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1.
Nature ; 587(7832): 139-144, 2020 11.
Artigo em Inglês | MEDLINE | ID: mdl-33116310

RESUMO

Zygotic genome activation (ZGA) is the first transcription event in life1. However, it is unclear how RNA polymerase is engaged in initiating ZGA in mammals. Here, by developing small-scale Tn5-assisted chromatin cleavage with sequencing (Stacc-seq), we investigated the landscapes of RNA polymerase II (Pol II) binding in mouse embryos. We found that Pol II undergoes 'loading', 'pre-configuration', and 'production' during the transition from minor ZGA to major ZGA. After fertilization, Pol II is preferentially loaded to CG-rich promoters and accessible distal regions in one-cell embryos (loading), in part shaped by the inherited parental epigenome. Pol II then initiates relocation to future gene targets before genome activation (pre-configuration), where it later engages in full transcription elongation upon major ZGA (production). Pol II also maintains low poising at inactive promoters after major ZGA until the blastocyst stage, coinciding with the loss of promoter epigenetic silencing factors. Notably, inhibition of minor ZGA impairs the Pol II pre-configuration and embryonic development, accompanied by aberrant retention of Pol II and ectopic expression of one-cell targets upon major ZGA. Hence, stepwise transition of Pol II occurs when mammalian life begins, and minor ZGA has a key role in the pre-configuration of transcription machinery and chromatin for genome activation.


Assuntos
Regulação da Expressão Gênica no Desenvolvimento/genética , Genoma/genética , RNA Polimerase II/metabolismo , Zigoto/metabolismo , Alelos , Animais , Cromatina/genética , Cromatina/metabolismo , Embrião de Mamíferos/citologia , Embrião de Mamíferos/enzimologia , Embrião de Mamíferos/metabolismo , Epigenoma/genética , Feminino , Masculino , Herança Materna/genética , Camundongos , Camundongos Endogâmicos C57BL , Oócitos/enzimologia , Oócitos/metabolismo , Regiões Promotoras Genéticas/genética , RNA Polimerase II/genética , Zigoto/citologia , Zigoto/enzimologia
2.
Nature ; 576(7787): 487-491, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31827285

RESUMO

Formation of the three primary germ layers during gastrulation is an essential step in the establishment of the vertebrate body plan and is associated with major transcriptional changes1-5. Global epigenetic reprogramming accompanies these changes6-8, but the role of the epigenome in regulating early cell-fate choice remains unresolved, and the coordination between different molecular layers is unclear. Here we describe a single-cell multi-omics map of chromatin accessibility, DNA methylation and RNA expression during the onset of gastrulation in mouse embryos. The initial exit from pluripotency coincides with the establishment of a global repressive epigenetic landscape, followed by the emergence of lineage-specific epigenetic patterns during gastrulation. Notably, cells committed to mesoderm and endoderm undergo widespread coordinated epigenetic rearrangements at enhancer marks, driven by ten-eleven translocation (TET)-mediated demethylation and a concomitant increase of accessibility. By contrast, the methylation and accessibility landscape of ectodermal cells is already established in the early epiblast. Hence, regulatory elements associated with each germ layer are either epigenetically primed or remodelled before cell-fate decisions, providing the molecular framework for a hierarchical emergence of the primary germ layers.


Assuntos
Metilação de DNA , Epigênese Genética , Gástrula/citologia , Gástrula/metabolismo , Gastrulação/genética , Regulação da Expressão Gênica no Desenvolvimento , RNA/genética , Análise de Célula Única , Animais , Diferenciação Celular/genética , Linhagem da Célula/genética , Cromatina/genética , Cromatina/metabolismo , Desmetilação , Corpos Embrioides/citologia , Endoderma/citologia , Endoderma/embriologia , Endoderma/metabolismo , Elementos Facilitadores Genéticos/genética , Epigenoma/genética , Eritropoese , Análise Fatorial , Gástrula/embriologia , Gastrulação/fisiologia , Mesoderma/citologia , Mesoderma/embriologia , Mesoderma/metabolismo , Camundongos , Células-Tronco Pluripotentes/citologia , Células-Tronco Pluripotentes/metabolismo , RNA/análise , Fatores de Tempo , Dedos de Zinco
3.
Mol Cell ; 63(6): 1066-79, 2016 09 15.
Artigo em Inglês | MEDLINE | ID: mdl-27635762

RESUMO

Polycomb group proteins and the related histone modification H3K27me3 can maintain the silencing of key developmental regulators and provide cellular memory. However, how such an epigenetic state is reprogrammed and inherited between generations is poorly understood. Using an ultra-sensitive approach, STAR ChIP-seq, we investigated H3K27me3 across 14 developmental stages along mouse gametogenesis and early development. Interestingly, highly pervasive H3K27me3 is found in regions depleted of transcription and DNA methylation in oocytes. Unexpectedly, we observed extensive loss of promoter H3K27me3 at Hox and other developmental genes upon fertilization. This is accompanied by global erasure of sperm H3K27me3 but inheritance of distal H3K27me3 from oocytes. The resulting allele-specific H3K27me3 patterns persist to blastocysts before being converted to canonical forms in postimplantation embryos, where both H3K4me3/H3K27me3 bivalent promoter marks are restored at developmental genes. Together, these data revealed widespread resetting of epigenetic memory and striking plasticity of epigenome during gametogenesis and early development.


