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1.
Cell ; 187(21): 6088-6103.e18, 2024 Oct 17.
Artigo em Inglês | MEDLINE | ID: mdl-39214079

RESUMO

5-Methylcytosine (5mC) is an established epigenetic mark in vertebrate genomic DNA, but whether its oxidation intermediates formed during TET-mediated DNA demethylation possess an instructive role of their own that is also physiologically relevant remains unresolved. Here, we reveal a 5-formylcytosine (5fC) nuclear chromocenter, which transiently forms during zygotic genome activation (ZGA) in Xenopus and mouse embryos. We identify this chromocenter as the perinucleolar compartment, a structure associated with RNA Pol III transcription. In Xenopus embryos, 5fC is highly enriched on Pol III target genes activated at ZGA, notably at oocyte-type tandem arrayed tRNA genes. By manipulating Tet and Tdg enzymes, we show that 5fC is required as a regulatory mark to promote Pol III recruitment as well as tRNA expression. Concordantly, 5fC modification of a tRNA transgene enhances its expression in vivo. The results establish 5fC as an activating epigenetic mark during zygotic reprogramming of Pol III gene expression.


Assuntos
Citosina , Epigênese Genética , RNA Polimerase III , Zigoto , Animais , Citosina/metabolismo , Citosina/análogos & derivados , Camundongos , Zigoto/metabolismo , RNA Polimerase III/metabolismo , RNA Polimerase III/genética , RNA de Transferência/metabolismo , RNA de Transferência/genética , Xenopus laevis/metabolismo , Xenopus laevis/embriologia , Xenopus laevis/genética , Xenopus/metabolismo , Xenopus/embriologia , Xenopus/genética , Feminino , Reprogramação Celular , Regulação da Expressão Gênica no Desenvolvimento , Oócitos/metabolismo
2.
Cell ; 187(13): 3284-3302.e23, 2024 Jun 20.
Artigo em Inglês | MEDLINE | ID: mdl-38843832

RESUMO

The cleavage of zygotes generates totipotent blastomeres. In human 8-cell blastomeres, zygotic genome activation (ZGA) occurs to initiate the ontogenesis program. However, capturing and maintaining totipotency in human cells pose significant challenges. Here, we realize culturing human totipotent blastomere-like cells (hTBLCs). We find that splicing inhibition can transiently reprogram human pluripotent stem cells into ZGA-like cells (ZLCs), which subsequently transition into stable hTBLCs after long-term passaging. Distinct from reported 8-cell-like cells (8CLCs), both ZLCs and hTBLCs widely silence pluripotent genes. Interestingly, ZLCs activate a particular group of ZGA-specific genes, and hTBLCs are enriched with pre-ZGA-specific genes. During spontaneous differentiation, hTBLCs re-enter the intermediate ZLC stage and further generate epiblast (EPI)-, primitive endoderm (PrE)-, and trophectoderm (TE)-like lineages, effectively recapitulating human pre-implantation development. Possessing both embryonic and extraembryonic developmental potency, hTBLCs can autonomously generate blastocyst-like structures in vitro without external cell signaling. In summary, our study provides key criteria and insights into human cell totipotency.


Assuntos
Diferenciação Celular , Spliceossomos , Animais , Humanos , Camundongos , Blastocisto/metabolismo , Blastocisto/citologia , Blastômeros/metabolismo , Blastômeros/citologia , Reprogramação Celular , Desenvolvimento Embrionário/genética , Camadas Germinativas/metabolismo , Camadas Germinativas/citologia , Células-Tronco Pluripotentes/metabolismo , Células-Tronco Pluripotentes/citologia , Splicing de RNA , Spliceossomos/metabolismo , Células-Tronco Totipotentes/metabolismo , Células-Tronco Totipotentes/citologia , Zigoto/metabolismo , Células Cultivadas , Modelos Moleculares , Estrutura Terciária de Proteína , Genoma Humano , Análise de Célula Única , Fator 15 de Diferenciação de Crescimento/química , Fator 15 de Diferenciação de Crescimento/genética , Fator 15 de Diferenciação de Crescimento/metabolismo , Epigenômica , Linhagem da Célula
3.
Cell ; 185(26): 4954-4970.e20, 2022 12 22.
Artigo em Inglês | MEDLINE | ID: mdl-36493774

