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      The Transcriptional and Epigenomic Foundations of Ground State Pluripotency

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          Summary

          Mouse embryonic stem (ES) cells grown in serum exhibit greater heterogeneity in morphology and expression of pluripotency factors than ES cells cultured in defined medium with inhibitors of two kinases (Mek and GSK3), a condition known as “2i” postulated to establish a naive ground state. We show that the transcriptome and epigenome profiles of serum- and 2i-grown ES cells are distinct. 2i-treated cells exhibit lower expression of lineage-affiliated genes, reduced prevalence at promoters of the repressive histone modification H3K27me3, and fewer bivalent domains, which are thought to mark genes poised for either up- or downregulation. Nonetheless, serum- and 2i-grown ES cells have similar differentiation potential. Precocious transcription of developmental genes in 2i is restrained by RNA polymerase II promoter-proximal pausing. These findings suggest that transcriptional potentiation and a permissive chromatin context characterize the ground state and that exit from it may not require a metastable intermediate or multilineage priming.

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          Highlights

          ► High-resolution genome-wide transcriptome and epigenome of naive pluripotency ► Reduced H3K27me3 at promoters and fewer bivalent domains in naive ES cells ► Reduced lineage priming and increased RNA polymerase II pausing in the naive state ► Naive ES cells show no delay in differentiation

          Abstract

          Ground state pluripotency is characterized by a permissive chromatin context, but gene expression is not promiscuous due to the high prevalence of promoter-proximal pausing of transcription.

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          Most cited references42

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          Ab initio reconstruction of transcriptomes of pluripotent and lineage committed cells reveals gene structures of thousands of lincRNAs

          RNA-Seq provides an unbiased way to study a transcriptome, including both coding and non-coding genes. To date, most RNA-Seq studies have critically depended on existing annotations, and thus focused on expression levels and variation in known transcripts. Here, we present Scripture, a method to reconstruct the transcriptome of a mammalian cell using only RNA-Seq reads and the genome sequence. We apply it to mouse embryonic stem cells, neuronal precursor cells, and lung fibroblasts to accurately reconstruct the full-length gene structures for the vast majority of known expressed genes. We identify substantial variation in protein-coding genes, including thousands of novel 5′-start sites, 3′-ends, and internal coding exons. We then determine the gene structures of over a thousand lincRNA and antisense loci. Our results open the way to direct experimental manipulation of thousands of non-coding RNAs, and demonstrate the power of ab initio reconstruction to render a comprehensive picture of mammalian transcriptomes.
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            Conversion of embryonic stem cells into neuroectodermal precursors in adherent monoculture.

            Mouse embryonic stem (ES) cells are competent for production of all fetal and adult cell types. However, the utility of ES cells as a developmental model or as a source of defined cell populations for pharmaceutical screening or transplantation is compromised because their differentiation in vitro is poorly controlled. Specification of primary lineages is not understood and consequently differentiation protocols are empirical, yielding variable and heterogeneous outcomes. Here we report that neither multicellular aggregation nor coculture is necessary for ES cells to commit efficiently to a neural fate. In adherent monoculture, elimination of inductive signals for alternative fates is sufficient for ES cells to develop into neural precursors. This process is not a simple default pathway, however, but requires autocrine fibroblast growth factor (FGF). Using flow cytometry quantitation and recording of individual colonies, we establish that the bulk of ES cells undergo neural conversion. The neural precursors can be purified to homogeneity by fluorescence activated cell sorting (FACS) or drug selection. This system provides a platform for defining the molecular machinery of neural commitment and optimizing the efficiency of neuronal and glial cell production from pluripotent mammalian stem cells.
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              BMP induction of Id proteins suppresses differentiation and sustains embryonic stem cell self-renewal in collaboration with STAT3.

              The cytokine leukemia inhibitory factor (LIF) drives self-renewal of mouse embryonic stem (ES) cells by activating the transcription factor STAT3. In serum-free cultures, however, LIF is insufficient to block neural differentiation and maintain pluripotency. Here, we report that bone morphogenetic proteins (BMPs) act in combination with LIF to sustain self-renewal and preserve multilineage differentiation, chimera colonization, and germline transmission properties. ES cells can be propagated from single cells and derived de novo without serum or feeders using LIF plus BMP. The critical contribution of BMP is to induce expression of Id genes via the Smad pathway. Forced expression of Id liberates ES cells from BMP or serum dependence and allows self-renewal in LIF alone. Upon LIF withdrawal, Id-expressing ES cells differentiate but do not give rise to neural lineages. We conclude that blockade of lineage-specific transcription factors by Id proteins enables the self-renewal response to LIF/STAT3.
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                Author and article information

                Journal
                Cell
                Cell
                Cell
                Cell Press
                0092-8674
                1097-4172
                27 April 2012
                27 April 2012
                : 149
                : 3
                : 590-604
                Affiliations
                [1 ]Department of Molecular Biology, Faculty of Science, Nijmegen Centre for Molecular Life Sciences (NCMLS), Radboud University, PO Box 9101, 6500 HB Nijmegen, The Netherlands
                [2 ]Wellcome Trust Centre for Stem Cell Research and Stem Cell Institute, University of Cambridge, Tennis Court Road, Cambridge CB2 1QR, UK
                [3 ]Department of Physiology, Development and Neuroscience, University of Cambridge, Tennis Court Road, Cambridge CB2 1QR, UK
                [4 ]Department of Biochemistry, University of Cambridge, Tennis Court Road, Cambridge CB2 1QR, UK
                [5 ]Genomics, BioInnovationsZentrum, Technische Universität Dresden, Am Tatzberg 47-51, D-01307 Dresden, Germany
                Author notes
                []Corresponding author austin.smith@ 123456cscr.cam.ac.uk
                [∗∗ ]Corresponding author h.stunnenberg@ 123456ncmls.ru.nl
                Article
                CELL6217
                10.1016/j.cell.2012.03.026
                3398752
                22541430
                2de0bd5f-3e20-4dd4-b277-96fe2f823a52
                © 2012 ELL & Excerpta Medica.

                This document may be redistributed and reused, subject to certain conditions.

                History
                : 9 July 2011
                : 26 December 2011
                : 6 March 2012
                Categories
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                Cell biology
                Cell biology

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