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• W2950402574 abstract "•Multi-omic maps of embryonic stem cells transitioning from naive to primed pluripotency•Phosphoproteome dynamics precede changes to epigenome, transcriptome, and proteome•ERK signaling is dispensable beyond the initial phase of exit from naive pluripotency•Comparative analysis of mouse and human naive and primed pluripotent states Pluripotency is highly dynamic and progresses through a continuum of pluripotent stem cell states. The two states that bookend the pluripotency continuum, naive and primed, are well characterized, but our understanding of the intermediate states and transitions between them remains incomplete. Here, we dissect the dynamics of pluripotent state transitions underlying pre- to post-implantation epiblast differentiation. Through comprehensive mapping of the proteome, phosphoproteome, transcriptome, and epigenome of embryonic stem cells transitioning from naive to primed pluripotency, we find that rapid, acute, and widespread changes to the phosphoproteome precede ordered changes to the epigenome, transcriptome, and proteome. Reconstruction of the kinase-substrate networks reveals signaling cascades, dynamics, and crosstalk. Distinct waves of global proteomic changes mark discrete phases of pluripotency, with cell-state-specific surface markers tracking pluripotent state transitions. Our data provide new insights into multi-layered control of the phased progression of pluripotency and a foundation for modeling mechanisms regulating pluripotent state transitions (www.stemcellatlas.org). Pluripotency is highly dynamic and progresses through a continuum of pluripotent stem cell states. The two states that bookend the pluripotency continuum, naive and primed, are well characterized, but our understanding of the intermediate states and transitions between them remains incomplete. Here, we dissect the dynamics of pluripotent state transitions underlying pre- to post-implantation epiblast differentiation. Through comprehensive mapping of the proteome, phosphoproteome, transcriptome, and epigenome of embryonic stem cells transitioning from naive to primed pluripotency, we find that rapid, acute, and widespread changes to the phosphoproteome precede ordered changes to the epigenome, transcriptome, and proteome. Reconstruction of the kinase-substrate networks reveals signaling cascades, dynamics, and crosstalk. Distinct waves of global proteomic changes mark discrete phases of pluripotency, with cell-state-specific surface markers tracking pluripotent state transitions. Our data provide new insights into multi-layered control of the phased progression of pluripotency and a foundation for modeling mechanisms regulating pluripotent state transitions (www.stemcellatlas.org). Pluripotency describes the developmental potential of a cell to give rise to derivatives of all three primary germ layers. Although pluripotency is ephemeral in vivo, pluripotent stem cells (PSCs), derived from various stages of early embryonic development, can self-renew indefinitely in vitro under defined culture conditions while retaining their pluripotent status (Nichols and Smith, 2009Nichols J. Smith A. Naive and primed pluripotent states.Cell Stem Cell. 2009; 4: 487-492Abstract Full Text Full Text PDF PubMed Scopus (880) Google Scholar). Studies of the early mouse embryo and PSCs in culture have led to the proposition that embryonic pluripotency is highly dynamic and proceeds through a continuum of pluripotent stem cell states (De Los Angeles et al., 2015De Los Angeles A. Ferrari F. Xi R. Fujiwara Y. Benvenisty N. Deng H. Hochedlinger K. Jaenisch R. Lee S. Leitch H.G. et al.Hallmarks of pluripotency.Nature. 