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1.
Mol Cell ; 77(4): 825-839.e7, 2020 02 20.
Artículo en Inglés | MEDLINE | ID: mdl-31837995

RESUMEN

In mammals, chromatin organization undergoes drastic reorganization during oocyte development. However, the dynamics of three-dimensional chromatin structure in this process is poorly characterized. Using low-input Hi-C (genome-wide chromatin conformation capture), we found that a unique chromatin organization gradually appears during mouse oocyte growth. Oocytes at late stages show self-interacting, cohesin-independent compartmental domains marked by H3K27me3, therefore termed Polycomb-associating domains (PADs). PADs and inter-PAD (iPAD) regions form compartment-like structures with strong inter-domain interactions among nearby PADs. PADs disassemble upon meiotic resumption from diplotene arrest but briefly reappear on the maternal genome after fertilization. Upon maternal depletion of Eed, PADs are largely intact in oocytes, but their reestablishment after fertilization is compromised. By contrast, depletion of Polycomb repressive complex 1 (PRC1) proteins attenuates PADs in oocytes, which is associated with substantial gene de-repression in PADs. These data reveal a critical role of Polycomb in regulating chromatin architecture during mammalian oocyte growth and early development.


Asunto(s)
Cromatina/química , Oocitos/crecimiento & desarrollo , Oogénesis/genética , Proteínas del Grupo Polycomb/fisiología , Animales , Blastocisto/química , Proteínas de Ciclo Celular/fisiología , Proteínas Cromosómicas no Histona/fisiología , Embrión de Mamíferos/química , Silenciador del Gen , Código de Histonas , Ratones , Oocitos/química , Transcripción Genética , Cohesinas
2.
PLoS Genet ; 19(8): e1010855, 2023 08.
Artículo en Inglés | MEDLINE | ID: mdl-37527244

RESUMEN

Establishment of a proper DNA methylation landscape in mammalian oocytes is important for maternal imprinting and embryonic development. De novo DNA methylation in oocytes is mediated by the DNA methyltransferase DNMT3A, which has an ATRX-DNMT3-DNMT3L (ADD) domain that interacts with histone H3 tail unmethylated at lysine-4 (H3K4me0). The domain normally blocks the methyltransferase domain via intramolecular interaction and binding to histone H3K4me0 releases the autoinhibition. However, H3K4me0 is widespread in chromatin and the role of the ADD-histone interaction has not been studied in vivo. We herein show that amino-acid substitutions in the ADD domain of mouse DNMT3A cause dwarfism. Oocytes derived from homozygous females show mosaic loss of CG methylation and almost complete loss of non-CG methylation. Embryos derived from such oocytes die in mid-to-late gestation, with stochastic and often all-or-none-type CG-methylation loss at imprinting control regions and misexpression of the linked genes. The stochastic loss is a two-step process, with loss occurring in cleavage-stage embryos and regaining occurring after implantation. These results highlight an important role for the ADD domain in efficient, and likely processive, de novo CG methylation and pose a model for stochastic inheritance of epigenetic perturbations in germ cells to the next generation.


Asunto(s)
Metilación de ADN , Histonas , Humanos , Femenino , Ratones , Masculino , Animales , Embarazo , Histonas/metabolismo , Metilación de ADN/genética , ADN (Citosina-5-)-Metiltransferasas/metabolismo , Cromosomas Humanos Y , ADN Metiltransferasa 3A , Mosaicismo , Oocitos/metabolismo , Factores de Transcripción/genética , Metilasas de Modificación del ADN , Mamíferos/genética
3.
BMC Genomics ; 25(1): 344, 2024 Apr 05.
Artículo en Inglés | MEDLINE | ID: mdl-38580899

