RESUMEN
Germ cells are vital for transmitting genetic information from one generation to the next and for maintaining the continuation of species. Here, we analyze the transcriptome of human primordial germ cells (PGCs) from the migrating stage to the gonadal stage at single-cell and single-base resolutions. Human PGCs show unique transcription patterns involving the simultaneous expression of both pluripotency genes and germline-specific genes, with a subset of them displaying developmental-stage-specific features. Furthermore, we analyze the DNA methylome of human PGCs and find global demethylation of their genomes. Approximately 10 to 11 weeks after gestation, the PGCs are nearly devoid of any DNA methylation, with only 7.8% and 6.0% of the median methylation levels in male and female PGCs, respectively. Our work paves the way toward deciphering the complex epigenetic reprogramming of the germline with the aim of restoring totipotency in fertilized oocytes.
Asunto(s)
Metilación de ADN , Células Germinativas/metabolismo , Transcriptoma , Movimiento Celular , Cromosomas Humanos X , Análisis por Conglomerados , Embrión de Mamíferos/metabolismo , Femenino , Histonas/metabolismo , Humanos , Masculino , Análisis de Componente Principal , Factores de Transcripción SOX/metabolismoRESUMEN
The developmental pathway of the neural retina (NR) and retinal pigment epithelium (RPE) has been revealed by extensive research in mice. However, the molecular mechanisms underlying the development of the human NR and RPE, as well as the interactions between these two tissues, have not been well defined. Here, we analyzed 2,421 individual cells from human fetal NR and RPE using single-cell RNA sequencing (RNA-seq) technique and revealed the tightly regulated spatiotemporal gene expression network of human retinal cells. We identified major cell classes of human fetal retina and potential crucial transcription factors for each cell class. We dissected the dynamic expression patterns of visual cycle- and ligand-receptor interaction-related genes in the RPE and NR. Moreover, we provided a map of disease-related genes for human fetal retinal cells and highlighted the importance of retinal progenitor cells as potential targets of inherited retinal diseases. Our findings captured the key in vivo features of the development of the human NR and RPE and offered insightful clues for further functional studies.
Asunto(s)
Regulación del Desarrollo de la Expresión Génica , Retina/metabolismo , Epitelio Pigmentado de la Retina/metabolismo , Análisis de Secuencia de ARN/métodos , Análisis de la Célula Individual/métodos , Transcriptoma , Adulto , Ciclo Celular/genética , Células Cultivadas , Análisis por Conglomerados , Perfilación de la Expresión Génica/métodos , Ontología de Genes , Humanos , Retina/citología , Retina/embriología , Enfermedades de la Retina/genética , Epitelio Pigmentado de la Retina/citología , Epitelio Pigmentado de la Retina/embriologíaRESUMEN
DNA methylation is a crucial element in the epigenetic regulation of mammalian embryonic development. However, its dynamic patterns have not been analysed at the genome scale in human pre-implantation embryos due to technical difficulties and the scarcity of required materials. Here we systematically profile the methylome of human early embryos from the zygotic stage through to post-implantation by reduced representation bisulphite sequencing and whole-genome bisulphite sequencing. We show that the major wave of genome-wide demethylation is complete at the 2-cell stage, contrary to previous observations in mice. Moreover, the demethylation of the paternal genome is much faster than that of the maternal genome, and by the end of the zygotic stage the genome-wide methylation level in male pronuclei is already lower than that in female pronuclei. The inverse correlation between promoter methylation and gene expression gradually strengthens during early embryonic development, reaching its peak at the post-implantation stage. Furthermore, we show that active genes, with the trimethylation of histone H3 at lysine 4 (H3K4me3) mark at the promoter regions in pluripotent human embryonic stem cells, are essentially devoid of DNA methylation in both mature gametes and throughout pre-implantation development. Finally, we also show that long interspersed nuclear elements or short interspersed nuclear elements that are evolutionarily young are demethylated to a milder extent compared to older elements in the same family and have higher abundance of transcripts, indicating that early embryos tend to retain higher residual methylation at the evolutionarily younger and more active transposable elements. Our work provides insights into the critical features of the methylome of human early embryos, as well as its functional relation to the regulation of gene expression and the repression of transposable elements.
