The metabolome regulates the epigenetic landscape during naive-to-primed human embryonic stem cell transition.
ABSTRACT: For nearly a century developmental biologists have recognized that cells from embryos can differ in their potential to differentiate into distinct cell types. Recently, it has been recognized that embryonic stem cells derived from both mice and humans exhibit two stable yet epigenetically distinct states of pluripotency: naive and primed. We now show that nicotinamide N-methyltransferase (NNMT) and the metabolic state regulate pluripotency in human embryonic stem cells (hESCs). Specifically, in naive hESCs, NNMT and its enzymatic product 1-methylnicotinamide are highly upregulated, and NNMT is required for low S-adenosyl methionine (SAM) levels and the H3K27me3 repressive state. NNMT consumes SAM in naive cells, making it unavailable for histone methylation that represses Wnt and activates the HIF pathway in primed hESCs. These data support the hypothesis that the metabolome regulates the epigenetic landscape of the earliest steps in human development.
Project description:The rate of glycolytic metabolism changes during differentiation of human embryonic stem cells (hESCs) and reprogramming of somatic cells to pluripotency. However, the functional contribution of glycolytic metabolism to the pluripotent state is unclear. Here we show that naive hESCs exhibit increased glycolytic flux, MYC transcriptional activity, and nuclear N-MYC localization relative to primed hESCs. This status is consistent with the inner cell mass of human blastocysts, where MYC transcriptional activity is higher than in primed hESCs and nuclear N-MYC levels are elevated. Reduction of glycolysis decreases self-renewal of naive hESCs and feeder-free primed hESCs, but not primed hESCs grown in feeder-supported conditions. Reduction of glycolysis in feeder-free primed hESCs also enhances neural specification. These findings reveal associations between glycolytic metabolism and human naive pluripotency and differences in the metabolism of feeder-/feeder-free cultured hESCs. They may also suggest methods for regulating self-renewal and initial cell fate specification of hESCs.
Project description:Until recently, human embryonic stem cells (hESCs) were shown to exist in a state of primed pluripotency, while mouse embryonic stem cells (mESCs) display a naive or primed pluripotent state. Here we show the rapid conversion of in-house-derived primed hESCs on mouse embryonic feeder layer (MEF) to a naive state within 5-6 days in naive conversion media (NCM-MEF), 6-10 days in naive human stem cell media (NHSM-MEF) and 14-20 days using the reverse-toggle protocol (RT-MEF). We further observe enhanced unbiased lineage-specific differentiation potential of naive hESCs converted in NCM-MEF, however, all naive hESCs fail to differentiate towards functional cell types. RNA-seq analysis reveals a divergent role of PI3K/AKT/mTORC signalling, specifically of the mTORC2 subunit, in the different naive hESCs. Overall, we demonstrate a direct evaluation of several naive culture conditions performed in the same laboratory, thereby contributing to an unbiased, more in-depth understanding of different naive hESCs.
Project description:Human embryonic stem cells (hESCs) typically exhibit "primed" pluripotency, analogous to stem cells derived from the mouse post-implantation epiblast. This has led to a search for growth conditions that support self-renewal of hESCs akin to hypomethylated naive epiblast cells in human pre-implantation embryos. We have discovered that reverting primed hESCs to a hypomethylated naive state or deriving a new hESC line under naive conditions results in the establishment of Stage Specific Embryonic Antigen 4 (SSEA4)-negative hESC lines with a transcriptional program resembling the human pre-implantation epiblast. In contrast, we discovered that the methylome of naive hESCs in vitro is distinct from that of the human epiblast in vivo with loss of DNA methylation at primary imprints and a lost "memory" of the methylation state of the human oocyte. This failure to recover the naive epiblast methylation landscape appears to be a consistent feature of self-renewing hypomethylated naive hESCs in vitro.
Project description:Recent progress has enabled the conversion of primed human embryonic stem cells (hESCs) to the naive state of pluripotency, resembling the well-characterized naive mouse ESCs (mESCs). However, a thorough histone epigenetic characterization of this conversion process is currently lacking, while its likeness to the mouse model has not been clearly established. Here, we profile the histone epigenome of hESCs during conversion in a time-resolved experimental design, using an untargeted mass spectrometry-based approach. In total, 23 histone post-translational modifications (hPTMs) changed significantly over time. H3K27Me3 was the most prominently increasing marker hPTM in naive hESCs. This is in line with previous reports in mouse, prompting us to compare all the shared hPTM fold changes between mouse and human, revealing a set of conserved hPTM markers for the naive state. Principally, we present the first roadmap of the changing human histone epigenome during the conversion of hESCs from the primed to the naive state. This further revealed similarities with mouse, which hint at a conserved mammalian epigenetic signature of the ground state of pluripotency.
Project description:Pluripotency is increasingly recognized as a spectrum of cell states defined by their growth conditions. Although naive and primed pluripotency states have been characterized molecularly, our understanding of events regulating state acquisition is wanting. Here, we performed comparative RNA sequencing of mouse embryonic stem cells (ESCs) and defined a pluripotent cell fate (PCF) gene signature associated with acquisition of naive and primed pluripotency. We identify Zfp281 as a key transcriptional regulator for primed pluripotency that also functions as a barrier toward achieving naive pluripotency in both mouse and human ESCs. Mechanistically, Zfp281 interacts with Tet1, but not Tet2, and its direct transcriptional target, miR-302/367, to negatively regulate Tet2 expression to establish and maintain primed pluripotency. Conversely, ectopic Tet2 alone, but not Tet1, efficiently reprograms primed cells toward naive pluripotency. Our study reveals a molecular circuitry in which opposing functions of Tet1 and Tet2 control acquisition of alternative pluripotent states.
