The metabolome regulates the epigenetic landscape during naive-to-primed human embryonic stem cell transition.

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

Quantitative phosphoproteomics reveal that mTOR regulates cell growth and proliferation by phosphorylating a functionally diverse set of substrates

The atypical Ser/Thr kinase target of rapamycin (TOR) is a central controller of cell growth and proliferation. TOR forms two distinct multiprotein complexes, TORC1 and TORC2, which are structurally and functionally conserved from yeast to humans. Four major inputs control mammalian TOR (mTOR): growth factors, such as insulin; cellular energy levels, such as the AMP:ATP ratio; stress, such as hypoxia; and nutrients, such as amino acids. mTOR controls cell growth by the positive and negative regulation of several anabolic and catabolic processes, respectively, that collectively regulate cell size and proliferation. These cellular processes include autophagy, cytoskeleton rearrangement, glycolysis, lipogenesis, nutrient transport, ribosome biogenesis, and translation. Dysregulation of the mTOR signaling network has been associated with aging, and a multitude of diseases including cancer, cardiovascular disease, diabetes, inflammation, immune dysfunctions, and neurodegeneration. However, relatively few direct substrates of either one of the two mTOR complexes, mTORC1 and mTORC2, are known. To determine downstream effectors of mammalian TOR (mTOR), we applied a functional, quantitative phosphoproteomics workflow to identify novel mTORC1 or mTORC2 regulated phosphorylations. Raptor and Rictor are essential components of mTORC1 and mTORC2, respectively. To distinguish phosphorylations regulated by mTORC1 or mTORC2, we specifically deleted Raptor or Rictor using an inducible gene knockout system in mouse embryonic fiberblasts (MEFs). We detected 4584 phosphorylation sites on 1398 proteins, and identified 335 novel mTOR effectors. Many of the novel effectors are implicated in cancer and metabolic diseases, but have no known links to mTOR. To distinguish direct mTOR substrates from indirect effectors, we combined peptide array in vitro kinase assays with phosphorylation motif analysis. This revealed that mTORC1 phosphorylates CAD in vivo and in vitro. CAD (carbamoyl-phosphate synthetase 2, aspartate transcarbamylase, and dihydroorotase) is the initial and rate limiting enzyme in de novo pyrimidine synthesis. The macrolide rapamycin, which forms a complex with FKBP12, binds and acutely inhibits mTORC1 but not mTORC2. Rapamycin treatment inhibited growth factor stimulated CAD phosphorylation and oligomerization, decreased de novo pyrimidine synthesis, and delayed progression of S-phase where CAD activity is essential. Thus mTORC1 phosphorylates CAD and thereby stimulates de novo pyrimidine synthesis to promote cell proliferation. Separately, we characterize the autophosphorylation of mTOR on Ser2481. Insulin stimulates the phosphorylation of mTOR at Ser2481 specifically in mTORC2. Knockout of Rictor, but not Raptor, abolished mTOR autophosphorylation at Ser2481. Prolonged treatment with rapamycin, which indirectly inhibits mTORC2 complex formation, inhibited Ser2481 phosphorylation. Surprisingly, mTORC2 autophosphorylation at Ser2481 temporally occurs after the insulin-induced phosphorylation of Akt/PKB and the SGK1 substrate NDGR1. Mutation of Ser2481 to aspartic acid rendered mTOR unable to phosphorylate Akt/PKB in vitro. However the function of mTOR-Ser2481 phosphorylation in vivo remains elusive, as mutation of mTOR-Ser2481 did not alter Akt/PKB phosphorylation in vivo. In summary, mTORC1 and mTORC2 regulate the phosphorylation of a functionally diverse set of substrates to control several anabolic and catabolic processes that determine cell size and proliferation. As a central controller of cell growth and proliferation, mTOR plays a key role in regulating development, whereas dysregulation of mTOR signaling has been linked to aging and diseases such as cancer and metabolic disorders

A Quantitative Proteomic Analysis of Hemogenic Endothelium Reveals Differential Regulation of Hematopoiesis by SOX17

