Systems analyses reveal shared and diverse attributes of Oct4 regulation in pluripotent cells
ABSTRACT: Epiblast stem cells (EpiSCs) are pluripotent cells that can be isolated and cultured from post implantation embryos. In contrast to embryonic stem cells (ESCs), systematic studies to investigate the genes that maintain pluripotency in EpiSCs have not been reported. Here we combine a genome-wide RNAi screen with genetic interaction, protein localization and protein-level dependency studies to delineate connectivity between factors that control Oct4 expression in EpiSCs and compare the role of these factors to their function in ES cells. We demonstrate the power of this integrative approach by the identification of Tox4 as an interactor of PP1 (Protein Phosphatase 1) and Paf1C, a complex that acts in multiple aspects of RNAPII regulation. Our results indicate that Tox4 cooperates with Paf1C and PP1 to influence the phosphorylation status of the RNAPII CTD tail during transcription and that this function is vital for maintenance of pluripotent cell identity. RNA-seq of Tox4 knockdown in mouse EpiSCs
Project description:Transcriptional profiling of XiGFP Epiblast Stem Cells (EpiSCs) overexpressing Klf2, Prdm14, Prdm14+Klf2, or vector control. Cells cultured in activin and bFGF (day 0) or on day 2 and day 4 after transfer to serum and LIF on feeder cells and sorting for dsRed expression to remove feeder cells.
Project description:Here we show that by simple modulation of extrinsic signaling pathways, a new class of pluripotent stem cells, referred to as region selective epiblast stem cells (rsEpiSCs), could be efficiently derived from different stages of the early embryo. rsEpiSCs share features of primed pluripotency yet are distinct from EpiSCs in their molecular characteristics and ability to colonize post-implantation embryos. We performed RNA-sequencing experiments and examined the global gene expression profiles of EpiSCs, rsEpiSCs, in vivo isolated four regions of E6.5 mouse epiblasts: AP (anterior-proximal), AD (anterior-distal), PP (posterior-proximal) and PD (posterior-distal), human H1 ESCs, H1 rsESCs, H9 ESCs, H9 rsESCs, rhesus monkey ORMES23 rsESCs, and chimpanzee rsiPSCs. Examination of global gene expression profiles in 2 pluripotent stem cell types across multiple species.
Project description:Embryonic Stem Cells (ESCs) and Epiblast Stem Cells (EpiSCs) are the in vitro representants of naïve and primed pluripotency, respectively. It is currently unclear how their epigenome underpin the phenotypic and molecular characteristics of these states of pluripotency. Here, we performed the first qualitative and quantitative comparison of DNA methylation between ESCs and EpiSCs. The global level and genomic distribution of DNA methylation were very similar between the two cell types. However, the analysis of promoter methylation patterns in EpiSCs revealed several distinct features: (i) the repression of germline-related genes by DNA methylation, a process already ongoing in ESCs but far more pronounced in EpiSCs; (ii) the hypermethylation of promoters (especially CpG rich) in EpiSC compared to both ESCs and dissected epiblasts from E6.5 and E7.5 embryos; (iii) the inability of hypomethylated (Dnmt-deficient) ESCs to be converted into EpiSCs despite their ability to self-renew. Altogether, our data show that DNA methylation is an important epigenetic regulator of gene expression in EpiSCs and suggest that it is essential for the obtention of EpiSCs. MethylCap-Seq (DNA methylation profiling) and RNA-Seq of 3 EpiStem Cells (EpiSCs) and 1 Embryonic Stem Cell line of which the data has been submitted to GEO before (part of GEO SuperSeries GSE23943 and GSE31343)
Project description:The PAF complex (Paf1C) has been shown to regulate chromatin modifications, gene transcription, and PolII elongation. Here, we provide the first genome-wide analysis of chromatin occupancy by the entire PAF complex in mammalian cells. We show that Paf1C is recruited not only to promoters and gene bodies, but also to regions downstream of cleavage/polyadenylation (pA) sites at 3’ ends, a profile that sharply contrasted with the yeast complex. Remarkably, our studies identified novel, subunit-specific links between Paf1C and regulation of alternative cleavage and polyadenylation (APA) and upstream antisense transcription. Moreover, we found that depletion of Paf1C subunits also resulted in the accumulation of RNA polymerase II (PolII) over gene bodies, which coincided with APA. Depletion of specific Paf1C subunits leads to global loss of histone H2B ubiquitylation, but surprisingly, there is little impact of Paf1C depletion on other histone modifications, including the tri-methylation of histone H3 on lysines 4 and 36 (H3K4me3 and H3K36me3), previously associated with this complex. Our results provide surprising differences with yeast, while unifying observations that link Paf1C with PolII elongation and RNA processing, and suggest that Paf1C could play a role in protecting transcripts from premature cleavage by preventing PolII accumulation at TSS-proximal pA sites. ChIP-seq, RNA-seq and 3'READS of Paf1C factors in mouse C2C12 myoblast cells
Project description:Carrying out both RNA-seq and Smad1/5 genome-wide chromatin immunoprecipitation and sequencing (ChIP-seq) analyses of mESCs in the naïve or primed states, we revisit the roles of BMP signaling in mESCs. RNA-seq analysis in 2 cell types; mESCs and ES-derived EpiSC (ESD-EpiSCs).
