Transcriptome of mouse preimplantation development [Rlim KO]
Ontology highlight
ABSTRACT: Purpose: The goals of this study are to establish a roadmap of XCI and compare the transcriptomes of WT and Rlim KO embryos during X chromosome inactivation. Methods: mRNA profiles of 175 preimplantation embryos WT and KO for Rlim were elucidated by RNA-seq at various stages. Trophoblasts isolated from blastocyst outgrowths were also included. The sequence reads that samples where gender could be determined and that passed quality filters were analyzed at the level of autosomes, X xhromosomes as well as single genes. Results: Using single cell RNA-seq technology on 175 whole preimplantation embryos, we obtained about 2.95 million sequence reads per sample. Reads were normalized to autosomal gene expression. Gender of each embryo was determined by expression of Y-linked genes and Xist. Data analysis showed normal expression profiles of marker genes for epiblast and trophoblast cell types during preimplantation development. Comparing Xist expression profiles in embryos WT and KO shows that Rlim is not required for initiation of Xist transcription but for upregulation of Xist expression. Moreover, our results identify two waves of XCI during preimplantation development: One that occurs at Morula stages that is Rlim-independent and one at blastocyst stages that in dependent on Rlim. Conclusions: Our study represents the first detailed mouse preimplantation transcriptome. Our results show that Rlim is required for a second wave of imprinted XCI that occurs in female embryos at blastocyst stages.
Project description:Purpose: The goals of this study are to establish a dynamic roadmap of imprinted X chromosome inactivation and the role of Xist by elucidation of the transcriptome of Xist KO embryos during mouse preimplantation development. Methods: mRNA profiles of the preimplantation embryos WT and KO for Xist were elucidated by RNA-seq at various stages. Trophoblasts isolated from blastocyst outgrowths were also included. The sequence reads for samples where gender could be determined and that passed quality filters were analyzed at the level of autosomes, X chromosome as well as single genes. Results: Female embryos fail to silence the X chromosome at late preimplantation development. General autosomal gene expression is not affected in embryos lacking Xist. Conclusions: Xist is crucial for iXCI. In preimplantation embryos, the main in vivo function of Xist is to regulate iXCI in females.
Project description:Purpose: The goals of this study are to establish a dynamic roadmap of imprinted X chromosome inactivation and the role of Xist by elucidation of the transcriptome of Xist KO embryos during mouse preimplantation development Methods: mRNA profiles of the preimplantation embryos WT and KO for Xist were elucidated by RNA-seq at various stages. Trophoblasts isolated from blastocyst outgrowths were also included. The sequence reads that samples where gender could be determined and that passed quality filters were analyzed at the level of autosomes, X chromosome as well as single genes. Effects of genetic background on the kinetics of iXCI was evaluated by RNA-seq on E3.5 embryos with a hybrid C57BL/6 x Cast background. Results: Female embryos fail to silence the X chromosome at late preimplantation development. General autosomal gene expression is not affected in embryos lacking Xist. Conclusions: Xist is crucial for iXCI. In preimplantation embryos the main in vivo function of Xist is to regulate iXCI in females. Genetic background does not significantly influence kinetics of iXCI.
Project description:Cloning mammals by somatic cell nuclear transfer (SCNT) is highly inefficient. Most SCNT-generated embryos die after implantation because of unidentified, complex epigenetic errors in the process of postimplantation embryonic development. Here, we identified the most upstream level of dysfunction leading to impaired development of clones by using RNA interference (RNAi) against Xist, a gene responsible for X chromosome inactivation (XCI). A prior injection of Xist-specific short interfering (si)RNA into reconstructed oocytes efficiently corrected the SCNT-specific aberrant Xist expression at the morula stage, but failed to do so thereafter at the blastocyst stage. However, we found that shortly after implantation this aberrant XCI status in cloned embryos had been corrected autonomously in both embryonic and extraembryonic tissues, probably through a newly established XCI control for postimplantation embryos. Embryo transfer experiments revealed that the siRNA-treated embryos showed 10 times higher survival than controls as early as embryonic day 5.5 and this high survival persisted until term, resulting in a remarkable improvement in cloning efficiency (12% vs. 1% in controls). Importantly, unlike control clones, these Xist-siRNA clones at birth showed only a limited dysregulation of their gene expression, indicating that correction of Xist expression in preimplantation embryos had a long-term effect on their postnatal normality. Thus, contrary to the general assumption, our results suggest that the fate of cloned embryos is determined almost exclusively before implantation by their XCI status. Furthermore, our strategy provides a promising breakthrough for mammalian SCNT cloning because RNAi treatment of oocytes is readily applicable to most mammal species. Gene expression were measured in mouse in vitro fertilized and somatic cell cloned embryos. Four biological replicates were performed in each group of Xist- or Control-siRNA.