2015; 525: 469-478Crossref PubMed Scopus (143) Google Scholar, Hackett and Surani, 2014Hackett J.A. Surani M.A. Regulatory principles of pluripotency: from the ground state up.Cell Stem Cell. 2014; 15: 416-430Abstract Full Text Full Text PDF PubMed Scopus (142) Google Scholar, Nichols and Smith, 2009Nichols J. Smith A. Naive and primed pluripotent states.Cell Stem Cell. 2009; 4: 487-492Abstract Full Text Full Text PDF PubMed Scopus (880) Google Scholar, Rossant and Tam, 2017Rossant J. Tam P.P.L. New insights into early human development: lessons for stem cell derivation and differentiation.Cell Stem Cell. 2017; 20: 18-28Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar, Shahbazi et al., 2017Shahbazi M.N. Scialdone A. Skorupska N. Weberling A. Recher G. Zhu M. Jedrusik A. Devito L.G. Noli L. Macaulay I.C. et al.Pluripotent state transitions coordinate morphogenesis in mouse and human embryos.Nature. 2017; 552: 239-243Crossref PubMed Scopus (23) Google Scholar, Weinberger et al., 2016Weinberger L. Ayyash M. Novershtern N. Hanna J.H. Dynamic stem cell states: naive to primed pluripotency in rodents and humans.Nat. Rev. Mol. Cell Biol. 2016; 17: 155-169Crossref PubMed Scopus (164) Google Scholar, Wu and Izpisua Belmonte, 2015Wu J. Izpisua Belmonte J.C. Dynamic pluripotent stem cell states and their applications.Cell Stem Cell. 2015; 17: 509-525Abstract Full Text Full Text PDF PubMed Google Scholar). At one end of this continuum is the naive pluripotent state (Nichols and Smith, 2009Nichols J. Smith A. Naive and primed pluripotent states.Cell Stem Cell. 2009; 4: 487-492Abstract Full Text Full Text PDF PubMed Scopus (880) Google Scholar), sometimes also referred to as the ground state (Hackett and Surani, 2014Hackett J.A. Surani M.A. Regulatory principles of pluripotency: from the ground state up.Cell Stem Cell. 2014; 15: 416-430Abstract Full Text Full Text PDF PubMed Scopus (142) Google Scholar, Marks et al., 2012Marks H. Kalkan T. Menafra R. Denissov S. Jones K. Hofemeister H. Nichols J. Kranz A. Stewart A.F. Smith A. et al.The transcriptional and epigenomic foundations of ground state pluripotency.Cell. 2012; 149: 590-604Abstract Full Text Full Text PDF PubMed Scopus (471) Google Scholar, Ying et al., 2008Ying Q.L. Wray J. Nichols J. Batlle-Morera L. Doble B. Woodgett J. Cohen P. Smith A. The ground state of embryonic stem cell self-renewal.Nature. 2008; 453: 519-523Crossref PubMed Scopus (1909) Google Scholar), representing the most unrestricted developmental potential that exists in the pre-implantation mouse embryo from approximately embryonic day 3.75 (E3.75) to E4.75 (Boroviak et al., 2014Boroviak T. Loos R. Bertone P. Smith A. Nichols J. The ability of inner-cell-mass cells to self-renew as embryonic stem cells is acquired following epiblast specification.Nat. Cell Biol. 2014; 16: 516-528Crossref PubMed Scopus (187) Google Scholar). At the other end of this continuum is the primed pluripotent state, representing pluripotent cells from post-implantation mouse epiblasts (E5.5–E8.25), which are lineage primed for differentiation. Embryonic stem cells (ESCs), derived from the inner cell mass (ICM) of pre-implantation mouse blastocysts (Figure 1A) and maintained under defined culture conditions known as 2i+LIF (Ying et al., 2008Ying Q.L. Wray J. Nichols J. Batlle-Morera L. Doble B. Woodgett J. Cohen P. Smith A. The ground state of embryonic stem cell self-renewal.Nature. 