RESUMO

Nuclear pore complexes (NPCs) are channels for nucleocytoplasmic transport of proteins and RNAs. However, it remains unclear whether composition, structure, and permeability of NPCs dynamically change during the cleavage period of vertebrate embryos and affect embryonic development. Here, we report that the comprehensive NPC maturity (CNM) controls the onset of zygotic genome activation (ZGA) during zebrafish early embryogenesis. We show that more nucleoporin proteins are recruited to and assembled into NPCs with development, resulting in progressive increase of NPCs in size and complexity. Maternal transcription factors (TFs) transport into nuclei more efficiently with increasing CNM. Deficiency or dysfunction of Nup133 or Ahctf1/Elys impairs NPC assembly, maternal TFs nuclear transport, and ZGA onset, while nup133 overexpression promotes these processes. Therefore, CNM may act as a molecular timer for ZGA by controlling nuclear transport of maternal TFs that reach nuclear concentration thresholds at a given time to initiate ZGA.


Assuntos
Poro Nuclear , Peixe-Zebra , Animais , Desenvolvimento Embrionário/genética , Regulação da Expressão Gênica no Desenvolvimento , Poro Nuclear/metabolismo , Complexo de Proteínas Formadoras de Poros Nucleares/genética , Complexo de Proteínas Formadoras de Poros Nucleares/metabolismo , Fatores de Transcrição/metabolismo , Peixe-Zebra/metabolismo , Zigoto/metabolismo , Genoma
4.
Cell ; 184(12): 3267-3280.e18, 2021 06 10.
Artigo em Inglês | MEDLINE | ID: mdl-34043941

RESUMO

Searching for factors to improve knockin efficiency for therapeutic applications, biotechnology, and generation of non-human primate models of disease, we found that the strand exchange protein RAD51 can significantly increase Cas9-mediated homozygous knockin in mouse embryos through an interhomolog repair (IHR) mechanism. IHR is a hallmark of meiosis but only occurs at low frequencies in somatic cells, and its occurrence in zygotes is controversial. Using multiple approaches, we provide evidence for an endogenous IHR mechanism in the early embryo that can be enhanced by RAD51. This process can be harnessed to generate homozygotes from wild-type zygotes using exogenous donors and to convert heterozygous alleles into homozygous alleles without exogenous templates. Furthermore, we identify additional IHR-promoting factors and describe features of IHR events. Together, our findings show conclusive evidence for IHR in mouse embryos and describe an efficient method for enhanced gene conversion.


Assuntos
Reparo do DNA/genética , Conversão Gênica , Rad51 Recombinase/metabolismo , Alelos , Animais , Sequência de Bases , Proteína 9 Associada à CRISPR/metabolismo , Proteínas de Ligação ao Cálcio/metabolismo , Proteínas de Ciclo Celular/metabolismo , Cromossomos de Mamíferos/genética , Quebras de DNA de Cadeia Dupla , Embrião de Mamíferos , Feminino , Loci Gênicos , Recombinação Homóloga/genética , Homozigoto , Humanos , Mutação INDEL/genética , Camundongos Endogâmicos C57BL , Mosaicismo , Proteínas Nucleares/metabolismo , Polimorfismo de Nucleotídeo Único/genética , Ribonucleoproteínas/metabolismo , Zigoto/metabolismo
5.
Cell ; 184(11): 2860-2877.e22, 2021 05 27.
Artigo em Inglês | MEDLINE | ID: mdl-33964210

