RESUMEN
The fate and function of epigenetic marks during the germline-to-embryo transition is a key issue in developmental biology, with relevance to stem cell programming and transgenerational inheritance. In zebrafish, DNA methylation patterns are programmed in transcriptionally quiescent cleavage embryos; paternally inherited patterns are maintained, whereas maternal patterns are reprogrammed to match the paternal. Here, we provide the mechanism by demonstrating that "Placeholder" nucleosomes, containing histone H2A variant H2A.Z(FV) and H3K4me1, virtually occupy all regions lacking DNA methylation in both sperm and cleavage embryos and reside at promoters encoding housekeeping and early embryonic transcription factors. Upon genome-wide transcriptional onset, genes with Placeholder become either active (H3K4me3) or silent (H3K4me3/K27me3). Notably, perturbations causing Placeholder loss confer DNA methylation accumulation, whereas acquisition/expansion of Placeholder confers DNA hypomethylation and improper gene activation. Thus, during transcriptionally quiescent gametic and embryonic stages, an H2A.Z(FV)/H3K4me1-containing Placeholder nucleosome deters DNA methylation, poising parental genes for either gene-specific activation or facultative repression.
Asunto(s)
Reprogramación Celular/genética , Metilación de ADN/genética , Embrión no Mamífero/metabolismo , Células Germinativas/metabolismo , Nucleosomas/metabolismo , Animales , Histonas/metabolismo , Masculino , Mutación/genética , Espermatozoides/metabolismo , Pez Cebra/genética , Proteínas de Pez Cebra/metabolismoRESUMEN
The transcription factor Oct4/Pou5f1 is a component of the regulatory circuitry governing pluripotency and is widely used to induce pluripotency from somatic cells. Here we used domain swapping and mutagenesis to study Oct4's reprogramming ability, identifying a redox-sensitive DNA binding domain, cysteine residue (Cys48), as a key determinant of reprogramming and differentiation. Oct4 Cys48 sensitizes the protein to oxidative inhibition of DNA binding activity and promotes oxidation-mediated protein ubiquitylation. Pou5f1 C48S point mutation has little effect on undifferentiated embryonic stem cells (ESCs) but upon retinoic acid (RA) treatment causes retention of Oct4 expression, deregulated gene expression, and aberrant differentiation. Pou5f1 C48S ESCs also form less differentiated teratomas and contribute poorly to adult somatic tissues. Finally, we describe Pou5f1 C48S (Janky) mice, which in the homozygous condition are severely developmentally restricted after E4.5. Rare animals bypassing this restriction appear normal at birth but are sterile. Collectively, these findings uncover a novel Oct4 redox mechanism involved in both entry into and exit from pluripotency.
Asunto(s)
Diferenciación Celular , Reprogramación Celular , Factor 3 de Transcripción de Unión a Octámeros , Oxidación-Reducción , Factor 3 de Transcripción de Unión a Octámeros/metabolismo , Factor 3 de Transcripción de Unión a Octámeros/genética , Animales , Ratones , Diferenciación Celular/genética , Reprogramación Celular/genética , Células Madre Embrionarias/citología , Células Madre Embrionarias/metabolismo , Tretinoina/farmacología , Tretinoina/metabolismo , Regulación del Desarrollo de la Expresión Génica/genética , HumanosRESUMEN
Cells utilize diverse ATP-dependent nucleosome-remodelling complexes to carry out histone sliding, ejection or the incorporation of histone variants, suggesting that different mechanisms of action are used by the various chromatin-remodelling complex subfamilies. However, all chromatin-remodelling complex subfamilies contain an ATPase-translocase 'motor' that translocates DNA from a common location within the nucleosome. In this Review, we discuss (and illustrate with animations) an alternative, unifying mechanism of chromatin remodelling, which is based on the regulation of DNA translocation. We propose the 'hourglass' model of remodeller function, in which each remodeller subfamily utilizes diverse specialized proteins and protein domains to assist in nucleosome targeting or to differentially detect nucleosome epitopes. These modules converge to regulate a common DNA translocation mechanism, to inform the conserved ATPase 'motor' on whether and how to apply DNA translocation, which together achieve the various outcomes of chromatin remodelling: nucleosome assembly, chromatin access and nucleosome editing.
