BMP-SMAD Signaling Regulates Lineage Priming, but Is Dispensable for Self-Renewal in Mouse Embryonic Stem Cells
Identifieur interne : 000431 ( Pmc/Corpus ); précédent : 000430; suivant : 000432BMP-SMAD Signaling Regulates Lineage Priming, but Is Dispensable for Self-Renewal in Mouse Embryonic Stem Cells
Auteurs : Maria Gomes Fernandes ; Ruben Dries ; Matthias S. Roost ; Stefan Semrau ; Ana De Melo Bernardo ; Richard P. Davis ; Ramprasad Ramakrishnan ; Karoly Szuhai ; Elke Maas ; Lieve Umans ; Vanesa Abon Escalona ; Daniela Salvatori ; Dieter Deforce ; Wim Van Criekinge ; Danny Huylebroeck ; Christine Mummery ; An Zwijsen ; Susana M. Chuva De Sousa LopesSource :
- Stem Cell Reports [ 2213-6711 ] ; 2015.
Abstract
Naive mouse embryonic stem cells (mESCs) are in a metastable state and fluctuate between inner cell mass- and epiblast-like phenotypes. Here, we show transient activation of the BMP-SMAD signaling pathway in mESCs containing a BMP-SMAD responsive reporter transgene. Activation of the BMP-SMAD reporter transgene in naive mESCs correlated with lower levels of genomic DNA methylation, high expression of 5-methylcytosine hydroxylases
Url:
DOI: 10.1016/j.stemcr.2015.11.012
PubMed: 26711875
PubMed Central: 4720007
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<record><TEI><teiHeader><fileDesc><titleStmt><title xml:lang="en">BMP-SMAD Signaling Regulates Lineage Priming, but Is Dispensable for Self-Renewal in Mouse Embryonic Stem Cells</title><author><name sortKey="Gomes Fernandes, Maria" sort="Gomes Fernandes, Maria" uniqKey="Gomes Fernandes M" first="Maria" last="Gomes Fernandes">Maria Gomes Fernandes</name><affiliation><nlm:aff id="aff1">Department Anatomy and Embryology, Leiden University Medical Center, Leiden 2333 ZC, the Netherlands</nlm:aff></affiliation></author><author><name sortKey="Dries, Ruben" sort="Dries, Ruben" uniqKey="Dries R" first="Ruben" last="Dries">Ruben Dries</name><affiliation><nlm:aff id="aff2">Department Development and Regeneration, Laboratory of Molecular Biology (Celgen), KU Leuven, Leuven 3000, Belgium</nlm:aff></affiliation><affiliation><nlm:aff id="aff3">Department of Cell Biology, Erasmus University Medical Center, Rotterdam 3015 CN, the Netherlands</nlm:aff></affiliation></author><author><name sortKey="Roost, Matthias S" sort="Roost, Matthias S" uniqKey="Roost M" first="Matthias S." last="Roost">Matthias S. Roost</name><affiliation><nlm:aff id="aff1">Department Anatomy and Embryology, Leiden University Medical Center, Leiden 2333 ZC, the Netherlands</nlm:aff></affiliation></author><author><name sortKey="Semrau, Stefan" sort="Semrau, Stefan" uniqKey="Semrau S" first="Stefan" last="Semrau">Stefan Semrau</name><affiliation><nlm:aff id="aff4">Leiden Institute of Physics, Leiden University, Leiden 2333 CA, the Netherlands</nlm:aff></affiliation></author><author><name sortKey="De Melo Bernardo, Ana" sort="De Melo Bernardo, Ana" uniqKey="De Melo Bernardo A" first="Ana" last="De Melo Bernardo">Ana De Melo Bernardo</name><affiliation><nlm:aff id="aff1">Department Anatomy and Embryology, Leiden University Medical Center, Leiden 2333 ZC, the Netherlands</nlm:aff></affiliation></author><author><name sortKey="Davis, Richard P" sort="Davis, Richard P" uniqKey="Davis R" first="Richard P." last="Davis">Richard P. Davis</name><affiliation><nlm:aff id="aff1">Department Anatomy and Embryology, Leiden University Medical Center, Leiden 2333 ZC, the Netherlands</nlm:aff></affiliation></author><author><name sortKey="Ramakrishnan, Ramprasad" sort="Ramakrishnan, Ramprasad" uniqKey="Ramakrishnan R" first="Ramprasad" last="Ramakrishnan">Ramprasad Ramakrishnan</name><affiliation><nlm:aff id="aff1">Department Anatomy and Embryology, Leiden University Medical Center, Leiden 2333 ZC, the Netherlands</nlm:aff></affiliation></author><author><name sortKey="Szuhai, Karoly" sort="Szuhai, Karoly" uniqKey="Szuhai K" first="Karoly" last="Szuhai">Karoly Szuhai</name><affiliation><nlm:aff id="aff5">Department Molecular Cell Biology, Leiden University Medical Center, Leiden 2333 ZC, the Netherlands</nlm:aff></affiliation></author><author><name sortKey="Maas, Elke" sort="Maas, Elke" uniqKey="Maas E" first="Elke" last="Maas">Elke Maas</name><affiliation><nlm:aff id="aff6">Department Human Genetics, VIB Center for the Biology of Disease, KU Leuven, Leuven 3000, Belgium</nlm:aff></affiliation></author><author><name sortKey="Umans, Lieve" sort="Umans, Lieve" uniqKey="Umans L" first="Lieve" last="Umans">Lieve Umans</name><affiliation><nlm:aff id="aff2">Department Development and Regeneration, Laboratory of Molecular Biology (Celgen), KU Leuven, Leuven 3000, Belgium</nlm:aff></affiliation><affiliation><nlm:aff id="aff3">Department of Cell Biology, Erasmus University Medical Center, Rotterdam 3015 CN, the Netherlands</nlm:aff></affiliation><affiliation><nlm:aff id="aff6">Department Human Genetics, VIB Center for the Biology of Disease, KU Leuven, Leuven 3000, Belgium</nlm:aff></affiliation></author><author><name sortKey="Abon Escalona, Vanesa" sort="Abon Escalona, Vanesa" uniqKey="Abon Escalona V" first="Vanesa" last="Abon Escalona">Vanesa Abon Escalona</name><affiliation><nlm:aff id="aff6">Department Human Genetics, VIB Center for the Biology of Disease, KU Leuven, Leuven 3000, Belgium</nlm:aff></affiliation></author><author><name sortKey="Salvatori, Daniela" sort="Salvatori, Daniela" uniqKey="Salvatori D" first="Daniela" last="Salvatori">Daniela Salvatori</name><affiliation><nlm:aff id="aff1">Department Anatomy and Embryology, Leiden University Medical Center, Leiden 2333 ZC, the Netherlands</nlm:aff></affiliation><affiliation><nlm:aff id="aff7">Center Laboratory Animal Facility, Leiden University Medical Center, Leiden 2333 ZC, the Netherlands</nlm:aff></affiliation></author><author><name sortKey="Deforce, Dieter" sort="Deforce, Dieter" uniqKey="Deforce D" first="Dieter" last="Deforce">Dieter Deforce</name><affiliation><nlm:aff id="aff8">Laboratory of Pharmaceutical Biotechnology, Faculty of Pharmaceutical Sciences, Ghent University, Ghent 9000, Belgium</nlm:aff></affiliation></author><author><name sortKey="Van Criekinge, Wim" sort="Van Criekinge, Wim" uniqKey="Van Criekinge W" first="Wim" last="Van Criekinge">Wim Van Criekinge</name><affiliation><nlm:aff id="aff9">Mathematical Modelling, Statistics and Bio-informatics, Faculty Bioscience Engineering, Ghent University, Ghent 9000, Belgium</nlm:aff></affiliation></author><author><name sortKey="Huylebroeck, Danny" sort="Huylebroeck, Danny" uniqKey="Huylebroeck D" first="Danny" last="Huylebroeck">Danny Huylebroeck</name><affiliation><nlm:aff id="aff2">Department Development and Regeneration, Laboratory of Molecular Biology (Celgen), KU Leuven, Leuven 3000, Belgium</nlm:aff></affiliation><affiliation><nlm:aff id="aff3">Department of Cell Biology, Erasmus University Medical Center, Rotterdam 3015 CN, the Netherlands</nlm:aff></affiliation></author><author><name sortKey="Mummery, Christine" sort="Mummery, Christine" uniqKey="Mummery C" first="Christine" last="Mummery">Christine Mummery</name><affiliation><nlm:aff id="aff1">Department Anatomy and Embryology, Leiden University Medical Center, Leiden 2333 ZC, the Netherlands</nlm:aff></affiliation></author><author><name sortKey="Zwijsen, An" sort="Zwijsen, An" uniqKey="Zwijsen A" first="An" last="Zwijsen">An Zwijsen</name><affiliation><nlm:aff id="aff6">Department Human Genetics, VIB Center for the Biology of Disease, KU Leuven, Leuven 3000, Belgium</nlm:aff></affiliation></author><author><name sortKey="Chuva De Sousa Lopes, Susana M" sort="Chuva De Sousa Lopes, Susana M" uniqKey="Chuva De Sousa Lopes S" first="Susana M." last="Chuva De Sousa Lopes">Susana M. Chuva De Sousa Lopes</name><affiliation><nlm:aff id="aff1">Department Anatomy and Embryology, Leiden University Medical Center, Leiden 2333 ZC, the Netherlands</nlm:aff></affiliation><affiliation><nlm:aff id="aff10">Department Reproductive Medicine, Ghent University Hospital, Ghent 9000, Belgium</nlm:aff></affiliation></author></titleStmt><publicationStmt><idno type="wicri:source">PMC</idno><idno type="pmid">26711875</idno><idno type="pmc">4720007</idno><idno type="url">http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4720007</idno><idno type="RBID">PMC:4720007</idno><idno type="doi">10.1016/j.stemcr.2015.11.012</idno><date when="2015">2015</date><idno type="wicri:Area/Pmc/Corpus">000431</idno></publicationStmt><sourceDesc><biblStruct><analytic><title