Asunto(s)
Metilación de ADN , Epigénesis Genética , Regulación del Desarrollo de la Expresión Génica , Animales , Elementos Transponibles de ADN/genética , Embrión de Mamíferos , Células Madre Embrionarias/fisiología , Femenino , Perfilación de la Expresión Génica , Células Germinativas/metabolismo , Histonas/metabolismo , Humanos , Elementos de Nucleótido Esparcido Largo/genética , Masculino , Ratones , Regiones Promotoras Genéticas/genética , Elementos de Nucleótido Esparcido Corto/genéticaRESUMEN
The epigenetic regulation of spatiotemporal gene expression is crucial for human development. Here, we present whole-genome chromatin immunoprecipitation followed by high throughput DNA sequencing (ChIP-seq) analyses of a wide variety of histone markers in the brain, heart, and liver of early human embryos shortly after their formation. We identified 40,181 active enhancers, with a large portion showing tissue-specific and developmental stage-specific patterns, pointing to their roles in controlling the ordered spatiotemporal expression of the developmental genes in early human embryos. Moreover, using sequential ChIP-seq, we showed that all three organs have hundreds to thousands of bivalent domains that are marked by both H3K4me3 and H3K27me3, probably to keep the progenitor cells in these organs ready for immediate differentiation into diverse cell types during subsequent developmental processes. Our work illustrates the potentially critical roles of tissue-specific and developmental stage-specific epigenomes in regulating the spatiotemporal expression of developmental genes during early human embryonic development.
Asunto(s)
Encéfalo/embriología , Cromatina/metabolismo , Epigénesis Genética , Corazón/embriología , Histonas/metabolismo , Hígado/embriología , Procesamiento Proteico-Postraduccional , Feto Abortado/metabolismo , Biomarcadores/metabolismo , Encéfalo/metabolismo , China , Cromatina/química , Bases de Datos de Ácidos Nucleicos , Regulación del Desarrollo de la Expresión Génica , Ontología de Genes , Biblioteca Genómica , Secuenciación de Nucleótidos de Alto Rendimiento , Histonas/genética , Humanos , Hígado/metabolismo , Lisina/metabolismo , Metilación , Miocardio/metabolismo , Especificidad de Órganos , Análisis de Secuencia de ADN , Especificidad de la EspecieRESUMEN
Studies of gene rearrangements and the consequent oncogenic fusion proteins have laid the foundation for targeted cancer therapy. To identify oncogenic fusions associated with glioma progression, we catalogued fusion transcripts by RNA-seq of 272 gliomas. Fusion transcripts were more frequently found in high-grade gliomas, in the classical subtype of gliomas, and in gliomas treated with radiation/temozolomide. Sixty-seven in-frame fusion transcripts were identified, including three recurrent fusion transcripts: FGFR3-TACC3, RNF213-SLC26A11, and PTPRZ1-MET (ZM). Interestingly, the ZM fusion was found only in grade III astrocytomas (1/13; 7.7%) or secondary GBMs (sGBMs, 3/20; 15.0%). In an independent cohort of sGBMs, the ZM fusion was found in three of 20 (15%) specimens. Genomic analysis revealed that the fusion arose from translocation events involving introns 3 or 8 of PTPRZ and intron 1 of MET. ZM fusion transcripts were found in GBMs irrespective of isocitrate dehydrogenase 1 (IDH1) mutation status. sGBMs harboring ZM fusion showed higher expression of genes required for PIK3CA signaling and lowered expression of genes that suppressed RB1 or TP53 function. Expression of the ZM fusion was mutually exclusive with EGFR overexpression in sGBMs. Exogenous expression of the ZM fusion in the U87MG glioblastoma line enhanced cell migration and invasion. Clinically, patients afflicted with ZM fusion harboring glioblastomas survived poorly relative to those afflicted with non-ZM-harboring sGBMs (P < 0.001). Our study profiles the shifting RNA landscape of gliomas during progression and reveled ZM as a novel, recurrent fusion transcript in sGBMs.