Assuntos
Epigênese Genética , Regulação da Expressão Gênica no Desenvolvimento , Histonas/genética , Oócitos/metabolismo , Proteínas do Grupo Polycomb/genética , Espermatozoides/metabolismo , Animais , Reprogramação Celular , Embrião de Mamíferos , Desenvolvimento Embrionário/genética , Feminino , Fertilização , Gametogênese/genética , Histonas/metabolismo , Padrões de Herança , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Oócitos/citologia , Oócitos/crescimento & desenvolvimento , Proteínas do Grupo Polycomb/metabolismo , Regiões Promotoras Genéticas , Espermatozoides/citologia , Espermatozoides/crescimento & desenvolvimento , Zigoto
4.
Nature ; 547(7662): 232-235, 2017 07 12.
Artigo em Inglês | MEDLINE | ID: mdl-28703188

RESUMO

In mammals, chromatin organization undergoes drastic reprogramming after fertilization. However, the three-dimensional structure of chromatin and its reprogramming in preimplantation development remain poorly understood. Here, by developing a low-input Hi-C (genome-wide chromosome conformation capture) approach, we examined the reprogramming of chromatin organization during early development in mice. We found that oocytes in metaphase II show homogeneous chromatin folding that lacks detectable topologically associating domains (TADs) and chromatin compartments. Strikingly, chromatin shows greatly diminished higher-order structure after fertilization. Unexpectedly, the subsequent establishment of chromatin organization is a prolonged process that extends through preimplantation development, as characterized by slow consolidation of TADs and segregation of chromatin compartments. The two sets of parental chromosomes are spatially separated from each other and display distinct compartmentalization in zygotes. Such allele separation and allelic compartmentalization can be found as late as the 8-cell stage. Finally, we show that chromatin compaction in preimplantation embryos can partially proceed in the absence of zygotic transcription and is a multi-level hierarchical process. Taken together, our data suggest that chromatin may exist in a markedly relaxed state after fertilization, followed by progressive maturation of higher-order chromatin architecture during early development.


Assuntos
Alelos , Montagem e Desmontagem da Cromatina/genética , Cromatina/química , Cromatina/genética , Cromossomos de Mamíferos/química , Cromossomos de Mamíferos/genética , Desenvolvimento Embrionário/genética , Animais , Blastocisto/metabolismo , Cromatina/metabolismo , Cromossomos de Mamíferos/metabolismo , Feminino , Fertilização , Regulação da Expressão Gênica no Desenvolvimento , Masculino , Camundongos , Transcrição Gênica , Zigoto/metabolismo
5.
Nucleic Acids Res ; 49(5): 2569-2582, 2021 03 18.
Artigo em Inglês | MEDLINE | ID: mdl-33621320

RESUMO

During oogenesis, oocytes gain competence and subsequently undergo meiotic maturation and prepare for embryonic development; trimethylated histone H3 on lysine-4 (H3K4me3) mediates a wide range of nuclear events during these processes. Oocyte-specific knockout of CxxC-finger protein 1 (CXXC1, also known as CFP1) impairs H3K4me3 accumulation and causes changes in chromatin configurations. This study investigated the changes in genomic H3K4me3 landscapes in oocytes with Cxxc1 knockout and the effects on other epigenetic factors such as the DNA methylation, H3K27me3, H2AK119ub1 and H3K36me3. H3K4me3 is overall decreased after knocking out Cxxc1, including both the promoter region and the gene body. CXXC1 and MLL2, which is another histone H3 methyltransferase, have nonoverlapping roles in mediating H3K4 trimethylation during oogenesis. Cxxc1 deletion caused a decrease in DNA methylation levels and affected H3K27me3 and H2AK119ub1 distributions, particularly at regions with high DNA methylation levels. The changes in epigenetic networks implicated by Cxxc1 deletion were correlated with the transcriptional changes in genes in the corresponding genomic regions. This study elucidates the epigenetic changes underlying the phenotypes and molecular defects in oocytes with deleted Cxxc1 and highlights the role of CXXC1 in orchestrating multiple factors that are involved in establishing the appropriate epigenetic states of maternal genome.