xml:lang="en" level="a" type="main">BMP-SMAD Signaling Regulates Lineage Priming, but Is Dispensable for Self-Renewal in Mouse Embryonic Stem Cells</title><author><name sortKey="Gomes Fernandes, Maria" sort="Gomes Fernandes, Maria" uniqKey="Gomes Fernandes M" first="Maria" last="Gomes Fernandes">Maria Gomes Fernandes</name><affiliation><nlm:aff id="aff1">Department Anatomy and Embryology, Leiden University Medical Center, Leiden 2333 ZC, the Netherlands</nlm:aff></affiliation></author><author><name sortKey="Dries, Ruben" sort="Dries, Ruben" uniqKey="Dries R" first="Ruben" last="Dries">Ruben Dries</name><affiliation><nlm:aff id="aff2">Department Development and Regeneration, Laboratory of Molecular Biology (Celgen), KU Leuven, Leuven 3000, Belgium</nlm:aff></affiliation><affiliation><nlm:aff id="aff3">Department of Cell Biology, Erasmus University Medical Center, Rotterdam 3015 CN, the Netherlands</nlm:aff></affiliation></author><author><name sortKey="Roost, Matthias S" sort="Roost, Matthias S" uniqKey="Roost M" first="Matthias S." last="Roost">Matthias S. Roost</name><affiliation><nlm:aff id="aff1">Department Anatomy and Embryology, Leiden University Medical Center, Leiden 2333 ZC, the Netherlands</nlm:aff></affiliation></author><author><name sortKey="Semrau, Stefan" sort="Semrau, Stefan" uniqKey="Semrau S" first="Stefan" last="Semrau">Stefan Semrau</name><affiliation><nlm:aff id="aff4">Leiden Institute of Physics, Leiden University, Leiden 2333 CA, the Netherlands</nlm:aff></affiliation></author><author><name sortKey="De Melo Bernardo, Ana" sort="De Melo Bernardo, Ana" uniqKey="De Melo Bernardo A" first="Ana" last="De Melo Bernardo">Ana De Melo Bernardo</name><affiliation><nlm:aff id="aff1">Department Anatomy and Embryology, Leiden University Medical Center, Leiden 2333 ZC, the Netherlands</nlm:aff></affiliation></author><author><name sortKey="Davis, Richard P" sort="Davis, Richard P" uniqKey="Davis R" first="Richard P." last="Davis">Richard P. Davis</name><affiliation><nlm:aff id="aff1">Department Anatomy and Embryology, Leiden University Medical Center, Leiden 2333 ZC, the Netherlands</nlm:aff></affiliation></author><author><name sortKey="Ramakrishnan, Ramprasad" sort="Ramakrishnan, Ramprasad" uniqKey="Ramakrishnan R" first="Ramprasad" last="Ramakrishnan">Ramprasad Ramakrishnan</name><affiliation><nlm:aff id="aff1">Department Anatomy and Embryology, Leiden University Medical Center, Leiden 2333 ZC, the Netherlands</nlm:aff></affiliation></author><author><name sortKey="Szuhai, Karoly" sort="Szuhai, Karoly" uniqKey="Szuhai K" first="Karoly" last="Szuhai">Karoly Szuhai</name><affiliation><nlm:aff id="aff5">Department Molecular Cell Biology, Leiden University Medical Center, Leiden 2333 ZC, the Netherlands</nlm:aff></affiliation></author><author><name sortKey="Maas, Elke" sort="Maas, Elke" uniqKey="Maas E" first="Elke" last="Maas">Elke Maas</name><affiliation><nlm:aff id="aff6">Department Human Genetics, VIB Center for the Biology of Disease, KU Leuven, Leuven 3000, Belgium</nlm:aff></affiliation></author><author><name sortKey="Umans, Lieve" sort="Umans, Lieve" uniqKey="Umans L" first="Lieve" last="Umans">Lieve Umans</name><affiliation><nlm:aff id="aff2">Department Development and Regeneration, Laboratory of Molecular Biology (Celgen), KU Leuven, Leuven 3000, Belgium</nlm:aff></affiliation><affiliation><nlm:aff id="aff3">Department of Cell Biology, Erasmus University Medical Center, Rotterdam 3015 CN, the Netherlands</nlm:aff></affiliation><affiliation><nlm:aff id="aff6">Department Human Genetics, VIB Center for the Biology of Disease, KU Leuven, Leuven 3000, Belgium</nlm:aff></affiliation></author><author><name sortKey="Abon Escalona, Vanesa" sort="Abon Escalona, Vanesa" uniqKey="Abon Escalona V" first="Vanesa" last="Abon Escalona">Vanesa Abon Escalona</name><affiliation><nlm:aff id="aff6">Department Human Genetics, VIB Center for the Biology of Disease, KU Leuven, Leuven 3000, Belgium</nlm:aff></affiliation></author><author><name sortKey="Salvatori, Daniela" sort="Salvatori, Daniela" uniqKey="Salvatori D" first="Daniela" last="Salvatori">Daniela Salvatori</name><affiliation><nlm:aff id="aff1">Department Anatomy and Embryology, Leiden University Medical Center, Leiden 2333 ZC, the Netherlands</nlm:aff></affiliation><affiliation><nlm:aff id="aff7">Center Laboratory Animal Facility, Leiden University Medical Center, Leiden 2333 ZC, the Netherlands</nlm:aff></affiliation></author><author><name sortKey="Deforce, Dieter" sort="Deforce, Dieter" uniqKey="Deforce D" first="Dieter" last="Deforce">Dieter Deforce</name><affiliation><nlm:aff id="aff8">Laboratory of Pharmaceutical Biotechnology, Faculty of Pharmaceutical Sciences, Ghent University, Ghent 9000, Belgium</nlm:aff></affiliation></author><author><name sortKey="Van Criekinge, Wim" sort="Van Criekinge, Wim" uniqKey="Van Criekinge W" first="Wim" last="Van Criekinge">Wim Van Criekinge</name><affiliation><nlm:aff id="aff9">Mathematical Modelling, Statistics and Bio-informatics, Faculty Bioscience Engineering, Ghent University, Ghent 9000, Belgium</nlm:aff></affiliation></author><author><name sortKey="Huylebroeck, Danny" sort="Huylebroeck, Danny" uniqKey="Huylebroeck D" first="Danny" last="Huylebroeck">Danny Huylebroeck</name><affiliation><nlm:aff id="aff2">Department Development and Regeneration, Laboratory of Molecular Biology (Celgen), KU Leuven, Leuven 3000, Belgium</nlm:aff></affiliation><affiliation><nlm:aff id="aff3">Department of Cell Biology, Erasmus University Medical Center, Rotterdam 3015 CN, the Netherlands</nlm:aff></affiliation></author><author><name sortKey="Mummery, Christine" sort="Mummery, Christine" uniqKey="Mummery C" first="Christine" last="Mummery">Christine Mummery</name><affiliation><nlm:aff id="aff1">Department Anatomy and Embryology, Leiden University Medical Center, Leiden 2333 ZC, the Netherlands</nlm:aff></affiliation></author><author><name sortKey="Zwijsen, An" sort="Zwijsen, An" uniqKey="Zwijsen A" first="An" last="Zwijsen">An Zwijsen</name><affiliation><nlm:aff id="aff6">Department Human Genetics, VIB Center for the Biology of Disease, KU Leuven, Leuven 3000, Belgium</nlm:aff></affiliation></author><author><name sortKey="Chuva De Sousa Lopes, Susana M" sort="Chuva De Sousa Lopes, Susana M" uniqKey="Chuva De Sousa Lopes S" first="Susana M." last="Chuva De Sousa Lopes">Susana M. Chuva De Sousa Lopes</name><affiliation><nlm:aff id="aff1">Department Anatomy and Embryology, Leiden University Medical Center, Leiden 2333 ZC, the Netherlands</nlm:aff></affiliation><affiliation><nlm:aff id="aff10">Department Reproductive Medicine, Ghent University Hospital, Ghent 9000, Belgium</nlm:aff></affiliation></author></analytic><series><title level="j">Stem Cell Reports</title><idno type="eISSN">2213-6711</idno><imprint><date when="2015">2015</date></imprint></series></biblStruct></sourceDesc></fileDesc><profileDesc><textClass></textClass></profileDesc></teiHeader><front><div type="abstract" xml:lang="en"><title>Summary</title><p>Naive mouse embryonic stem cells (mESCs) are in a metastable state and fluctuate between inner cell mass- and epiblast-like phenotypes. Here, we show transient activation of the BMP-SMAD signaling pathway in mESCs containing a BMP-SMAD responsive reporter transgene. Activation of the BMP-SMAD reporter transgene in naive mESCs correlated with lower levels of genomic DNA methylation, high expression of 5-methylcytosine hydroxylases <italic>Tet1/2</italic> and low levels of DNA methyltransferases <italic>Dnmt3a/b</italic>. Moreover, naive mESCs, in which the BMP-SMAD reporter transgene was activated, showed higher resistance to differentiation. Using double <italic>Smad1;Smad5</italic> knockout mESCs, we showed that BMP-SMAD signaling is dispensable for self-renewal in both naive and ground state. These mutant mESCs were still pluripotent, but they exhibited higher levels of DNA methylation than their wild-type counterparts and had a higher propensity to differentiate. We showed that BMP-SMAD signaling modulates lineage priming in mESCs, by transiently regulating the enzymatic machinery responsible for DNA methylation.