RESUMO
Nucleosome positioning can alter the accessibility of DNA-binding proteins to their cognate DNA elements, and thus its precise control is essential for cell identity and function. Mammalian preimplantation embryos undergo temporal changes in gene expression and cell potency, suggesting the involvement of dynamic epigenetic control during this developmental phase. However, the dynamics of nucleosome organization during early development are poorly understood. In this study, using a low-input MNase-seq method, we show that nucleosome positioning is globally obscure in zygotes but becomes well defined during subsequent development. Down-regulation of the chromatin assembly in embryonic stem cells can partially reverse nucleosome organization into a zygote-like pattern, suggesting a possible link between the chromatin assembly pathway and fuzzy nucleosomes in zygotes. We also reveal that YY1, a zinc finger-containing transcription factor expressed upon zygotic genome activation, regulates the de novo formation of well-positioned nucleosome arrays at the regulatory elements through identifying YY1-binding sites in eight-cell embryos. The YY1-binding regions acquire H3K27ac enrichment around the eight-cell and morula stages, and YY1 depletion impairs the morula-to-blastocyst transition. Thus, our study delineates the remodeling of nucleosome organization and its underlying mechanism during early mouse development.
Assuntos
Nucleossomos , Fatores de Transcrição , Animais , Camundongos , Cromatina , Montagem e Desmontagem da Cromatina/genética , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Regulação da Expressão Gênica , Mamíferos/genética , Nucleossomos/genética , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
Pluripotent stem cells can be classified into two distinct states, naïve and primed, which show different degrees of potency. One difficulty in stem cell research is the inability to distinguish these states in live cells. Studies on female mice have shown that reactivation of inactive X chromosomes occurs in the naïve state, while one of the X chromosomes is inactivated in the primed state. Therefore, we aimed to distinguish the two states by monitoring X chromosome reactivation. Thus far, X chromosome reactivation has been analysed using fixed cells; here, we inserted different fluorescent reporter gene cassettes (mCherry and eGFP) into each X chromosome. Using these knock-in 'Momiji' mice, we detected X chromosome reactivation accurately in live embryos, and confirmed that the pluripotent states of embryos were stable ex vivo, as represented by embryonic and epiblast stem cells in terms of X chromosome reactivation. Thus, Momiji mice provide a simple and accurate method for identifying stem cell status based on X chromosome reactivation.
Assuntos
Embrião de Mamíferos/metabolismo , Células-Tronco Pluripotentes/metabolismo , Inativação do Cromossomo X/fisiologia , Cromossomo X/metabolismo , Animais , Feminino , Camadas Germinativas/citologia , Camadas Germinativas/metabolismo , Humanos , Imuno-Histoquímica , Hibridização in Situ Fluorescente , Camundongos , Camundongos Mutantes , Fosfoglicerato Quinase/genética , Fosfoglicerato Quinase/metabolismo , Células-Tronco Pluripotentes/citologia , Cromossomo X/genética , Inativação do Cromossomo X/genéticaRESUMO
Errors during cell division in oocytes and early embryos are linked to birth defects in mammals. Bipolar spindle assembly in early mouse embryos is unique in that three or more acentriolar microtubule-organizing centers (MTOCs) are initially formed and are then clustered into two spindle poles. Using a knockout mouse and live imaging of spindles in embryos, we demonstrate that MTOC clustering during the blastocyst stage requires augmin, a critical complex for MT-dependent MT nucleation within the spindle. Functional analyses in cultured cells with artificially increased numbers of centrosomes indicate that the lack of intra-spindle MT nucleation, but not loss of augmin per se or overall reduction of spindle MTs, is the cause of clustering failure. These data suggest that onset of mitosis with three or more MTOCs is turned into a typical bipolar division through augmin-dependent intra-spindle MT assembly.
Assuntos
Blastocisto/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Centro Organizador dos Microtúbulos/metabolismo , Microtúbulos/metabolismo , Animais , Blastocisto/ultraestrutura , Células Cultivadas , Camundongos , Proteínas Associadas aos Microtúbulos/genética , Centro Organizador dos Microtúbulos/ultraestrutura , MitoseRESUMO
During early embryogenesis, before the conceptus forms the placenta, maternal nutrients as well as signaling molecules must reach the embryo proper through a tightly sealed epithelial tissue, the visceral endoderm (VE). The VE serves as a signaling center for embryogenesis, where exocytic and endocytic processes integrate signal production, perception and termination. However, the endocytic process in this important tissue has not been well characterized. We show that endocytic delivery to the lysosomes occurs via RAB7-dependent microautophagy. This process is essential for early mammalian development.