Asunto(s)
Adenosina Trifosfato/metabolismo , Ensamble y Desensamble de Cromatina/fisiología , ADN/metabolismo , Nucleosomas/metabolismo , Animales , Ensamble y Desensamble de Cromatina/genética , HumanosRESUMEN
Early vertebrate embryos must achieve totipotency and prepare for zygotic genome activation (ZGA). To understand this process, we determined the DNA methylation (DNAme) profiles of zebrafish gametes, embryos at different stages, and somatic muscle and compared them to gene activity and histone modifications. Sperm chromatin patterns are virtually identical to those at ZGA. Unexpectedly, the DNA of many oocyte genes important for germline functions (i.e., piwil1) or early development (i.e., hox genes) is methylated, but the loci are demethylated during zygotic cleavage stages to precisely the state observed in sperm, even in parthenogenetic embryos lacking a replicating paternal genome. Furthermore, this cohort constitutes the genes and loci that acquire DNAme during development (i.e., ZGA to muscle). Finally, DNA methyltransferase inhibition experiments suggest that DNAme silences particular gene and chromatin cohorts at ZGA, preventing their precocious expression. Thus, zebrafish achieve a totipotent chromatin state at ZGA through paternal genome competency and maternal genome DNAme reprogramming.
Asunto(s)
Metilación de ADN , Embrión no Mamífero/metabolismo , Pez Cebra/genética , Animales , Epigénesis Genética , Femenino , Fertilización , Masculino , Oocitos/metabolismo , Espermatozoides/metabolismo , Sitio de Iniciación de la Transcripción , Transcripción GenéticaRESUMEN
SWI/SNF-family remodelers (BAF/PBAF in mammals) are essential chromatin regulators, and mutations in human BAF/PBAF components are associated with â¼20% of cancers. Cancer-associated missense mutations in human BRG1 (encoding the catalytic ATPase) have been characterized previously as conferring loss-of-function. Here, we show that cancer-associated missense mutations in BRG1, when placed into the orthologous Sth1 ATPase of the yeast RSC remodeler, separate into two categories: loss-of-function enzymes, or instead, gain-of-function enzymes that greatly improve DNA translocation efficiency and nucleosome remodeling in vitro. Our work identifies a structural "hub," formed by the association of several Sth1 domains, that regulates ATPase activity and DNA translocation efficiency. Remarkably, all gain-of-function cancer-associated mutations and all loss-of-function mutations physically localize to distinct adjacent regions in the hub, which specifically regulate and implement DNA translocation, respectively. In vivo, only gain-of-function cancer-associated mutations conferred precocious chromatin accessibility. Taken together, we provide a structure-function mechanistic basis for cancer-associated hyperactivity.
Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Ensamble y Desensamble de Cromatina , Proteínas Cromosómicas no Histona/metabolismo , Mutación con Ganancia de Función , Neoplasias/patología , Proteínas Nucleares/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas Cromosómicas no Histona/genética , ADN Helicasas/genética , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Humanos , Neoplasias/genética , Proteínas Nucleares/genética , Nucleosomas , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crecimiento & desarrollo , Proteínas de Saccharomyces cerevisiae/genética , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Translocación GenéticaRESUMEN
Although genome-wide hypomethylation is a hallmark of many cancers, roles for active DNA demethylation during tumorigenesis are unknown. Here, loss of the APC tumor suppressor gene causes upregulation of a DNA demethylase system and the concomitant hypomethylation of key intestinal cell fating genes. Notably, this hypomethylation maintained zebrafish intestinal cells in an undifferentiated state that was released upon knockdown of demethylase components. Mechanistically, the demethylase genes are directly activated by Pou5f1 and Cebpß and are indirectly repressed by retinoic acid, which antagonizes Pou5f1 and Cebpß. Apc mutants lack retinoic acid as a result of the transcriptional repression of retinol dehydrogenase l1 via a complex that includes Lef1, Groucho2, Ctbp1, Lsd1, and Corest. Our findings imply a model wherein APC controls intestinal cell fating through a switch in DNA methylation dynamics. Wild-type APC and retinoic acid downregulate demethylase components, thereby promoting DNA methylation of key genes and helping progenitors commit to differentiation.
