The role of Myc in the ground state of pluripotency

Pluripotency in the early embryo is defined as the capacity of a single cell to generate all lineages of the adult organism. In vivo this property is possessed only transiently by the cells of the epiblast, but it can be indefinitely “captured” in vitro by deriving embryonic stem (ES) cells from the inner cell mass of the blastocyst. A second property that characterizes ES cells is self-renewal, the capacity to generate more stem cells. Self-renewal requires the coordination of cell proliferation and cell-fate choice (Orford and Scadden, 2008). Originally, mouse ES cells were expanded on mitotically inactivated fibroblasts, in a culture medium containing fetal bovine serum and the leukemia inhibitory factor (LIF). Under these conditions ES cells are metastable, since they show heterogeneity both in morphology and expression of the core pluripotency factors Oct4 and Nanog due to differentiation signals coming from the serum components (Nichols and Smith, 2009). In serum + LIF medium, ES cells depend on the transcription factor and proto‐oncogene Myc for pluripotency and self‐renewal (Smith et al., 2010). Myc proteins have been shown to repress the expression of the primitive endoderm master regulator Gata6, and control the cell cycle by regulating the mir-17-92 miRNA cluster. The laboratory of Austin Smith has recently shown that the culture in the absence of serum but in the presence of two inhibitors (2i + LIF) of the Erk and glycogen synthase kinase-3 (GSK3) pathways is sufficient to stabilize ES cells in a more naïve, so-called ground state of pluripotency, which more closely resembles the status of the inner cell mass of the blastocyst (Ying et al., 2008). Since different culture environments impose distinctive transcriptional and epigenetic properties on mouse ES cells (Marks et al., 2012), we re-evaluated the expression and requirement of the Myc proteins in the naïve state of pluripotency. Making use of ES cells expressing a Myc-GFP reporter from the endogenous c-myc locus, we found that c-Myc protein expression is significantly lower in naïve ES cells compared to cells cultured in serum + LIF. When both c- and N-myc genes are conditionally deleted (Myc dKO) using the Cre-loxP system, naïve mouse ES cells undergo cell cycle arrest. Although this type of cell cycle behavior with a decreased rate of cell division is often associated with differentiation, Myc dKO ES cells form smaller but undifferentiated colonies. Most surprisingly, Myc dKO ES cells maintain the expression of the core stem cell factor network including Oct4, Nanog and Sox2 both at the RNA and the protein level. To determine the molecular properties of Myc dKO ES cells, we performed RNA‐seq analysis 24 and 96 hours after Cre induction. Our results show that Myc is not exclusively regulating the cell cycle machinery but is directly and indirectly involved in multiple aspects of ES cell metabolism. In the absence of both Myc proteins, expression of genes controlling and executing ribosomal biogenesis as well as protein and DNA synthesis is strongly down-regulated, leading to a state of “metabolic dormancy”. The state of “metabolic dormancy” of ES cells in vitro resembled the state of embryonic diapause in vivo. Diapause is a poorly understood phenomenon of reversible arrest of embryonic development prior to implantation. In mice, facultative diapause occurs to delay implantation of newly formed embryos when the mother is feeding a previous litter. When we compared our data to reported global gene expression profiling of diapause embryos (Hamatani et al., 2004), we observed that dKO Myc ES cells possess surprising similarity with the dormant blastocyst, characterized by reduced DNA synthesis and cell division, low metabolic activity and activation of the insulin pathway (Given, 1988; Hamatani et al., 2004). These data suggest that, in the absence of differentiation cues, c-Myc and N-Myc control the cell cycle of ES cells and the transcriptional networks responsible for the entire metabolism and biosynthesis pathways while the pluripotency network is controlled by other means. Loss of Myc activity has dramatic consequences on the metabolic status of ES cells promoting their entry into a state of likely reversible dormancy closely resembling arrested diapause embryos

Convergence of cMyc and β-catenin on Tcf7l1 enables endoderm specification.

The molecular machinery that directs formation of definitive endoderm from pluripotent stem cells is not well understood. Wnt/β-catenin and Nodal signalling have been implicated, but the requirements for lineage specification remain incompletely defined. Here, we demonstrate a potent effect of inhibiting glycogen synthase kinase 3 (GSK3) on definitive endoderm production. We find that downstream of GSK3 inhibition, elevated cMyc and β-catenin act in parallel to reduce transcription and DNA binding, respectively, of the transcriptional repressor Tcf7l1. Tcf7l1 represses FoxA2, a pioneer factor for endoderm specification. Deletion of Tcf7l1 is sufficient to allow upregulation of FoxA2 in the presence of Activin. In wild-type cells, cMyc contributes by reducing Tcf7l1 mRNA, while β-catenin acts on Tcf7l1 protein. GSK3 inhibition is further required for consolidation of endodermal fate via upregulation of Sox17, highlighting sequential roles for Wnt signalling. The identification of a cMyc/β-catenin-Tcf7l1-FoxA2 axis reveals a de-repression mechanism underlying endoderm induction that may be recapitulated in other developmental and patho-logical contexts.This study was funded by the Juvenile Diabetes Research Foundation International, the European Commission FP7 project BetaCellTherapy (agreement No. 241883), a core support grant from the Wellcome Trust and MRC to the Wellcome Trust – Medical Research Council Cambridge Stem Cell Institute, and a University of Edinburgh Chancellor’s Fellowship awarded to GM. GM was a JDRF advanced postdoctoral fellow. AS is a Medical Research Council Professor.This is the final version of the article. It was first available from Wiley via http://dx.doi.org/10.15252/embj.20159211