SummaryThe in vitro derivation of hematopoietic stem cells (HSCs) from pluripotent stem cells (PSCs) is complicated by the existence of multiple overlapping embryonic blood cell programs called primitive, erythromyeloid progenitor (EMP), and definitive. As HSCs are only generated during the definitive stage of hematopoiesis, deciphering the regulatory pathways that control the emergence of this program and identifying markers that distinguish it from the other programs are essential. To identify definitive specific pathways and marker sets, we used label-free proteomics to determine the proteome of embryo-derived and mouse embryonic stem cell-derived VE-CADHERIN+CD45− definitive hematopoietic progenitors. With this approach, we identified Stat1 as a marker that distinguishes the definitive erythroid lineage from the primitive- and EMP-derived lineages. Additionally, we provide evidence that the generation of the Stat1+ definitive lineage is dependent on Sox17. These findings establish an approach for monitoring the emergence of definitive hematopoiesis in the PSC differentiation cultures

Ramping up mitosis : an AMPKα2-regulated signaling network promotes mitotic progression

In the December 23rd issue of Molecular Cell, Banko et al. (2011) describe a chemical genetic screen that identified 28 novel AMPKα2 direct substrates. A subset of these substrates comprise a signaling network by which AMPK, seemingly independent of cellular energy status, promotes mitotic progression

Sestd1 Encodes a Developmentally Dynamic Synapse Protein That Complexes With BCR Rac1-GAP to Regulate Forebrain Dendrite, Spine and Synapse Formation.

In figure 4 the first sentence of the caption has been corrected to "Loss of Sestd1 does not affect β-catenin but increases activity of Rac1 and RhoA in the neonatal and mature hippocampus." The new caption text has been corrected online and in print. The author regrets this error

Sestd1 Encodes a Developmentally Dynamic Synapse Protein That Complexes With BCR Rac1-GAP to Regulate Forebrain Dendrite, Spine and Synapse Formation.

In figure 4 the first sentence of the caption has been corrected to "Loss of Sestd1 does not affect β-catenin but increases activity of Rac1 and RhoA in the neonatal and mature hippocampus." The new caption text has been corrected online and in print. The author regrets this error

mTORC1 Directly Phosphorylates and Regulates Human MAF1▿

mTORC1 is a central regulator of growth in response to nutrient availability, but few direct targets have been identified. RNA polymerase (pol) III produces a number of essential RNA molecules involved in protein synthesis, RNA maturation, and other processes. Its activity is highly regulated, and deregulation can lead to cell transformation. The human phosphoprotein MAF1 becomes dephosphorylated and represses pol III transcription after various stresses, but neither the significance of the phosphorylations nor the kinase involved is known. We find that human MAF1 is absolutely required for pol III repression in response to serum starvation or TORC1 inhibition by rapamycin or Torin1. The protein is phosphorylated mainly on residues S60, S68, and S75, and this inhibits its pol III repression function. The responsible kinase is mTORC1, which phosphorylates MAF1 directly. Our results describe molecular mechanisms by which mTORC1 controls human MAF1, a key repressor of RNA polymerase III transcription, and add a new branch to the signal transduction cascade immediately downstream of TORC1

Quantitative phosphoproteomics reveal mTORC1 activates de novo pyrimidine synthesis

The Ser-Thr kinase mammalian target of rapamycin (mTOR) controls cell growth and metabolism by stimulating glycolysis and synthesis of proteins and lipids. To further understand the central role of mTOR in cell physiology, we used quantitative phosphoproteomics to identify substrates or downstream effectors of the two mTOR complexes. mTOR controlled the phosphorylation of 335 proteins, including CAD (carbamoyl-phosphate synthetase 2, aspartate transcarbamylase, and dihydroorotase). CAD catalyzes the first three steps in de novo pyrimidine synthesis. mTORC1 indirectly phosphorylated CAD-S1859 through S6 kinase (S6K). CAD-S1859 phosphorylation promoted CAD oligomerization and thereby stimulated de novo synthesis of pyrimidines and progression through S phase of the cell cycle in mammalian cells. Thus, mTORC1 also stimulates the synthesis of nucleotides to control cell proliferation