Asunto(s)
Neoplasias Encefálicas/genética , Glioblastoma/genética , Glioma/genética , Proteínas de Fusión Oncogénica/genética , Proteínas Proto-Oncogénicas c-met/genética , Proteínas Tirosina Fosfatasas Clase 5 Similares a Receptores/genética , Adolescente , Adulto , Anciano , Antineoplásicos Alquilantes , Western Blotting , Neoplasias Encefálicas/patología , Neoplasias Encefálicas/secundario , Línea Celular Tumoral , Quimioradioterapia , Dacarbazina/análogos & derivados , Dacarbazina/uso terapéutico , Femenino , Regulación Neoplásica de la Expresión Génica , Glioblastoma/secundario , Glioma/patología , Glioma/terapia , Células HEK293 , Humanos , Intrones/genética , Masculino , Persona de Mediana Edad , Proteínas de Fusión Oncogénica/metabolismo , Proteínas Proto-Oncogénicas c-met/metabolismo , Proteínas Tirosina Fosfatasas Clase 5 Similares a Receptores/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Análisis de Secuencia de ARN , Temozolomida , Translocación Genética , Adulto JovenRESUMEN
CBX4, a component of polycomb repressive complex 1 (PRC1), plays important roles in the maintenance of cell identity and organ development through gene silencing. However, whether CBX4 regulates human stem cell homeostasis remains unclear. Here, we demonstrate that CBX4 counteracts human mesenchymal stem cell (hMSC) aging via the maintenance of nucleolar homeostasis. CBX4 protein is downregulated in aged hMSCs, whereas CBX4 knockout in hMSCs results in destabilized nucleolar heterochromatin, enhanced ribosome biogenesis, increased protein translation, and accelerated cellular senescence. CBX4 maintains nucleolar homeostasis by recruiting nucleolar protein fibrillarin (FBL) and heterochromatin protein KRAB-associated protein 1 (KAP1) at nucleolar rDNA, limiting the excessive expression of rRNAs. Overexpression of CBX4 alleviates physiological hMSC aging and attenuates the development of osteoarthritis in mice. Altogether, our findings reveal a critical role of CBX4 in counteracting cellular senescence by maintaining nucleolar homeostasis, providing a potential therapeutic target for aging-associated disorders.
Asunto(s)
Nucléolo Celular/fisiología , Senescencia Celular/fisiología , Homeostasis , Ligasas/fisiología , Células Madre Mesenquimatosas/fisiología , Osteoartritis/terapia , Proteínas del Grupo Polycomb/fisiología , Animales , Proteínas Cromosómicas no Histona/metabolismo , Técnicas de Inactivación de Genes , Terapia Genética , Células HEK293 , Humanos , Ligasas/genética , Masculino , Ratones Endogámicos C57BL , Ratones Endogámicos NOD , Proteínas del Grupo Polycomb/genéticaRESUMEN
Primary germ layers have the potential to form all tissues in the mature organism, and their formation during gastrulation requires precise epigenetic modulation of both proximal and distal regulatory elements. Previous studies indicated that spatial and temporal patterns of gene expression in the gastrula predispose individual regions to distinct cell fates. However, the underlying epigenetic mechanisms remain largely unexplored. Here, we profile the spatiotemporal landscape of the epigenome and transcriptome of the mouse gastrula. We reveal the asynchronous dynamics of proximal chromatin states during germ layer formation as well as unique gastrula-specific epigenomic features of regulatory elements, which have strong usage turnover dynamics and clear germ layer-specific signatures. Importantly, we also find that enhancers around organogenetic genes, which are weakly expressed at the gastrulation stage, are frequently pre-marked by histone H3 lysine 27 acetylation (H3K27ac) in the gastrula. By using the transgenic mice and genome editing system, we demonstrate that a pre-marked enhancer, which is located in the intron of a brain-specific gene 2510009E07Rik, exhibits specific enhancer activity in the ectoderm and future brain tissue, and also executes important function during mouse neural differentiation. Taken together, our study provides the comprehensive epigenetic information for embryonic patterning during mouse gastrulation, demonstrates the importance of gastrula pre-marked enhancers in regulating the correct development of the mouse embryo, and thus broadens the current understanding of mammalian embryonic development and related diseases.