Project description:Recently, (in vitro) pluripotent EpiSCs were derived from the post-implantation egg cylinder stage epiblasts of mouse and rat. These EpiSCs resemble and correspond very closely to the conventional human embryonic stem cells (hESCs) in the colony morphology and culture/signaling requirements for maintaining pluripotency, but exhibit a range of significant phenotypic and signaling response differences from the conventional mouse ES cells (mESCs). These observations strongly support the notion that EpiSCs and hESCs are intrinsically similar, and raise an attractive hypothesis: as mESCs and EpiSCs/hESCs represent two distinct pluripotency states: the mESC-like state representing the ICM of pre-implantation blastcyst and the EpiSC-like state representing the post-implantation epiblasts, whether the epiblast state (including conventional hESCs) can be converted back to the ICM state. Despite studies providing evidence that epiblast-like cells exist and transition back and forth within colony of conventional mESCs; mESCs and EpiSCs share substantial set of pluripotency transcriptional factors, including Oct4, Sox2 and Nanog; and mESCs are more stable in culture, in the present study we found that EpiSCs differentiated rapidly under mESC culture conditions and no “spontaneously” converted mESC could be readily identified and isolated over serial passages at the population or clonal level. Remarkably, we found that blockage of the TGFβ pathway or inhibition of the H3K4 demethylase LSD1 with small molecule inhibitors induced dramatic morphological changes of EpiSCs towards mESC phenotypes with activation of some ICM-specific gene expression. However, full conversion of EpiSCs to a mESC-like state with competence to chimeric contribution can only be readily generated with a combination of inhibitors of LSD1 and ALK. These observations underscore a powerful and direct induction of reprogramming from the developmentally later-stage EpiSCs to a mESC-like stage by a synergy of signaling and direct epigenetic modulations. It also highlights a significant role for TGFβ pathway inhibition in promoting reprogramming to and sustaining true pluripotency, which further supports our recent studies in generating chimerism-competent rat pluripotent cells. Collectively, our studies provide a proof-of-concept demonstration that pluripotency-restricted EpiSCs can be readily converted to a mESC-like state in the absence of any genetic manipulation by precise pharmacological control of signaling pathways that distinguish the two pluripotency states and an epigenetic target simultaneously, and offer a convenient experimental system to further study the mechanism. Such method and concept may also provide an avenue for generating a new type of mESC-like human pluripotent cell. Global gene-expression analyses of the parnate/mAMFGi cells
Project description:Pluripotent cell identity comprises a spectrum of cell states including naive and primed states, which are typified by mouse embryonic stem cells (ESCs) and epiblast-derived stem cells (EpiSCs), respectively. Here we define a pluripotent cell fate (PCF) gene signature based on RNA-seq analysis associated with naive and primed pluripotency acquisition, and identify Zfp281 as a key transcriptional regulator for primed pluripotency and also as a barrier to achieve the naive pluripotency of both mouse and human ESCs. RNA sequencing analysis was performed in WT and Zfp281 null mouse embryonic stem cells under different pluripotent culture conditions. RNA-seq Experiments were carry out in two biological replciates. Genome binding/occupancy profiling of Zfp281 was performed in mouse embryonic stem cells by ChIP sequencing.
Project description:Mammalian primed pluripotent stem cells have been shown to be highly susceptible to cell death stimuli due to their low apoptotic threshold, but how this threshold is regulated remains largely unknown. Here we identify miRNA-mediated regulation as a key mechanism controlling apoptosis in the post-implantation epiblast. Moreover, we find that three miRNA families, miR-20, miR-92 and miR-302, control the mitochondrial apoptotic machinery by fine-tuning the levels of expression of the pro-apoptotic protein BIM. These families therefore represent an essential buffer needed to maintain cell survival in stem cells that are not only primed for differentiation but also for cell death. We used microarrays to compare the gene expression profiles of Dicer conditional Epiblast stem cells (Dicer fx/fx EpiSCs, used as control cells) and Dicer deleted epiblast stem cells (Dicer-/- EpiSCs) five days after the induction of Dicer deletion Dicer fx/fx EpiSCs were left untreated (control cells) or treated with 0.3uM of 4-OH-Tamoxifen for three days and without Tamoxifen for two further days, until day 5 when RNA was extracted and used for microarray analysis. Three independent deletion experiments including a Dicer fx/fx sample and a Dicer -/- sample were analyzed as biological replicates.
Project description:Here we show that by simple modulation of extrinsic signaling pathways, a new class of pluripotent stem cells, referred to as region selective epiblast stem cells (rsEpiSCs), could be efficiently derived from different stages of the early embryo. rsEpiSCs share features of primed pluripotency yet are distinct from EpiSCs in their molecular characteristics and ability to colonize post-implantation embryos. We performed ChIP-seqencing experiments using antibodies against H3K4me3 and H3K27me3, comparing conventional EpiSCs and rsEpiSCs, as well as comparing conventional human H1 ESCs and human H1 ESCs cultured in mouse rsEpiSCs based culture conditions (H1 rsESCs). Examination of 2 different histone modifications (H3K4me3, H3K27me3) in 2 pluripotent stem cell types in mouse and human.