RESUMO

Most human embryos are aneuploid. Aneuploidy frequently arises during the early mitotic divisions of the embryo, but its origin remains elusive. Human zygotes that cluster their nucleoli at the pronuclear interface are thought to be more likely to develop into healthy euploid embryos. Here, we show that the parental genomes cluster with nucleoli in each pronucleus within human and bovine zygotes, and clustering is required for the reliable unification of the parental genomes after fertilization. During migration of intact pronuclei, the parental genomes polarize toward each other in a process driven by centrosomes, dynein, microtubules, and nuclear pore complexes. The maternal and paternal chromosomes eventually cluster at the pronuclear interface, in direct proximity to each other, yet separated. Parental genome clustering ensures the rapid unification of the parental genomes on nuclear envelope breakdown. However, clustering often fails, leading to chromosome segregation errors and micronuclei, incompatible with healthy embryo development.


Assuntos
Embrião de Mamíferos/metabolismo , Desenvolvimento Embrionário/genética , Aneuploidia , Animais , Bovinos , Nucléolo Celular/metabolismo , Núcleo Celular/metabolismo , Centrossomo/metabolismo , Segregação de Cromossomos/fisiologia , Cromossomos/metabolismo , Fertilização/genética , Humanos , Masculino , Microtúbulos/metabolismo , Mitose , Oócitos/metabolismo , Espermatozoides/metabolismo , Zigoto/metabolismo
6.
Annu Rev Biochem ; 89: 695-715, 2020 06 20.
Artigo em Inglês | MEDLINE | ID: mdl-32569527

RESUMO

The zona pellucida (ZP) is an extracellular matrix that surrounds all mammalian oocytes, eggs, and early embryos and plays vital roles during oogenesis, fertilization, and preimplantation development. The ZP is composed of three or four glycosylated proteins, ZP1-4, that are synthesized, processed, secreted, and assembled into long, cross-linked fibrils by growing oocytes. ZP proteins have an immunoglobulin-like three-dimensional structure and a ZP domain that consists of two subdomains, ZP-N and ZP-C, with ZP-N of ZP2 and ZP3 required for fibril assembly. A ZP2-ZP3 dimer is located periodically along ZP fibrils that are cross-linked by ZP1, a protein with a proline-rich N terminus. Fibrils in the inner and outer regions of the ZP are oriented perpendicular and parallel to the oolemma, respectively, giving the ZP a multilayered appearance. Upon fertilization of eggs, modification of ZP2 and ZP3 results in changes in the ZP's physical and biological properties that have important consequences. Certain structural features of ZP proteins suggest that they may be amyloid-like proteins.


Assuntos
Proteínas Amiloidogênicas/química , Glicoproteínas da Zona Pelúcida/química , Zigoto/metabolismo , Proteínas Amiloidogênicas/genética , Proteínas Amiloidogênicas/metabolismo , Animais , Embrião de Mamíferos/metabolismo , Embrião de Mamíferos/ultraestrutura , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Humanos , Oócitos/crescimento & desenvolvimento , Oócitos/metabolismo , Oócitos/ultraestrutura , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Multimerização Proteica , Zona Pelúcida/metabolismo , Zona Pelúcida/ultraestrutura , Glicoproteínas da Zona Pelúcida/genética , Glicoproteínas da Zona Pelúcida/metabolismo , Zigoto/crescimento & desenvolvimento , Zigoto/ultraestrutura
7.
Cell ; 183(7): 1757-1771.e18, 2020 12 23.
Artigo em Inglês | MEDLINE | ID: mdl-33271061

RESUMO

CRISPR-Cas defense systems have been coopted multiple times in nature for guide RNA-directed transposition by Tn7-like elements. Prototypic Tn7 uses dedicated proteins for two targeting pathways: one targeting a neutral and conserved attachment site in the chromosome and a second directing transposition into mobile plasmids facilitating cell-to-cell transfer. We show that Tn7-CRISPR-Cas elements evolved a system of guide RNA categorization to accomplish the same two-pathway lifestyle. Multiple mechanisms allow functionally distinct guide RNAs for transposition: a conventional system capable of acquiring guide RNAs to new plasmid and phage targets and a second providing long-term memory for access to chromosomal sites upon entry into a new host. Guide RNAs are privatized to be recognized only by the transposon-adapted system via sequence specialization, mismatch tolerance, and selective regulation to avoid toxic self-targeting by endogenous CRISPR-Cas defense systems. This information reveals promising avenues to engineer guide RNAs for enhanced CRISPR-Cas functionality for genome modification.


