RESUMEN
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RESUMEN
Single-cell RNA sequencing of embryos can resolve the transcriptional landscape of development at unprecedented resolution. To date, single-cell RNA-sequencing studies of mammalian embryos have focused exclusively on eutherian species. Analysis of mammalian outgroups has the potential to identify deeply conserved lineage specification and pluripotency factors, and can extend our understanding of X dosage compensation. Metatherian (marsupial) mammals diverged from eutherians around 160 million years ago. They exhibit distinctive developmental features, including late implantation1 and imprinted X chromosome inactivation2, which is associated with expression of the XIST-like noncoding RNA RSX3. Here we perform a single-cell RNA-sequencing analysis of embryogenesis and X chromosome inactivation in a marsupial, the grey short-tailed opossum (Monodelphis domestica). We resolve the developmental trajectory and transcriptional signatures of the epiblast, primitive endoderm and trophectoderm, and identify deeply conserved lineage-specific markers that pre-date the eutherian-marsupial divergence. RSX coating and inactivation of the X chromosome occurs early and rapidly. This observation supports the hypothesis that-in organisms with early X chromosome inactivation-imprinted X chromosome inactivation prevents biallelic X silencing. We identify XSR, an RSX antisense transcript expressed from the active X chromosome, as a candidate for the regulator of imprinted X chromosome inactivation. Our datasets provide insights into the evolution of mammalian embryogenesis and X dosage compensation.
Asunto(s)
Embrión de Mamíferos/citología , Desarrollo Embrionario/genética , Monodelphis/embriología , Monodelphis/genética , Análisis de la Célula Individual , Transcriptoma/genética , Inactivación del Cromosoma X/genética , Animales , Linaje de la Célula/genética , Embrión de Mamíferos/embriología , Femenino , Estratos Germinativos/citología , Estratos Germinativos/embriología , Masculino , Monodelphis/clasificación , ARN sin Sentido/genética , ARN no Traducido/genética , Regulación hacia Arriba , Cromosoma X/genéticaRESUMEN
DNA double-strand breaks (DSBs) are toxic to mammalian cells. However, during meiosis, more than 200 DSBs are generated deliberately, to ensure reciprocal recombination and orderly segregation of homologous chromosomes. If left unrepaired, meiotic DSBs can cause aneuploidy in gametes and compromise viability in offspring. Oocytes in which DSBs persist are therefore eliminated by the DNA-damage checkpoint. Here we show that the DNA-damage checkpoint eliminates oocytes via the pro-apoptotic BCL-2 pathway members Puma, Noxa and Bax. Deletion of these factors prevents oocyte elimination in recombination-repair mutants, even when the abundance of unresolved DSBs is high. Remarkably, surviving oocytes can extrude a polar body and be fertilised, despite chaotic chromosome segregation at the first meiotic division. Our findings raise the possibility that allelic variants of the BCL-2 pathway could influence the risk of embryonic aneuploidy.
Asunto(s)
Mutación , Oocitos/metabolismo , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Reparación del ADN por Recombinación/genética , Aneuploidia , Animales , Apoptosis , Proteínas Reguladoras de la Apoptosis/deficiencia , Proteínas Reguladoras de la Apoptosis/genética , Proteínas Reguladoras de la Apoptosis/metabolismo , Proteínas de Ciclo Celular/deficiencia , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Segregación Cromosómica , Roturas del ADN de Doble Cadena , Proteínas de Unión al ADN/deficiencia , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Endodesoxirribonucleasas/deficiencia , Endodesoxirribonucleasas/genética , Endodesoxirribonucleasas/metabolismo , Femenino , Fertilización , Genes bcl-2 , Meiosis/genética , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Oocitos/citología , Proteínas de Unión a Fosfato/deficiencia , Proteínas de Unión a Fosfato/genética , Proteínas de Unión a Fosfato/metabolismo , Proteínas Proto-Oncogénicas c-bcl-2/deficiencia , Proteínas Proto-Oncogénicas c-bcl-2/genética , Transducción de Señal , Proteínas Supresoras de Tumor/deficiencia , Proteínas Supresoras de Tumor/genética , Proteínas Supresoras de Tumor/metabolismo , Proteína X Asociada a bcl-2/deficiencia , Proteína X Asociada a bcl-2/genética , Proteína X Asociada a bcl-2/metabolismoRESUMEN
Meiotic synapsis and recombination ensure correct homologous segregation and genetic diversity. Asynapsed homologs are transcriptionally inactivated by meiotic silencing, which serves a surveillance function and in males drives meiotic sex chromosome inactivation. Silencing depends on the DNA damage response (DDR) network, but how DDR proteins engage repressive chromatin marks is unknown. We identify the histone H3-lysine-9 methyltransferase SETDB1 as the bridge linking the DDR to silencing in male mice. At the onset of silencing, X chromosome H3K9 trimethylation (H3K9me3) enrichment is downstream of DDR factors. Without Setdb1, the X chromosome accrues DDR proteins but not H3K9me3. Consequently, sex chromosome remodeling and silencing fail, causing germ cell apoptosis. Our data implicate TRIM28 in linking the DDR to SETDB1 and uncover additional factors with putative meiotic XY-silencing functions. Furthermore, we show that SETDB1 imposes timely expression of meiotic and post-meiotic genes. Setdb1 thus unites the DDR network, asynapsis, and meiotic chromosome silencing.