Asunto(s)
Proteína de la Poliposis Adenomatosa del Colon/metabolismo , Poliposis Adenomatosa del Colon/metabolismo , Metilación de ADN , Intestinos/embriología , Pez Cebra/embriología , Poliposis Adenomatosa del Colon/patología , Oxidorreductasas de Alcohol/metabolismo , Animales , Encéfalo/citología , Proteína beta Potenciadora de Unión a CCAAT/metabolismo , Línea Celular Tumoral , Proliferación Celular , Proteínas Co-Represoras/metabolismo , Neoplasias del Colon/metabolismo , Humanos , Mucosa Intestinal/metabolismo , Intestinos/citología , Factor 3 de Transcripción de Unión a Octámeros/metabolismo , Factores de Transcripción/metabolismo , Transcripción Genética , Tretinoina/metabolismoRESUMEN
The packaging of chromosomal DNA by nucleosomes condenses and organizes the genome, but occludes many regulatory DNA elements. However, this constraint also allows nucleosomes and other chromatin components to actively participate in the regulation of transcription, chromosome segregation, DNA replication, and DNA repair. To enable dynamic access to packaged DNA and to tailor nucleosome composition in chromosomal regions, cells have evolved a set of specialized chromatin remodeling complexes (remodelers). Remodelers use the energy of ATP hydrolysis to move, destabilize, eject, or restructure nucleosomes. Here, we address many aspects of remodeler biology: their targeting, mechanism, regulation, shared and unique properties, and specialization for particular biological processes. We also address roles for remodelers in development, cancer, and human syndromes.
Asunto(s)
Ensamble y Desensamble de Cromatina , Cromatina/metabolismo , Animales , Humanos , Nucleosomas/metabolismo , Plantas/química , Plantas/genética , Plantas/metabolismoRESUMEN
Chromatin architecture mapping in 3D formats has increased our understanding of how regulatory sequences and gene expression are connected and regulated in a genome. The 3D chromatin genome shows extensive remodeling during embryonic development, and although the cleavage-stage embryos of most species lack structure before zygotic genome activation (pre-ZGA), zebrafish has been reported to have structure. Here, we aimed to determine the chromosomal architecture in paternal/sperm zebrafish gamete cells to discern whether it either resembles or informs early pre-ZGA zebrafish embryo chromatin architecture. First, we assessed the higher-order architecture through advanced low-cell in situ Hi-C. The structure of zebrafish sperm, packaged by histones, lacks topological associated domains and instead displays "hinge-like" domains of â¼150 kb that repeat every 1-2 Mbs, suggesting a condensed repeating structure resembling mitotic chromosomes. The pre-ZGA embryos lacked chromosomal structure, in contrast to prior work, and only developed structure post-ZGA. During post-ZGA, we find chromatin architecture beginning to form at small contact domains of a median length of â¼90 kb. These small contact domains are established at enhancers, including super-enhancers, and chemical inhibition of Ep300a (p300) and Crebbpa (CBP) activity, lowering histone H3K27ac, but not transcription inhibition, diminishes these contacts. Together, this study reveals hinge-like domains in histone-packaged zebrafish sperm chromatin and determines that the initial formation of high-order chromatin architecture in zebrafish embryos occurs after ZGA primarily at enhancers bearing high H3K27ac.