Asunto(s)
Elementos de Facilitación Genéticos/fisiología , Epigénesis Genética , Gástrula/embriología , Gastrulación/genética , Regulación del Desarrollo de la Expresión Génica , Estratos Germinativos/embriología , Animales , Encéfalo/embriología , Encéfalo/metabolismo , Células Cultivadas , Embrión de Mamíferos , Células Madre Embrionarias , Femenino , Gástrula/citología , Gástrula/metabolismo , Estratos Germinativos/citología , Estratos Germinativos/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Neurogénesis/genética , TranscriptomaRESUMEN
BACKGROUND: Organogenesis is crucial for proper organ formation during mammalian embryonic development. However, the similarities and shared features between different organs and the cellular heterogeneity during this process at single-cell resolution remain elusive. RESULTS: We perform single-cell RNA sequencing analysis of 1916 individual cells from eight organs and tissues of E9.5 to E11.5 mouse embryos, namely, the forebrain, hindbrain, skin, heart, somite, lung, liver, and intestine. Based on the regulatory activities rather than the expression patterns, all cells analyzed can be well classified into four major groups with epithelial, mesodermal, hematopoietic, and neuronal identities. For different organs within the same group, the similarities and differences of their features and developmental paths are revealed and reconstructed. CONCLUSIONS: We identify mutual interactions between epithelial and mesenchymal cells and detect epithelial cells with prevalent mesenchymal features during organogenesis, which are similar to the features of intermediate epithelial/mesenchymal cells during tumorigenesis. The comprehensive transcriptome at single-cell resolution profiled in our study paves the way for future mechanistic studies of the gene-regulatory networks governing mammalian organogenesis.
Asunto(s)
Epitelio/metabolismo , Mesodermo/metabolismo , Organogénesis/genética , Transcriptoma , Animales , Células Epiteliales/metabolismo , Epitelio/embriología , Perfilación de la Expresión Génica , Células Madre Hematopoyéticas/metabolismo , Mesodermo/embriología , Ratones , Neuronas/metabolismo , Análisis de Secuencia de ARN , Análisis de la Célula IndividualRESUMEN
In the version of this Resource originally published, owing to a technical error an incorrect file was used for Supplementary Table 2; this has now been replaced with the correct file.
RESUMEN
DNA methylation, chromatin states and their interrelationships represent critical epigenetic information, but these are largely unknown in human early embryos. Here, we apply single-cell chromatin overall omic-scale landscape sequencing (scCOOL-seq) to generate a genome-wide map of DNA methylation and chromatin accessibility at single-cell resolution during human preimplantation development. Unlike in mice, the chromatin of the paternal genome is already more open than that of the maternal genome at the mid-zygote stage in humans, and this state is maintained until the 4-cell stage. After fertilization, genes with high variations in DNA methylation, and those with high variations in chromatin accessibility, tend to be two different sets. Furthermore, 1,797 out of 5,155 (35%) widely open chromatin regions in promoters closed when transcription activity was inhibited, indicating a feedback mechanism between transcription and open chromatin maintenance. Our work paves the way for dissecting the complex, yet highly coordinated, epigenetic reprogramming during human preimplantation development.
Asunto(s)
Blastocisto/fisiología , Ensamble y Desensamble de Cromatina , Metilación de ADN , Epigénesis Genética , Regulación del Desarrollo de la Expresión Génica , Genómica/métodos , Análisis de la Célula Individual/métodos , Células Cultivadas , Cromosomas Humanos X , Técnicas de Cultivo de Embriones , Femenino , Células Madre Embrionarias Humanas/fisiología , Humanos , Masculino , Conformación de Ácido Nucleico , Regiones Promotoras Genéticas , Inyecciones de Esperma Intracitoplasmáticas , Transcripción GenéticaRESUMEN
Loss of organelle homeostasis is a hallmark of aging. However, it remains elusive how this occurs at gene expression level. Here, we report that human mesenchymal stem cell (hMSC) aging is associated with dysfunction of double-membrane organelles and downregulation of transcription factor ATF6. CRISPR/Cas9-mediated inactivation of ATF6 in hMSCs, not in human embryonic stem cells and human adipocytes, results in premature cellular aging, characteristic of loss of endomembrane homeostasis. Transcriptomic analyses uncover cell type-specific constitutive and stress-induced ATF6-regulated genes implicated in various layers of organelles' homeostasis regulation. FOS was characterized as a constitutive ATF6 responsive gene, downregulation of which contributes to hMSC aging. Our study unravels the first ATF6-regulated gene expression network related to homeostatic regulation of membrane organelles, and provides novel mechanistic insights into aging-associated attrition of human stem cells.
