a link between the lysine methyltransferase EHMT2 (also known as G9A and KMT1C) and DNA methylation in the mouse. Here, we used a model of knockout mice to explore the role of EHMT2 in DNA methylation during mouse embryogenesis. The Ehmt2 gene is expressed in epiblast cells but is dispensable for global DNA methylation in embryogenesis. In contrast, EHMT2 regulates DNA methylation at specific sequences that include CpG-rich promoters of germline-specific genes. These loci are bound by EHMT2 in embryonic cells, are marked by H3K9 dimethylation, and have strongly reduced DNA methylation in Ehmt2(-/-) embryos. EHMT2 also plays a role in the maintenance of germline-derived DNA methylation at one imprinted locus, the Slc38a4 gene. Finally, we show that DNA methylation is instrumental for EHMT2-mediated gene silencing in embryogenesis. Our findings identify EHMT2 as a critical factor that facilitates repressive DNA methylation at specific genomic loci during mammalian development.
Asunto(s)
Metilación de ADN , Silenciador del Gen , N-Metiltransferasa de Histona-Lisina/fisiología , Sistema de Transporte de Aminoácidos A/genética , Animales , Células Cultivadas , Embrión de Mamíferos/metabolismo , Femenino , Expresión Génica , Regulación del Desarrollo de la Expresión Génica , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Células Madre Embrionarias de Ratones/fisiología , Análisis de Secuencia de ADNRESUMEN
Functional genomic elements are marked by characteristic DNA and histone modification signatures. How combinatorial chromatin modification states are recognized by epigenetic reader proteins and how this is linked to their biological function is largely unknown. Here we provide a detailed molecular analysis of chromatin recognition by the lysine demethylase KDM2A. Using biochemical approaches we identify a nucleosome interaction module within KDM2A consisting of a CXXC type zinc finger, a PHD domain and a newly identified Heterochromatin Protein 1 (HP1) interaction motif that mediates direct binding between KDM2A and HP1. This nucleosome interaction module enables KDM2A to decode nucleosomal H3K9me3 modification in addition to CpG methylation signals. The multivalent engagement with DNA and HP1 results in a nucleosome binding circuit in which KDM2A can be recruited to H3K9me3-modified chromatin through HP1, and HP1 can be recruited to unmodified chromatin by KDM2A. A KDM2A mutant deficient in HP1-binding is inactive in an in vivo overexpression assay in zebrafish embryos demonstrating that the HP1 interaction is essential for KDM2A function. Our results reveal a complex regulation of chromatin binding for both KDM2A and HP1 that is modulated by DNA- and H3K9-methylation, and suggest a direct role for KDM2A in chromatin silencing.
Asunto(s)
Proteínas Cromosómicas no Histona/química , Proteínas Cromosómicas no Histona/metabolismo , Proteínas F-Box/química , Proteínas F-Box/metabolismo , Histona Demetilasas con Dominio de Jumonji/química , Histona Demetilasas con Dominio de Jumonji/metabolismo , Animales , Animales Modificados Genéticamente , Línea Celular , Homólogo de la Proteína Chromobox 5 , Proteínas Cromosómicas no Histona/genética , Islas de CpG , Cricetinae , Metilación de ADN , Proteínas F-Box/genética , Células HEK293 , Células HeLa , Histonas/metabolismo , Humanos , Histona Demetilasas con Dominio de Jumonji/genética , Modelos Genéticos , Mutación , Neoplasias/genética , Neoplasias/metabolismo , Nucleosomas/genética , Nucleosomas/metabolismo , Dominios y Motivos de Interacción de Proteínas , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Técnicas del Sistema de Dos Híbridos , Pez Cebra , Dedos de ZincRESUMEN
The INK4B-ARF-INK4A (INK/ARF) locus is composed of three tumor suppressor genes, which are kept silenced by DNA methylation in different cancer types. In addition, a non-coding RNA (ANRIL) is transcribed in the anti-sense orientation upstream of the ARF gene. The resulting divergent promoter region is bound by the chromatin insulator protein CTCF in association with histone H3 tri-methylated on lysine 4, irrespective of transcription of ANRIL and ARF. Methylation of the overlapping CpG island abolishes CTCF binding and the associated modification, which can be restored by 5-Aza-2'-deoxycytidine (5-Aza-dC) treatment. shRNA knock down of CTCF expression dramatically reduces the induction of ANRIL and ARF, but also that of INK4A and INK4B expression by 5-Aza-dC. We propose that CTCF is an essential factor for transcription of the INK/ARF locus and that abrogation of its binding by DNA methylation contributes to the permanent silencing of several genes of the locus in tumors.