2008; 453: 519-523Crossref PubMed Scopus (1909) Google Scholar), most closely resemble naive epiblasts of the pre-implantation embryo (Boroviak et al., 2014Boroviak T. Loos R. Bertone P. Smith A. Nichols J. The ability of inner-cell-mass cells to self-renew as embryonic stem cells is acquired following epiblast specification.Nat. Cell Biol. 2014; 16: 516-528Crossref PubMed Scopus (187) Google Scholar, 2015). Hence, ESCs are considered to capture the naive pluripotent state. Epiblast stem cells (EpiSCs), isolated from pre-gastrulation (E5.5) to late-bud (E8.25) stage post-implantation mouse epiblasts (Brons et al., 2007Brons I.G. Smithers L.E. Trotter M.W. Rugg-Gunn P. Sun B. Chuva de Sousa Lopes S.M. Howlett S.K. Clarkson A. Ahrlund-Richter L. Pedersen R.A. et al.Derivation of pluripotent epiblast stem cells from mammalian embryos.Nature. 2007; 448: 191-195Crossref PubMed Scopus (1229) Google Scholar, Tesar et al., 2007Tesar P.J. Chenoweth J.G. Brook F.A. Davies T.J. Evans E.P. Mack D.L. Gardner R.L. McKay R.D.G. New cell lines from mouse epiblast share defining features with human embryonic stem cells.Nature. 2007; 448: 196-199Crossref PubMed Scopus (1364) Google Scholar) (Figure 1A), are developmentally comparable to the late-gastrulation stage (E7.0) embryo, irrespective of the original developmental stage (E5.5–E8.25) of their source tissue (Kojima et al., 2014Kojima Y. Kaufman-Francis K. Studdert J.B. Steiner K.A. Power M.D. Loebel D.A. Jones V. Hor A. de Alencastro G. Logan G.J. et al.The transcriptional and functional properties of mouse epiblast stem cells resemble the anterior primitive streak.Cell Stem Cell. 2014; 14: 107-120Abstract Full Text Full Text PDF PubMed Scopus (124) Google Scholar); these cells are considered an archetypal representative of the primed pluripotent state. Interestingly, conventional human ESCs (hESCs), derived from pre-implantation human blastocysts, exhibit molecular and morphological characteristics that are more similar to primed EpiSCs than to naive ESCs (Davidson et al., 2015Davidson K.C. Mason E.A. Pera M.F. The pluripotent state in mouse and human.Development. 2015; 142: 3090-3099Crossref PubMed Scopus (53) Google Scholar, De Los Angeles et al., 2015De Los Angeles A. Ferrari F. Xi R. Fujiwara Y. Benvenisty N. Deng H. Hochedlinger K. Jaenisch R. Lee S. Leitch H.G. et al.Hallmarks of pluripotency.Nature. 2015; 525: 469-478Crossref PubMed Scopus (143) Google Scholar, Hackett and Surani, 2014Hackett J.A. Surani M.A. Regulatory principles of pluripotency: from the ground state up.Cell Stem Cell. 2014; 15: 416-430Abstract Full Text Full Text PDF PubMed Scopus (142) Google Scholar, Rossant and Tam, 2017Rossant J. Tam P.P.L. New insights into early human development: lessons for stem cell derivation and differentiation.Cell Stem Cell. 2017; 20: 18-28Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar, Weinberger et al., 2016Weinberger L. Ayyash M. Novershtern N. Hanna J.H. Dynamic stem cell states: naive to primed pluripotency in rodents and humans.Nat. Rev. Mol. Cell Biol. 2016; 17: 155-169Crossref PubMed Scopus (164) Google Scholar, Wu and Izpisua Belmonte, 2015Wu J. Izpisua Belmonte J.C. Dynamic pluripotent stem cell states and their applications.Cell Stem Cell. 2015; 17: 509-525Abstract Full Text Full Text PDF PubMed Google Scholar). Several protocols that reprogram hESCs back to the ground state have been proposed (Chan et al., 2013Chan Y.S. Göke J. Ng J.H. Lu X. Gonzales K.A. Tan C.P. Tng W.Q. Hong Z.Z. Lim Y.S. Ng H.H. Induction of a human pluripotent state with distinct regulatory circuitry that resembles preimplantation epiblast.Cell Stem Cell. 2013; 13: 663-675Abstract Full Text Full Text PDF PubMed Scopus (203) Google Scholar, Gafni et al., 2013Gafni O. Weinberger L. Mansour A.A. Manor Y.S. Chomsky E. Ben-Yosef D. Kalma Y. Viukov S. Maza I. Zviran A. et al.Derivation of novel human ground state naive pluripotent stem cells.Nature. 