RESUMEN
Open or accessible chromatin typifies euchromatic regions and helps define cell type-specific transcription programs. DNA replication massively disorders chromatin composition and structure, and how accessible regions are affected by and recover from this disruption has been unclear. Here, we present repli-ATAC-seq, a protocol to profile accessible chromatin genome-wide on replicated DNA starting from 100,000 cells. In this method, replicated DNA is labeled with a short 5-ethynyl-2'-deoxyuridine (EdU) pulse in cultured cells and isolated from a population of tagmented fragments for amplification and next-generation sequencing. Repli-ATAC-seq provides high-resolution information on chromatin dynamics after DNA replication and reveals new insights into the interplay between DNA replication, transcription, and the chromatin landscape.
Asunto(s)
Secuenciación de Inmunoprecipitación de Cromatina , Cromatina , Análisis de Secuencia de ADN/métodos , ADN/genética , Secuenciación de Nucleótidos de Alto Rendimiento/métodosRESUMEN
Chromatin landscapes are disrupted during DNA replication and must be restored faithfully to maintain genome regulation and cell identity. The histone H3-H4 modification landscape is restored by parental histone recycling and modification of new histones. How DNA replication impacts on histone H2A-H2B is currently unknown. Here, we measure H2A-H2B modifications and H2A.Z during DNA replication and across the cell cycle using quantitative genomics. We show that H2AK119ub1, H2BK120ub1, and H2A.Z are recycled accurately during DNA replication. Modified H2A-H2B are segregated symmetrically to daughter strands via POLA1 on the lagging strand, but independent of H3-H4 recycling. Post-replication, H2A-H2B modification and variant landscapes are quickly restored, and H2AK119ub1 guides accurate restoration of H3K27me3. This work reveals epigenetic transmission of parental H2A-H2B during DNA replication and identifies cross talk between H3-H4 and H2A-H2B modifications in epigenome propagation. We propose that rapid short-term memory of recycled H2A-H2B modifications facilitates restoration of stable H3-H4 chromatin states.
Asunto(s)
Cromatina , Memoria a Corto Plazo , Ciclo Celular , Replicación del ADN , Histonas/metabolismo , Nucleosomas , Animales , Ratones , ConejosRESUMEN
DNA methylation is a critical epigenetic mark in mammalian cells. Many aspects of DNA methylation maintenance have been characterized; however, the exact kinetics of post-replicative methylation maintenance remain a subject of debate. Here we develop isolation of DNA by 5-ethynyl-deoxyuridine labelling for mass spectrometry (iDEMS), a highly sensitive, quantitative mass spectrometry-based method for measuring DNA modifications on metabolically labelled DNA. iDEMS reveals an unexpectedly hemi-methylated landscape on nascent DNA. Combining iDEMS with metabolic labelling reveals that methylation maintenance is outpaced by cell division in mouse embryonic stem cells. Our approach shows that hydroxymethylation is perpetually asymmetric between sister strands in favour of the parental, template strand. iDEMS can be coupled with immunoprecipitation of chromatin proteins, revealing features of DNA methylation-histone modification crosstalk and suggesting a model for interplay between methylation and nucleosome assembly. iDEMS therefore elucidates long-standing questions about DNA modification propagation and provides an important orthogonal technology to understanding this process in dynamic cellular contexts.
Asunto(s)
Metilación de ADN , ADN , Animales , Ratones , ADN/genética , ADN/metabolismo , Células Madre Embrionarias de Ratones/metabolismo , Ensamble y Desensamble de Cromatina , Procesamiento Proteico-Postraduccional , Epigénesis Genética , Mamíferos/metabolismoRESUMEN
Propagation of the chromatin landscape across cell divisions is central to epigenetic cell memory. Mechanistic analysis of the interplay between DNA replication, the cell cycle, and the epigenome has provided insights into replication-coupled chromatin assembly and post-replicative chromatin maintenance. These breakthroughs are critical for defining how proliferation impacts the epigenome during cell identity changes in development and disease. Here we review these findings in the broader context of epigenetic inheritance across mitotic cell division.
Asunto(s)
Replicación del ADN , Epigénesis Genética , Epigenoma , Nucleosomas , Ciclo Celular/genética , Ensamble y Desensamble de Cromatina , Histonas/genética , Histonas/metabolismo , Humanos , Patrón de Herencia , Procesamiento Proteico-PostraduccionalRESUMEN
DNA replication is highly disruptive to chromatin, leading to eviction of nucleosomes, RNA polymerase, and regulatory factors. When and how transcription resumes on DNA following DNA replication is unknown. Here we develop a replication-coupled assay for transposase-accessible chromatin (repli-ATAC-seq) to investigate active chromatin restoration post-replication in mouse embryonic stem cells. We find that nascent chromatin is inaccessible and transcriptionally silenced, with accessibility and RNA polymerase occupancy re-appearing within 30 minutes. Chromatin accessibility restores differentially genome wide, with super enhancers regaining transcription factor occupancy faster than other genomic features. We also identify opportunistic and transiently accessible chromatin within gene bodies after replication. Systematic inhibition of transcription shows that transcription restart is required to re-establish active chromatin states genome wide and resolve opportunistic binding events resulting from DNA replication. Collectively, this establishes a central role for transcription in overcoming the genome-wide chromatin inaccessibility imposed by DNA replication every cell division.