Asunto(s)
Cromatina , Pez Cebra , Animales , Cromatina/genética , Cromatina/metabolismo , Cromosomas/genética , Desarrollo Embrionario/genética , Regulación del Desarrollo de la Expresión Génica , Masculino , Espermatozoides/metabolismo , Pez Cebra/genética , CigotoRESUMEN
Chromatin looping plays an important role in genome regulation. However, because ChIP-seq and loop-resolution Hi-C (DNA-DNA proximity ligation) are extremely challenging in mammalian early embryos, the developmental stage at which cohesin-mediated loops form remains unknown. Here, we study early development in medaka (the Japanese killifish, Oryzias latipes) at 12 time points before, during, and after gastrulation (the onset of cell differentiation) and characterize transcription, protein binding, and genome architecture. We find that gastrulation is associated with drastic changes in genome architecture, including the formation of the first loops between sites bound by the insulator protein CTCF and a large increase in the size of contact domains. In contrast, the binding of the CTCF is fixed throughout embryogenesis. Loops form long after genome-wide transcriptional activation, and long after domain formation seen in mouse embryos. These results suggest that, although loops may play a role in differentiation, they are not required for zygotic transcription. When we repeated our experiments in zebrafish, loops did not emerge until gastrulation, that is, well after zygotic genome activation. We observe that loop positions are highly conserved in synteny blocks of medaka and zebrafish, indicating that the 3D genome architecture has been maintained for >110-200 million years of evolution.
Asunto(s)
Oryzias , Animales , Factor de Unión a CCCTC/genética , Factor de Unión a CCCTC/metabolismo , Proteínas de Ciclo Celular/genética , Cromatina/genética , Gastrulación/genética , Ratones , Oryzias/genética , Pez Cebra/genéticaRESUMEN
The RSC chromatin remodeler slides and ejects nucleosomes, utilizing a catalytic subunit (Sth1) with DNA translocation activity, which can pump DNA around the nucleosome. A central question is whether and how DNA translocation is regulated to achieve sliding versus ejection. Here, we report the regulation of DNA translocation efficiency by two domains residing on Sth1 (Post-HSA and Protrusion 1) and by actin-related proteins (ARPs) that bind Sth1. ARPs facilitated sliding and ejection by improving "coupling"-the amount of DNA translocation by Sth1 relative to ATP hydrolysis. We also identified and characterized Protrusion 1 mutations that promote "coupling," and Post-HSA mutations that improve ATP hydrolysis; notably, the strongest mutations conferred efficient nucleosome ejection without ARPs. Taken together, sliding-to-ejection involves a continuum of DNA translocation efficiency, consistent with higher magnitudes of ATPase and coupling activities (involving ARPs and Sth1 domains), enabling the simultaneous rupture of multiple histone-DNA contacts facilitating ejection.
Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Ensamble y Desensamble de Cromatina , ADN de Hongos/genética , ADN de Hongos/metabolismo , Proteínas de Unión al ADN/metabolismo , Proteínas Nucleares/metabolismo , Nucleosomas/enzimología , Nucleosomas/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/enzimología , Saccharomyces cerevisiae/genética , Factores de Transcripción/metabolismo , Adenosina Trifosfato/metabolismo , Sitios de Unión , Transporte Biológico , Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/genética , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/genética , Hidrólisis , Proteínas de Microfilamentos/genética , Proteínas de Microfilamentos/metabolismo , Mutación , Proteínas Nucleares/química , Proteínas Nucleares/genética , Unión Proteica , Dominios y Motivos de Interacción de Proteínas , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Factores de Tiempo , Factores de Transcripción/química , Factores de Transcripción/genéticaRESUMEN
Evidence for active DNA demethylation in vertebrates is accumulating, but the mechanisms and enzymes remain unclear. Using zebrafish embryos we provide evidence for 5-methylcytosine (5-meC) removal in vivo via the coupling of a 5-meC deaminase (AID, which converts 5-meC to thymine) and a G:T mismatch-specific thymine glycosylase (Mbd4). The injection of methylated DNA into embryos induced a potent DNA demethylation activity, which was attenuated by depletion of AID or the non enzymatic factor Gadd45. Remarkably, overexpression of the deaminase/glycosylase pair AID/Mbd4 in vivo caused demethylation of the bulk genome and injected methylated DNA fragments, likely involving a G:T intermediate. Furthermore, AID or Mbd4 knockdown caused the remethylation of a set of common genes. Finally, Gadd45 promoted demethylation and enhanced functional interactions between deaminase/glycosylase pairs. Our results provide evidence for a coupled mechanism of 5-meC demethylation, whereby AID deaminates 5-meC, followed by thymine base excision by Mbd4, promoted by Gadd45.