2013; 504: 282-286Crossref PubMed Scopus (511) Google Scholar, Takashima et al., 2014Takashima Y. Guo G. Loos R. Nichols J. Ficz G. Krueger F. Oxley D. Santos F. Clarke J. Mansfield W. et al.Resetting transcription factor control circuitry toward ground-state pluripotency in human.Cell. 2014; 158: 1254-1269Abstract Full Text Full Text PDF PubMed Scopus (0) Google Scholar, Theunissen et al., 2014Theunissen T.W. Powell B.E. Wang H. Mitalipova M. Faddah D.A. Reddy J. Fan Z.P. Maetzel D. Ganz K. Shi L. et al.Systematic identification of culture conditions for induction and maintenance of naive human pluripotency.Cell Stem Cell. 2014; 15: 471-487Abstract Full Text Full Text PDF PubMed Google Scholar, Ware et al., 2014Ware C.B. Nelson A.M. Mecham B. Hesson J. Zhou W. Jonlin E.C. Jimenez-Caliani A.J. Deng X. Cavanaugh C. Cook S. et al.Derivation of naive human embryonic stem cells.Proc. Natl. Acad. Sci. USA. 2014; 111: 4484-4489Crossref PubMed Scopus (234) Google Scholar), but they each generate “naive” hESCs with distinct transcriptional profiles (Davidson et al., 2015Davidson K.C. Mason E.A. Pera M.F. The pluripotent state in mouse and human.Development. 2015; 142: 3090-3099Crossref PubMed Scopus (53) Google Scholar, Huang et al., 2014Huang K. Maruyama T. Fan G. The naive state of human pluripotent stem cells: a synthesis of stem cell and preimplantation embryo transcriptome analyses.Cell Stem Cell. 2014; 15: 410-415Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar) and fail to recover the naive epiblast methylation landscape (Pastor et al., 2016Pastor W.A. Chen D. Liu W. Kim R. Sahakyan A. Lukianchikov A. Plath K. Jacobsen S.E. Clark A.T. Naive human pluripotent cells feature a methylation landscape devoid of blastocyst or germline memory.Cell Stem Cell. 2016; 18: 323-329Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar). Although these purported naive hESCs satisfy some features of mouse criteria for the naive pluripotent state, whether they can be considered equivalent to naive mouse ESCs remains an open question. ESCs are highly competent to form high-contribution mouse chimeras with germline transmission, following microinjection into pre-implantation embryos. EpiSCs, however, do not integrate well into host blastocysts, likely because they correspond to a developmentally advanced stage compared to the host pre-implantation environment and thus, contribute poorly or not at all to blastocyst chimeras (Dejosez and Zwaka, 2012Dejosez M. Zwaka T.P. Pluripotency and nuclear reprogramming.Annu. Rev. Biochem. 2012; 81: 737-765Crossref PubMed Scopus (28) Google Scholar, Hackett and Surani, 2014Hackett J.A. Surani M.A. Regulatory principles of pluripotency: from the ground state up.Cell Stem Cell. 2014; 15: 416-430Abstract Full Text Full Text PDF PubMed Scopus (142) Google Scholar, Han et al., 2010Han D.W. Tapia N. Joo J.Y. Greber B. Araúzo-Bravo M.J. Bernemann C. Ko K. Wu G. Stehling M. Do J.T. et al.Epiblast stem cell subpopulations represent mouse embryos of distinct pregastrulation stages.Cell. 2010; 143: 617-627Abstract Full Text Full Text PDF PubMed Scopus (143) Google Scholar, Nichols and Smith, 