RESUMEN

BACKGROUND: Genome-wide DNA demethylation occurs in mammalian primordial germ cells (PGCs) as part of the epigenetic reprogramming important for gametogenesis and resetting the epigenetic information for totipotency. Dppa3 (also known as Stella or Pgc7) is highly expressed in mouse PGCs and oocytes and encodes a factor essential for female fertility. It prevents excessive DNA methylation in oocytes and ensures proper gene expression in preimplantation embryos: however, its role in PGCs is largely unexplored. In the present study, we investigated whether or not DPPA3 has an impact on CG methylation/demethylation in mouse PGCs. RESULTS: We show that DPPA3 plays a role in genome-wide demethylation in PGCs even before sex differentiation. Dppa3 knockout female PGCs show aberrant hypermethylation, most predominantly at H3K9me3-marked retrotransposons, which persists up to the fully-grown oocyte stage. DPPA3 works downstream of PRDM14, a master regulator of epigenetic reprogramming in embryonic stem cells and PGCs, and independently of TET1, an enzyme that hydroxylates 5-methylcytosine. CONCLUSIONS: The results suggest that DPPA3 facilitates DNA demethylation through a replication-coupled passive mechanism in PGCs. Our study identifies DPPA3 as a novel epigenetic reprogramming factor in mouse PGCs.


Asunto(s)
Proteínas Cromosómicas no Histona , Desmetilación del ADN , Epigénesis Genética , Animales , Femenino , Ratones , Proteínas Cromosómicas no Histona/metabolismo , Metilación de ADN , Genoma , Células Germinativas/metabolismo , Mamíferos/genética
4.
BMC Bioinformatics ; 23(1): 371, 2022 Sep 12.
Artículo en Inglés | MEDLINE | ID: mdl-36096737

RESUMEN

BACKGROUND: Epigenetic modifications established in mammalian gametes are largely reprogrammed during early development, however, are partly inherited by the embryo to support its development. In this study, we examine CpG island (CGI) sequences to predict whether a mouse blastocyst CGI inherits oocyte-derived DNA methylation from the maternal genome. Recurrent neural networks (RNNs), including that based on gated recurrent units (GRUs), have recently been employed for variable-length inputs in classification and regression analyses. One advantage of this strategy is the ability of RNNs to automatically learn latent features embedded in inputs by learning their model parameters. However, the available CGI dataset applied for the prediction of oocyte-derived DNA methylation inheritance are not large enough to train the neural networks. RESULTS: We propose a GRU-based model called CMIC (CGI Methylation Inheritance Classifier) to augment CGI sequence by converting it into variable-length k-mers, where the length k is randomly selected from the range [Formula: see text] to [Formula: see text], N times, which were then used as neural network input. N was set to 1000 in the default setting. In addition, we proposed a new embedding vector generator for k-mers called splitDNA2vec. The randomness of this procedure was higher than the previous work, dna2vec. CONCLUSIONS: We found that CMIC can predict the inheritance of oocyte-derived DNA methylation at CGIs in the maternal genome of blastocysts with a high F-measure (0.93). We also show that the F-measure can be improved by increasing the parameter N, that is, the number of sequences of variable-length k-mers derived from a single CGI sequence. This implies the effectiveness of augmenting input data by converting a DNA sequence to N sequences of variable-length k-mers. This approach can be applied to different DNA sequence classification and regression analyses, particularly those involving a small amount of data.


Asunto(s)
Metilación de ADN , Bases de Datos Genéticas , Animales , Carbazoles , Islas de CpG , Patrón de Herencia , Mamíferos/genética , Ratones
5.
BMC Bioinformatics ; 22(1): 341, 2021 Jun 23.
Artículo en Inglés | MEDLINE | ID: mdl-34162326

RESUMEN

BACKGROUND: Epigenetic modifications, including CG methylation (a major form of DNA methylation) and histone modifications, interact with each other to shape their genomic distribution patterns. However, the entire picture of the epigenetic crosstalk regulating the CG methylation pattern is unknown especially in cells that are available only in a limited number, such as mammalian oocytes. Most machine learning approaches developed so far aim at finding DNA sequences responsible for the CG methylation patterns and were not tailored for studying the epigenetic crosstalk. RESULTS: We built a machine learning model named epiNet to predict CG methylation patterns based on other epigenetic features, such as histone modifications, but not DNA sequence. Using epiNet, we identified biologically relevant epigenetic crosstalk between histone H3K36me3, H3K4me3, and CG methylation in mouse oocytes. This model also predicted the altered CG methylation pattern of mutant oocytes having perturbed histone modification, was applicable to cross-species prediction of the CG methylation pattern of human oocytes, and identified the epigenetic crosstalk potentially important in other cell types. CONCLUSIONS: Our findings provide insight into the epigenetic crosstalk regulating the CG methylation pattern in mammalian oocytes and other cells. The use of epiNet should help to design or complement biological experiments in epigenetics studies.