Assuntos
Epigênese Genética , Oócitos/metabolismo , Transativadores/fisiologia , Animais , Células Cultivadas , Metilação de DNA , Feminino , Deleção de Genes , Genoma , Código das Histonas , Histonas/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Regiões Promotoras Genéticas , Transativadores/genética , Transcrição Gênica
6.
Nature ; 537(7621): 553-557, 2016 09 22.
Artigo em Inglês | MEDLINE | ID: mdl-27626382

RESUMO

Histone modifications are fundamental epigenetic regulators that control many crucial cellular processes. However, whether these marks can be passed on from mammalian gametes to the next generation is a long-standing question that remains unanswered. Here, by developing a highly sensitive approach, STAR ChIP-seq, we provide a panoramic view of the landscape of H3K4me3, a histone hallmark for transcription initiation, from developing gametes to post-implantation embryos. We find that upon fertilization, extensive reprogramming occurs on the paternal genome, as H3K4me3 peaks are depleted in zygotes but are readily observed after major zygotic genome activation at the late two-cell stage. On the maternal genome, we unexpectedly find a non-canonical form of H3K4me3 (ncH3K4me3) in full-grown and mature oocytes, which exists as broad peaks at promoters and a large number of distal loci. Such broad H3K4me3 peaks are in contrast to the typical sharp H3K4me3 peaks restricted to CpG-rich regions of promoters. Notably, ncH3K4me3 in oocytes overlaps almost exclusively with partially methylated DNA domains. It is then inherited in pre-implantation embryos, before being erased in the late two-cell embryos, when canonical H3K4me3 starts to be established. The removal of ncH3K4me3 requires zygotic transcription but is independent of DNA replication-mediated passive dilution. Finally, downregulation of H3K4me3 in full-grown oocytes by overexpression of the H3K4me3 demethylase KDM5B is associated with defects in genome silencing. Taken together, these data unveil inheritance and highly dynamic reprogramming of the epigenome in early mammalian development.


Assuntos
Alelos , Metilação de DNA , Embrião de Mamíferos/embriologia , Embrião de Mamíferos/metabolismo , Inativação Gênica , Histonas/metabolismo , Lisina/metabolismo , Animais , Reprogramação Celular/genética , Imunoprecipitação da Cromatina , Ilhas de CpG/genética , Replicação do DNA , Proteínas de Ligação a DNA/metabolismo , Feminino , Fertilização/genética , Genoma/genética , Histonas/química , Histona Desmetilases com o Domínio Jumonji/metabolismo , Masculino , Metilação , Camundongos , Oócitos/metabolismo , Regiões Promotoras Genéticas , Análise de Sequência de DNA , Iniciação da Transcrição Genética , Zigoto/metabolismo
7.
Nature ; 534(7609): 652-7, 2016 06 30.
Artigo em Inglês | MEDLINE | ID: mdl-27309802

RESUMO

In mammals, extensive chromatin reorganization is essential for reprogramming terminally committed gametes to a totipotent state during preimplantation development. However, the global chromatin landscape and its dynamics in this period remain unexplored. Here we report a genome-wide map of accessible chromatin in mouse preimplantation embryos using an improved assay for transposase-accessible chromatin with high throughput sequencing (ATAC-seq) approach with CRISPR/Cas9-assisted mitochondrial DNA depletion. We show that despite extensive parental asymmetry in DNA methylomes, the chromatin accessibility between the parental genomes is globally comparable after major zygotic genome activation (ZGA). Accessible chromatin in early embryos is widely shaped by transposable elements and overlaps extensively with putative cis-regulatory sequences. Unexpectedly, accessible chromatin is also found near the transcription end sites of active genes. By integrating the maps of cis-regulatory elements and single-cell transcriptomes, we construct the regulatory network of early development, which helps to identify the key modulators for lineage specification. Finally, we find that the activities of cis-regulatory elements and their associated open chromatin diminished before major ZGA. Surprisingly, we observed many loci showing non-canonical, large open chromatin domains over the entire transcribed units in minor ZGA, supporting the presence of an unusually permissive chromatin state. Together, these data reveal a unique spatiotemporal chromatin configuration that accompanies early mammalian development.


Assuntos
Blastocisto/metabolismo , Montagem e Desmontagem da Cromatina , Cromatina/genética , Cromatina/metabolismo , Alelos , Animais , Linhagem da Célula/genética , Reprogramação Celular , Metilação de DNA , Desenvolvimento Embrionário/genética , Feminino , Regulação da Expressão Gênica no Desenvolvimento/genética , Redes Reguladoras de Genes/genética , Genoma/genética , Histonas/metabolismo , Masculino , Camundongos , Sequências Reguladoras de Ácido Nucleico/genética , Análise de Célula Única , Transcriptoma/genética , Transposases/metabolismo , Zigoto/metabolismo
8.
Cell Mol Life Sci ; 77(15): 2997-3012, 2020 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31676962