2009Nichols J. Smith A. Naive and primed pluripotent states.Cell Stem Cell. 2009; 4: 487-492Abstract Full Text Full Text PDF PubMed Scopus (880) Google Scholar, Weinberger et al., 2016Weinberger L. Ayyash M. Novershtern N. Hanna J.H. Dynamic stem cell states: naive to primed pluripotency in rodents and humans.Nat. Rev. Mol. Cell Biol. 2016; 17: 155-169Crossref PubMed Scopus (164) Google Scholar, Wu and Izpisua Belmonte, 2015Wu J. Izpisua Belmonte J.C. Dynamic pluripotent stem cell states and their applications.Cell Stem Cell. 2015; 17: 509-525Abstract Full Text Full Text PDF PubMed Google Scholar). Conversely, when grafted into post-implantation (E7.5) embryos in whole-embryo culture, EpiSCs but not ESCs efficiently incorporate into the host and contribute to all three germ layers (Huang et al., 2012Huang Y. Osorno R. Tsakiridis A. Wilson V. In vivo differentiation potential of epiblast stem cells revealed by chimeric embryo formation.Cell Rep. 2012; 2: 1571-1578Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar). Consequently, primed EpiSCs are considered to be functionally and developmentally distinct from naive epiblasts and ESCs (De Los Angeles et al., 2015De Los Angeles A. Ferrari F. Xi R. Fujiwara Y. Benvenisty N. Deng H. Hochedlinger K. Jaenisch R. Lee S. Leitch H.G. et al.Hallmarks of pluripotency.Nature. 2015; 525: 469-478Crossref PubMed Scopus (143) Google Scholar, Weinberger et al., 2016Weinberger L. Ayyash M. Novershtern N. Hanna J.H. Dynamic stem cell states: naive to primed pluripotency in rodents and humans.Nat. Rev. Mol. Cell Biol. 2016; 17: 155-169Crossref PubMed Scopus (164) Google Scholar). While the naive and primed states, which bookend the pluripotency continuum, are well characterized (Kojima et al., 2014Kojima Y. Kaufman-Francis K. Studdert J.B. Steiner K.A. Power M.D. Loebel D.A. Jones V. Hor A. de Alencastro G. Logan G.J. et al.The transcriptional and functional properties of mouse epiblast stem cells resemble the anterior primitive streak.Cell Stem Cell. 2014; 14: 107-120Abstract Full Text Full Text PDF PubMed Scopus (124) Google Scholar, Marks et al., 2012Marks H. Kalkan T. Menafra R. Denissov S. Jones K. Hofemeister H. Nichols J. Kranz A. Stewart A.F. Smith A. et al.The transcriptional and epigenomic foundations of ground state pluripotency.Cell. 2012; 149: 590-604Abstract Full Text Full Text PDF PubMed Scopus (471) Google Scholar), our understanding of the intermediate pluripotent states and the transitions between them remains incomplete. Cell signaling underlies transcriptional and/or epigenetic control of a vast majority of cell fate decisions during early embryonic development (Dejosez and Zwaka, 2012Dejosez M. Zwaka T.P. Pluripotency and nuclear reprogramming.Annu. Rev. Biochem. 2012; 81: 737-765Crossref PubMed Scopus (28) Google Scholar, Hackett and Surani, 2014Hackett J.A. Surani M.A. Regulatory principles of pluripotency: from the ground state up.Cell Stem Cell. 2014; 15: 416-430Abstract Full Text Full Text PDF PubMed Scopus (142) Google Scholar, Rossant and Tam, 2017Rossant J. Tam P.P.L. New insights into early human development: lessons for stem cell derivation and differentiation.Cell Stem Cell. 2017; 20: 18-28Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar, Weinberger et al., 2016Weinberger L. Ayyash M. Novershtern N. Hanna J.H. Dynamic stem cell states: naive to primed pluripotency in rodents and humans.Nat. Rev. Mol. Cell Biol. 2016; 17: 155-169Crossref PubMed Scopus (164) Google Scholar). Yet, our understanding of the signaling dynamics during pluripotent state transitions and how they instruct epigenetic and/or transcriptional programs controlling ICM to post-implantation epiblast differentiation remains poorly understood. Recent advances in mass-spectrometry (MS)-based proteomics now allow for near-comprehensive characterization of proteomes (Aebersold and Mann, 2016Aebersold R. Mann M. Mass-spectrometric exploration of proteome structure and function.Nature. 