</p></div></front><back><div1 type="bibliography"><listBibl><biblStruct><analytic><author><name sortKey="Boroviak, T" uniqKey="Boroviak T">T. Boroviak</name></author><author><name sortKey="Loos, R" uniqKey="Loos R">R. Loos</name></author><author><name sortKey="Bertone, P" uniqKey="Bertone P">P. Bertone</name></author><author><name sortKey="Smith, A" uniqKey="Smith A">A. Smith</name></author><author><name sortKey="Nichols, J" uniqKey="Nichols J">J. Nichols</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Chen, X" uniqKey="Chen X">X. Chen</name></author><author><name sortKey="Xu, H" uniqKey="Xu H">H. Xu</name></author><author><name sortKey="Yuan, P" uniqKey="Yuan P">P. Yuan</name></author><author><name sortKey="Fang, F" uniqKey="Fang F">F. 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<front><journal-meta><journal-id journal-id-type="nlm-ta">Stem Cell Reports</journal-id><journal-id journal-id-type="iso-abbrev">Stem Cell Reports</journal-id><journal-title-group><journal-title>Stem Cell Reports</journal-title></journal-title-group><issn pub-type="epub">2213-6711</issn><publisher><publisher-name>Elsevier</publisher-name></publisher></journal-meta><article-meta><article-id pub-id-type="pmid">26711875</article-id><article-id pub-id-type="pmc">4720007</article-id><article-id pub-id-type="publisher-id">S2213-6711(15)00348-3</article-id><article-id pub-id-type="doi">10.1016/j.stemcr.2015.11.012</article-id><article-categories><subj-group subj-group-type="heading"><subject>Article</subject></subj-group></article-categories><title-group><article-title>BMP-SMAD Signaling Regulates Lineage Priming, but Is Dispensable for Self-Renewal in Mouse Embryonic Stem Cells</article-title></title-group><contrib-group><contrib contrib-type="author"><name><surname>Gomes Fernandes</surname><given-names>Maria</given-names></name><xref rid="aff1" ref-type="aff">1</xref></contrib><contrib contrib-type="author"><name><surname>Dries</surname><given-names>Ruben</given-names></name><xref rid="aff2" ref-type="aff">2</xref><xref rid="aff3" ref-type="aff">3</xref></contrib><contrib contrib-type="author"><name><surname>Roost</surname><given-names>Matthias S.</given-names></name><xref rid="aff1" ref-type="aff">1</xref></contrib><contrib contrib-type="author"><name><surname>Semrau</surname><given-names>Stefan</given-names></name><xref rid="aff4" ref-type="aff">4</xref></contrib><contrib contrib-type="author"><name><surname>de Melo Bernardo</surname><given-names>Ana</given-names></name><xref rid="aff1" ref-type="aff">1</xref></contrib><contrib contrib-type="author"><name><surname>Davis</surname><given-names>Richard P.</given-names></name><xref rid="aff1" ref-type="aff">1</xref></contrib><contrib contrib-type="author"><name><surname>Ramakrishnan</surname><given-names>Ramprasad</given-names></name><xref rid="aff1" ref-type="aff">1</xref></contrib><contrib contrib-type="author"><name><surname>Szuhai</surname><given-names>Karoly</given-names></name><xref rid="aff5" ref-type="aff">5</xref></contrib><contrib contrib-type="author"><name><surname>Maas</surname><given-names>Elke</given-names></name><xref rid="aff6" ref-type="aff">6</xref></contrib><contrib contrib-type="author"><name><surname>Umans</surname><given-names>Lieve</given-names></name><xref rid="aff2" ref-type="aff">2</xref><xref rid="aff3" ref-type="aff">3</xref><xref rid="aff6" ref-type="aff">6</xref></contrib><contrib contrib-type="author"><name><surname>Abon Escalona</surname><given-names>Vanesa</given-names></name><xref rid="aff6" ref-type="aff">6</xref></contrib><contrib contrib-type="author"><name><surname>Salvatori</surname><given-names>Daniela</given-names></name><xref rid="aff1" ref-type="aff">1</xref><xref rid="aff7" ref-type="aff">7</xref></contrib><contrib contrib-type="author"><name><surname>Deforce</surname><given-names>Dieter</given-names></name><xref rid="aff8" ref-type="aff">8</xref></contrib><contrib contrib-type="author"><name><surname>Van Criekinge</surname><given-names>Wim</given-names></name><xref rid="aff9" ref-type="aff">9</xref></contrib><contrib contrib-type="author"><name><surname>Huylebroeck</surname><given-names>Danny</given-names></name><xref rid="aff2" ref-type="aff">2</xref><xref rid="aff3" ref-type="aff">3</xref></contrib><contrib contrib-type="author"><name><surname>Mummery</surname><given-names>Christine</given-names></name><xref rid="aff1" ref-type="aff">1</xref></contrib><contrib contrib-type="author"><name><surname>Zwijsen</surname><given-names>An</given-names></name><xref rid="aff6" ref-type="aff">6</xref></contrib><contrib contrib-type="author"><name><surname>Chuva de Sousa Lopes</surname><given-names>Susana M.</given-names></name><email>lopes@lumc.nl</email><xref rid="aff1" ref-type="aff">1</xref><xref rid="aff10" ref-type="aff">10</xref><xref rid="cor1" ref-type="corresp">∗</xref></contrib></contrib-group><aff id="aff1"><label>1</label>Department Anatomy and Embryology, Leiden University Medical Center, Leiden 2333 ZC, the Netherlands</aff><aff id="aff2"><label>2</label>Department Development and Regeneration, Laboratory of Molecular Biology (Celgen), KU Leuven, Leuven 3000, Belgium</aff><aff id="aff3"><label>3</label>Department of Cell Biology, Erasmus University Medical Center, Rotterdam 3015 CN, the Netherlands</aff><aff id="aff4"><label>4</label>Leiden Institute of Physics, Leiden University, Leiden 2333 CA, the Netherlands</aff><aff id="aff5"><label>5</label>Department Molecular Cell Biology, Leiden University Medical Center, Leiden 2333 ZC, the Netherlands</aff><aff id="aff6"><label>6</label>Department Human Genetics, VIB Center for the Biology of Disease, KU Leuven, Leuven 3000, Belgium</aff><aff id="aff7"><label>7</label>Center Laboratory Animal Facility, Leiden University Medical Center, Leiden 2333 ZC, the Netherlands</aff><aff id="aff8"><label>8</label>Laboratory of Pharmaceutical Biotechnology, Faculty of Pharmaceutical Sciences, Ghent University, Ghent 9000, Belgium</aff><aff id="aff9"><label>9</label>Mathematical Modelling, Statistics and Bio-informatics, Faculty Bioscience Engineering, Ghent University, Ghent 9000, Belgium</aff><aff id="aff10"><label>10</label>Department Reproductive Medicine, Ghent University Hospital, Ghent 9000, Belgium</aff><author-notes><corresp id="cor1"><label>∗</label>Corresponding author <email>lopes@lumc.nl</email></corresp></author-notes><pub-date pub-type="pmc-release"><day>17</day><month>12</month><year>2015</year></pub-date><pmc-comment> PMC Release delay is 0 months and 0 days and was based on .</pmc-comment>
<pub-date pub-type="collection"><day>12</day><month>1</month><year>2016</year></pub-date><pub-date pub-type="epub"><day>17</day><month>12</month><year>2015</year></pub-date><volume>6</volume><issue>1</issue><fpage>85</fpage><lpage>94</lpage><history><date date-type="received"><day>24</day><month>8</month><year>2014</year></date><date date-type="rev-recd"><day>16</day><month>11</month><year>2015</year></date><date date-type="accepted"><day>18</day><month>11</month><year>2015</year></date></history><permissions><copyright-statement>© 2016 The Authors</copyright-statement><copyright-year>2016</copyright-year><license license-type="CC BY-NC-ND" xlink:href="http://creativecommons.org/licenses/by-nc-nd/4.0/"><license-p>This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).</license-p></license></permissions><abstract><title>Summary</title><p>Naive mouse embryonic stem cells (mESCs) are in a metastable state and fluctuate between inner cell mass- and epiblast-like phenotypes. Here, we show transient activation of the BMP-SMAD signaling pathway in mESCs containing a BMP-SMAD responsive reporter transgene. Activation of the BMP-SMAD reporter transgene in naive mESCs correlated with lower levels of genomic DNA methylation, high expression of 5-methylcytosine hydroxylases <italic>Tet1/2</italic> and low levels of DNA methyltransferases <italic>Dnmt3a/b</italic>. Moreover, naive mESCs, in which the BMP-SMAD reporter transgene was activated, showed higher resistance to differentiation. Using double <italic>Smad1;Smad5</italic> knockout mESCs, we showed that BMP-SMAD signaling is dispensable for self-renewal in both naive and ground state. These mutant mESCs were still pluripotent, but they exhibited higher levels of DNA methylation than their wild-type counterparts and had a higher propensity to differentiate. We showed that BMP-SMAD signaling modulates lineage priming in mESCs, by transiently regulating the enzymatic machinery responsible for DNA methylation.