Project description:Xist represents a paradigm for long non-coding RNA function in epigenetic regulation, although how it mediates X-chromosome inactivation (XCI) remains largely unexplained. Multiple Xist-RNA binding proteins have recently been identified, including SPEN/SHARP, whose knockdown has been associated with deficient XCI at multiple loci. Here we demonstrate that SPEN is a key orchestrator of XCI in vivo and unravel its mechanism of action. We show that SPEN is essential for initiating gene silencing on the X chromosome in preimplantation mouse embryos and embryonic stem cells. On the other hand, SPEN is dispensable for maintenance of XCI in neural progenitor cells, although it significantly dampens expression of genes that escape from XCI. During initiation of XCI, we show by live-cell imaging and CUT&RUN approaches that SPEN is immediately recruited to the X chromosome upon Xist up-regulation, where it is targeted to enhancers and promoters of actively transcribed genes. SPEN rapidly disengages from chromatin once silencing is accomplished, implying a need for active transcription to tether it to chromatin. We define SPEN’s SPOC (SPEN paralog and ortholog C-terminal) domain as a major effector of SPEN’s gene silencing function, and show that artificial tethering of SPOC to Xist RNA is sufficient to mediate X-linked gene silencing. We identify SPOC’s protein partners which include NCOR/SMRT, the m6A RNA methylation machinery, the NuRD complex, RNA polymerase II and factors involved in regulation of transcription initiation and elongation. We propose that SPEN acts as a molecular integrator for initiation of XCI, bridging Xist RNA with the transcription machinery as well as nucleosome remodelers and histone deacetylases, at active enhancers and promoters.
Project description:Cloning mammals by somatic cell nuclear transfer (SCNT) is highly inefficient. Most SCNT-generated embryos die after implantation because of unidentified, complex epigenetic errors in the process of postimplantation embryonic development. Here, we identified the most upstream level of dysfunction leading to impaired development of clones by using RNA interference (RNAi) against Xist, a gene responsible for X chromosome inactivation (XCI). A prior injection of Xist-specific short interfering (si)RNA into reconstructed oocytes efficiently corrected the SCNT-specific aberrant Xist expression at the morula stage, but failed to do so thereafter at the blastocyst stage. However, we found that shortly after implantation this aberrant XCI status in cloned embryos had been corrected autonomously in both embryonic and extraembryonic tissues, probably through a newly established XCI control for postimplantation embryos. Embryo transfer experiments revealed that the siRNA-treated embryos showed 10 times higher survival than controls as early as embryonic day 5.5 and this high survival persisted until term, resulting in a remarkable improvement in cloning efficiency (12% vs. 1% in controls). Importantly, unlike control clones, these Xist-siRNA clones at birth showed only a limited dysregulation of their gene expression, indicating that correction of Xist expression in preimplantation embryos had a long-term effect on their postnatal normality. Thus, contrary to the general assumption, our results suggest that the fate of cloned embryos is determined almost exclusively before implantation by their XCI status. Furthermore, our strategy provides a promising breakthrough for mammalian SCNT cloning because RNAi treatment of oocytes is readily applicable to most mammal species.
Project description:Genomic imprinting is essential for mammalian development. Recent studies have revealed that maternal histone H3 lysine 27 tri-methylation (H3K27me3) can mediate DNA methylation-independent genomic imprinting. However, the regulatory mechanisms and functions of this new imprinting mechanism are largely unknown. Here we demonstrate that maternal Eed, an essential component of the Polycomb group complex 2 (PRC2), is required for establishing H3K27me3 imprinting. We found that all H3K27me3 imprinted genes, including Xist, lose their imprinted expression in Eed maternal KO (matKO) embryos, resulting in male-biased lethality. Surprisingly, although maternal X chromosome inactivation (XmCI) occurs in Eed matKO embryos at preimplantation due to loss of Xist imprinting, it is resolved at peri-implantation. Ultimately, both X chromosomes are reactivated in the embryonic cell lineage prior to random XCI, and only a single X chromosome undergoes random XCI in the extra-embryonic cell lineage. Thus, our study not only demonstrates an essential role of Eed in H3K27me3 imprinting establishment but also reveals a unique XCI dynamics in the absence of Xist imprinting.
Project description:In this project, we have studied the role of Chd8 in Xist regulation and XCI initiation by means of Chd8 Knock-Downs (KD) and Knock-Out (KO).