2016; 537: 347-355Crossref PubMed Scopus (754) Google Scholar) and deep phosphoproteome coverage (Needham et al., 2019Needham E.J. Parker B.L. Burykin T. James D.E. Humphrey S.J. Illuminating the dark phosphoproteome.Sci. Signal. 2019; 12Crossref PubMed Scopus (3) Google Scholar). To elucidate the signaling and molecular dynamics that underlie pluripotent state transitions, here we generated comprehensive high-temporal-resolution maps of the phosphoproteome, proteome, transcriptome, and epigenome of ESCs transitioning from naive to primed pluripotency. Our data provide new insights into the multi-layered control of the phased progression of pluripotency and a foundation for investigating mechanisms underlying ICM to post-implantation epiblast differentiation. To elucidate the temporal dynamics of the phosphoproteome, proteome, epigenome, and transcriptome during the transition from naive to primed pluripotency, we employed a previously validated system to induce naive mouse ESCs to post-implantation pre-gastrulating epiblast-like cells (EpiLCs) (Buecker et al., 2014Buecker C. Srinivasan R. Wu Z. Calo E. Acampora D. Faial T. Simeone A. Tan M. Swigut T. Wysocka J. Reorganization of enhancer patterns in transition from naive to primed pluripotency.Cell Stem Cell. 2014; 14: 838-853Abstract Full Text Full Text PDF PubMed Scopus (178) Google Scholar, Hayashi et al., 2011Hayashi K. Ohta H. Kurimoto K. Aramaki S. Saitou M. Reconstitution of the mouse germ cell specification pathway in culture by pluripotent stem cells.Cell. 2011; 146: 519-532Abstract Full Text Full Text PDF PubMed Scopus (570) Google Scholar, Kurimoto et al., 2015Kurimoto K. Yabuta Y. Hayashi K. Ohta H. Kiyonari H. Mitani T. Moritoki Y. Kohri K. Kimura H. Yamamoto T. et al.Quantitative dynamics of chromatin remodeling during germ cell specification from mouse embryonic stem cells.Cell Stem Cell. 2015; 16: 517-532Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar, Shirane et al., 2016Shirane K. Kurimoto K. Yabuta Y. Yamaji M. Satoh J. Ito S. Watanabe A. Hayashi K. Saitou M. Sasaki H. Global landscape and regulatory principles of DNA methylation reprogramming for germ cell specification by mouse pluripotent stem cells.Dev. Cell. 2016; 39: 87-103Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar), which more closely resemble the early post-implantation epiblast (E5.5–E6.5) compared to EpiSCs (Hayashi et al., 2011Hayashi K. Ohta H. Kurimoto K. Aramaki S. Saitou M. Reconstitution of the mouse germ cell specification pathway in culture by pluripotent stem cells.Cell. 2011; 146: 519-532Abstract Full Text Full Text PDF PubMed Scopus (570) Google Scholar). EpiLCs were induced by plating naive ESCs, grown in the ground state under serum-free 2i+LIF medium, onto fibronectin-coated plates in N2B27 medium containing activin A, bFGF, and knockout serum replacement (KOSR, 1%) (Hayashi et al., 2011Hayashi K. Ohta H. Kurimoto K. Aramaki S. Saitou M. Reconstitution of the mouse germ cell specification pathway in culture by pluripotent stem cells.Cell. 2011; 146: 519-532Abstract Full Text Full Text PDF PubMed Scopus (570) Google Scholar). Consistent