</p></abstract><abstract abstract-type="graphical"><title>Graphical Abstract</title><fig id="undfig1" position="anchor"><graphic xlink:href="fx1"></graphic></fig></abstract><abstract abstract-type="author-highlights"><title>Highlights</title><p><list list-type="simple"><list-item id="u0010"><label>•</label><p>BMP-SMAD signaling in mESCs is more prominent in naive than ground state</p></list-item><list-item id="u0015"><label>•</label><p>BMP-SMAD signaling is dispensable for pluripotency in mESCs</p></list-item><list-item id="u0020"><label>•</label><p>BMP-SMAD signaling facilitates lineage priming in mESCs</p></list-item><list-item id="u0025"><label>•</label><p>BMP-SMAD signaling regulates <italic>Dnmt3b</italic> and hence levels of DNA methylation</p></list-item></list></p></abstract><abstract abstract-type="teaser"><p>In this article, Chuva de Sousa Lopes and colleagues show that the BMP-SMAD signaling is dispensable for the derivation, maintenance, and self-renewal of mESCs both in “serum” and/or “2i” pluripotency states. The BMP-SMAD signaling plays a role regulating the levels of DNA methylation (via Dnmt3a/b and Tet1/2) and hence lineage priming in pluripotent mESCs.</p></abstract></article-meta><notes><p id="misc0010">Published: December 17, 2015</p></notes></front><body><sec id="sec1"><title>Introduction</title><p>Culture conditions affect features of mouse embryonic stem cells (mESCs), such as their proliferation, gene expression, epigenetic status, self-renewal, and capacity for multi-lineage differentiation (<xref rid="bib19" ref-type="bibr">Marks et al., 2012</xref>, <xref rid="bib34" ref-type="bibr">Tesar et al., 2007</xref>). In culture medium with fetal calf serum, naive mESCs grown on mouse embryonic fibroblast feeder cells (here abbreviated as “serum”) transit between inner cell mass (ICM)-like and epiblast-like pluripotency states (<xref rid="bib28" ref-type="bibr">Sasai et al., 2013</xref>, <xref rid="bib38" ref-type="bibr">Trott and Martinez Arias, 2013</xref>). However, when cultured in serum-free conditions with inhibitors of mitogen-activated protein kinase and glycogen synthase kinase 3 signaling, also called “2i” medium, mESCs become more homogeneous and adopt the more ICM-like or “ground” state (<xref rid="bib19" ref-type="bibr">Marks et al., 2012</xref>, <xref rid="bib23" ref-type="bibr">Nichols et al., 2009</xref>, <xref rid="bib40" ref-type="bibr">Ying et al., 2003</xref>). The observation that naive mESCs interconvert between pluripotent states while remaining uncommitted has raised the suggestion that such heterogeneity may allow the cells to respond differently to environmental cues. In agreement, subpopulations of naive mESCs show different potentials to differentiate (<xref rid="bib7" ref-type="bibr">Graf and Stadtfeld, 2008</xref>, <xref rid="bib11" ref-type="bibr">Hanna et al., 2009</xref>, <xref rid="bib12" ref-type="bibr">Hayashi et al., 2008</xref>). How the metastable transcriptional and epigenetic diversity of cultured mESCs is regulated and maintained has remained elusive.</p><p>The two notable characteristics of mESCs are their capacity to self-renew and differentiate into all embryonic lineages (<xref rid="bib24" ref-type="bibr">Niwa et al., 1998</xref>). In mESCs, pluripotency is maintained by a core network of regulatory transcription factors, including <italic>Pou5f1</italic>, <italic>Sox2</italic>, and <italic>Nanog</italic> (<xref rid="bib13" ref-type="bibr">Kashyap et al., 2009</xref>, <xref rid="bib14" ref-type="bibr">Kim et al., 2008</xref>, <xref rid="bib20" ref-type="bibr">Marson et al., 2008</xref>, <xref rid="bib22" ref-type="bibr">Navarro et al., 2012</xref>); the balance between self-renewal and differentiation is regulated by protein-encoding genes that include <italic>Id1</italic> and <italic>Dusp9</italic>, both downstream targets of the bone morphogenetic protein (BMP) signaling pathway (<xref rid="bib18" ref-type="bibr">Li and Chen, 2013</xref>). Moreover, it has been shown that both the BMP and TGFβ (via NODAL) SMAD-mediated signaling pathways are involved in maintaining heterogeneity of NANOG in naive mESCs (<xref rid="bib5" ref-type="bibr">Galvin-Burgess et al., 2013</xref>). Conversely, NANOG may attenuate BMP signaling via a feedback loop that involves titration of phosphorylated (P)SMAD1 by direct NANOG-SMAD1 interaction (<xref rid="bib32" ref-type="bibr">Suzuki et al., 2006</xref>). However, the functional role of BMP-SMAD signaling in the metastable state of naive pluripotency has not been investigated.</p><p>Here, we report the derivation and characterization of transgenic mESCs that allow a real-time readout of SMAD-mediated BMP signaling activity. This transgenic <italic>BRE:gfp</italic> reporter mESC line expresses a well-characterized BMP responsive element (BRE) containing several PSMAD1/5 DNA-binding sites isolated from the <italic>Id1</italic> promoter to drive GFP expression (<xref rid="bib16" ref-type="bibr">Korchynskyi and ten Dijke, 2002</xref>, <xref rid="bib21" ref-type="bibr">Monteiro et al., 2008</xref>). Activation of the BMP-SMAD reporter transgene was heterogeneous in serum mESCs (±50% GFP + cells) and 2i mESCs (±4% GFP + cells). By genetic abrogation of the core BMP pathway components SMAD1 and SMAD5, we demonstrated that BMP-SMAD signaling is dispensable for the maintenance and self-renewal of mESCs both in serum and 2i states, but that it regulates the levels of DNA methylation (via <italic>Dnmt3a/b</italic> and <italic>Tet1/2</italic>) and hence lineage priming in pluripotent mESCs.</p></sec><sec id="sec2"><title>Results</title><sec id="sec2.1"><title>BMP-SMAD Signaling Is Activated during the Acquisition of Pluripotency</title><p>BMP signaling plays key roles in patterning of post-implantation mouse embryos (<xref rid="bib15" ref-type="bibr">Kishigami and Mishina, 2005</xref>, <xref rid="bib33" ref-type="bibr">Tam and Loebel, 2007</xref>). However, a role during pre-implantation development has been less evident because genetic ablation of single members of the BMP-SMAD pathway showed no evidence of a phenotype during the pre-implantation period (<xref rid="bib6" ref-type="bibr">Goumans and Mummery, 2000</xref>, <xref rid="bib8" ref-type="bibr">Graham et al., 2014</xref>, <xref rid="bib26" ref-type="bibr">Reyes de Mochel et al., 2015</xref>, <xref rid="bib41" ref-type="bibr">Zhao, 2003</xref>). We investigated whether the BMP-SMAD signaling pathway was active in pre-implantation embryos by examining <italic>BRE:gfp</italic> blastocysts at E3.5. We were unable to detect GFP at this stage (data not shown). As the BMP-SMAD pathway has been shown to play dual roles in self-renewal and differentiation of mESCs (<xref rid="bib18" ref-type="bibr">Li and Chen, 2013</xref>), we monitored GFP during the derivation of mESCs from <italic>BRE:gfp</italic> blastocysts into the naive state (serum) and the ground state (2i). One day after plating (D1), GFP was still undetectable in blastocysts in either culture condition (<xref rid="fig1" ref-type="fig">Figure 1</xref>A); however, by D4, GFP+ cells were evident within the ICM-like cells of <italic>BRE:gfp</italic> blastocyst outgrowths in both serum and 2i (<xref rid="fig1" ref-type="fig">Figure 1</xref>A). This suggested that the BMP-SMAD pathway was activated during the acquisition of pluripotency in vitro.</p></sec><sec id="sec2.2"><title>BMP-SMAD Signaling Activation in Serum and 2i mESCs</title><p>Once <italic>BRE:gfp</italic> mESCs lines had been established (<xref rid="fig1" ref-type="fig">Figures 1</xref>A and 1B) and karyotyped (<xref rid="mmc1" ref-type="supplementary-material">Figure S1</xref>A), a striking difference was observed between the two conditions: serum <italic>BRE:gfp</italic> mESCs exhibited an heterogeneous pattern of GFP expression with about 50% of the cells being GFP+, whereas in 2i <italic>BRE:gfp</italic> mESCs less than 4% of cells were GFP+ (<xref rid="fig1" ref-type="fig">Figure 1</xref>B). In serum <italic>BRE:gfp</italic> mESCs, the GFP+ cells produced ID1 (<xref rid="fig1" ref-type="fig">Figure 1</xref>C), confirming that GFP expression corresponded to the activation of BMP-SMADs. The promoter of <italic>Id1</italic> contains the PSMAD1/5 DNA-binding sites that were used to generate the <italic>BRE:gfp</italic> transgene (<xref rid="mmc1" ref-type="supplementary-material">Figure S1</xref>B). Most 2i <italic>BRE:gfp</italic> mESCs showed no GFP and consequently no/low ID1 (<xref rid="fig1" ref-type="fig">Figure 1</xref>C). POU5F1 and NANOG were detected in both serum and 2i <italic>BRE:gfp</italic> mESCs. Quantification of NANOG suggested that it was more homogeneously expressed in GFP− cells per colony (<xref rid="fig1" ref-type="fig">Figure 1</xref>D) and this difference was statistically significant (n = 16; p < 0.05).