RESUMEN
DNA methylation is a crucial layer of epigenetic regulation during mammalian embryonic development 1-3 . Although the DNA methylome of early human embryos has been analyzed 4-6 , some of the key features have not been addressed thus far. Here we performed single-cell DNA methylome sequencing for human preimplantation embryos and found that tens of thousands of genomic loci exhibited de novo DNA methylation. This finding indicates that genome-wide DNA methylation reprogramming during preimplantation development is a dynamic balance between strong global demethylation and drastic focused remethylation. Furthermore, demethylation of the paternal genome is much faster and thorough than that of the maternal genome. From the two-cell to the postimplantation stage, methylation of the paternal genome is consistently lower than that of the maternal genome. We also show that the genetic lineage of early blastomeres can be traced by DNA methylation analysis. Our work paves the way for deciphering the secrets of DNA methylation reprogramming in early human embryos.
Asunto(s)
Blastocisto/metabolismo , Metilación de ADN , Islas de CpG , Femenino , Humanos , Masculino , Análisis de Secuencia de ADN , Análisis de la Célula IndividualRESUMEN
Although genomic instability, epigenetic abnormality, and gene expression dysregulation are hallmarks of colorectal cancer, these features have not been simultaneously analyzed at single-cell resolution. Using optimized single-cell multiomics sequencing together with multiregional sampling of the primary tumor and lymphatic and distant metastases, we developed insights beyond intratumoral heterogeneity. Genome-wide DNA methylation levels were relatively consistent within a single genetic sublineage. The genome-wide DNA demethylation patterns of cancer cells were consistent in all 10 patients whose DNA we sequenced. The cancer cells' DNA demethylation degrees clearly correlated with the densities of the heterochromatin-associated histone modification H3K9me3 of normal tissue and those of repetitive element long interspersed nuclear element 1. Our work demonstrates the feasibility of reconstructing genetic lineages and tracing their epigenomic and transcriptomic dynamics with single-cell multiomics sequencing.
Asunto(s)
Neoplasias Colorrectales/genética , Epigenómica/métodos , Análisis de la Célula Individual/métodos , Metilación de ADN , Femenino , Estudio de Asociación del Genoma Completo , Histonas , Humanos , Masculino , Análisis de Secuencia de ADNRESUMEN
Spermatogenesis generates mature male gametes and is critical for the proper transmission of genetic information between generations. However, the developmental landscapes of human spermatogenesis remain unknown. Here, we performed single-cell RNA sequencing (scRNA-seq) analysis for 2,854 testicular cells from donors with normal spermatogenesis and 174 testicular cells from one nonobstructive azoospermia (NOA) donor. A hierarchical model was established, which was characterized by the sequential and stepwise development of three spermatogonia subtypes, seven spermatocyte subtypes, and four spermatid subtypes. Further analysis identified several stage-specific marker genes of human germ cells, such as HMGA1, PIWIL4, TEX29, SCML1, and CCDC112. Moreover, we identified altered gene expression patterns in the testicular somatic cells of one NOA patient via scRNA-seq analysis, paving the way for further diagnosis of male infertility. Our work allows for the reconstruction of transcriptional programs inherent to sequential cell fate transition during human spermatogenesis and has implications for deciphering male-related reproductive disorders.