RESUMO

CxxC-finger protein 1 (CFP1)-mediated trimethylated histone H3 at lysine-4 (H3K4me3) during oocyte development enables the oocyte genome to establish the competence to generate a new organism. Nevertheless, it remains unclear to which extent this epigenetic modification forms an instructive component of ovarian follicle development. We investigated the ovarian functions using an oocyte-specific Cxxc1 knockout mouse model, in which the H3K4me3 accumulation is downregulated in oocytes of developing follicles. CFP1-dependent H3K4 trimethylation in oocytes was necessary to maintain the expression of key paracrine factors and to facilitate the communication between an oocyte and the surrounding granulosa cells. The distinct gene expression patterns in cumulus cells within preovulatory follicles were disrupted by the Cxxc1 deletion in oocytes. Both follicle growth and ovulation were compromised after CFP1 deletion, because Cxxc1 deletion in oocytes indirectly impaired essential signaling pathways in granulosa cells that mediate the functions of follicle-stimulating hormone and luteinizing hormone. Therefore, CFP1-regulated epigenetic modification of the oocyte genome influences the responses of ovarian follicles to gonadotropin in a cell-nonautonomous manner.


Assuntos
Histonas/metabolismo , Oócitos/metabolismo , Folículo Ovariano/metabolismo , Transativadores/metabolismo , Animais , Células do Cúmulo/metabolismo , Feminino , Hormônio Foliculoestimulante/metabolismo , Células da Granulosa/citologia , Células da Granulosa/metabolismo , Hormônio Luteinizante/metabolismo , Metilação , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Folículo Ovariano/crescimento & desenvolvimento , Ovulação , Comunicação Parácrina , Fosfatidilinositol 3-Quinases/química , Fosfatidilinositol 3-Quinases/metabolismo , Transdução de Sinais , Transativadores/deficiência , Transativadores/genética
9.
J Biol Chem ; 292(4): 1438-1448, 2017 01 27.
Artigo em Inglês | MEDLINE | ID: mdl-27994054

RESUMO

Trp-Asp (WD) repeat domain 1 (WDR1) is a highly conserved actin-binding protein across all eukaryotes and is involved in numerous actin-based processes by accelerating Cofilin severing actin filament. However, the function and the mechanism of WDR1 in mammalian early development are still largely unclear. We now report that WDR1 is essential for mouse peri-implantation development and regulates Cofilin phosphorylation in mouse cells. The disruption of maternal WDR1 does not obviously affect ovulation and female fertility. However, depletion of zygotic WDR1 results in embryonic lethality at the peri-implantation stage. In WDR1 knock-out cells, we found that WDR1 regulates Cofilin phosphorylation. Interestingly, WDR1 is overdosed to regulate Cofilin phosphorylation in mouse cells. Furthermore, we showed that WDR1 interacts with Lim domain kinase 1 (LIMK1), a well known phosphorylation kinase of Cofilin. Altogether, our results provide new insights into the role and mechanism of WDR1 in physiological conditions.


Assuntos
Fatores de Despolimerização de Actina/metabolismo , Implantação do Embrião , Embrião de Mamíferos/embriologia , Desenvolvimento Embrionário , Quinases Lim/metabolismo , Proteínas dos Microfilamentos/metabolismo , Fatores de Despolimerização de Actina/genética , Animais , Perda do Embrião/genética , Perda do Embrião/metabolismo , Feminino , Quinases Lim/genética , Camundongos , Camundongos Knockout , Proteínas dos Microfilamentos/genética , Fosforilação
10.
Development ; 142(22): 3943-53, 2015 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-26428007

RESUMO

Mammalian early embryos maintain accurate genome integrity for proper development within a programmed timeline despite constant assaults on their DNA by replication, DNA demethylation and genetic defects transmitted from germ cells. However, how genome integrity is safeguarded during mammalian early embryonic development remains unclear. BCAS2 (breast carcinoma amplified sequence 2), a core component of the PRP19 complex involved in pre-mRNA splicing, plays an important role in the DNA damage response through the RPA complex, a key regulator in the maintenance of genome integrity. Currently, the physiological role of BCAS2 in mammals is unknown. We now report that BCAS2 responds to endogenous and exogenous DNA damage in mouse zygotes. Maternal depletion of BCAS2 compromises the DNA damage response in early embryos, leading to developmental arrest at the two- to four-cell stage accompanied by the accumulation of damaged DNA and micronuclei. Furthermore, BCAS2 mutants that are unable to bind RPA1 fail in DNA repair during the zygotic stage. In addition, phosphorylated RPA2 cannot localise to the DNA damage sites in mouse zygotes with disrupted maternal BCAS2. These data suggest that BCAS2 might function through the RPA complex during DNA repair in zygotes. Together, our results reveal that maternal BCAS2 maintains the genome integrity of early embryos and is essential for female mouse fertility.