Assuntos
Sistemas CRISPR-Cas/genética , Elementos de DNA Transponíveis/genética , RNA Guia de Cinetoplastídeos/genética , Proteínas de Bactérias/metabolismo , Sequência de Bases , Gammaproteobacteria/metabolismo , Filogenia , Regiões Promotoras Genéticas/genética , Fatores de Transcrição/metabolismo , Zigoto/metabolismo
8.
Cell ; 180(6): 1212-1227.e14, 2020 03 19.
Artigo em Inglês | MEDLINE | ID: mdl-32169215

RESUMO

The paternal genome undergoes a massive exchange of histone with protamine for compaction into sperm during spermiogenesis. Upon fertilization, this process is potently reversed, which is essential for parental genome reprogramming and subsequent activation; however, it remains poorly understood how this fundamental process is initiated and regulated. Here, we report that the previously characterized splicing kinase SRPK1 initiates this life-beginning event by catalyzing site-specific phosphorylation of protamine, thereby triggering protamine-to-histone exchange in the fertilized oocyte. Interestingly, protamine undergoes a DNA-dependent phase transition to gel-like condensates and SRPK1-mediated phosphorylation likely helps open up such structures to enhance protamine dismissal by nucleoplasmin (NPM2) and enable the recruitment of HIRA for H3.3 deposition. Remarkably, genome-wide assay for transposase-accessible chromatin sequencing (ATAC-seq) analysis reveals that selective chromatin accessibility in both sperm and MII oocytes is largely erased in early pronuclei in a protamine phosphorylation-dependent manner, suggesting that SRPK1-catalyzed phosphorylation initiates a highly synchronized reorganization program in both parental genomes.


Assuntos
Cromatina/metabolismo , Protaminas/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Animais , Proteínas de Ciclo Celular/metabolismo , Núcleo Celular/metabolismo , Cromatina/fisiologia , Montagem e Desmontagem da Cromatina/genética , Montagem e Desmontagem da Cromatina/fisiologia , Fertilização/genética , Histonas/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , Oócitos/metabolismo , Oócitos/fisiologia , Fosforilação , Protamina Quinase/genética , Protamina Quinase/metabolismo , Protaminas/genética , Proteínas Serina-Treonina Quinases/fisiologia , Splicing de RNA/genética , Splicing de RNA/fisiologia , Espermatozoides/metabolismo , Fatores de Transcrição/metabolismo , Zigoto/metabolismo
9.
Cell ; 182(1): 127-144.e23, 2020 07 09.
Artigo em Inglês | MEDLINE | ID: mdl-32502394

RESUMO

Before zygotic genome activation (ZGA), the quiescent genome undergoes reprogramming to transition into the transcriptionally active state. However, the mechanisms underlying euchromatin establishment during early embryogenesis remain poorly understood. Here, we show that histone H4 lysine 16 acetylation (H4K16ac) is maintained from oocytes to fertilized embryos in Drosophila and mammals. H4K16ac forms large domains that control nucleosome accessibility of promoters prior to ZGA in flies. Maternal depletion of MOF acetyltransferase leading to H4K16ac loss causes aberrant RNA Pol II recruitment, compromises the 3D organization of the active genomic compartments during ZGA, and causes downregulation of post-zygotically expressed genes. Germline depletion of histone deacetylases revealed that other acetyl marks cannot compensate for H4K16ac loss in the oocyte. Moreover, zygotic re-expression of MOF was neither able to restore embryonic viability nor onset of X chromosome dosage compensation. Thus, maternal H4K16ac provides an instructive function to the offspring, priming future gene activation.