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Linaje de la Célula , Análisis de Secuencia de ARN , Análisis de la Célula Individual , Espermatogénesis/genética , Espermatozoides/citología , Espermatozoides/metabolismo , Células Cultivadas , Biología Computacional , Humanos , MasculinoRESUMEN
Single-cell epigenome sequencing techniques have recently been developed. However, the combination of different layers of epigenome sequencing in an individual cell has not yet been achieved. Here, we developed a single-cell multi-omics sequencing technology (single-cell COOL-seq) that can analyze the chromatin state/nucleosome positioning, DNA methylation, copy number variation and ploidy simultaneously from the same individual mammalian cell. We used this method to analyze the reprogramming of the chromatin state and DNA methylation in mouse preimplantation embryos. We found that within < 12 h of fertilization, each individual cell undergoes global genome demethylation together with the rapid and global reprogramming of both maternal and paternal genomes to a highly opened chromatin state. This was followed by decreased openness after the late zygote stage. Furthermore, from the late zygote to the 4-cell stage, the residual DNA methylation is preferentially preserved on intergenic regions of the paternal alleles and intragenic regions of maternal alleles in each individual blastomere. However, chromatin accessibility is similar between paternal and maternal alleles in each individual cell from the late zygote to the blastocyst stage. The binding motifs of several pluripotency regulators are enriched at distal nucleosome depleted regions from as early as the 2-cell stage. This indicates that the cis-regulatory elements of such target genes have been primed to an open state from the 2-cell stage onward, long before pluripotency is eventually established in the ICM of the blastocyst. Genes may be classified into homogeneously open, homogeneously closed and divergent states based on the chromatin accessibility of their promoter regions among individual cells. This can be traced to step-wise transitions during preimplantation development. Our study offers the first single-cell and parental allele-specific analysis of the genome-scale chromatin state and DNA methylation dynamics at single-base resolution in early mouse embryos and provides new insights into the heterogeneous yet highly ordered features of epigenomic reprogramming during this process.
Asunto(s)
Blastocisto/metabolismo , Cromatina , Metilación de ADN/fisiología , Regulación del Desarrollo de la Expresión Génica/fisiología , Genómica , Células Madre Embrionarias de Ratones/metabolismo , Animales , Blastocisto/citología , Línea Celular , Cromatina/genética , Cromatina/metabolismo , Femenino , Masculino , Ratones , Células Madre Embrionarias de Ratones/citologíaRESUMEN
We described a novel single-cell RNA-seq technique called MR-seq (measure a single-cell transcriptome repeatedly), which permits statistically assessing the technical variation and identifying the differentially expressed genes between just two single cells by measuring each single cell twice. We demonstrated that MR-seq gave sensitivity and reproducibility similar to the standard single-cell RNA-seq and increased the positive predicate value. Application of MR-seq to early mouse embryos identified hundreds of candidate intra-embryonic heterogeneous genes among mouse 2-, 4- and 8-cell stage embryos. MR-seq should be useful for detecting differentially expressed genes among a small number of cells.
RESUMEN
Chromatin remodeling is important for the epigenetic reprogramming of human primordial germ cells. However, the comprehensive chromatin state has not yet been analyzed for human fetal germ cells (FGCs). Here we use nucleosome occupancy and methylation sequencing method to analyze both the genome-wide chromatin accessibility and DNA methylome at a series of crucial time points during fetal germ cell development in both human and mouse. We find 116 887 and 137 557 nucleosome-depleted regions (NDRs) in human and mouse FGCs, covering a large set of germline-specific and highly dynamic regulatory genomic elements, such as enhancers. Moreover, we find that the distal NDRs are enriched specifically for binding motifs of the pluripotency and germ cell master regulators such as NANOG, SOX17, AP2γ and OCT4 in human FGCs, indicating the existence of a delicate regulatory balance between pluripotency-related genes and germ cell-specific genes in human FGCs, and the functional significance of these genes for germ cell development in vivo. Our work offers a comprehensive and high-resolution roadmap for dissecting chromatin state transition dynamics during the epigenomic reprogramming of human and mouse FGCs.
