Systems-level dynamic analyses of fate change in murine embryonic stem cells. Nature

Department of Molecular Biology, Princeton University, Princeton, New Jersey 08544, USA.
Nature (Impact Factor: 41.46). 11/2009; 462(7271):358-62. DOI: 10.1038/nature08575
Source: PubMed


Molecular regulation of embryonic stem cell (ESC) fate involves a coordinated interaction between epigenetic, transcriptional and translational mechanisms. It is unclear how these different molecular regulatory mechanisms interact to regulate changes in stem cell fate. Here we present a dynamic systems-level study of cell fate change in murine ESCs following a well-defined perturbation. Global changes in histone acetylation, chromatin-bound RNA polymerase II, messenger RNA (mRNA), and nuclear protein levels were measured over 5 days after downregulation of Nanog, a key pluripotency regulator. Our data demonstrate how a single genetic perturbation leads to progressive widespread changes in several molecular regulatory layers, and provide a dynamic view of information flow in the epigenome, transcriptome and proteome. We observe that a large proportion of changes in nuclear protein levels are not accompanied by concordant changes in the expression of corresponding mRNAs, indicating important roles for translational and post-translational regulation of ESC fate. Gene-ontology analysis across different molecular layers indicates that although chromatin reconfiguration is important for altering cell fate, it is preceded by transcription-factor-mediated regulatory events. The temporal order of gene expression alterations shows the order of the regulatory network reconfiguration and offers further insight into the gene regulatory network. Our studies extend the conventional systems biology approach to include many molecular species, regulatory layers and temporal series, and underscore the complexity of the multilayer regulatory mechanisms responsible for changes in protein expression that determine stem cell fate.

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Available from: Edoardo M Airoldi
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    • "A growing body of evidence underscores the importance of pluripotency factors during differentiation. Human and mouse ES cell studies have demonstrated that the core pluripotency transcription factors, SOX2, OCT4, and NANOG, play distinct roles in coordinating ES cell lineage commitment (Lu et al., 2009; Thomson et al., 2011; Wang et al., 2012). NANOG promotes definitive endoderm (DE) formation by coordinating with the activation of the TGF-b signaling pathway through the induction of EOMES (Teo et al., 2011). "
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    ABSTRACT: We demonstrate that the pluripotency gene OCT4 has a role in regulating differentiation via Wnt signaling. OCT4 expression levels in human embryonic stem cells increases transiently during the first 24 hr of in vitro differentiation, with OCT4 occupancy increasing at endoderm regulators such as SOX17 and FOXA2. This increased occupancy correlates with loss of the PRC2 complex and the inhibitory histone mark H3K27me3. Knockdown of OCT4 during differentiation inhibits mesendoderm formation and removal of the H3K27me3 mark from the SOX17 promoter, suggesting that OCT4 acts to induce removal of the PRC2 complex. Furthermore, OCT4 and β-catenin can be co-immunoprecipitated upon differentiation, and Wnt stimulation is required for the enhanced OCT4 occupancy and loss of the PRC2 complex from the SOX17 promoter. In conclusion, our study reveals that OCT4, a master regulator of pluripotency, may also collaborate with Wnt signaling to drive endoderm induction by pre-patterning epigenetic markers on endodermal promoters.
    Full-text · Article · Sep 2015 · Stem Cell Reports
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    • "This dependence conforms to the bell-shaped curve as well as gene expression in single cells (compare F and G). identities in the two LIF subpopulations were established using published data (Hailesellasse Sene et al., 2007; Lu et al., 2009; Tang et al., 2010). Cells in one subpopulation matched, by their expression profiles, cells from the E2.5 morula/E3.5 "
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    ABSTRACT: Analyses of gene expression in single mouse embryonic stem cells (mESCs) cultured in serum and LIF revealed the presence of two distinct cell subpopulations with individual gene expression signatures. Comparisons with published data revealed that cells in the first subpopulation are phenotypically similar to cells isolated from the inner cell mass (ICM). In contrast, cells in the second subpopulation appear to be more mature. Pluripotency Gene Regulatory Network (PGRN) reconstruction based on single-cell data and published data suggested antagonistic roles for Oct4 and Nanog in the maintenance of pluripotency states. Integrated analyses of published genomic binding (ChIP) data strongly supported this observation. Certain target genes alternatively regulated by OCT4 and NANOG, such as Sall4 and Zscan10, feed back into the top hierarchical regulator Oct4. Analyses of such incoherent feedforward loops with feedback (iFFL-FB) suggest a dynamic model for the maintenance of mESC pluripotency and self-renewal. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.
    Full-text · Article · Aug 2015 · Stem Cell Reports
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    • "While the transcriptional, signaling, and epigenetic regulation of these cells have been the primary focus of research efforts in recent years (reviewed in Ng and Surani, 2011; Young, 2011; Watanabe et al., 2013), posttranscriptional and translational mechanisms of control remain relatively unexplored, despite evidence that they play a dominant role in driving ESC fate decisions . Indeed, posttranscriptional regulation has been reported to account for nearly 75% of the changes in protein levels after differentiation induced by knockdown of the transcription factor Nanog (Lu et al., 2009), and it was recently demonstrated that control over translational initiation by the eIF4e binding proteins dramatically influences the efficiency of reprogramming somatic cells to induced pluripotent stem cells (iPSCs) (Tahmasebi et al., 2014). The cell controls protein levels posttranscriptionally using a large collection of tools that includes noncoding RNAs and RNA binding proteins (RBPs). "
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    ABSTRACT: Establishment, maintenance, and exit from pluripotency require precise coordination of a cell’s molecular machinery. Substantial headway has been made in deciphering many aspects of this elaborate system, particularly with respect to epigenetics, transcription, and noncoding RNAs. Less attention has been paid to posttranscriptional regulatory processes such as alternative splicing, RNA processing and modification, nuclear export, regulation of transcript stability, and translation. Here, we introduce the RNA binding proteins that enable the posttranscriptional regulation of gene expression, summarizing current and ongoing research on their roles at different regulatory points and discussing how they help script the fate of pluripotent stem cells.
    Full-text · Article · Sep 2014 · Cell Stem Cell
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