Project description:Naive human embryonic stem cells (hESCs) can be derived from primed hESCs or directly from blastocysts, but their X chromosome state has remained unresolved. Here, we show that the inactive X chromosome (Xi) of primed hESCs was reactivated in naive culture conditions. Like cells of the blastocyst, the resulting naive cells contained two active X chromosomes with XIST expression and chromosome-wide transcriptional dampening and initiated XIST-mediated X inactivation upon differentiation. Both establishment of and exit from the naive state (differentiation) happened via an XIST-negative XaXa intermediate. Together, these findings identify a cell culture system for functionally exploring the two X chromosome dosage compensation processes in early human development: X dampening and X inactivation. However, remaining differences between naive hESCs and embryonic cells related to mono-allelic XIST expression and non-random X inactivation highlight the need for further culture improvement. As the naive state resets Xi abnormalities seen in primed hESCs, it may provide cells better suited for downstream applications.
Project description:Polycomb repressive complex 2 and the epigenetic mark that it deposits, H3K27me3, are evolutionarily conserved and play critical roles in development and cancer. However, their roles in cell fate decisions in early embryonic development remain poorly understood. Here we report that knockout of polycomb repressive complex 2 genes in human embryonic stem cells causes pluripotency loss and spontaneous differentiation toward a meso-endoderm fate, owing to de-repression of BMP signalling. Moreover, human embryonic stem cells with deletion of EZH1 or EZH2 fail to differentiate into ectoderm lineages. We further show that polycomb repressive complex 2-deficient mouse embryonic stem cells also release Bmp4 but retain their pluripotency. However, when converted into a primed state, they undergo spontaneous differentiation similar to that of hESCs. In contrast, polycomb repressive complex 2 is dispensable for pluripotency when human embryonic stem cells are converted into the naive state. Our studies reveal both lineage- and pluripotent state-specific roles of polycomb repressive complex 2 in cell fate decisions.Polycomb repressive complex 2 (PRC2) plays an essential role in development by modifying chromatin but what this means at a cellular level is unclear. Here, the authors show that ablation of PRC2 genes in human embryonic stem cells and in mice results in changes in pluripotency and the primed state of cells.
Project description:Nicotinamide N-methyltransferase (NNMT) transfers the methyl from S-adenosyl-L-methionine (SAM) to nicotinamide (NA) to produce S-adenosyl-L-homocysteine (SAH) and 1-methylnicotinamide (MeN). NNMT has been implicated in a variety of diseases; however, the role of NNMT in drug addiction is largely unknown. Here, we found that the expression of Nnmt was significantly upregulated in the dorsal striatum (DS) of cocaine-conditioned mice. Cocaine significantly decreased SAM/SAH ratio levels in the DS, which was accompanied with the decreased activities of Rac1 and RhoA. Lentivirus-mediated knockdown of Nnmt in the dorsomedial striatum (DMS) attenuated cocaine conditioned place preference (CPP) reward, but increased striatal SAM/SAH ratio levels as well as Rac1 and RhoA activities. In addition, pharmacological inhibition of NNMT through intra-DMS infusion of MeN attenuated cocaine CPP and the activities of Rac1 and RhoA, but increased SAM/SAH ratio. These results suggest that NNMT-dependent transmethylation is involved in the activation of Rac1 and RhoA, which utilize SAM as a methyl donor cofactor. Co-immunoprecipitation assay using a RhoGDI? antibody indirectly captured Rac1 or RhoA that were bound to RhoGDI?. The results showed that cocaine increased the association of RhoGDI? with Rac1 or RhoA, whereas such effect was inhibited by Nnmt knockdown. Collectively, our findings show that NNMT regulates cocaine CPP through SAM-mediated modification of Rac1 and RhoA.
Project description:Nicotinamide N-methyl transferase (NNMT) transfers a methyl group from S-adenosyl-L-methionine (SAM) to nicotinamide (NAM), producing 1-methylnicotinamide (1MNA). NNMT has been implicated in several cancer types and recently in metabolism, but its role in autophagy regulation has not yet been investigated. In this study, we determined that NNMT negatively regulated autophagy at the stage of ULK1 activation through protein phosphatase 2A (PP2A) activity. Specifically, NNMT knockdown increased PP2A methylation and subsequently enhanced phosphatase activity. Consequent p-ULK1 (S638) dephosphorylation derepressed ULK1 activity, resulting in autophagy induction. Accordingly, NNMT downregulation rescued tumor cells under nutrient deficiency in vivo, which was alleviated by ULK1 inhibitor treatment. In summary, our results suggest a novel mechanism by which tumor cells protect themselves against nutrient deprivation through NNMT suppression to accelerate autophagy.
Project description:Conventional generation of stem cells from human blastocysts produces a developmentally advanced, or primed, stage of pluripotency. In vitro resetting to a more naive phenotype has been reported. However, whether the reset culture conditions of selective kinase inhibition can enable capture of naive epiblast cells directly from the embryo has not been determined. Here, we show that in these specific conditions individual inner cell mass cells grow into colonies that may then be expanded over multiple passages while retaining a diploid karyotype and naive properties. The cells express hallmark naive pluripotency factors and additionally display features of mitochondrial respiration, global gene expression, and genome-wide hypomethylation distinct from primed cells. They transition through primed pluripotency into somatic lineage differentiation. Collectively these attributes suggest classification as human naive embryonic stem cells. Human counterparts of canonical mouse embryonic stem cells would argue for conservation in the phased progression of pluripotency in mammals.