Assuntos
Reparo do DNA/fisiologia , Desenvolvimento Embrionário/fisiologia , Fertilidade/fisiologia , Instabilidade Genômica/fisiologia , Complexos Multiproteicos/metabolismo , Proteínas de Neoplasias/metabolismo , Animais , Western Blotting , Reparo do DNA/genética , Desenvolvimento Embrionário/genética , Feminino , Fertilidade/genética , Marcação de Genes , Instabilidade Genômica/genética , Marcação In Situ das Extremidades Cortadas , Camundongos , Microscopia de Fluorescência , Proteínas Associadas à Matriz Nuclear/metabolismo , Gravidez , Fatores de Processamento de RNA , Reação em Cadeia da Polimerase em Tempo Real , Proteína de Replicação A/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa
11.
Dev Cell ; 59(4): 465-481.e6, 2024 Feb 26.
Artigo em Inglês | MEDLINE | ID: mdl-38237590

RESUMO

The progression from naive through formative to primed in vitro pluripotent stem cell states recapitulates epiblast development in vivo during the peri-implantation period of mouse embryo development. Activation of the de novo DNA methyltransferases and reorganization of transcriptional and epigenetic landscapes are key events that occur during these pluripotent state transitions. However, the upstream regulators that coordinate these events are relatively underexplored. Here, using Zfp281 knockout mouse and degron knockin cell models, we identify the direct transcriptional activation of Dnmt3a/3b by ZFP281 in pluripotent stem cells. Chromatin co-occupancy of ZFP281 and DNA hydroxylase TET1, which is dependent on the formation of R-loops in ZFP281-targeted gene promoters, undergoes a "high-low-high" bimodal pattern regulating dynamic DNA methylation and gene expression during the naive-formative-primed transitions. ZFP281 also safeguards DNA methylation in maintaining primed pluripotency. Our study demonstrates a previously unappreciated role for ZFP281 in coordinating DNMT3A/3B and TET1 functions to promote pluripotent state transitions.


Assuntos
Epigênese Genética , Células-Tronco Pluripotentes , Animais , Camundongos , Metilação de DNA/genética , Cromatina/metabolismo , DNA/metabolismo , Diferenciação Celular/genética , Camadas Germinativas/metabolismo , Fatores de Transcrição/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismo
12.
Biochim Biophys Acta Mol Cell Res ; 1871(7): 119768, 2024 Jun 04.
Artigo em Inglês | MEDLINE | ID: mdl-38838858

RESUMO

The regulatory mechanism of the transcription factor GATA3 in the differentiation and maturation process of extravillous trophoblasts (EVT) in early pregnancy placenta, as well as its relevance to the occurrence of pregnancy disorders, remains poorly understood. This study leveraged single-cell RNA sequencing data from placental organoid models and placental tissue to explore the dynamic changes in GATA3 expression during EVT maturation. The expression pattern exhibited an initial upregulation followed by subsequent downregulation, with aberrant GATA3 localization observed in cases of recurrent miscarriage (RM). By identifying global targets regulated by GATA3 in primary placental EVT cells, JEG3, and HTR8/SVneo cell lines, this study offered insights into its regulatory mechanisms across different EVT cell models. Shared regulatory targets among these cell types and activation of trophoblast cell marker genes emphasized the importance of GATA3 in EVT differentiation and maturation. Knockdown of GATA3 in JEG3 cells led to repression of GATA3-induced epithelial-mesenchymal transition (EMT), as evidenced by changes in marker gene expression levels and enhanced migration ability. Additionally, interference with GATA3 accelerated cellular senescence, as indicated by reduced proliferation rates and increased activity levels for senescence-associated ß-galactosidase enzyme, along with elevated expression levels for senescence-associated genes. This study provides comprehensive insights into the dual role of GATA3 in regulating EMT and cellular senescence during EVT differentiation, shedding light on the dynamic changes in GATA3 expression in normal and pathological placental conditions.

13.
Nat Cell Biol ; 26(6): 962-974, 2024 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-38839978

RESUMO

Dynamic epigenomic reprogramming occurs during mammalian oocyte maturation and early development. However, the underlying transcription circuitry remains poorly characterized. By mapping cis-regulatory elements using H3K27ac, we identified putative enhancers in mouse oocytes and early embryos distinct from those in adult tissues, enabling global transitions of regulatory landscapes around fertilization and implantation. Gene deserts harbour prevalent putative enhancers in fully grown oocytes linked to oocyte-specific genes and repeat activation. Embryo-specific enhancers are primed before zygotic genome activation and are restricted by oocyte-inherited H3K27me3. Putative enhancers in oocytes often manifest H3K4me3, bidirectional transcription, Pol II binding and can drive transcription in STARR-seq and a reporter assay. Finally, motif analysis of these elements identified crucial regulators of oogenesis, TCF3 and TCF12, the deficiency of which impairs activation of key oocyte genes and folliculogenesis. These data reveal distinctive regulatory landscapes and their interacting transcription factors that underpin the development of mammalian oocytes and early embryos.


Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos , Elementos Facilitadores Genéticos , Regulação da Expressão Gênica no Desenvolvimento , Oócitos , Oogênese , Animais , Oócitos/metabolismo , Feminino , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Oogênese/genética , Camundongos , Histonas/metabolismo , Histonas/genética , Embrião de Mamíferos/metabolismo , Camundongos Endogâmicos C57BL , Desenvolvimento Embrionário/genética , Folículo Ovariano/metabolismo , Camundongos Knockout
14.
bioRxiv ; 2023 Mar 25.
Artigo em Inglês | MEDLINE | ID: mdl-36993548

RESUMO

The progression from naive through formative to primed in vitro pluripotent stem cell states recapitulates the development of the epiblast in vivo during the peri-implantation period of mammalian development. Activation of the de novo DNA methyltransferases and reorganization of transcriptional and epigenetic landscapes are key events occurring during these pluripotent state transitions. However, the upstream regulators that coordinate these events are relatively underexplored. Here, using Zfp281 knockout mouse and degron knock-in cell models, we uncover the direct transcriptional activation of Dnmt3a/3b by ZFP281 in pluripotent stem cells. Chromatin co-occupancy of ZFP281 and DNA hydroxylase TET1, dependent on the formation of R loops in ZFP281-targeted gene promoters, undergoes a "high-low-high" bimodal pattern regulating dynamic DNA methylation and gene expression during the naïive-formative-primed transitions. ZFP281 also safeguards DNA methylation in maintaining primed pluripotency. Our study demonstrates a previously unappreciated role for ZFP281 in coordinating DNMT3A/3B and TET1 functions to promote pluripotent state transitions.

15.
Science ; 382(6676): eadi5516, 2023 12 15.
Artigo em Inglês | MEDLINE | ID: mdl-38096290

RESUMO

Pioneer transcription factors (TFs), such as OCT4 and SOX2, play crucial roles in pluripotency regulation. However, the master TF-governed pluripotency regulatory circuitry was largely inferred from cultured cells. In this work, we investigated SOX2 binding from embryonic day 3.5 (E3.5) to E7.5 in the mouse. In E3.5 inner cell mass (ICM), SOX2 regulates the ICM-trophectoderm program but is dispensable for opening global enhancers. Instead, SOX2 occupies preaccessible enhancers in part opened by early-stage expressing TFs TFAP2C and NR5A2. SOX2 then widely redistributes when cells adopt naive and formative pluripotency by opening enhancers or poising them for rapid future activation. Hence, multifaceted pioneer TF-enhancer interaction underpins pluripotency progression in embryos, including a distinctive state in E3.5 ICM that bridges totipotency and pluripotency.


Assuntos
Blastocisto , Linhagem da Célula , Cromatina , Elementos Facilitadores Genéticos , Regulação da Expressão Gênica no Desenvolvimento , Fatores de Transcrição SOXB1 , Animais , Camundongos , Blastocisto/citologia , Blastocisto/metabolismo , Células Cultivadas , Cromatina/metabolismo , Fator 3 de Transcrição de Octâmero/genética , Fator 3 de Transcrição de Octâmero/metabolismo , Fatores de Transcrição SOXB1/genética , Fatores de Transcrição SOXB1/metabolismo , Diferenciação Celular/genética , Linhagem da Célula/genética
16.
Biochem Biophys Res Commun ; 408(4): 595-601, 2011 May 20.
Artigo em Inglês | MEDLINE | ID: mdl-21530494

RESUMO

A defective ratio between DNA damage and repair may result in the occurrence of a malignant phenotype. Previous studies have found that many genetic alterations in DNA repair genes occur frequently in lung cancer. However, the epigenetic mechanisms underlying this tumorigenesis are not clear. Herein, we have used a chemical-induced rat lung carcinogenesis model to study the evolution of methylation alterations of DNA repair genes BRCA1, ERCC1, XRCC1, and MLH1. Methylation-specific PCR and immunohistochemistry were used to analyze gene methylation status and protein expression during the progression of lung carcinogenesis. Promoter hypermethylation of BRCA1 was only detected in three samples of infiltrating carcinoma. CpG island hypermethylation of ERCC1, XRCC1, and MLH1 was found to increase gradually throughout lung carcinogenesis progression. Both the prevalence of at least one methylated gene and the average number of methylated genes were heightened in squamous metaplasia and dysplasia compared with normal tissue and hyperplasia, and was further increased in carcinoma in situ (CIS) and infiltrating carcinoma. Immunohistochemical analysis showed that BRCA1 and MLH1 protein expression decreased progressively during the stages of lung carcinogenesis, whereas ERCC1 and XRCC1 expression were only found in later stages. Although methylation levels were elevated for ERCC1 and XRCC1 during carcinogenesis, an inverse correlation with protein expression was found only for BRCA1 and MLH1. These results suggest that a continuous accumulation of DNA repair gene hypermethylation and the consequent protein alterations might be a vital molecular mechanism during the process of multistep chemical-induced rat lung carcinogenesis.


