RESUMEN
In female eutherian mammal development, X-chromosome inactivation (XCI) of one of the two X chromosomes is initiated early. Understanding the relationship between the initiation of XCI and cell fate is critical for understanding early female development and requires a system that can monitor XCI in single living cells. Traditional embryonic stem cells (ESCs) used for XCI studies often lose X chromosomes spontaneously during culture and differentiation, making accurate monitoring difficult. Additionally, most XCI assessment methods necessitate cell disruption, hindering cell fate tracking. We developed the Momiji (version 2) ESC line to address these difficulties, enabling real-time monitoring of X-chromosome activity via fluorescence. We inserted green and red fluorescent reporter genes and neomycin and puromycin resistance genes into the two X chromosomes of PGK12.1 ESCs, creating a female ESC line that retains two X chromosomes more faithfully during differentiation. Momiji (version 2) ESCs exhibit a more stable XX karyotype than other ESC lines, including the parental PGK12.1 line. This new tool offers valuable insights into the relationship between XCI and cell fate, improving our understanding of early female development.
Asunto(s)
Imagen de Lapso de Tiempo , Inactivación del Cromosoma X , Inactivación del Cromosoma X/genética , Animales , Femenino , Ratones , Imagen de Lapso de Tiempo/métodos , Diferenciación Celular/genética , Análisis de la Célula Individual/métodos , Línea Celular , Células Madre Embrionarias/metabolismo , Células Madre Embrionarias/citología , Cromosoma X/genética , Genes ReporterosRESUMEN
In eukaryotes, DNA is housed within the cell nucleus. Molecules required for the formation of a nucleus have been identified using in vitro systems with frog egg extracts and in vivo imaging of somatic cells. However, little is known about the physicochemical factors and conditions required for nuclear formation in mouse oocytes. In this study, using a reconstitution approach with purified DNA, we aimed to determine factors, such as the amount and timing of DNA introduction, required for the formation of nuclei with nuclear transport activity in mouse oocytes. T4 phage DNA (~166 kbp) was microinjected into strontium-activated oocytes to evaluate the conditions appropriate for nuclear formation. Microinjection of 100-500 ng/µL of T4 DNA, but not 20 ng/µL, was sufficient for the formation of nucleus-like structures. Furthermore, microinjection of DNA during metaphase II to telophase II, but not during interphase, was sufficient. Electron and fluorescence microscopy showed that T4 DNA-induced nucleus-like structures had nuclear lamina and nuclear pore complex structures similar to those of natural nuclei, as well as nuclear import activity. These results suggest that exogenous DNA can form artificial nuclei with nuclear transport functions in mouse oocytes, regardless of the sequence or source of the DNA.
RESUMEN
Histone H3 lysine36 dimethylation (H3K36me2) is generally distributed in the gene body and euchromatic intergenic regions. However, we found that H3K36me2 is enriched in pericentromeric heterochromatin in some mouse cell lines. We here revealed the mechanism of heterochromatin targeting of H3K36me2. Among several H3K36 methyltransferases, NSD2 was responsible for inducing heterochromatic H3K36me2. Depletion and overexpression analyses of NSD2-associating proteins revealed that NSD2 recruitment to heterochromatin was mediated through the imitation switch (ISWI) chromatin remodeling complexes, such as BAZ1B-SMARCA5 (WICH), which directly binds to AT-rich DNA via a BAZ1B domain-containing AT-hook-like motifs. The abundance and stoichiometry of NSD2, SMARCA5, and BAZ1B could determine the localization of H3K36me2 in different cell types. In mouse embryos, H3K36me2 heterochromatin localization was observed at the two- to four-cell stages, suggesting its physiological relevance.
Asunto(s)
Ensamble y Desensamble de Cromatina , Heterocromatina , N-Metiltransferasa de Histona-Lisina , Histonas , Proteínas Represoras , Animales , Humanos , Ratones , Adenosina Trifosfatasas , Proteínas que Contienen Bromodominio/genética , Proteínas que Contienen Bromodominio/metabolismo , Centrómero/metabolismo , Centrómero/genética , Proteínas Cromosómicas no Histona/metabolismo , Proteínas Cromosómicas no Histona/genética , Heterocromatina/metabolismo , Heterocromatina/genética , N-Metiltransferasa de Histona-Lisina/metabolismo , N-Metiltransferasa de Histona-Lisina/genética , Histonas/metabolismo , Histonas/genética , Metilación , Proteínas Represoras/metabolismo , Proteínas Represoras/genética , Factores de Transcripción/metabolismo , Factores de Transcripción/genéticaRESUMEN
Zygotes require two accurate sets of parental chromosomes, one each from the mother and the father, to undergo normal embryogenesis. However, upon egg-sperm fusion in vertebrates, the zygote has three sets of chromosomes, one from the sperm and two from the egg. The zygote therefore eliminates one set of maternal chromosomes (but not the paternal chromosomes) into the polar body through meiosis, but how the paternal chromosomes are protected from maternal meiosis has been unclear. Here we report that RanGTP and F-actin dynamics prevent egg-sperm fusion in proximity to maternal chromosomes. RanGTP prevents the localization of Juno and CD9, egg membrane proteins that mediate sperm fusion, at the cell surface in proximity to maternal chromosomes. Following egg-sperm fusion, F-actin keeps paternal chromosomes away from maternal chromosomes. Disruption of these mechanisms causes the elimination of paternal chromosomes during maternal meiosis. This study reveals a novel critical mechanism that prevents aneuploidy in zygotes.