Asunto(s)
ADN (Citosina-5-)-Metiltransferasas/metabolismo , ADN Glicosilasas/metabolismo , Metilación de ADN , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Timina ADN Glicosilasa/metabolismo , Proteínas de Pez Cebra/metabolismo , Pez Cebra/metabolismo , Animales , Línea Celular , Citidina Desaminasa/metabolismo , Embrión no Mamífero/metabolismo , Humanos , Neuropéptidos/metabolismo , Regulación hacia Arriba , Proteinas GADD45RESUMEN
Paternal cigarette smoke (CS) exposure is associated with increased risk of behavioral disorders and cancer in offspring, but the mechanism has not been identified. Here we use mouse models to investigate mechanisms and impacts of paternal CS exposure. We demonstrate that CS exposure induces sperm DNAme changes that are partially corrected within 28 days of removal from CS exposure. Additionally, paternal smoking is associated with changes in prefrontal cortex DNAme and gene expression patterns in offspring. Remarkably, the epigenetic and transcriptional effects of CS exposure that we observed in wild type mice are partially recapitulated in Nrf2-/- mice and their offspring, independent of smoking status. Nrf2 is a central regulator of antioxidant gene transcription, and mice lacking Nrf2 consequently display elevated oxidative stress, suggesting that oxidative stress may underlie CS-induced heritable epigenetic changes. Importantly, paternal sperm DNAme changes do not overlap with DNAme changes measured in offspring prefrontal cortex, indicating that the observed DNAme changes in sperm are not directly inherited. Additionally, the changes in sperm DNAme associated with CS exposure were not observed in sperm of unexposed offspring, suggesting the effects are likely not maintained across multiple generations.
Asunto(s)
Epigénesis Genética , Factor 2 Relacionado con NF-E2/genética , Exposición Paterna , Contaminación por Humo de Tabaco/efectos adversos , Animales , Metilación de ADN , Femenino , Masculino , Ratones , Ratones Endogámicos C57BL , Corteza Prefrontal/metabolismo , Espermatozoides/metabolismoRESUMEN
Postnatal spermatogonial stem cells (SSCs) progress through proliferative and developmental stages to populate the testicular niche prior to productive spermatogenesis. To better understand, we conducted extensive genomic profiling at multiple postnatal stages on subpopulations enriched for particular markers (THY1, KIT, OCT4, ID4, or GFRa1). Overall, our profiles suggest three broad populations of spermatogonia in juveniles: (1) epithelial-like spermatogonia (THY1(+); high OCT4, ID4, and GFRa1), (2) more abundant mesenchymal-like spermatogonia (THY1(+); moderate OCT4 and ID4; high mesenchymal markers), and (3) (in older juveniles) abundant spermatogonia committing to gametogenesis (high KIT(+)). Epithelial-like spermatogonia displayed the expected imprinting patterns, but, surprisingly, mesenchymal-like spermatogonia lacked imprinting specifically at paternally imprinted loci but fully restored imprinting prior to puberty. Furthermore, mesenchymal-like spermatogonia also displayed developmentally linked DNA demethylation at meiotic genes and also at certain monoallelic neural genes (e.g., protocadherins and olfactory receptors). We also reveal novel candidate receptor-ligand networks involving SSCs and the developing niche. Taken together, neonates/juveniles contain heterogeneous epithelial-like or mesenchymal-like spermatogonial populations, with the latter displaying extensive DNA methylation/chromatin dynamics. We speculate that this plasticity helps SSCs proliferate and migrate within the developing seminiferous tubule, with proper niche interaction and membrane attachment reverting mesenchymal-like spermatogonial subtype cells back to an epithelial-like state with normal imprinting profiles.