Assuntos
Transformação Celular Neoplásica/genética , Metilação de DNA/genética , Reparo do DNA/genética , Regulação Neoplásica da Expressão Gênica , Neoplasias Pulmonares/genética , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Proteína BRCA1/genética , Transformação Celular Neoplásica/induzido quimicamente , Enzimas Reparadoras do DNA/genética , Proteínas de Ligação a DNA/genética , Feminino , Neoplasias Pulmonares/induzido quimicamente , Masculino , Proteína 1 Homóloga a MutL , Proteínas Nucleares/genética , Biossíntese de Proteínas/genética , Ratos , Ratos Wistar , Proteína 1 Complementadora Cruzada de Reparo de Raio-X
17.
Toxicol Appl Pharmacol ; 251(1): 70-8, 2011 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-21163286

RESUMO

To evaluate the significance of alterations in cell adhesion-related genes methylation during lung multistep carcinogenesis induced by the genotoxic carcinogens 3-methylcholanthrene (MCA) and diethylnitrosamine (DEN), tissue samples microdissected from MCA/DEN-induced rat lung carcinogenesis model were subjected to methylation-specific PCR to evaluate the DNA methylation status of CADM1, TIMP3, E-cadherin and N-cadherin. Immunohistochemistry was used to determine protein expression of CADM1, TIMP3, N-cadherin and the DNA methyltransferases (DNMTs) 1, 3a and 3b. E-cadherin hypermethylation was not detected in any tissue. CADM1, TIMP3 and N-cadherin hypermethylation was correlated with the loss of their protein expression during the progression of pathologic lesions. The prevalence of DNA methylation of at least one gene and the average number of methylated genes increased with the histological progression. DNMT1 and DNMT3a protein expression increased progressively during the stages of lung carcinogenesis, whereas DNMT3b overexpression was only found in several samples. Furthermore, DNMT1 protein expression levels were correlated with CADM1 methylation, and DNMT3a protein expression levels were correlated with CADM1, TIMP3 and N-cadherin methylation. The average number of methylated genes during carcinogenesis was significantly correlated with DNMT1 and DNMT3a protein expression levels. Moreover, mRNA expression of CADM1 significantly increased after treatment with DNMT inhibitor 5-aza-2'-deoxycytidine in CADM1-methylated primary tumor cell lines. Our findings suggest that an accumulation of hypermethylation accounts for cell adhesion-related gene silencing is associated with dynamic changes in the progression of MCA/DEN-induced rat lung carcinogenesis. We suggest that DNMT1 and DNMT3a protein overexpression may be responsible for this aberrant DNA methylation.


Assuntos
Moléculas de Adesão Celular/genética , Adesão Celular/genética , Transformação Celular Neoplásica/genética , DNA (Citosina-5-)-Metiltransferases/metabolismo , Metilação de DNA , Inativação Gênica , Neoplasias Pulmonares/genética , Animais , Azacitidina/análogos & derivados , Azacitidina/farmacologia , Caderinas/genética , Adesão Celular/efeitos dos fármacos , Moléculas de Adesão Celular/metabolismo , Transformação Celular Neoplásica/induzido quimicamente , Transformação Celular Neoplásica/patologia , DNA (Citosina-5-)-Metiltransferase 1 , DNA (Citosina-5-)-Metiltransferases/antagonistas & inibidores , DNA (Citosina-5-)-Metiltransferases/genética , Metilação de DNA/efeitos dos fármacos , DNA Metiltransferase 3A , Decitabina , Dietilnitrosamina , Modelos Animais de Doenças , Inibidores Enzimáticos/farmacologia , Feminino , Inativação Gênica/efeitos dos fármacos , Imunoglobulinas/genética , Neoplasias Pulmonares/induzido quimicamente , Neoplasias Pulmonares/enzimologia , Neoplasias Pulmonares/patologia , Masculino , Metilcolantreno , Ratos , Ratos Wistar , Inibidor Tecidual de Metaloproteinase-3/genética , Regulação para Cima , DNA Metiltransferase 3B
18.
Sci Adv ; 7(48): eabi6178, 2021 Nov 26.
Artigo em Inglês | MEDLINE | ID: mdl-34818044