Assuntos
Histonas/metabolismo , Lisina/metabolismo , Ativação Transcricional/genética , Acetilação , Animais , Sequência de Bases , Segregação de Cromossomos/genética , Sequência Conservada , Mecanismo Genético de Compensação de Dose , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/embriologia , Drosophila melanogaster/genética , Embrião não Mamífero/metabolismo , Evolução Molecular , Feminino , Genoma , Histona Acetiltransferases/genética , Histona Acetiltransferases/metabolismo , Masculino , Mamíferos/genética , Camundongos , Mutação/genética , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Nucleossomos/metabolismo , Oócitos/metabolismo , Regiões Promotoras Genéticas , RNA Polimerase II/metabolismo , Cromossomo X/metabolismo , Zigoto/metabolismo
10.
Cell ; 177(4): 799-801, 2019 05 02.
Artigo em Inglês | MEDLINE | ID: mdl-31051102

RESUMO

Deneke et al. (2019) discover that dynamic interactions of cell cycle and actomyosin contractility systems synchronize nuclear cleavages, generating a cytoplasmic flow that results in a spatially uniform distribution of zygotic nuclei in the early Drosophila embryo. This work underscores the importance of self-organizing mechanisms before the onset of zygotic transcription.


Assuntos
Drosophila melanogaster , Drosophila , Animais , Ciclo Celular , Física , Zigoto
11.
Cell ; 177(6): 1463-1479.e18, 2019 05 30.
Artigo em Inglês | MEDLINE | ID: mdl-31080065

RESUMO

Segregation of maternal determinants within the oocyte constitutes the first step in embryo patterning. In zebrafish oocytes, extensive ooplasmic streaming leads to the segregation of ooplasm from yolk granules along the animal-vegetal axis of the oocyte. Here, we show that this process does not rely on cortical actin reorganization, as previously thought, but instead on a cell-cycle-dependent bulk actin polymerization wave traveling from the animal to the vegetal pole of the oocyte. This wave functions in segregation by both pulling ooplasm animally and pushing yolk granules vegetally. Using biophysical experimentation and theory, we show that ooplasm pulling is mediated by bulk actin network flows exerting friction forces on the ooplasm, while yolk granule pushing is achieved by a mechanism closely resembling actin comet formation on yolk granules. Our study defines a novel role of cell-cycle-controlled bulk actin polymerization waves in oocyte polarization via ooplasmic segregation.


Assuntos
Actinas/metabolismo , Ciclo Celular/fisiologia , Oócitos/metabolismo , Actinas/fisiologia , Animais , Polaridade Celular/fisiologia , Citoplasma/metabolismo , Gema de Ovo/fisiologia , Polimerização , Peixe-Zebra/embriologia , Peixe-Zebra/metabolismo , Proteínas de Peixe-Zebra/metabolismo , Zigoto
12.
Annu Rev Cell Dev Biol ; 36: 411-440, 2020 10 06.
Artigo em Inglês | MEDLINE | ID: mdl-33021826

RESUMO

Understanding human embryology has historically relied on comparative approaches using mammalian model organisms. With the advent of low-input methods to investigate genetic and epigenetic mechanisms and efficient techniques to assess gene function, we can now study the human embryo directly. These advances have transformed the investigation of early embryogenesis in nonrodent species, thereby providing a broader understanding of conserved and divergent mechanisms. Here, we present an overview of the major events in human preimplantation development and place them in the context of mammalian evolution by comparing these events in other eutherian and metatherian species. We describe the advances of studies on postimplantation development and discuss stem cell models that mimic postimplantation embryos. A comparative perspective highlights the importance of analyzing different organisms with molecular characterization and functional studies to reveal the principles of early development. This growing field has a fundamental impact in regenerative medicine and raises important ethical considerations.