</p><p>To measure BMP-SMAD signaling activation, we investigated the levels of PSMAD1/5/8, which were low in 2i medium in serum mESCs and high in 2i after 1 hr of stimulation with 25 ng/ml BMP4; in agreement, faint GFP was observed in 2i compared with serum <italic>BRE:gfp</italic> mESCs (<xref rid="fig1" ref-type="fig">Figure 1</xref>E). In addition, we examined the number of GFP+ cells present in 2i and showed that this increased in response to BMP4 but not to Activin A (which activates the NODAL pathway) (<xref rid="fig1" ref-type="fig">Figure 1</xref>F), and that <italic>BRE:gfp</italic> mESCs could be interconverted to adopt the GFP pattern associated with each culture medium within four cell passages (<xref rid="fig1" ref-type="fig">Figure 1</xref>G).</p></sec><sec id="sec2.3"><title>In Serum, GFP+ <italic>BRE:gfp</italic> mESCs Correlated with Low Levels of <italic>Dnmt3b</italic> and Lower DNA Methylation</title><p>To further understand the role of BMP-SMAD signaling activation in pluripotency, fluorescence-activated cell sorting (FACS) sorted subpopulations of serum (GFP++, GFP+, GFP−) and 2i (GFP+, GFP−) <italic>BRE:gfp</italic> mESCs (<xref rid="fig2" ref-type="fig">Figures 2</xref>A and <xref rid="mmc1" ref-type="supplementary-material">S2</xref>A) were analyzed by qPCR (<xref rid="fig2" ref-type="fig">Figures 2</xref>B and <xref rid="mmc1" ref-type="supplementary-material">S2</xref>B). In serum, the sorted GFP++ mESCs (N = 3) exhibited lower levels of <italic>Dnmt3a/b</italic>, in particular <italic>Dnmt3b</italic>, and higher levels of <italic>Tet1/2</italic>, but similar high transcriptional levels of pluripotency genes (<xref rid="fig2" ref-type="fig">Figure 2</xref>B). A direct comparison between 2i and serum is provided in <xref rid="mmc1" ref-type="supplementary-material">Figure S2</xref>B. Comparing whole transcriptome RNA sequencing (RNAseq) data of three independent serum GFP++ and GFP− mESC samples, we confirmed that <italic>Dnmt3b</italic> as well as <italic>Tet1/2</italic> were among the few statistically significant differentially regulated genes observed (n = 315; p < 0.05), mostly protein-coding genes (<xref rid="fig2" ref-type="fig">Figures 2</xref>C, <xref rid="mmc1" ref-type="supplementary-material">S2</xref>C, and S2D; <xref rid="mmc2" ref-type="supplementary-material">Table S1</xref>). Next, using available single-cell RNAseq data (<xref rid="bib27" ref-type="bibr">Sasagawa et al., 2013</xref>), we performed a hierarchical clustering of 38 individual cells from naive mESCs based on the expression of 30 selected genes. Interestingly, the cluster with the lowest transcriptional levels of <italic>Dnmt3b</italic> and high levels of <italic>Tet1</italic> (Group 1) did not correlate with the cell clusters showing high transcriptional levels of <italic>Id1</italic>/<italic>Bmp4</italic> (group 2/3) (<xref rid="mmc1" ref-type="supplementary-material">Figure S2</xref>E). This is in agreement with our qPCR (<xref rid="fig2" ref-type="fig">Figure 2</xref>B) and RNAseq results (<italic>Id1</italic> is not differentially expressed) (<xref rid="fig2" ref-type="fig">Figure 2</xref>C; <xref rid="mmc2" ref-type="supplementary-material">Table S1</xref>) and suggests a clear discrepancy between the cells expressing ID1 protein (and GFP protein) and <italic>Id1</italic> transcript. This discrepancy in the co-expression of proteins and transcripts is a well-known confounding but intrinsic property of cells, including mESCs (<xref rid="bib36" ref-type="bibr">Torres-Padilla and Chambers, 2014</xref>).</p><p>We performed reduced-representation bisulfite sequencing (RRBS) of GFP++ and GFP− <italic>BRE:gfp</italic> mESCs and observed that DNA methylation levels were in general lower in mESCs with activation of the BMP-SMAD reporter transgene than in mESCs without reporter activity, as illustrated by the significant shifts toward lower DNA methylation at all genomic regions in GFP++ cells (<xref rid="fig2" ref-type="fig">Figures 2</xref>D–2F; <xref rid="mmc3" ref-type="supplementary-material">Table S2</xref>). This is in agreement with the reduced levels of <italic>Dnmt3b</italic> expression in GFP++ cells.</p></sec><sec id="sec2.4"><title>BMP-SMAD Signaling Is Dispensable for Self-Renewal of mESCs</title><p>To clarify the role of BMP-SMAD signaling in the maintenance of the naive and ground state, we derived <italic>Smad1</italic> and <italic>Smad5</italic> double-knockout (<italic>S1</italic><sup><italic>−/−</italic></sup><italic>S5</italic><sup><italic>−/−</italic></sup>) mESC lines in 2i from double homozygous floxed <italic>Smad1;Smad5</italic> mESC lines (<italic>S1</italic><sup><italic>fl/fl</italic></sup><italic>S5</italic><sup><italic>fl/fl</italic></sup>) (<xref rid="bib37" ref-type="bibr">Tremblay et al., 2001</xref>, <xref rid="bib39" ref-type="bibr">Umans et al., 2003</xref>) that were hemizygous for the R26R Cre-reporter transgene (<xref rid="bib31" ref-type="bibr">Soriano, 1999</xref>) using Cre recombinase (<xref rid="mmc1" ref-type="supplementary-material">Figures S3</xref>A and S3B). We derived the <italic>S1</italic><sup><italic>−/−</italic></sup><italic>S5</italic><sup><italic>−/−</italic></sup> mESC in 2i because BMP-SMAD signaling activation was less prominent in 2i and therefore the chance of deriving pluripotent <italic>S1</italic><sup>−<italic>/−</italic></sup><italic>S5</italic><sup><italic>−/−</italic></sup> mESCs was higher. The pluripotency of the <italic>S1</italic><sup><italic>−/−</italic></sup><italic>S5</italic><sup><italic>−/−</italic></sup> mESCs was confirmed by showing its contribution to the three germ layers in <italic>S1</italic><sup><italic>−/−</italic></sup><italic>S5</italic><sup><italic>−/−</italic></sup> <> wild-type chimeric embryos (<xref rid="mmc1" ref-type="supplementary-material">Figure S3</xref>C), as well as in teratoma formation assays (<xref rid="mmc1" ref-type="supplementary-material">Figure S3</xref>D) in independent lines with a normal karyotype (<xref rid="mmc1" ref-type="supplementary-material">Figure S4</xref>A). Moreover, we showed that <italic>Smad8</italic> was not upregulated in response to the deletion of <italic>Smad1</italic> and <italic>Smad5</italic>, and that <italic>Id1</italic> and <italic>Id2</italic> were upregulated after stimulation with BMP4 only in the <italic>S1</italic><sup><italic>fl/fl</italic></sup><italic>S5</italic><sup><italic>fl/fl</italic></sup> parental line, as expected (<xref rid="mmc1" ref-type="supplementary-material">Figure S4</xref>B). The 2i <italic>S1</italic><sup><italic>−/−</italic></sup><italic>S5</italic><sup><italic>−/−</italic></sup> mESCs self-renewed at the same rate as the parental <italic>S1</italic><sup><italic>fl/fl</italic></sup><italic>S5</italic><sup><italic>fl/fl</italic></sup> mESCs (<xref rid="fig3" ref-type="fig">Figure 3</xref>A) and showed comparable alkaline phosphatase activity (<xref rid="fig3" ref-type="fig">Figure 3</xref>B). Unexpectedly, when <italic>S1</italic><sup><italic>−/−</italic></sup><italic>S5</italic><sup><italic>−/−</italic></sup> mESCs were switched from 2i to serum, after an initial period of adaptation the cells continued to self-renew at similar rates as the parental <italic>S1</italic><sup><italic>fl/fl</italic></sup><italic>S5</italic><sup><italic>fl/fl</italic></sup> mESCs (<xref rid="mmc1" ref-type="supplementary-material">Figure S4</xref>C) instead of differentiating. In general, the expression level of pluripotency genes remained high in the parental <italic>S1</italic><sup><italic>fl/fl</italic></sup><italic>S5</italic><sup><italic>fl/fl</italic></sup> and <italic>S1</italic><sup><italic>−/−</italic></sup><italic>S5</italic><sup><italic>−/−</italic></sup> mESCs in 2i (<xref rid="fig3" ref-type="fig">Figure 3</xref>C) and serum (<xref rid="mmc1" ref-type="supplementary-material">Figure S4</xref>D). Our results demonstrated that BMP-SMAD signaling is dispensable for self-renewal of mESCs.