Overexpression of the Hsa21 Transcription Factor RUNX1 Modulates the Extracellular Matrix in Trisomy 21 Cells

Down syndrome is a neurodevelopmental disorder frequently characterized by other developmental defects, such as congenital heart disease. Analysis of gene expression profiles of hearts from trisomic fetuses have shown upregulation of extracellular matrix (ECM) genes. The aim of this work was to identify genes on chromosome 21 potentially responsible for the upregulation of ECM genes and to pinpoint any functional consequences of this upregulation. By gene set enrichment analysis of public data sets, we identified the transcription factor RUNX1, which maps to chromosome 21, as a possible candidate for regulation of ECM genes. We assessed that approximately 80% of ECM genes overexpressed in trisomic hearts have consensus sequences for RUNX1 in their promoters. We found that in human fetal fibroblasts with chromosome 21 trisomy there is increased expression of both RUNX1 and several ECM genes, whether located on chromosome 21 or not. SiRNA silencing of RUNX1 reduced the expression of 11 of the 14 ECM genes analyzed. In addition, collagen IV, an ECM protein secreted in high concentrations in the culture media of trisomic fibroblasts, was modulated by RUNX1 silencing. Attenuated expression of RUNX1 increased the migratory capacity of trisomic fibroblasts, which are characterized by a reduced migratory capacity compared to euploid controls

Destabilisation, aggregation, toxicity and cytosolic mislocalisation of nucleophosmin regions associated with acute myeloid leukemia

Nucleophosmin (NPM1) is a multifunctional protein that is implicated in the pathogenesis of several human malignancies. To gain insight into the role of isolated fragments of NPM1 in its biological activities, we dissected the C-terminal domain (CTD) into its helical fragments. Here we focus the attention on the third helix of the NPM1-CTD in its wild-type (H3 wt) and AML-mutated (H3 mutA and H3 mutE) sequences. Conformational studies, by means of CD and NMR spectroscopies, showed that the H3 wt peptide was partially endowed with an a-helical structure, but the AML-sequences exhibited a lower content of this conformation, particularly the H3 mutA peptide. Thioflavin T assays showed that the H3 mutE and the H3 mutA peptides displayed a significant aggregation propensity that was confirmed by CD and DLS assays. In addition, we found that the H3 mutE and H3 mutA peptides, unlike the H3 wt, were moderately and highly toxic, respectively, when exposed to human neuroblastoma cells. Cellular localization experiments confirmed that the mutated sequences hamper their nucleolar accumulation, and more importantly, that the helical conformation of the H3 region is crucial for such a localization

Destabilisation, aggregation, toxicity and cytosolic mislocalisation of nucleophosmin regions associated with acute myeloid leukemia

Nucleophosmin (NPM1) is a multifunctional protein that is implicated in the pathogenesis of several human malignancies. To gain insight into the role of isolated fragments of NPM1 in its biological activities, we dissected the C-terminal domain (CTD) into its helical fragments. Here we focus the attention on the third helix of the NPM1-CTD in its wild-type (H3 wt) and AML-mutated (H3 mutA and H3 mutE) sequences. Conformational studies, by means of CD and NMR spectroscopies, showed that the H3 wt peptide was partially endowed with an a-helical structure, but the AML-sequences exhibited a lower content of this conformation, particularly the H3 mutA peptide. Thioflavin T assays showed that the H3 mutE and the H3 mutA peptides displayed a significant aggregation propensity that was confirmed by CD and DLS assays. In addition, we found that the H3 mutE and H3 mutA peptides, unlike the H3 wt, were moderately and highly toxic, respectively, when exposed to human neuroblastoma cells. Cellular localization experiments confirmed that the mutated sequences hamper their nucleolar accumulation, and more importantly, that the helical conformation of the H3 region is crucial for such a localization

Improved HSC reconstitution and protection from inflammatory stress and chemotherapy in mice lacking granzyme B.

The serine protease granzyme B (GzmB) is stored in the granules of cytotoxic T and NK cells and facilitates immune-mediated destruction of virus-infected cells. In this study, we use genetic tools to report novel roles for GzmB as an important regulator of hematopoietic stem cell (HSC) function in response to stress. HSCs lacking the GzmB gene show improved bone marrow (BM) reconstitution associated with increased HSC proliferation and mitochondrial activity. In addition, recipients deficient in GzmB support superior engraftment of wild-type HSCs compared with hosts with normal BM niches. Stimulation of mice with lipopolysaccharide strongly induced GzmB protein expression in HSCs, which was mediated by the TLR4-TRIF-p65 NF-κB pathway. This is associated with increased cell death and GzmB secretion into the BM environment, suggesting an extracellular role of GzmB in modulating HSC niches. Moreover, treatment with the chemotherapeutic agent 5-fluorouracil (5-FU) also induces GzmB production in HSCs. In this situation GzmB is not secreted, but instead causes cell-autonomous apoptosis. Accordingly, GzmB-deficient mice are more resistant to serial 5-FU treatments. Collectively, these results identify GzmB as a negative regulator of HSC function that is induced by stress and chemotherapy in both HSCs and their niches. Blockade of GzmB production may help to improve hematopoiesis in various situations of BM stress

Multiple large osteolytic lesions in a patient with systemic mastocytosis: a challenging diagnosis

Patients with advanced variants of Systemic Mastocytosis may develop destructive bone lesions when massive mast cell (MC) infiltrates are present. Finding of large osteolyses in indolent systemic mastocytosis, typically characterized by low MC burden, should prompt investigations for an alternative explanation