Asunto(s)
Inhibidor p15 de las Quinasas Dependientes de la Ciclina/genética , Metilación de ADN , Regulación Neoplásica de la Expresión Génica , Genes p16 , Neoplasias/genética , Proteínas Represoras/metabolismo , Proteína p14ARF Supresora de Tumor/genética , Factor de Unión a CCCTC , Línea Celular Tumoral , Sitios Genéticos , Humanos , Regiones Promotoras Genéticas , Activación TranscripcionalRESUMEN
The involvement of epigenetic processes in the origin and progression of cancer is now widely appreciated. Consequently, targeting the enzymatic machinery that controls the epigenetic regulation of the genome has emerged as an attractive new strategy for therapeutic intervention. The development of epigenetic drugs requires a detailed knowledge of the processes that govern chromatin regulation. Over the recent years, mass spectrometry (MS) has become an indispensable tool in epigenetics research. In this review, we will give an overview of the applications of MS-based proteomics in studying various aspects of chromatin biology. We will focus on the use of MS in the discovery and mapping of histone modifications and how novel proteomic approaches are being utilized to identify and study chromatin-associated proteins and multi-subunit complexes. Finally, we will discuss the application of proteomic methods in the diagnosis and prognosis of cancer based on epigenetic biomarkers and comment on their future impact on cancer epigenetics.
Asunto(s)
Epigenómica/métodos , Neoplasias/genética , Neoplasias/metabolismo , Proteómica/métodos , Animales , Humanos , Espectrometría de MasasRESUMEN
Methylated DNA immunoprecipitation (MeDIP) is an immunocapturing approach for unbiased enrichment of DNA that is methylated on cytosines. The principle is that genomic DNA is randomly sheared by sonication and immunoprecipitated with an antibody that specifically recognizes 5-methylcytidine (5mC), which can be combined with PCR or high-throughput analysis (microarrays, deep sequencing). The MeDIP technique has been originally used to generate DNA methylation profiles on a genome scale in mammals and plants. Here we provide an optimized version of the MeDIP protocol suitable for low amounts of DNA, which can be used to study DNA methylation in cellular populations available in small quantities.
Asunto(s)
Recuento de Células , Metilación de ADN , ADN/química , ADN/aislamiento & purificación , Inmunoprecipitación/métodos , Animales , ADN/genética , Genoma/genética , Humanos , Ratones , Técnicas de Amplificación de Ácido Nucleico , SonicaciónRESUMEN
DNA methylation is extensively reprogrammed during the early phases of mammalian development, yet genomic targets of this process are largely unknown. We optimized methylated DNA immunoprecipitation for low numbers of cells and profiled DNA methylation during early development of the mouse embryonic lineage in vivo. We observed a major epigenetic switch during implantation at the transition from the blastocyst to the postimplantation epiblast. During this period, DNA methylation is primarily targeted to repress the germline expression program. DNA methylation in the epiblast is also targeted to promoters of lineage-specific genes such as hematopoietic genes, which are subsequently demethylated during terminal differentiation. De novo methylation during early embryogenesis is catalyzed by Dnmt3b, and absence of DNA methylation leads to ectopic gene activation in the embryo. Finally, we identify nonimprinted genes that inherit promoter DNA methylation from parental gametes, suggesting that escape of post-fertilization DNA methylation reprogramming is prevalent in the mouse genome.