Asunto(s)
Cromatina/metabolismo , Metilación de ADN/genética , Feto/citología , Células Germinativas/metabolismo , Animales , Reprogramación Celular/genética , Impresión Genómica , Humanos , Masculino , Mamíferos/genética , Ratones , Nucleosomas/metabolismo , Especificidad de Órganos/genética , Regiones Promotoras Genéticas/genética , Secuencias Repetitivas de Ácidos Nucleicos/genéticaRESUMEN
Human fetal germ cells (FGCs) are precursors to sperm and eggs and are crucial for maintenance of the species. However, the developmental trajectories and heterogeneity of human FGCs remain largely unknown. Here we performed single-cell RNA-seq analysis of over 2,000 FGCs and their gonadal niche cells in female and male human embryos spanning several developmental stages. We found that female FGCs undergo four distinct sequential phases characterized by mitosis, retinoic acid signaling, meiotic prophase, and oogenesis. Male FGCs develop through stages of migration, mitosis, and cell-cycle arrest. Individual embryos of both sexes simultaneously contain several subpopulations, highlighting the asynchronous and heterogeneous nature of FGC development. Moreover, we observed reciprocal signaling interactions between FGCs and their gonadal niche cells, including activation of the bone morphogenic protein (BMP) and Notch signaling pathways. Our work provides key insights into the crucial features of human FGCs during their highly ordered mitotic, meiotic, and gametogenetic processes in vivo.
Asunto(s)
División Celular/fisiología , Células Germinales Embrionarias/metabolismo , Feto/metabolismo , Gónadas/enzimología , Transducción de Señal/fisiología , Nicho de Células Madre/fisiología , Proteínas Morfogenéticas Óseas/metabolismo , Células Germinales Embrionarias/citología , Femenino , Feto/citología , Gónadas/citología , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Masculino , Receptores Notch/metabolismoRESUMEN
Single-cell genome, DNA methylome, and transcriptome sequencing methods have been separately developed. However, to accurately analyze the mechanism by which transcriptome, genome and DNA methylome regulate each other, these omic methods need to be performed in the same single cell. Here we demonstrate a single-cell triple omics sequencing technique, scTrio-seq, that can be used to simultaneously analyze the genomic copy-number variations (CNVs), DNA methylome, and transcriptome of an individual mammalian cell. We show that large-scale CNVs cause proportional changes in RNA expression of genes within the gained or lost genomic regions, whereas these CNVs generally do not affect DNA methylation in these regions. Furthermore, we applied scTrio-seq to 25 single cancer cells derived from a human hepatocellular carcinoma tissue sample. We identified two subpopulations within these cells based on CNVs, DNA methylome, or transcriptome of individual cells. Our work offers a new avenue of dissecting the complex contribution of genomic and epigenomic heterogeneities to the transcriptomic heterogeneity within a population of cells.
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Carcinoma Hepatocelular/genética , Variaciones en el Número de Copia de ADN , Epigenómica/métodos , Perfilación de la Expresión Génica/métodos , Genómica/métodos , Neoplasias Hepáticas/genética , Animales , Metilación de ADN , Epigénesis Genética , Células Hep G2 , Humanos , Masculino , Ratones , Persona de Mediana Edad , Análisis de Secuencia de ADN , Análisis de la Célula Individual , TranscriptomaRESUMEN
The mRNAs stored in oocytes undergo general decay during the maternal-zygotic transition (MZT), and their stability is tightly interconnected with meiotic cell-cycle progression. However, the factors that trigger decay of maternal mRNA and couple this event to oocyte meiotic maturation remain elusive. Here, we identified B-cell translocation gene-4 (BTG4) as an MZT licensing factor in mice. BTG4 bridged CNOT7, a catalytic subunit of the CCR4-NOT deadenylase, to eIF4E, a key translation initiation factor, and facilitated decay of maternal mRNA. Btg4-null females produced morphologically normal oocytes but were infertile, owing to early developmental arrest. The intrinsic MAP kinase cascade in oocytes triggered translation of Btg4 mRNA stored in fully grown oocytes by targeting the 3' untranslated region, thereby coupling CCR4-NOT deadenylase-mediated decay of maternal mRNA with oocyte maturation and fertilization. This is a key step in oocyte cytoplasmic maturation that determines the developmental potential of mammalian embryos.