Assuntos
Desenvolvimento Embrionário , Animais , Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/metabolismo , Humanos , Modelos Biológicos , Filogenia , Zigoto/metabolismo
13.
Cell ; 173(1): 248-259.e15, 2018 03 22.
Artigo em Inglês | MEDLINE | ID: mdl-29526463

RESUMO

The dynamics of the chromatin regulatory landscape during human early embryogenesis remains unknown. Using DNase I hypersensitive site (DHS) sequencing, we report that the chromatin accessibility landscape is gradually established during human early embryogenesis. Interestingly, the DHSs with OCT4 binding motifs are enriched at the timing of zygotic genome activation (ZGA) in humans, but not in mice. Consistently, OCT4 contributes to ZGA in humans, but not in mice. We further find that lower CpG promoters usually establish DHSs at later stages. Similarly, younger genes tend to establish promoter DHSs and are expressed at later embryonic stages, while older genes exhibit these features at earlier stages. Moreover, our data show that human active transposons SVA and HERV-K harbor DHSs and are highly expressed in early embryos, but not in differentiated tissues. In summary, our data provide an evolutionary developmental view for understanding the regulation of gene and transposon expression.


Assuntos
Cromatina/metabolismo , Embrião de Mamíferos/metabolismo , Evolução Molecular , Animais , Sítios de Ligação , Ilhas de CpG , Metilação de DNA , Elementos de DNA Transponíveis/genética , Desoxirribonuclease I/metabolismo , Regulação para Baixo , Desenvolvimento Embrionário , Humanos , Camundongos , Fator 3 de Transcrição de Octâmero/antagonistas & inibidores , Fator 3 de Transcrição de Octâmero/genética , Fator 3 de Transcrição de Octâmero/metabolismo , Regiões Promotoras Genéticas , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Zigoto/metabolismo
14.
Cell ; 169(2): 184-185, 2017 04 06.
Artigo em Inglês | MEDLINE | ID: mdl-28388402

RESUMO

The striking correlation between genome topology and transcriptional activity has for decades made researchers revisit the question, "Does form follow function, or does function follow form?" In a new study, Hug et al. address this question by comparing the timing of zygotic genome activation to the emergence of genome structures during Drosophila embryogenesis.


Assuntos
Drosophila melanogaster/genética , Regulação da Expressão Gênica no Desenvolvimento , Animais , Desenvolvimento Embrionário , Genoma , Zigoto
15.
Cell ; 168(1-2): 210-223.e11, 2017 Jan 12.
Artigo em Inglês | MEDLINE | ID: mdl-28086092

RESUMO

Transcriptional control requires epigenetic changes directed by mitochondrial tricarboxylic acid (TCA) cycle metabolites. In the mouse embryo, global epigenetic changes occur during zygotic genome activation (ZGA) at the 2-cell stage. Pyruvate is essential for development beyond this stage, which is at odds with the low activity of mitochondria in this period. We now show that a number of enzymatically active mitochondrial enzymes associated with the TCA cycle are essential for epigenetic remodeling and are transiently and partially localized to the nucleus. Pyruvate is essential for this nuclear localization, and a failure of TCA cycle enzymes to enter the nucleus correlates with loss of specific histone modifications and a block in ZGA. At later stages, however, these enzymes are exclusively mitochondrial. In humans, the enzyme pyruvate dehydrogenase is transiently nuclear at the 4/8-cell stage coincident with timing of human embryonic genome activation, suggesting a conserved metabolic control mechanism underlying early pre-implantation development.