Asunto(s)
Células Madre Adultas/citología , Células Madre Adultas/fisiología , Diferenciación Celular , Regulación del Desarrollo de la Expresión Génica , Impresión Genómica/genética , Factores de Transcripción/genética , Animales , Cadherinas/genética , Células Cultivadas , Metilación de ADN , Epigenómica , Gametogénesis/genética , Perfilación de la Expresión Génica , Masculino , Ratones , Receptores Odorantes/genética , Transducción de Señal/genética , Antígenos Thy-1/metabolismoRESUMEN
Chromatin transactions are typically studied in vivo, or in vitro using artificial chromatin lacking the epigenetic complexity of the natural material. Attempting to bridge the gap between these approaches, we established a system for isolating the yeast genome as a library of mononucleosomes harboring the natural epigenetic signature, suitable for biochemical manipulation. Combined with deep sequencing, this library was used to investigate the stability of individual nucleosomes and, as proof of principle, the nucleosome preference of the chromatin remodeling complex, RSC. This approach uncovered a distinct preference of RSC for nucleosomes derived from regions with a high density of histone variant H2AZ, and this preference is indeed markedly diminished using nucleosomes from cells lacking H2AZ. The preference for H2AZ remodeling/nucleosome ejection can also be reconstituted with recombinant nucleosome arrays. Together, our data indicate that, despite being separated from their genomic context, individual nucleosomes can retain their original identity as promoter- or transcription start site (TSS)-nucleosomes. Besides shedding new light on substrate preference of the chromatin remodeler RSC, the simple experimental system outlined here should be generally applicable to the study of chromatin transactions.
Asunto(s)
Ensamble y Desensamble de Cromatina , Cromatina/genética , Cromatina/metabolismo , Estudio de Asociación del Genoma Completo , Histonas/metabolismo , Nucleosomas/metabolismo , Transcripción Genética , Regulación Fúngica de la Expresión Génica , Genoma Fúngico , Unión Proteica , Levaduras/genética , Levaduras/metabolismoRESUMEN
Polycomb group (PcG) proteins are transcriptional repressors that are important regulators of cell fate during embryonic development. Among them, Ezh2 is responsible for catalyzing the epigenetic repressive mark H3K27me3 and is essential for animal development. The ability of zebrafish embryos lacking both maternal and zygotic ezh2 to form a normal body plan provides a unique model for comprehensively studying Ezh2 function during early development in vertebrates. By using a multi-omics approach, we found that Ezh2 is required for the deposition of H3K27me3 and is essential for proper recruitment of Polycomb group protein Rnf2. However, despite the complete absence of PcG-associated epigenetic mark and proteins, only minor changes in H3K4me3 deposition and gene and protein expression occur. These changes were mainly due to local dysregulation of transcription factors outside their normal expression boundaries. Altogether, our results in zebrafish show that Polycomb-mediated gene repression is important immediately after the body plan is formed to maintain spatially restricted expression profiles of transcription factors, and we highlight the differences that exist in the timing of PcG protein action between vertebrate species.