Asunto(s)
Emparejamiento Cromosómico , Daño del ADN , Silenciador del Gen , Código de Histonas , N-Metiltransferasa de Histona-Lisina/metabolismo , Animales , Apoptosis , Reparación del ADN , N-Metiltransferasa de Histona-Lisina/genética , Histonas/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Proteína 28 que Contiene Motivos Tripartito/genética , Proteína 28 que Contiene Motivos Tripartito/metabolismoRESUMEN
Sex chromosomes are advantageous to mammals, allowing them to adopt a genetic rather than environmental sex determination system. However, sex chromosome evolution also carries a burden, because it results in an imbalance in gene dosage between females (XX) and males (XY). This imbalance is resolved by X dosage compensation, which comprises both X chromosome inactivation and X chromosome upregulation. X dosage compensation has been well characterized in the soma, but not in the germ line. Germ cells face a special challenge, because genome wide reprogramming erases epigenetic marks responsible for maintaining the X dosage compensated state. Here we explain how evolution has influenced the gene content and germ line specialization of the mammalian sex chromosomes. We discuss new research uncovering unusual X dosage compensation states in germ cells, which we postulate influence sexual dimorphisms in germ line development and cause infertility in individuals with sex chromosome aneuploidy.
Asunto(s)
Compensación de Dosificación (Genética)/genética , Dosificación de Gen/genética , Células Germinativas/fisiología , Mamíferos/genética , Cromosoma X/genética , Animales , Humanos , Caracteres Sexuales , Regulación hacia Arriba/genéticaRESUMEN
Somatic X dosage compensation requires two mechanisms: X inactivation balances X gene output between males (XY) and females (XX), while X upregulation, hypothesized by Ohno and documented in vivo, balances X gene with autosomal gene output. Whether X dosage compensation occurs in germ cells is unclear. We show that mouse and human germ cells exhibit non-canonical X dosage states that differ from the soma and between the sexes. Prior to genome-wide reprogramming, X upregulation is present, consistent with Ohno's hypothesis. Subsequently, however, it is erased. In females, erasure follows loss of X inactivation, causing X dosage excess. Conversely, in males, erasure leads to permanent X dosage decompensation. Sex chromosomally abnormal models exhibit a "sex-reversed" X dosage state: XX males, like XX females, develop X dosage excess, while XO females, like XY males, develop X dosage decompensation. Thus, germline X dosage compensation states are determined by X chromosome number, not phenotypic sex. These unexpected differences in X dosage compensation states between germline and soma offer unique perspectives on sex chromosome infertility.
Asunto(s)
Cromosomas Humanos X/genética , Compensación de Dosificación (Genética) , Células Germinativas/metabolismo , Caracteres Sexuales , Cromosoma X/genética , Animales , Reprogramación Celular/genética , Bases de Datos Genéticas , Femenino , Perfilación de la Expresión Génica , Regulación de la Expresión Génica , Células Germinativas/citología , Gónadas/citología , Gónadas/metabolismo , Humanos , Masculino , Ratones , Modelos Genéticos , Análisis de Secuencia de ARN , Regulación hacia Arriba/genéticaRESUMEN
In female (XX) mammals, one of the two X chromosomes is inactivated to ensure an equal dose of X-linked genes with males (XY). X-chromosome inactivation in eutherian mammals is mediated by the non-coding RNA Xist. Xist is not found in metatherians (marsupials), and how X-chromosome inactivation is initiated in these mammals has been the subject of speculation for decades. Using the marsupial Monodelphis domestica, here we identify Rsx (RNA-on-the-silent X), an RNA that has properties consistent with a role in X-chromosome inactivation. Rsx is a large, repeat-rich RNA that is expressed only in females and is transcribed from, and coats, the inactive X chromosome. In female germ cells, in which both X chromosomes are active, Rsx is silenced, linking Rsx expression to X-chromosome inactivation and reactivation. Integration of an Rsx transgene on an autosome in mouse embryonic stem cells leads to gene silencing in cis. Our findings permit comparative studies of X-chromosome inactivation in mammals and pose questions about the mechanisms by which X-chromosome inactivation is achieved in eutherians.
Asunto(s)
Monodelphis/genética , Monodelphis/metabolismo , ARN/genética , ARN/metabolismo , Inactivación del Cromosoma X , Cromosoma X/genética , Cromosoma X/metabolismo , Animales , Femenino , Regulación de la Expresión Génica , Silenciador del Gen , Ratones , TransgenesRESUMEN
How the replication machinery is loaded at origins of DNA replication is poorly understood. Here, we implicate in this process the Xenopus laevis homolog (xRTS) of the RECQL4 helicase mutated in Rothmund-Thomson syndrome. xRTS, which bears homology to the yeast replication factors Sld2/DRC1, is essential for DNA replication in egg extracts. xRTS can be replaced in extracts by its human homolog, while RECQL4 depletion from mammalian cells induces proliferation failure, suggesting an evolutionarily conserved function. xRTS accumulates on chromatin during replication initiation, after prereplication-complex (pre-RC) proteins, Cut5, Sld5, or Cdc45 but before replicative polymerases. xRTS depletion suppresses the loading of RPA, the ssDNA binding protein that marks unwound origins before polymerase recruitment. However, xRTS is unaffected by xRPA depletion. Thus, xRTS functions after pre-RC formation to promote loading of replication factors at origins, a previously unrecognized activity necessary for initiation. This role connects defective replication initiation to a chromosome-fragility disorder.