Assuntos
Ciclo do Ácido Cítrico , Genoma , Zigoto/metabolismo , Animais , Blastocisto/metabolismo , Núcleo Celular/metabolismo , Epigênese Genética , Glicosilação , Histonas/metabolismo , Cetona Oxirredutases/metabolismo , Camundongos , Mitocôndrias/enzimologia , Mitocôndrias/metabolismo , Ácido Pirúvico/metabolismo
16.
Cell ; 169(2): 216-228.e19, 2017 04 06.
Artigo em Inglês | MEDLINE | ID: mdl-28388407

RESUMO

Chromatin architecture is fundamental in regulating gene expression. To investigate when spatial genome organization is first established during development, we examined chromatin conformation during Drosophila embryogenesis and observed the emergence of chromatin architecture within a tight time window that coincides with the onset of transcription activation in the zygote. Prior to zygotic genome activation, the genome is mostly unstructured. Early expressed genes serve as nucleation sites for topologically associating domain (TAD) boundaries. Activation of gene expression coincides with the establishment of TADs throughout the genome and co-localization of housekeeping gene clusters, which remain stable in subsequent stages of development. However, the appearance of TAD boundaries is independent of transcription and requires the transcription factor Zelda for locus-specific TAD boundary insulation. These results offer insight into when spatial organization of the genome emerges and identify a key factor that helps trigger this architecture.


Assuntos
Cromatina/metabolismo , Drosophila melanogaster/embriologia , Drosophila melanogaster/genética , Genoma de Inseto , Ativação Transcricional , Zigoto/metabolismo , Animais , Proteínas de Drosophila/metabolismo , Embrião não Mamífero/metabolismo , Genes Essenciais , Proteínas Nucleares , RNA Polimerase II/metabolismo , Fatores de Tempo , Fatores de Transcrição/metabolismo , Transcrição Gênica
17.
Cell ; 170(2): 367-381.e20, 2017 Jul 13.
Artigo em Inglês | MEDLINE | ID: mdl-28709003

RESUMO

High-order chromatin structure plays important roles in gene expression regulation. Knowledge of the dynamics of 3D chromatin structures during mammalian embryo development remains limited. We report the 3D chromatin architecture of mouse gametes and early embryos using an optimized Hi-C method with low-cell samples. We find that mature oocytes at the metaphase II stage do not have topologically associated domains (TADs). In sperm, extra-long-range interactions (>4 Mb) and interchromosomal interactions occur frequently. The high-order structures of both the paternal and maternal genomes in zygotes and two-cell embryos are obscure but are gradually re-established through development. The establishment of the TAD structure requires DNA replication but not zygotic genome activation. Furthermore, unmethylated CpGs are enriched in A compartment, and methylation levels are decreased to a greater extent in A compartment than in B compartment in embryos. In summary, the global reprogramming of chromatin architecture occurs during early mammalian development.


Assuntos
Cromatina/metabolismo , Embrião de Mamíferos/metabolismo , Desenvolvimento Embrionário , Animais , Cromatina/química , Ilhas de CpG , Metilação de DNA , Replicação do DNA , Embrião de Mamíferos/química , Epigênese Genética , Feminino , Células Germinativas/metabolismo , Masculino , Metáfase , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos DBA , Oócitos/citologia , Espermatozoides/metabolismo , Zigoto/metabolismo
18.
Annu Rev Cell Dev Biol ; 34: 381-403, 2018 10 06.
Artigo em Inglês | MEDLINE | ID: mdl-30028643

RESUMO

Fertilizable eggs develop from diploid precursor cells termed oocytes. Once every menstrual cycle, an oocyte matures into a fertilizable egg in the ovary. To this end, the oocyte eliminates half of its chromosomes into a small cell termed a polar body. The egg is then released into the Fallopian tube, where it can be fertilized. Upon fertilization, the egg completes the second meiotic division, and the mitotic division of the embryo starts. This review highlights recent work that has shed light on the cytoskeletal structures that drive the meiotic divisions of the oocyte in mammals. In particular, we focus on how mammalian oocytes assemble a microtubule spindle in the absence of centrosomes, how they position the spindle in preparation for polar body extrusion, and how the spindle segregates the chromosomes. We primarily focus on mouse oocytes as a model system but also highlight recent insights from human oocytes.


