RESUMEN
Sexual reproduction culminates in a totipotent zygote with the potential to produce a whole organism. Sperm chromatin reorganization and epigenetic reprogramming that alter DNA and histone modifications generate a totipotent embryo. Active DNA demethylation of the paternal genome has been proposed to involve base excision and DNA repair-based mechanisms. The nature and consequence of DNA lesions generated during reprogramming are not known. Using mouse genetics and chemical biology, we discovered that Tet3-dependent zygotic reprogramming generates paternal DNA lesions that are monitored by a surveillance mechanism. In vivo structure-function rescue assays revealed that cohesin-dependent repair of paternal DNA lesions prevents activation of a Chk1-dependent checkpoint that delays mitotic entry. Culturing conditions affect checkpoint stringency, which has implications for human in vitro fertilization. We propose the zygotic checkpoint senses DNA lesions generated during paternal DNA demethylation and ensures reprogrammed loci are repaired before mitosis to prevent chromosome fragmentation, embryo loss, and infertility.
Asunto(s)
Reparación del ADN , Cigoto/citología , Cigoto/metabolismo , Animales , Proteínas de Ciclo Celular/metabolismo , Reprogramación Celular , Proteínas Cromosómicas no Histona/metabolismo , Metilación de ADN , Proteínas de Unión al ADN/metabolismo , Dioxigenasas , Embrión de Mamíferos/metabolismo , Femenino , Masculino , Ratones , Ratones Endogámicos C57BL , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Proteína 1 de Reparación por Escisión del Grupo de Complementación Cruzada de las Lesiones por Rayos X , CohesinasRESUMEN
Fertilization triggers assembly of higher-order chromatin structure from a condensed maternal and a naïve paternal genome to generate a totipotent embryo. Chromatin loops and domains have been detected in mouse zygotes by single-nucleus Hi-C (snHi-C), but not bulk Hi-C. It is therefore unclear when and how embryonic chromatin conformations are assembled. Here, we investigated whether a mechanism of cohesin-dependent loop extrusion generates higher-order chromatin structures within the one-cell embryo. Using snHi-C of mouse knockout embryos, we demonstrate that the zygotic genome folds into loops and domains that critically depend on Scc1-cohesin and that are regulated in size and linear density by Wapl. Remarkably, we discovered distinct effects on maternal and paternal chromatin loop sizes, likely reflecting differences in loop extrusion dynamics and epigenetic reprogramming. Dynamic polymer models of chromosomes reproduce changes in snHi-C, suggesting a mechanism where cohesin locally compacts chromatin by active loop extrusion, whose processivity is controlled by Wapl. Our simulations and experimental data provide evidence that cohesin-dependent loop extrusion organizes mammalian genomes over multiple scales from the one-cell embryo onward.
Asunto(s)
Proteínas Portadoras/metabolismo , Proteínas de Ciclo Celular/metabolismo , Cromatina/genética , Proteínas Cromosómicas no Histona/metabolismo , Genoma/genética , Proteínas Nucleares/metabolismo , Fosfoproteínas/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Animales , Proteínas Portadoras/genética , Proteínas de Ciclo Celular/genética , Proteínas Cromosómicas no Histona/genética , Cromosomas/genética , Proteínas de Unión al ADN , Epigenómica , Femenino , Técnicas de Inactivación de Genes , Masculino , Ratones , Ratones Endogámicos C57BL , Proteínas Nucleares/genética , Fosfoproteínas/genética , Proteínas Proto-Oncogénicas/genética , Cigoto , CohesinasRESUMEN
The significance of transient neuropeptide expression during postnatal brain development is unknown. Here, we show that galanin expression in the ventrobasal thalamus of infant mice coincides with whisker map development and modulates subcortical circuit wiring. Time-resolved neuroanatomy and single-nucleus RNA-seq identified complementary galanin (Gal) and galanin receptor 1 (Galr1) expression in the ventrobasal thalamus and the principal sensory nucleus of the trigeminal nerve (Pr5), respectively. Somatodendritic galanin release from the ventrobasal thalamus was time-locked to the first postnatal week, when Gal1R+ Pr5 afferents form glutamatergic (Slc17a6+) synapses for the topographical whisker map to emerge. RNAi-mediated silencing of galanin expression disrupted glutamatergic synaptogenesis, which manifested as impaired whisker-dependent exploratory behaviors in infant mice, with behavioral abnormalities enduring into adulthood. Pharmacological probing of receptor selectivity in vivo corroborated that target recognition and synaptogenesis in the thalamus, at least in part, are reliant on agonist-induced Gal1R activation in inbound excitatory axons. Overall, we suggest a neuropeptide-dependent developmental mechanism to contribute to the topographical specification of a fundamental sensory neurocircuit in mice.
Asunto(s)
Galanina , Vibrisas , Animales , Humanos , Ratones , Axones/metabolismo , Encéfalo/metabolismo , Galanina/metabolismo , Tálamo/metabolismo , Vibrisas/fisiologíaRESUMEN
Life begins with a switch in genetic control from the maternal to the embryonic genome during zygotic genome activation (ZGA). Despite its importance, the essential regulators of ZGA remain largely unknown in mammals. On the basis of de novo motif searches, we identified the orphan nuclear receptor Nr5a2 as a key activator of major ZGA in mouse two-cell embryos. Nr5a2 is required for progression beyond the two-cell stage. It binds to its motif within SINE B1/Alu retrotransposable elements found in cis-regulatory regions of ZGA genes. Chemical inhibition suggests that 72% of ZGA genes are regulated by Nr5a2 and potentially other orphan nuclear receptors. Nr5a2 promotes chromatin accessibility during ZGA and binds nucleosomal DNA in vitro. We conclude that Nr5a2 is an essential pioneer factor that regulates ZGA.