Asunto(s)
Tipificación del Cuerpo/genética , Regulación del Desarrollo de la Expresión Génica , Proteínas del Grupo Polycomb/metabolismo , Proteínas Represoras/metabolismo , Vertebrados/embriología , Vertebrados/genética , Animales , Embrión no Mamífero/metabolismo , Epigénesis Genética , Histonas/metabolismo , Lisina/metabolismo , Metilación , Mutación/genética , Proteoma/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Factores de Transcripción/metabolismo , Transcriptoma/genética , Pez Cebra/embriología , Pez Cebra/genética , Cigoto/metabolismoRESUMEN
The breadth and importance of RNA modifications are growing rapidly as modified ribonucleotides can impact the sequence, structure, function, stability, and fate of RNAs and their interactions with other molecules. Therefore, knowing cellular RNA modifications at single-base resolution could provide important information regarding cell status and fate. A current major limitation is the lack of methods that allow the reproducible profiling of multiple modifications simultaneously, transcriptome-wide and at single-base resolution. Here we developed RBS-Seq, a modification of RNA bisulfite sequencing that enables the sensitive and simultaneous detection of m5C, Ψ, and m1A at single-base resolution transcriptome-wide. With RBS-Seq, m5C and m1A are accurately detected based on known signature base mismatches and are detected here simultaneously along with Ψ sites that show a 1-2 base deletion. Structural analyses revealed the mechanism underlying the deletion signature, which involves Ψ-monobisulfite adduction, heat-induced ribose ring opening, and Mg2+-assisted reorientation, causing base-skipping during cDNA synthesis. Detection of each of these modifications through a unique chemistry allows high-precision mapping of all three modifications within the same RNA molecule, enabling covariation studies. Application of RBS-Seq on HeLa RNA revealed almost all known m5C, m1A, and ψ sites in tRNAs and rRNAs and provided hundreds of new m5C and Ψ sites in noncoding RNAs and mRNAs. However, our results diverge greatly from earlier work, suggesting â¼10-fold fewer m5C sites in noncoding and coding RNAs and the absence of substantial m1A in mRNAs. Taken together, the approaches and refined datasets in this work will greatly enable future epitranscriptome studies.
Asunto(s)
Perfilación de la Expresión Génica/métodos , Procesamiento Postranscripcional del ARN/fisiología , ARN Mensajero , ARN Ribosómico , ARN de Transferencia , Análisis de Secuencia de ARN/métodos , Células HeLa , Humanos , ARN Mensajero/genética , ARN Mensajero/metabolismo , ARN Ribosómico/genética , ARN Ribosómico/metabolismo , ARN de Transferencia/genética , ARN de Transferencia/metabolismoRESUMEN
Chromatin-remodelling complexes (CRCs) mobilize nucleosomes to mediate the access of DNA-binding factors to their sites in vivo. These CRCs contain a catalytic subunit that bears an ATPase/DNA-translocase domain and flanking regions that bind nucleosomal epitopes. A central question is whether and how these flanking regions regulate ATP hydrolysis or the coupling of hydrolysis to DNA translocation, to affect nucleosome-sliding efficiency. ISWI-family CRCs contain the protein ISWI, which uses its ATPase/DNA-translocase domain to pump DNA around the histone octamer to enable sliding. ISWI is positively regulated by two 'activating' nucleosomal epitopes: the 'basic patch' on the histone H4 tail, and extranucleosomal (linker) DNA. Previous work defined the HAND-SANT-SLIDE (HSS) domain at the ISWI carboxy terminus that binds linker DNA, needed for ISWI activity. Here we define two new, conserved and separate regulatory regions on Drosophila ISWI, termed AutoN and NegC, which negatively regulate ATP hydrolysis (AutoN) or the coupling of ATP hydrolysis to productive DNA translocation (NegC). The two aforementioned nucleosomal epitopes promote remodelling indirectly by preventing the negative regulation of AutoN and NegC. Notably, mutation or removal of AutoN and NegC enables marked nucleosome sliding without the H4 basic patch or extranucleosomal DNA, or the HSS domain, conferring on ISWI the biochemical attributes normally associated with SWI/SNF-family ATPases. Thus, the ISWI ATPase catalytic core is an intrinsically active DNA translocase that conducts nucleosome sliding, onto which selective 'inhibition-of-inhibition' modules are placed, to help ensure that remodelling occurs only in the presence of proper nucleosomal epitopes. This supports a general concept for the specialization of chromatin-remodelling ATPases, in which specific regulatory modules adapt an ancient active DNA translocase to conduct particular tasks only on the appropriate chromatin landscape.