Assuntos
Meiose/genética , Oócitos/crescimento & desenvolvimento , Fuso Acromático/genética , Zigoto/crescimento & desenvolvimento , Animais , Centrossomo , Cromossomos/genética , Feminino , Fertilização/genética , Humanos , Camundongos , Microtúbulos/genética
19.
Annu Rev Biochem ; 85: 659-83, 2016 Jun 02.
Artigo em Inglês | MEDLINE | ID: mdl-27145846

RESUMO

Life depends on cell proliferation and the accurate segregation of chromosomes, which are mediated by the microtubule (MT)-based mitotic spindle and ∼200 essential MT-associated proteins. Yet, a mechanistic understanding of how the mitotic spindle is assembled and achieves chromosome segregation is still missing. This is mostly due to the density of MTs in the spindle, which presumably precludes their direct observation. Recent insight has been gained into the molecular building plan of the metaphase spindle using bulk and single-molecule measurements combined with computational modeling. MT nucleation was uncovered as a key principle of spindle assembly, and mechanistic details about MT nucleation pathways and their coordination are starting to be revealed. Lastly, advances in studying spindle assembly can be applied to address the molecular mechanisms of how the spindle segregates chromosomes.


Assuntos
Centrossomo/metabolismo , Cinetocoros/metabolismo , Metáfase , Microtúbulos/metabolismo , Fuso Acromático/metabolismo , Animais , Centrossomo/ultraestrutura , Segregação de Cromossomos , Drosophila melanogaster/citologia , Drosophila melanogaster/metabolismo , Regulação da Expressão Gênica , Humanos , Cinesinas/genética , Cinesinas/metabolismo , Cinetocoros/ultraestrutura , Proteínas Associadas aos Microtúbulos/genética , Proteínas Associadas aos Microtúbulos/metabolismo , Microtúbulos/ultraestrutura , Transdução de Sinais , Fuso Acromático/ultraestrutura , Tubulina (Proteína)/genética , Tubulina (Proteína)/metabolismo , Proteínas de Xenopus/genética , Proteínas de Xenopus/metabolismo , Xenopus laevis/genética , Xenopus laevis/metabolismo , Zigoto/citologia , Zigoto/metabolismo
20.
Genes Dev ; 38(3-4): 131-150, 2024 03 22.
Artigo em Inglês | MEDLINE | ID: mdl-38453481

RESUMO

Maternal inactivation of genes encoding components of the subcortical maternal complex (SCMC) and its associated member, PADI6, generally results in early embryo lethality. In humans, SCMC gene variants were found in the healthy mothers of children affected by multilocus imprinting disturbances (MLID). However, how the SCMC controls the DNA methylation required to regulate imprinting remains poorly defined. We generated a mouse line carrying a Padi6 missense variant that was identified in a family with Beckwith-Wiedemann syndrome and MLID. If homozygous in female mice, this variant resulted in interruption of embryo development at the two-cell stage. Single-cell multiomic analyses demonstrated defective maturation of Padi6 mutant oocytes and incomplete DNA demethylation, down-regulation of zygotic genome activation (ZGA) genes, up-regulation of maternal decay genes, and developmental delay in two-cell embryos developing from Padi6 mutant oocytes but little effect on genomic imprinting. Western blotting and immunofluorescence analyses showed reduced levels of UHRF1 in oocytes and abnormal localization of DNMT1 and UHRF1 in both oocytes and zygotes. Treatment with 5-azacytidine reverted DNA hypermethylation but did not rescue the developmental arrest of mutant embryos. Taken together, this study demonstrates that PADI6 controls both nuclear and cytoplasmic oocyte processes that are necessary for preimplantation epigenetic reprogramming and ZGA.


Assuntos
Oócitos , Zigoto , Animais , Criança , Feminino , Humanos , Camundongos , Proteínas Estimuladoras de Ligação a CCAAT/genética , Citoplasma/genética , Citoplasma/metabolismo , Metilação de DNA/genética , Desenvolvimento Embrionário/genética , Impressão Genômica/genética , Ubiquitina-Proteína Ligases/metabolismo
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