Asunto(s)
Desarrollo Embrionario , Cigoto , Ratones , Animales , Desarrollo Embrionario/genética , Cigoto/metabolismo , Cromatina/genética , Cromatina/metabolismo , Genoma , Regulación del Desarrollo de la Expresión Génica , Mamíferos/genética , Receptores Citoplasmáticos y Nucleares/genéticaRESUMEN
Cohesin is essential for genome folding and inheritance. In somatic cells, these functions are both mediated by Scc1-cohesin, which in mitosis is released from chromosomes by Wapl and separase. In mammalian oocytes, cohesion is mediated by Rec8-cohesin. Scc1 is expressed but neither required nor sufficient for cohesion, and its function remains unknown. Likewise, it is unknown whether Wapl regulates one or both cohesin complexes and chromosome segregation in mature oocytes. Here, we show that Wapl is required for accurate meiosis I chromosome segregation, predominantly releases Scc1-cohesin from chromosomes, and promotes production of euploid eggs. Using single-nucleus Hi-C, we found that Scc1 is essential for chromosome organization in oocytes. Increasing Scc1 residence time on chromosomes by Wapl depletion leads to vermicelli formation and intra-loop structures but, unlike in somatic cells, does not increase loop size. We conclude that distinct cohesin complexes generate loops and cohesion in oocytes and propose that the same principle applies to all cell types and species.
Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Proteínas Cromosómicas no Histona/metabolismo , Cromosomas de los Mamíferos/metabolismo , Proteínas de Unión al ADN/metabolismo , Oocitos/metabolismo , Proteínas/metabolismo , Animales , Células Cultivadas , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , CohesinasRESUMEN
The early embryo is the natural prototype for the acquisition of totipotency, which is the potential of a cell to produce a whole organism. Generation of a totipotent embryo involves chromatin reorganization and epigenetic reprogramming that alter DNA and histone modifications. Understanding embryonic chromatin architecture and how this is related to the epigenome and transcriptome will provide invaluable insights into cell fate decisions. Recently emerging low-input genomic assays allow the exploration of regulatory networks in the sparsely available mammalian embryo. Thus, the field of developmental biology is transitioning from microscopy to genome-wide chromatin descriptions. Ultimately, the prototype becomes a unique model for studying fundamental principles of development, epigenetic reprogramming, and cellular plasticity. In this review, we discuss chromatin reprogramming in the early mouse embryo, focusing on DNA methylation, chromatin accessibility, and higher-order chromatin structure.
Asunto(s)
Reprogramación Celular , Cromatina/química , Epigénesis Genética , Regulación del Desarrollo de la Expresión Génica , Genoma , Histonas/genética , Animales , Diferenciación Celular , Cromatina/metabolismo , ADN/genética , ADN/metabolismo , Metilación de ADN , Embrión de Mamíferos , Desarrollo Embrionario , Células Germinativas/citología , Células Germinativas/metabolismo , Código de Histonas , Histonas/metabolismo , RatonesRESUMEN
The cohesin complex is essential for chromosome segregation in mitosis and meiosis. Cohesin is a tripartite protein complex that holds sister chromatids together from DNA replication until anaphase. In mammals, meiotic DNA replication occurs in oogonia of embryos and chromosome segregation occurs in oocytes of sexually mature females. Sister chromatid cohesion establishment and chromosome segregation are thus separated by months in the mouse and decades in the human. The meiotic cohesin complex that maintains sister chromatid cohesion must therefore hold replicated sisters together for a long time in oocytes. Remarkably, this is achieved by establishing cohesion exclusively in prenatal oocytes. Meiotic cohesion in females is maintained without detectable turnover and cohesin is therefore thought to be a long-lived protein complex. Nevertheless, the lifespan of cohesin molecules is limited as chromosomal cohesin levels decline with maternal age. The age-related loss of cohesin and weakened cohesion correlate with an age-related increase in chromosome missegregation of meiosis I oocytes that results in aneuploid eggs. Therefore, loss of chromosomal cohesin has been proposed to be a leading cause of the maternal age effect. To better understand cohesin deterioration in oocytes, it is crucial to gain insights into mammalian cohesion establishment and maintenance mechanisms by manipulating cohesin in live oocytes.This chapter describes techniques that address the manipulation of meiotic cohesin levels in mouse oocytes. First, we describe how cohesin can be efficiently removed from meiotic chromosomes by injecting mRNA encoding TEV protease in live oocytes expressing cohesin with engineered TEV recognition sites, followed by imaging. Secondly, we describe how cohesin expression can be induced during different stages of oocyte development using genetically modified mouse strains. In particular, we describe how to determine the deletion timing of germline-specific Cre recombinases using ß-galactosidase staining of fetal ovaries. Lastly, we provide guidance on how to quantify cohesin levels on metaphase I chromosome spreads.