Asunto(s)
Adenosina Trifosfatasas/genética , Adenosina Trifosfatasas/metabolismo , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Epítopos/metabolismo , Regulación de la Expresión Génica , Nucleosomas/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Adenosina Trifosfatasas/química , Adenosina Trifosfato/metabolismo , Secuencia de Aminoácidos , Animales , Ensamble y Desensamble de Cromatina , Proteínas de Drosophila/química , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/enzimología , Hidrólisis , Modelos Moleculares , Datos de Secuencia Molecular , Mutación , Unión Proteica , Estructura Terciaria de Proteína , Secuencias Reguladoras de Ácidos Nucleicos/genética , Alineación de Secuencia , Factores de Transcripción/químicaRESUMEN
Recent evidence demonstrates a role for paternal aging on offspring disease susceptibility. It is well established that various neuropsychiatric disorders (schizophrenia, autism, etc.), trinucleotide expansion associated diseases (myotonic dystrophy, Huntington's, etc.) and even some forms of cancer have increased incidence in the offspring of older fathers. Despite strong epidemiological evidence that these alterations are more common in offspring sired by older fathers, in most cases the mechanisms that drive these processes are unclear. However, it is commonly believed that epigenetics, and specifically DNA methylation alterations, likely play a role. In this study we have investigated the impact of aging on DNA methylation in mature human sperm. Using a methylation array approach we evaluated changes to sperm DNA methylation patterns in 17 fertile donors by comparing the sperm methylome of 2 samples collected from each individual 9-19 years apart. With this design we have identified 139 regions that are significantly and consistently hypomethylated with age and 8 regions that are significantly hypermethylated with age. A representative subset of these alterations have been confirmed in an independent cohort. A total of 117 genes are associated with these regions of methylation alterations (promoter or gene body). Intriguingly, a portion of the age-related changes in sperm DNA methylation are located at genes previously associated with schizophrenia and bipolar disorder. While our data does not establish a causative relationship, it does raise the possibility that the age-associated methylation of the candidate genes that we observe in sperm might contribute to the increased incidence of neuropsychiatric and other disorders in the offspring of older males. However, further study is required to determine whether, and to what extent, a causative relationship exists.
Asunto(s)
Envejecimiento/genética , Metilación de ADN/genética , Epigénesis Genética , Espermatozoides/patología , Adulto , Envejecimiento/patología , Animales , Trastorno Autístico/genética , Trastorno Bipolar/genética , Trastorno Bipolar/patología , Predisposición Genética a la Enfermedad , Humanos , Masculino , Persona de Mediana Edad , Edad Paterna , Esquizofrenia/genética , Esquizofrenia/patología , Espermatozoides/metabolismoRESUMEN
Histone H3 lysine 4 (H3K4) methylation is a dynamic modification. In budding yeast, H3K4 methylation is catalyzed by the Set1-COMPASS methyltransferase complex and is removed by Jhd2, a JMJC domain family demethylase. The catalytic JmjC and JmjN domains of Jhd2 have the ability to remove all three degrees (mono-, di-, and tri-) of H3K4 methylation. Jhd2 also contains a plant homeodomain (PHD) finger required for its chromatin association and H3K4 demethylase functions. The Jhd2 PHD finger associates with chromatin independent of H3K4 methylation and the H3 N-terminal tail. Therefore, how Jhd2 associates with chromatin to perform H3K4 demethylation has remained unknown. We report a novel interaction between the Jhd2 PHD finger and histone H2A. Two residues in H2A (Phe-26 and Glu-57) serve as a binding site for Jhd2 in vitro and mediate its chromatin association and H3K4 demethylase functions in vivo. Using RNA sequencing, we have identified the functional target genes for Jhd2 and the H2A Phe-26 and Glu-57 residues. We demonstrate that H2A Phe-26 and Glu-57 residues control chromatin association and H3K4 demethylase functions of Jhd2 during positive or negative regulation of transcription at target genes. Importantly, we show that H2B Lys-123 ubiquitination blocks Jhd2 from accessing its binding site on chromatin, and thereby, we have uncovered a second mechanism by which H2B ubiquitination contributes to the trans-histone regulation of H3K4 methylation. Overall, our study provides novel insights into the chromatin binding dynamics and H3K4 demethylase functions of Jhd2.