Asunto(s)
Replicación del ADN/genética , ADN/genética , Genoma/genética , Transcripción Genética , Animales , División Celular/genética , Cromatina/genética , ADN/química , ARN Polimerasas Dirigidas por ADN/química , ARN Polimerasas Dirigidas por ADN/genética , Regulación de la Expresión Génica/genética , Secuenciación de Nucleótidos de Alto Rendimiento , Ratones , Células Madre Embrionarias de Ratones/química , Nucleosomas/química , Nucleosomas/genética , Regiones Promotoras Genéticas/genética , Análisis de Secuencia de ADN , Sitio de Iniciación de la Transcripción , Transposasas/química , Transposasas/genéticaRESUMEN
DNA methyltransferases (DNMTs) deposit DNA methylation, which regulates gene expression and is essential for mammalian development. Histone post-translational modifications modulate the recruitment and activity of DNMTs. The PWWP domains of DNMT3A and DNMT3B are posited to interact with histone 3 lysine 36 trimethylation (H3K36me3); however, the functionality of this interaction for DNMT3A remains untested in vivo. Here we present a mouse model carrying a D329A point mutation in the DNMT3A PWWP domain. The mutation causes dominant postnatal growth retardation. At the molecular level, it results in progressive DNA hypermethylation across domains marked by H3K27me3 and bivalent chromatin, and de-repression of developmental regulatory genes in adult hypothalamus. Evaluation of non-CpG methylation, a marker of de novo methylation, further demonstrates the altered recruitment and activity of DNMT3AD329A at bivalent domains. This work provides key molecular insights into the function of the DNMT3A-PWWP domain and role of DNMT3A in regulating postnatal growth.
Asunto(s)
Cromatina/metabolismo , ADN (Citosina-5-)-Metiltransferasas/genética , Metilación de ADN/fisiología , Regulación del Desarrollo de la Expresión Génica , Trastornos del Crecimiento/genética , Animales , Animales Recién Nacidos , ADN Metiltransferasa 3A , Modelos Animales de Enfermedad , Femenino , Mutación con Ganancia de Función/fisiología , Trastornos del Crecimiento/patología , Histonas/metabolismo , Humanos , Hipotálamo/metabolismo , Hipotálamo/patología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Mutación Puntual/fisiología , Unión Proteica/genética , Dominios Proteicos/genética , Procesamiento Proteico-Postraduccional/fisiologíaRESUMEN
Chromatin organization is disrupted genome-wide during DNA replication. On newly synthesized DNA, nucleosomes are assembled from new naive histones and old modified histones. It remains unknown whether the landscape of histone post-translational modifications (PTMs) is faithfully copied during DNA replication or the epigenome is perturbed. Here we develop chromatin occupancy after replication (ChOR-seq) to determine histone PTM occupancy immediately after DNA replication and across the cell cycle. We show that H3K4me3, H3K36me3, H3K79me3, and H3K27me3 positional information is reproduced with high accuracy on newly synthesized DNA through histone recycling. Quantitative ChOR-seq reveals that de novo methylation to restore H3K4me3 and H3K27me3 levels occurs across the cell cycle with mark- and locus-specific kinetics. Collectively, this demonstrates that accurate parental histone recycling preserves positional information and allows PTM transmission to daughter cells while modification of new histones gives rise to complex epigenome fluctuations across the cell cycle that could underlie cell-to-cell heterogeneity.
Asunto(s)
Replicación del ADN/genética , Histonas/genética , Ciclo Celular/genética , Línea Celular Tumoral , Cromatina/genética , Epigénesis Genética/genética , Femenino , Células HeLa , Humanos , Metilación , Nucleosomas/genética , Procesamiento Proteico-Postraduccional/genéticaRESUMEN
BACKGROUND: Gametogenesis in mammals entails profound re-patterning of the epigenome. In the female germline, DNA methylation is acquired late in oogenesis from an essentially unmethylated baseline and is established largely as a consequence of transcription events. Molecular and functional studies have shown that imprinted genes become methylated at different times during oocyte growth; however, little is known about the kinetics of methylation gain genome wide and the reasons for asynchrony in methylation at imprinted loci. RESULTS: Given the predominant role of transcription, we sought to investigate whether transcription timing is rate limiting for de novo methylation and determines the asynchrony of methylation events. Therefore, we generated genome-wide methylation and transcriptome maps of size-selected, growing oocytes to capture the onset and progression of methylation. We find that most sequence elements, including most classes of transposable elements, acquire methylation at similar rates overall. However, methylation of CpG islands (CGIs) is delayed compared with the genome average and there are reproducible differences amongst CGIs in onset of methylation. Although more highly transcribed genes acquire methylation earlier, the major transitions in the oocyte transcriptome occur well before the de novo methylation phase, indicating that transcription is generally not rate limiting in conferring permissiveness to DNA methylation. Instead, CGI methylation timing negatively correlates with enrichment for histone 3 lysine 4 (H3K4) methylation and dependence on the H3K4 demethylases KDM1A and KDM1B, implicating chromatin remodelling as a major determinant of methylation timing. We also identified differential enrichment of transcription factor binding motifs in CGIs acquiring methylation early or late in oocyte growth. By combining these parameters into multiple regression models, we were able to account for about a fifth of the variation in methylation timing of CGIs. Finally, we show that establishment of non-CpG methylation, which is prevalent in fully grown oocytes, and methylation over non-transcribed regions, are later events in oogenesis. CONCLUSIONS: These results do not support a major role for transcriptional transitions in the time of onset of DNA methylation in the oocyte, but suggest a model in which sequences least dependent on chromatin remodelling are the earliest to become permissive for methylation.