Asunto(s)
Metilación de ADN , Epigénesis Genética , Animales , Epigenómica , Código de Histonas , Ratones , Redes Neurales de la Computación
6.
Genes Cells ; 25(1): 54-64, 2020 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-31733167

RESUMEN

Monoallelic gene expression occurs in various mammalian cells and can be regulated genetically, epigenetically and/or stochastically. We identified 145 monoallelically expressed genes (MoEGs), including seven known imprinted genes, in mouse embryonic stem cells (ESCs) derived from reciprocal F1 hybrid blastocysts and cultured in 2i/LIF. As all MoEGs except for the imprinted genes were expressed in a genetic-origin-dependent manner, we focused on this class of MoEGs for mechanistic studies. We showed that a majority of the genetic-origin-dependent MoEGs identified in 2i/LIF ESCs remain monoallelically expressed in serum/LIF ESCs, but become more relaxed or even biallelically expressed upon differentiation. These MoEGs and their regulatory regions were highly enriched for single nucleotide polymorphisms. In addition, some MoEGs were associated with retrotransposon insertions/deletions, consistent with the fact that certain retrotransposons act as regulatory elements in pluripotent stem cells. Interestingly, most MoEGs showed allelic differences in enrichment of histone H3K27me and H3K4me marks, linking allelic epigenetic differences and monoallelic expression. In contrast, there was little or no allelic difference in CpG methylation or H3K9me. Taken together, our study highlights the impact of genetic variation including single nucleotide polymorphisms and retrotransposon insertions/deletions on monoallelic epigenetic marks and expression in ESCs.


Asunto(s)
Regulación del Desarrollo de la Expresión Génica/genética , Células Madre Embrionarias de Ratones/metabolismo , Transcriptoma/genética , Alelos , Animales , Diferenciación Celular/genética , Línea Celular , Metilación de ADN/genética , Células Madre Embrionarias/metabolismo , Epigénesis Genética/genética , Epigenómica/métodos , Femenino , Expresión Génica/genética , Perfilación de la Expresión Génica/métodos , Impresión Genómica/genética , Masculino , Ratones , Ratones Endogámicos , Células Madre Pluripotentes/metabolismo
7.
Methods Mol Biol ; 2509: 233-250, 2022.
Artículo en Inglés | MEDLINE | ID: mdl-35796967

RESUMEN

Whole genome bisulfite sequencing (WGBS) is a high-throughput DNA sequencing-based technique that is used to determine genome-wide DNA methylation patterns at base resolution. Library construction by post-bisulfite adaptor tagging (PBAT ) extends the application of WGBS to several hundred cells and minimizes the required number of library amplification cycles. We herein describe a PBAT protocol to prepare WGBS libraries from 200 cells and introduce the outline of a downstream bioinformatic analysis. The prepared library can typically generate 800 million sequencing reads, which is sufficient to cover the human and mouse genomes approximately 15 times, using the Illumina NovaSeq 6000 sequencing system.


Asunto(s)
Metilación de ADN , Sulfitos , Animales , ADN/genética , Biblioteca de Genes , Genoma , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Ratones , Análisis de Secuencia de ADN/métodos , Secuenciación Completa del Genoma/métodos
8.
Nat Cell Biol ; 23(9): 1002-1012, 2021 09.
Artículo en Inglés | MEDLINE | ID: mdl-34489571

RESUMEN

Many animals have a conserved adaptive genome defence system known as the Piwi-interacting RNA (piRNA) pathway, which is essential for germ cell development and function. Disruption of individual mouse Piwi genes results in male but not female sterility, leading to the assumption that PIWI genes play little or no role in mammalian oocytes. Here, we report the generation of PIWI-defective golden hamsters, which have defects in the production of functional oocytes. The mechanisms involved vary among the hamster PIWI genes, whereby the lack of PIWIL1 has a major impact on gene expression, including hamster-specific young transposon de-silencing, whereas PIWIL3 deficiency has little impact on gene expression in oocytes, although DNA methylation was reduced to some extent in PIWIL3-deficient oocytes. Our findings serve as the foundation for developing useful models to study the piRNA pathway in mammalian oocytes, including humans.


