Inhibition of the NNMT regulator, STAT3, in na?ve hESCs increases H3K27me3 repressive marks in developmental and metabolic genes, including Wnt signaling and the HIF1 repressor, prolyl hydroxylase EGLN1. the body. AZD3463 These cells hold promise for understanding early human development as well as developing therapies in regenerative medicine. Recent findings have revealed that pluripotency does not represent a single defined state; diverse states of pluripotency, with differences in measurable characteristics relating to gene expression, epigenetics and cellular phenotype, provide an experimental system for studying potential key regulators that constrain or expand the developmental capacity of pluripotent cells1C4. Two stable pluripotent states have been derived in the mouse, and now in humans; preimplantation na?ve and postimplantation primed ESC states5C12 . Since na?ve, preimplantation human embryonic stem cells (hESCs) AZD3463 show higher developmental potential than postimplantation, primed hESCs8,12, it is critical to understand the key molecular differences between these pluripotent cell types. Metabolic signatures are highly characteristic for a cell and may act as a leading cause for cell fate changes13C20. Recent data have shown that pluripotent stem cells have a unique metabolic pattern. The na?ve to primed mouse ESC transition accompanies a dramatic metabolic switch from bivalent to highly glycolytic state20. However, primed state of inert mitochondria rapidly changes to highly respiring mitochondria during further differentiation. It is not yet understood AZD3463 how and why the pluripotent cells enter the highly glycolytic, metabolically cancer-like (Warburg effect) state and how a differentiating cell leaves this state. In mouse embryonic stem cells (mESCs) threonine and S-adenosyl methionine (SAM) metabolism are coupled resulting in regulation of histone methylation marks21. Methionine and SAM are also required for the Rabbit polyclonal to DYKDDDDK Tag conjugated to HRP self renewal of hESCs, since depletion of SAM leads AZD3463 to reduced H3K4me3 marks and defects in maintenance of the hESC state22. SAM therefore is shown to be a key regulator for maintaining ESC undifferentiated state and regulating their differentiation. However, little is known about SAM levels or its regulation during the transition between na?ve and primed human embryonic states. Recent derivation of na?ve human ESCs allows a deeper analysis of the human na?ve to primed transition6C12. These studies have already revealed that the epigenetic landscape changes from the na?ve to primed state through increased H3K27me3 repressive methylation marks. However, the regulation of this process or the metabolomics of this transition have not been dissected. We now show that the upregulation of H3K27me3 repressive epigenetic marks during na?ve to primed hESC transition is controlled by the metabolic enzyme, NNMT. Knockdown of NNMT in na?ve hESCs increased H3K27me3 repressive marks in developmental as well as key metabolic genes that regulate the metabolic switch in na?ve to primed transition. CRISPR-Cas9 based NNMT KO na?ve hESC lines show upregulation of SAM, H3K27me3 marks, HIF activation, Wnt repression and a general gene expression shift towards primed stage. These data show that NNMT consumes SAM in na?ve cells, making it unavailable for histone methylation. Histone methylation further regulates the key signaling pathways important for the metabolic changes that are necessary for early human development. RESULTS A dramatic metabolic switch occurs in mouse ESCs between pre-implantation (na?ve) and post-implantation (primed) state20. Human na?ve counterpart has been recently toggled or derived from embryos. Principal component analysis (PCA) of the expression signatures of these new cell types confirmed that all derived human na?ve hESCs are in a significantly earlier stage than primed hESCs6,8C10,23(Fig.1ACB, Suppl.Fig.1ACC, Suppl.Table.1A). To assess the metabolic profiles of the human.