</p></sec><sec id="sec2.5"><title><italic>S1</italic><sup><italic>−/−</italic></sup><italic>S5</italic><sup><italic>−/−</italic></sup> mESCs Have High Levels of <italic>Dnmt3b</italic> and High Levels of DNA Methylation</title><p>Next, we investigated the SMAD1/5-responsive genes using RNAseq (<xref rid="fig3" ref-type="fig">Figure 3</xref>D) and found that most differentially expressed genes (DEGs) between <italic>S1</italic><sup><italic>−/−</italic></sup><italic>S5</italic><sup><italic>−/−</italic></sup> and <italic>S1</italic><sup><italic>fl/fl</italic></sup><italic>S5</italic><sup><italic>fl/fl</italic></sup> mESCs were protein-coding genes (<xref rid="mmc1" ref-type="supplementary-material">Figure S4</xref>E). Interestingly, about half of the DEGs (including protein-coding, pseudogenes, and long non-coding RNAs) were upregulated (n = 781; p < 0.01) and half of the genes were downregulated (n = 854; p < 0.01) in <italic>S1</italic><sup><italic>−/−</italic></sup><italic>S5</italic><sup><italic>−/−</italic></sup> mESCs (<xref rid="fig3" ref-type="fig">Figure 3</xref>E; <xref rid="mmc2" ref-type="supplementary-material">Table S1</xref>).</p><p>To investigate whether the observed expression changes were consistent with direct transcriptional regulation, we integrated our RNAseq dataset with a list of direct SMAD1/5 targets (n = 562) identified by ChIP (<xref rid="bib3" ref-type="bibr">Fei et al., 2010</xref>). Using gene set enrichment analysis, we found a significant enrichment of SMAD1/5 targets in genes that were downregulated in <italic>S1</italic><sup><italic>−/−</italic></sup><italic>S5</italic><sup><italic>−/−</italic></sup> mESCs (p < 1 × 10<sup>−4</sup>) (<xref rid="mmc1" ref-type="supplementary-material">Figure S4</xref>F).</p><p>Moreover, the great majority of the DEGs contained the sequence motifs GCCG and/or GGCGCC, well-characterized SMAD1/5 binding sites (<xref rid="bib16" ref-type="bibr">Korchynskyi and ten Dijke, 2002</xref>), in their promoters, defined as ±2 kb from the transcriptional start site (TSS) (<xref rid="fig3" ref-type="fig">Figure 3</xref>E; <xref rid="mmc4" ref-type="supplementary-material">Table S3</xref>). By contrast, genome-wide occurrence of GGCGCC and GCCG motifs at such promoters (including protein-coding, pseudogenes, and long non-coding RNAs) was not, or much less, enriched (<xref rid="mmc1" ref-type="supplementary-material">Figure S4</xref>G), and significantly different from the enrichment observed at DEGs (p < 2.2 × 10<sup>−16</sup>). As an example, <italic>Dnmt3b</italic> was significantly upregulated in <italic>S1</italic><sup><italic>−/−</italic></sup><italic>S5</italic><sup><italic>−/−</italic></sup> mESCs and contained 21x GCCG and 5x GGCGCC in the promoter region, suggesting direct (co-)regulation by DNA-binding BMP-SMADs. The DEGs were significantly enriched for gene ontology (GO) categories such as “regulation of developmental process,” “regulation of cell development,” and “regulation of cell differentiation” (<xref rid="fig3" ref-type="fig">Figure 3</xref>F), compatible with BMP-SMAD signaling not being involved in self-renewal of mESC, but rather predisposing mESCs to differentiate. The downregulation of <italic>Dnmt3b</italic> and enrichment in developmental genes in <italic>S1</italic><sup><italic>−/−</italic></sup><italic>S5</italic><sup><italic>−/−</italic></sup> mESCs, led us to investigate the levels of DNA methylation by RRBS on several independent <italic>S1</italic><sup><italic>fl/fl</italic></sup><italic>S5</italic><sup><italic>fl/fl</italic></sup> and <italic>S1</italic><sup><italic>−/−</italic></sup><italic>S5</italic><sup><italic>−/−</italic></sup> mESC lines (<xref rid="mmc3" ref-type="supplementary-material">Table S2</xref>). <italic>S1</italic><sup><italic>−/−</italic></sup><italic>S5</italic><sup><italic>−/−</italic></sup> mESCs displayed a significant shift toward higher levels of DNA methylation at all genomic regions analyzed when compared with <italic>S1</italic><sup><italic>fl/fl</italic></sup><italic>S5</italic><sup><italic>fl/fl</italic></sup> mESCs (<xref rid="fig3" ref-type="fig">Figures 3</xref>G–3I), suggesting that the enrichment in developmental genes is caused by the higher levels of DNA methylation.</p></sec><sec id="sec2.6"><title>mESCs Differentiated More Efficiently to Mesendoderm or Neurectoderm in the Absence of BMP-SMAD Signaling</title><p>Finally, we examined the differentiation capacity of <italic>S1</italic><sup><italic>−/−</italic></sup><italic>S5</italic><sup><italic>−/−</italic></sup> mESCs in both serum and 2i and found that they formed endoderm (<italic>Sox17</italic>), mesoderm (<italic>T</italic>), and ectoderm (<italic>Pax6</italic> and <italic>Sox1</italic>) more efficiently than the parental line (<xref rid="fig4" ref-type="fig">Figures 4</xref>A–4C) in the monolayer using differentiation protocols for either the mesendoderm (ME) or neuroectoderm (NE) lineages (<xref rid="bib35" ref-type="bibr">Thomson et al., 2011</xref>). In addition, we investigated the capacity of the FACS-sorted subpopulations of serum <italic>BRE:gfp</italic> mESCs to differentiate to ME and NE and showed that GFP++ mESCs had lower levels of ME and NE early differentiation markers than GFP− mESCs (<xref rid="fig4" ref-type="fig">Figure 4</xref>D), demonstrating that GFP++ mESCs were less prone to differentiate. In agreement, GFP++ mESCs retained higher levels of pluripotency markers, at least after 4 days of differentiation to ME (<xref rid="fig4" ref-type="fig">Figure 4</xref>E). Our data showed that transient BMP-SMAD signaling activation tilted mESCs to a less differentiation-prone state, whereas in the absence of BMP-SMAD signaling the balance was shifted toward an increased predisposition to differentiate.</p></sec></sec><sec id="sec3"><title>Discussion</title><p>A recent study reported the absence of <italic>Bmp4</italic> and <italic>Id1</italic> in (embryonic day) E3.5 ICMs and a high transient upregulation in E4.5 epiblasts, followed by downregulation of <italic>Bmp4</italic> and <italic>Id3</italic> expression during the next 6 days of the derivation of mESCs and their further maintenance in 2i (<xref rid="bib1" ref-type="bibr">Boroviak et al., 2014</xref>). We now show this in real-time using <italic>BRE:gfp</italic> blastocysts to derive mESCs. Moreover, we demonstrated that BMP-SMAD signaling is not functionally implicated in self-renewal, in agreement with studies that have mapped genome-wide the genes that are directly regulated by SMAD1/5 (<xref rid="bib2" ref-type="bibr">Chen et al., 2008</xref>, <xref rid="bib3" ref-type="bibr">Fei et al., 2010</xref>). They showed that the genes regulated by SMAD1/5 were involved in fate determination, rather than self-renewal. Here, we provide functional evidence that SMAD1/5 are not necessary for mESC self-renewal in either naive (serum) or ground (2i) state.</p><p>Specific levels of DNA methylation and associated enzymes have been associated with the different pluripotency states (ground, naive, primed) (<xref rid="bib9" ref-type="bibr">Habibi et al., 2013</xref>, <xref rid="bib10" ref-type="bibr">Hackett et al., 2013</xref>, <xref rid="bib29" ref-type="bibr">Smallwood et al., 2014</xref>), as well as with different levels of GFP in <italic>Nanog:gfp</italic> naive mESCs (<xref rid="bib4" ref-type="bibr">Ficz et al., 2013</xref>). This reflects faithfully the rapid loss of genomic DNA methylation that the embryo undergoes in vivo during pre-implantation development, and the gain of DNA methylation during the transition between ICM and epiblast (<xref rid="bib30" ref-type="bibr">Smith et al., 2012</xref>). Therefore, it is perhaps not surprising that the machinery to regulate rapid switches in genomic DNA methylation is present in pluripotent stem cells derived from ICM and epiblast. A role for BMP-SMAD signaling in LIF-dependent conversion between EpiSCs and ESCs has been reported (<xref rid="bib25" ref-type="bibr">Onishi et al., 2014</xref>), but the association with changes in DNA methylation between EpiSCs and ESCs remains to be investigated.</p><p>Finally, it has been suggested that the epigenetic variation observed in pluripotent cells is stochastic and results in a diversity of predispositions to acquire specific cell fates when the cells are triggered to differentiate (<xref rid="bib17" ref-type="bibr">Lee et al., 2014</xref>). Our data provide evidence that the cellular diversity of both serum and 2i mESCs regarding DNA methylation and associated enzymes is not a stochastic process as previously thought, but is in fact regulated by cell-cell signaling interactions involving the BMP-SMAD signaling pathway.