Asunto(s)
Mesocricetus/metabolismo , Oocitos/metabolismo , ARN Interferente Pequeño/genética , Testículo/metabolismo , Animales , Proteínas Argonautas/genética , Cricetinae , Metilación de ADN/fisiología , Expresión Génica/fisiología , Células Germinativas/metabolismo , Masculino
9.
Nat Commun ; 11(1): 5417, 2020 10 27.
Artículo en Inglés | MEDLINE | ID: mdl-33110091

RESUMEN

De novo DNA methylation (DNAme) during mammalian spermatogenesis yields a densely methylated genome, with the exception of CpG islands (CGIs), which are hypomethylated in sperm. While the paternal genome undergoes widespread DNAme loss before the first S-phase following fertilization, recent mass spectrometry analysis revealed that the zygotic paternal genome is paradoxically also subject to a low level of de novo DNAme. However, the loci involved, and impact on transcription were not addressed. Here, we employ allele-specific analysis of whole-genome bisulphite sequencing data and show that a number of genomic regions, including several dozen CGI promoters, are de novo methylated on the paternal genome by the 2-cell stage. A subset of these promoters maintains DNAme through development to the blastocyst stage. Consistent with paternal DNAme acquisition, many of these loci are hypermethylated in androgenetic blastocysts but hypomethylated in parthenogenetic blastocysts. Paternal DNAme acquisition is lost following maternal deletion of Dnmt3a, with a subset of promoters, which are normally transcribed from the paternal allele in blastocysts, being prematurely transcribed at the 4-cell stage in maternal Dnmt3a knockout embryos. These observations uncover a role for maternal DNMT3A activity in post-fertilization epigenetic reprogramming and transcriptional silencing of the paternal genome.


Asunto(s)
Blastocisto/metabolismo , ADN (Citosina-5-)-Metiltransferasas/metabolismo , Genoma , Herencia Materna , Herencia Paterna , Alelos , Animales , Islas de CpG , ADN (Citosina-5-)-Metiltransferasas/genética , Metilación de ADN , ADN Metiltransferasa 3A , Epigenómica , Femenino , Regulación del Desarrollo de la Expresión Génica , Masculino , Ratones Endogámicos DBA , Oocitos/metabolismo , Espermatozoides/metabolismo
10.
Cell Rep ; 27(1): 282-293.e4, 2019 04 02.
Artículo en Inglés | MEDLINE | ID: mdl-30943408

RESUMEN

Mammalian histone methyltransferase G9a (also called EHMT2) deposits H3K9me2 on chromatin and is essential for postimplantation development. However, its role in oogenesis and preimplantation development remains poorly understood. We show that H3K9me2-enriched chromatin domains in mouse oocytes are generally depleted of CG methylation, contrasting with their association in embryonic stem and somatic cells. Oocyte-specific disruption of G9a results in reduced H3K9me2 enrichment and impaired reorganization of heterochromatin in oocytes, but only a modest reduction in CG methylation is detected. Furthermore, in both oocytes and 2-cell embryos, G9a depletion has limited impact on the expression of genes and retrotransposons. Although their CG methylation is minimally affected, preimplantation embryos derived from such oocytes show abnormal chromosome segregation and frequent developmental arrest. Our findings illuminate the functional importance of G9a independent of CG methylation in preimplantation development and call into question the proposed role for H3K9me2 in CG methylation protection in zygotes.


Asunto(s)
Blastocisto/metabolismo , Metilación de ADN , Código de Histonas , N-Metiltransferasa de Histona-Lisina/metabolismo , Oocitos/metabolismo , Animales , Segregación Cromosómica , Femenino , N-Metiltransferasa de Histona-Lisina/genética , Histonas/química , Histonas/metabolismo , Ratones , Ratones Endogámicos C57BL
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