RESUMO

While mouse remains the most popular model, the conservation of parental-to-embryonic epigenetic transition across mammals is poorly defined. Through analysis of oocytes and early embryos in human, bovine, porcine, rat, and mouse, we revealed remarkable species-specific innovations as no single animal model fully recapitulates the human epigenetic transition. In rodent oocytes, transcription-dependent DNA methylation allows methylation of maternal imprints but not intergenic paternal imprints. Unexpectedly, prevalent DNA hypermethylation, paralleled by H3K36me2/3, also occurs in nontranscribed regions in porcine and bovine oocytes, except for megabase-long "CpG continents (CGCs)" where imprinting control regions preferentially reside. Broad H3K4me3 and H3K27me3 domains exist in nonhuman oocytes, yet only rodent H3K27me3 survives beyond genome activation. Coincidently, regulatory elements preferentially evade H3K27me3 in rodent oocytes, and failure to do so causes aberrant embryonic gene repression. Hence, the diverse mammalian innovations of parental-to-embryonic transition center on a delicate "to-methylate-or-not" balance in establishing imprints while protecting other regulatory regions.

19.
Cell Res ; 31(5): 526-541, 2021 05.
Artigo em Inglês | MEDLINE | ID: mdl-33608671

RESUMO

The pluripotency of mammalian early and late epiblast could be recapitulated by naïve embryonic stem cells (ESCs) and primed epiblast stem cells (EpiSCs), respectively. However, these two states of pluripotency may not be sufficient to reflect the full complexity and developmental potency of the epiblast during mammalian early development. Here we report the establishment of self-renewing formative pluripotent stem cells (fPSCs) which manifest features of epiblast cells poised for gastrulation. fPSCs can be established from different mouse ESCs, pre-/early-gastrula epiblasts and induced PSCs. Similar to pre-/early-gastrula epiblasts, fPSCs show the transcriptomic features of formative pluripotency, which are distinct from naïve ESCs and primed EpiSCs. fPSCs show the unique epigenetic states of E6.5 epiblast, including the super-bivalency of a large set of developmental genes. Just like epiblast cells immediately before gastrulation, fPSCs can efficiently differentiate into three germ layers and primordial germ cells (PGCs) in vitro. Thus, fPSCs highlight the feasibility of using PSCs to explore the development of mammalian epiblast.


Assuntos
Gastrulação , Células-Tronco Pluripotentes , Animais , Diferenciação Celular , Células-Tronco Embrionárias , Camadas Germinativas , Camundongos
20.
Biochem Biophys Res Commun ; 402(3): 507-14, 2010 Nov 19.
Artigo em Inglês | MEDLINE | ID: mdl-20970405

RESUMO

The epigenetic mechanisms underlying the tumorigenesis caused by polycyclic aromatic hydrocarbons and nitrosamine compounds such as 3-methylcholanthrene (MCA) and diethylnitrosamine (DEN) are currently unknown. We reported previously that dynamic changes in DNA methylation occurred during MCA/DEN-induced rat lung carcinogenesis. Here, we used the same animal model to further study the evolution of methylation alterations in tumor suppressor genes (TSGs) DAPK1, FHIT, RASSF1A, and SOCS-3. We found that none of these genes were methylated in either normal or hyperplasia tissue. However, as the severity of the cancer progressed through squamous metaplasia and dysplasia to carcinoma in situ (CIS) and infiltrating carcinoma, so methylation became more prevalent. Particularly dramatic increases in the level of methylation, the average number of methylated genes, and the incidence of concurrent methylation in three genes were observed in CIS and infiltrating carcinoma. Similar but less profound changes were seen in squamous metaplasia and dysplasia. Furthermore, methylation status was closely correlated to loss of protein expression for these genes, with protein levels markedly declining along the continuum of carcinogenesis. These results suggest that progressive CpG island hypermethylation leading to inactivation of TSGs might be a vital molecular mechanism in the pathogenesis of MCA/DEN-induced multistep rat lung carcinogenesis.


Assuntos
Ilhas de CpG , Metilação de DNA , Regulação Neoplásica da Expressão Gênica , Genes Supressores de Tumor , Neoplasias Pulmonares/genética , Hidrolases Anidrido Ácido/genética , Hidrolases Anidrido Ácido/metabolismo , Animais , Proteínas Reguladoras de Apoptose/genética , Proteínas Reguladoras de Apoptose/metabolismo , Proteínas Quinases Dependentes de Cálcio-Calmodulina/genética , Proteínas Quinases Dependentes de Cálcio-Calmodulina/metabolismo , Carcinógenos/toxicidade , Proteínas Quinases Associadas com Morte Celular , Dietilnitrosamina/toxicidade , Feminino , Neoplasias Pulmonares/induzido quimicamente , Masculino , Metilcolantreno/toxicidade , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Ratos , Ratos Wistar , Proteína 3 Supressora da Sinalização de Citocinas , Proteínas Supressoras da Sinalização de Citocina/genética , Proteínas Supressoras da Sinalização de Citocina/metabolismo , Proteínas Supressoras de Tumor/genética , Proteínas Supressoras de Tumor/metabolismo
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