</p></sec><sec id="sec4"><title>Experimental Procedures</title><sec id="sec4.1"><title>mESCs Derivation and Culture</title><p>Derivation of <italic>BRE:gfp</italic> mESCs in 2i and serum and the conditional knockout mESCs for <italic>Smad1</italic> and <italic>Smad5</italic> (<italic>S1</italic><sup><italic>fl/fl</italic></sup><italic>S5</italic><sup><italic>fl/fl</italic></sup>) in 2i, as well as the Cre-recombination of <italic>S1</italic><sup><italic>fl/fl</italic></sup><italic>S5</italic><sup><italic>fl/fl</italic></sup> mESCs, are described in the <xref rid="mmc1" ref-type="supplementary-material">Supplemental Experimental Procedures</xref>. Genotyping of the <italic>BRE:gfp</italic> mESCs was performed as described (<xref rid="bib21" ref-type="bibr">Monteiro et al., 2008</xref>). E14 mESCs were cultured in either 2i or serum. Stimulation (1 hr) with BMP4 (R&D Systems) or Activin A (R&D Systems) was followed by FACS analysis or western blotting (see <xref rid="mmc1" ref-type="supplementary-material">Supplemental Experimental Procedures</xref>). Details about generation of chimeric embryos, the teratoma formation assay, RNAseq, and RRBS are provided in the <xref rid="mmc1" ref-type="supplementary-material">Supplemental Experimental Procedures</xref>.</p></sec><sec id="sec4.2"><title>mESCs Differentiation and Proliferation</title><p>mESCs were differentiated to ME or NE as described (<xref rid="bib35" ref-type="bibr">Thomson et al., 2011</xref>). Briefly, mESCs (10,000 cells/cm<sup>2</sup>) were grown in N2B27 medium without supplements for 48 hr, after which either 3 μM CHIR99021 or 500 nM all-<italic>trans</italic> retinoic acid (RA) (Sigma-Aldrich) was added to the N2B27 medium for an additional 48 hr. Cells were then collected for immunofluorescence or qPCR (see <xref rid="mmc1" ref-type="supplementary-material">Supplemental Experimental Procedures</xref>). For the proliferation assay, the total number of serum and 2i mESCs was monitored during each passage for 26 days of culture. Serum mESCs were pre-plated prior to counting.</p></sec><sec id="sec4.3"><title>Statistics</title><sec id="sec4.3.1"><title>Quantification of NANOG-Positive Cells</title><p>Whole <italic>BRE:gf</italic>p mESC colonies (total n = 16) from three independent experiments (N = 3, 5–6 colonies per experiment) were manually counted three times and averaged. N refers to the number of independent experiments; n refers to total number or colonies counted. Statistical analysis was performed using the Student t-test (two-tailed, unequal variance), <sup>∗</sup>p ≤ 0.05.</p></sec><sec id="sec4.3.2"><title>qPCR</title><p>In qPCR, each bar represents the average of technical triplicates. N refers to the number of independent experiments; n refers to total replicates. Statistical analysis was performed using the Student t-test (two-tailed, unequal variance), <sup>∗</sup>p ≤ 0.05; <sup>∗∗</sup>p ≤ 0.01.</p></sec><sec id="sec4.3.3"><title>RNAseq Expression Data</title><p>To determine significantly DEGs between GFP++ and GFP− or S1<sup>−/−</sup>S5<sup>−/−</sup> and <sup>S1fl/fl</sup>S5<sup>fl/fl</sup> mESCs, we applied a cut-off of 0.01 and/or 0.05 on the p values adjusted for multiple testing hypothesis. N refers to the number of independent experiments; n refers to the number of genes.</p></sec><sec id="sec4.3.4"><title>RNAseq GO</title><p>Enrichment analysis for GO terms was done with the R package topGO based on DEGs (p < 0.05) and utilizing Fisher’s exact test.</p></sec><sec id="sec4.3.5"><title>RNAseq Motif Sequence Analysis</title><p>One-sided Fisher’s exact was used to determine significant over-representation of the analyzed motifs in promoter regions of DEGs relative to the genome-wide promoter regions. n refers to the number of genes.</p></sec><sec id="sec4.3.6"><title>SMAD1/5 ChIP-on-chip Data</title><p>To calculate the enrichment of SMAD1/5 targets identified p values were calculated by permuting genes. n refers to the number of genes.</p></sec><sec id="sec4.3.7"><title>RRBS Global Methylation Profile</title><p>To quantitatively assess global DNA methylation changes, we created histograms for tiles (methylation change >20%) and performed a one-sided two-sample Kolmogorov-Smirnov test to determine significant distribution differences between populations.</p></sec></sec></sec><sec id="sec5"><title>Author Contributions</title><p>M.G.F. designed and performed the experiments, analyzed the data, and wrote the manuscript. 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Figures S1–S4, Table S4, and Supplemental Experimental Procedures</title></caption><media xlink:href="mmc1.pdf"></media></supplementary-material><supplementary-material content-type="local-data" id="mmc2"><caption><title>Table S1. Differentially Expressed Genes between GFP++ and GFP− BRE:gfp mESCs and between S1fl/flS5fl/fl and S1−/−S5−/− mESCs</title></caption><media xlink:href="mmc2.xlsx"></media></supplementary-material><supplementary-material content-type="local-data" id="mmc3"><caption><title>Table S2. Differential DNA Methylation between GFP++ and GFP− BRE:gfp mESCs and between S1fl/flS5fl/fl and S1−/−S5−/− mESCs</title></caption><media xlink:href="mmc3.xlsx"></media></supplementary-material><supplementary-material content-type="local-data" id="mmc4"><caption><title>Table S3. Counts of SMAD Binding Motifs GGCGCC/GCCG in the Promoters of Differentially Expressed Genes between S1fl/flS5fl/fl and S1−/−S5−/− mESCs</title></caption><media xlink:href="mmc4.xlsx"></media></supplementary-material><supplementary-material content-type="local-data" id="mmc5"><caption><title>Document S2. Article plus Supplemental Information</title></caption><media xlink:href="mmc5.pdf"></media></supplementary-material></p></sec><ack id="ack0010"><title>Acknowledgments</title><p>We acknowledge S. Kobayakawa and M. Bialecka for discussions, S. Mendes and C. Visseren for technical support, M. Bouma for technical help with teratomas, and Z. Zhang for generation of mouse chimeras using equipment provided by InfraMouse (KU Leuven-VIB) through a Hercules type 3 project (ZW09-03). This manuscript is dedicated to Cheryl Visseren, one of our most talented students, who carried out the initial experiments but tragically passed away on July 4, 2014. This work was supported by the <funding-source id="gs1">Interuniversity Attraction Poles</funding-source>-Phase VII [IUAP/PAI P7/14] (to S.C.dL., C.M., A.Z., and D.H.) and individual grants by the <funding-source id="gs2">Fundação para a Ciência e Tecnologia</funding-source> (FCT) [SFRH/BD/78689/2011 and SFRH/BD/94387/2013] to M.M.G.F. and A.dM.B., respectively, the <funding-source id="gs3">Netherlands organization of Scientific Research</funding-source> (NWO) [ASPASIA 015.007.037] to S.M.C.dS.L., and the <funding-source id="gs4">Bontius Stichting</funding-source> [PANCREAS] to M.S.R. The bio-informatics work in the D.H. team was also supported by Fund for <funding-source id="gs5">Scientific Research-Flanders</funding-source> (FWO-V) GA.0941.11 and G.0782.14.</p></ack><fn-group><fn id="d32e450"><p id="ccnp0005">This is an open access article under the CC BY-NC-ND license (<ext-link ext-link-type="uri" xlink:href="http://creativecommons.org/licenses/by-nc-nd/4.0/" id="ccintref0005">http://creativecommons.org/licenses/by-nc-nd/4.0/</ext-link>).</p></fn><fn id="app2" fn-type="supplementary-material"><p>Supplemental Information includes Supplemental Experimental Procedures, four figures, and four tables and can be found with this article online at <ext-link ext-link-type="doi" xlink:href="10.1016/j.stemcr.2015.11.012" id="intref0015">http://dx.doi.org/10.1016/j.stemcr.2015.11.012</ext-link>.</p></fn></fn-group></back><floats-group><fig id="fig1"><label>Figure 1</label><caption><p>BMP-SMAD Signaling Activation in Serum and 2i Culture Conditions</p><p>(A) Derivation of <italic>BRE:gfp</italic> mESCs in serum and 2i conditions. D1, 1 day after blastocyst collection; D4, D1 plus 3 days after blastocyst plating, P3 mESCs, passage 3 of the derived mESCs. Scale bars represent 100 μm.</p><p>(B) Established serum and 2i <italic>BRE:gfp</italic> mESCs and their respective GFP expression profiles by FACS analysis. Scale bars represent 100 μm.</p><p>(C) Immunofluorescence of serum and 2i <italic>BRE:gfp</italic> mESCs for ID1, POU5F1, and NANOG. Scale bars represent 20 μm.</p><p>(D) Percentage (%) of NANOG-positive cells in the GFP+ and GFP− cells per colony <italic>BRE:gfp</italic> mESCs.</p><p>(E) Western blotting for PSMAD1/5/8, SMAD1/5/8, GFP and Tubulin in serum and 2i <italic>BRE:gfp</italic> and E14 mESCs as well as 2i E14 stimulated 1 hr with 25 ng/ml BMP4.