Project description:X chromosome inactivation (XCI) triggers a drastic reprogramming of gene activities and chromosome architecture. However, how the 3D organization of the inactive X chromosome (Xi) is de novo established in vivo in mammals remains poorly understood. By comprehensive stage- and lineage- specific Hi-C mapping, we identified a unique Xist-separated megadomain structure (X-megadomains) on the Xi in mouse early embryos. X-megadomains emerge in extraembryonic lineages during imprinted XCI, in derived extraembryonic endoderm (XEN) cells, and transiently in the embryonic lineages during random XCI, before Dxz4-delineated megadomains (D-megadomains) occur at later stages in a strain-specific manner. Mechanistically, the emergence of X-megadomain boundary coincides with developmentally regulated enhancer activities and cohesin binding in a regulatory region near Xist (XRR). We pinpointed a subregion XRRa that is critical for the X-megadomain boundary. X-megadomains are impaired when XRRa is removed or cohesin is degraded in XEN cells. Importantly, this is accompanied by ectopic activation of regulatory elements and genes near Xist, suggesting that cohesin loading at regulatory elements promotes X-megadomains and confines local gene activities. Finally, the knockout of XRRa in mouse preimplantation embryos severely impairs the activation of Xist and the initiation of XCI. Hence, our data not only reveal stepwise chromosome folding during de novo XCI in vivo, but also support a model that regulatory element-dependent gene activation and cohesin loading simultaneously promote essential transcription activities and subsequent self-insulation amid global silencing during the early stage of XCI.
Project description:X chromosome inactivation (XCI) triggers a drastic reprogramming of gene activities and chromosome architecture. However, how the 3D organization of the inactive X chromosome (Xi) is de novo established in vivo in mammals remains poorly understood. By comprehensive stage- and lineage- specific Hi-C mapping, we identified a unique Xist-separated megadomain structure (X-megadomains) on the Xi in mouse early embryos. X-megadomains emerge in extraembryonic lineages during imprinted XCI, in derived extraembryonic endoderm (XEN) cells, and transiently in the embryonic lineages during random XCI, before Dxz4-delineated megadomains (D-megadomains) occur at later stages in a strain-specific manner. Mechanistically, the emergence of X-megadomain boundary coincides with developmentally regulated enhancer activities and cohesin binding in a regulatory region near Xist (XRR). We pinpointed a subregion XRRa that is critical for the X-megadomain boundary. X-megadomains are impaired when XRRa is removed or cohesin is degraded in XEN cells. Importantly, this is accompanied by ectopic activation of regulatory elements and genes near Xist, suggesting that cohesin loading at regulatory elements promotes X-megadomains and confines local gene activities. Finally, the knockout of XRRa in mouse preimplantation embryos severely impairs the activation of Xist and the initiation of XCI. Hence, our data not only reveal stepwise chromosome folding during de novo XCI in vivo, but also support a model that regulatory element-dependent gene activation and cohesin loading simultaneously promote essential transcription activities and subsequent self-insulation amid global silencing during the early stage of XCI.
Project description:X chromosome inactivation (XCI) triggers a drastic reprogramming of gene activities and chromosome architecture. However, how the 3D organization of the inactive X chromosome (Xi) is de novo established in vivo in mammals remains poorly understood. By comprehensive stage- and lineage- specific Hi-C mapping, we identified a unique Xist-separated megadomain structure (X-megadomains) on the Xi in mouse early embryos. X-megadomains emerge in extraembryonic lineages during imprinted XCI, in derived extraembryonic endoderm (XEN) cells, and transiently in the embryonic lineages during random XCI, before Dxz4-delineated megadomains (D-megadomains) occur at later stages in a strain-specific manner. Mechanistically, the emergence of X-megadomain boundary coincides with developmentally regulated enhancer activities and cohesin binding in a regulatory region near Xist (XRR). We pinpointed a subregion XRRa that is critical for the X-megadomain boundary. X-megadomains are impaired when XRRa is removed or cohesin is degraded in XEN cells. Importantly, this is accompanied by ectopic activation of regulatory elements and genes near Xist, suggesting that cohesin loading at regulatory elements promotes X-megadomains and confines local gene activities. Finally, the knockout of XRRa in mouse preimplantation embryos severely impairs the activation of Xist and the initiation of XCI. Hence, our data not only reveal stepwise chromosome folding during de novo XCI in vivo, but also support a model that regulatory element-dependent gene activation and cohesin loading simultaneously promote essential transcription activities and subsequent self-insulation amid global silencing during the early stage of XCI.