</p><p>(F) Percentage (%) of GFP+ and GFP− cells in 2i <italic>BRE:gfp</italic> mESCs after 1 hr treatment with Activin A or BMP4. Bars represent the mean ± SD (N = 3).</p><p>(G) Percentage (%) of GFP+ and GFP− cells in 2i <italic>BRE:gfp</italic> mESCs switched to serum and serum <italic>BRE:gfp</italic> mESCs switched to 2i and cultured for four consecutive passages (P1–P4). See also <xref rid="mmc1" ref-type="supplementary-material">Figure S1</xref>.</p></caption><graphic xlink:href="gr1"></graphic></fig><fig id="fig2"><label>Figure 2</label><caption><p>Transcriptome and Methylome in Subsets of Serum <italic>BRE:gfp</italic> mESCs</p><p>(A) Gatings used to FACS sort three subpopulations (GFP−, GFP+, GFP++) of serum <italic>BRE:gfp</italic> mESCs and the profile of the individual cell groups.</p><p>(B) Relative expression of several genes in the three subpopulations (GFP−, GFP+, GFP++) of serum <italic>BRE:gfp</italic> mESCs compared with the GFP− cells. Each bar represents the mean ± SD of technical triplicates and the three bars of the same color represent independent experiments (n = 9, N = 3).</p><p>(C) Volcano plot showing –log10 p values versus log2 fold transcriptional changes between GFP++ and GFP− fractions of serum <italic>BRE:gfp</italic> mESCs. Differentially expressed genes (DEGs) with p < 0.05 are blue, and genes with p > 0.05 are red; some highlighted DEGs are black.</p><p>(D) Scatterplot depicting a comparison of the percentage of DNA methylation in each 600-bp tile (dot) between GFP++ and GFP− fractions of serum <italic>BRE:gfp</italic> mESCs. Each tile was classified into a biotype category according to the nearest TSS. The red line represents no difference; the inner and outer blue lines represent borders for 10% and 20% change in methylation levels, respectively.</p><p>(E) Distribution of DNA methylation at specific genomic regions in GFP++ (in blue) and GFP− fractions of serum <italic>BRE:gfp</italic> (in red) mESCs. p Values were calculated with the two-sample Kolmogorov-Smirnov test. HCP, high CpG-content promoters; LCP, low CpG-content promoters; Enh, enhancers; NA, no annotation.</p><p>(F) Number of (600-bp tile) counts showing loss of methylation (LOM) or gain of methylation (GOM) in GFP++ compared with GFP− serum <italic>BRE:gfp</italic> mESC. See also <xref rid="mmc1" ref-type="supplementary-material">Figure S2</xref> and <xref rid="mmc2" ref-type="supplementary-material">Tables S1</xref> and <xref rid="mmc5" ref-type="supplementary-material">S2</xref>.</p></caption><graphic xlink:href="gr2"></graphic></fig><fig id="fig3"><label>Figure 3</label><caption><p>Transcriptome and Methylome in <italic>S1</italic><sup><italic>−/−</italic></sup><italic>S5</italic><sup><italic>−/−</italic></sup> versus <italic>S1</italic><sup><italic>fl/fl</italic></sup><italic>S5</italic><sup><italic>fl/fl</italic></sup> mESCs</p><p>(A) Growth of <italic>S1</italic><sup><italic>fl/fl</italic></sup><italic>S5</italic><sup><italic>fl/fl</italic></sup> mESCs and three independent <italic>S1</italic><sup><italic>−/−</italic></sup><italic>S5</italic><sup><italic>−/−</italic></sup> mESCs lines in 2i during 26 days. Means ± SD are depicted.</p><p>(B) Alkaline phosphatase activity in 2i <italic>S1</italic><sup><italic>fl/fl</italic></sup><italic>S5</italic><sup><italic>fl/fl</italic></sup> and <italic>S1</italic><sup><italic>−/−</italic></sup><italic>S5</italic><sup><italic>−/−</italic></sup> mESC. Scale bars represent 100 μm.</p><p>(C) Expression of <italic>Sox2</italic>, <italic>Zfp42</italic>, <italic>Nanog</italic>, and <italic>Pou5f1</italic> in transcripts per million (TPM) in 2i <italic>S1</italic><sup><italic>fl/fl</italic></sup><italic>S5</italic><sup><italic>fl/fl</italic></sup> (FL) and <italic>S1</italic><sup><italic>−/−</italic></sup><italic>S5</italic><sup><italic>−/−</italic></sup> (KO) mESC.</p><p>(D) Volcano plot showing –log10 p values versus log2 fold transcriptional changes between <italic>S1</italic><sup><italic>fl/fl</italic></sup><italic>S5</italic><sup><italic>fl/fl</italic></sup> and <italic>S1</italic><sup><italic>−/−</italic></sup><italic>S5</italic><sup><italic>−/−</italic></sup> mESCs in 2i. DEGs with p < 0.05 are blue, and genes with p > 0.05 are red; some highlighted DEGs are black.</p><p>(E) Percentage of DEGs (p < 0.01) (n = 781 upregulated in 2i <italic>S1</italic><sup><italic>−/−</italic></sup><italic>S5</italic><sup><italic>−/−</italic></sup>; n = 854 downregulated in 2i <italic>S1</italic><sup><italic>−/−</italic></sup><italic>S5</italic><sup><italic>−/−</italic></sup>) showing putative SMAD1/5 binding sites (GGCGCC/GCCG) in the promoter region.</p><p>(F) Top ten GO terms associated with biological processes (p < 0.05) in DEGs in 2i <italic>S1</italic><sup><italic>−/−</italic></sup><italic>S5</italic><sup><italic>−/−</italic></sup> mESCs.</p><p>(G) Distribution of DNA methylation levels at specific genomic regions in 2i <italic>S1</italic><sup><italic>fl/fl</italic></sup><italic>S5</italic><sup><italic>fl/fl</italic></sup> (in red) and <italic>S1</italic><sup><italic>−/−</italic></sup><italic>S5</italic><sup><italic>−/−</italic></sup> mESCs (in blue). p Values were calculated with two-sample Kolmogorov-Smirnov test. HCP, high CpG-content promoters; LCP, low CpG-content promoters; Enh, enhancers; NA, no annotation.</p><p>(H) Number of (600-bp tile) counts showing LOM or GOM in 2i <italic>S1</italic><sup><italic>fl/fl</italic></sup><italic>S5</italic><sup><italic>fl/fl</italic></sup> compared with <italic>S1</italic><sup><italic>−/−</italic></sup><italic>S5</italic><sup><italic>−/−</italic></sup> mESCs.</p><p>(I) Scatterplot depicting a comparison of the percentage of DNA methylation in each 600-bp tile (dot) between 2i <italic>S1</italic><sup><italic>fl/fl</italic></sup><italic>S5</italic><sup><italic>fl/fl</italic></sup> and <italic>S1</italic><sup><italic>−/−</italic></sup><italic>S5</italic><sup><italic>−/−</italic></sup> mESCs. Each tile was classified into a biotype category according to the nearest TSS. The red line represents no difference, and the inner and outer blue lines represent borders for 10% and 20% change in methylation levels, respectively. See also <xref rid="mmc1" ref-type="supplementary-material">Figures S3</xref>, <xref rid="mmc1" ref-type="supplementary-material">S4</xref> and <xref rid="mmc2" ref-type="supplementary-material">Tables S1</xref>, <xref rid="mmc3" ref-type="supplementary-material">S2</xref>, and <xref rid="mmc4" ref-type="supplementary-material">S3</xref>.</p></caption><graphic xlink:href="gr3"></graphic></fig><fig id="fig4"><label>Figure 4</label><caption><p>BMP-SMAD Signaling during mESC Differentiation to Mesendoderm and Neurectoderm</p><p>(A) Schematic representation of the protocol to differentiate mESCs to mesendoderm (3 μM CHIR) or neurectoderm (500 nM retinoic acid [RA]).</p><p>(B) Relative expression of early lineage markers in differentiated serum and 2i <italic>S1</italic><sup><italic>−/−</italic></sup><italic>S5</italic><sup><italic>−/−</italic></sup> and <italic>S1</italic><sup><italic>fl/fl</italic></sup><italic>S5</italic><sup><italic>fl/fl</italic></sup> mESCs.</p><p>(C) Immunofluorescence of differentiated serum <italic>S1</italic><sup><italic>fl/fl</italic></sup><italic>S5</italic><sup><italic>fl/fl</italic></sup> and <italic>S1</italic><sup><italic>−/−</italic></sup><italic>S5</italic><sup><italic>−/−</italic></sup> mESCs for NANOG, SOX17, T, and SOX1. Scale bars represent 100 μm.</p><p>(D) Relative expression of early lineage markers in differentiated subpopulations (GFP−, GFP+, GFP++) of serum <italic>BRE:gfp</italic> mESCs compared with GFP− cells.</p><p>(E) Relative expression of pluripotency genes in differentiated subpopulations (GFP−, GFP+, GFP++) of serum <italic>BRE:gfp</italic> mESCs compared with GFP− cells.</p><p>Each bar represents the mean ± SD of technical triplicates and bars of the same color represent independent experiments (n = 9, N = 3) in (B) and independent experiments (n = 6, N = =2) in (D) and (E). Statistical analysis was performed on technical triplicates of independent experiments (n = 9, N = 3), <sup>∗</sup>p ≤ 0.05, <sup>∗∗</sup>p ≤ 0.01.</p></caption><graphic xlink:href="gr4"></graphic></fig></floats-group></pmc></record>Pour manipuler ce document sous Unix (Dilib)
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