with previous reports, within 48 h of EpiLC induction, morphological transformation in the form of flattened epithelial structures resembling epiblasts was evident (Figure S1A). RNA analysis using quantitative RT-PCR confirmed the downregulation of naive pluripotency- and/or ICM-associated genes (Nanog, Klf4, and Prdm14) accompanied by the induction of post-implantation epiblast-associated genes (Fgf5, Otx2, and Pou3f1/Oct6) (Figure S1B) (Buecker et al., 2014Buecker C. Srinivasan R. Wu Z. Calo E. Acampora D. Faial T. Simeone A. Tan M. Swigut T. Wysocka J. Reorganization of enhancer patterns in transition from naive to primed pluripotency.Cell Stem Cell. 2014; 14: 838-853Abstract Full Text Full Text PDF PubMed Scopus (178) Google Scholar, Hayashi et al., 2011Hayashi K. Ohta H. Kurimoto K. Aramaki S. Saitou M. Reconstitution of the mouse germ cell specification pathway in culture by pluripotent stem cells.Cell. 2011; 146: 519-532Abstract Full Text Full Text PDF PubMed Scopus (570) Google Scholar, Kalkan et al., 2017Kalkan T. Olova N. Roode M. Mulas C. Lee H.J. Nett I. Marks H. Walker R. Stunnenberg H.G. Lilley K.S. et al.Tracking the embryonic stem cell transition from ground state pluripotency.Development. 2017; 144: 1221-1234Crossref PubMed Scopus (54) Google Scholar). Although no dramatic changes in transcript levels of these marker genes were evident after 48 h post induction, we included the 72-h time point in our analyses to capture changes to the proteome that may lag changes to the transcriptome. Using advances in MS-based proteomics (Kulak et al., 2014Kulak N.A. Pichler G. Paron I. Nagaraj N. Mann M. Minimal, encapsulated proteomic-sample processing applied to copy-number estimation in eukaryotic cells.Nat. Methods. 2014; 11: 319-324Crossref PubMed Scopus (486) Google Scholar) and our EasyPhos workflow (Humphrey et al., 2015Humphrey S.J. Azimifar S.B. Mann M. High-throughput phosphoproteomics reveals in vivo insulin signaling dynamics.Nat. Biotechnol. 2015; 33: 990-995Crossref PubMed Google Scholar, Humphrey et al., 2018Humphrey S.J. Karayel O. James D.E. Mann M. High-throughput and high-sensitivity phosphoproteomics with the EasyPhos platform.Nat. Protoc. 2018; 13: 1897-1916Crossref PubMed Scopus (2) Google Scholar), together with next-generation sequencing, we generated maps of the phosphoproteome, proteome, transcriptome, and epigenome of cells at various time points during the 72-h ESC to EpiLC transition (Figure 1B; http://www.stemcellatlas.org). To capture the earliest signaling responses, we profiled the phosphoproteome of transitioning cells at high temporal resolution within the first hour post induction (Figure 1B). All MS experiments were performed in biological quadruplicates. In addition, to enhance coverage of the proteome measurements, we pooled the four biological replicates from each time point and performed StageTip-based strong cation exchange (SCX) fractionation (Ishihama et al., 2006Ishihama Y. Rappsilber J. Mann M. Modular stop and go extraction tips with stacked disks for parallel and multidimensional peptide fractionation in proteomics.J. Proteome Res. 2006; 5: 988-994Crossref PubMed Scopus (173) Google Scholar) of this pooled sample for the proteome runs (Figures 1C, S2A, and S2B). All MS data were analyzed using the MaxQuant computational platform (Cox and Mann, 2008Cox J. Mann M. MaxQuant enables high peptide identification rates, individualized p.p.b.-range mass accuracies and proteome-wide protein quantification.Nat. Biotechnol. 2008; 26: 1367-1372Crossref PubMed Scopus (5038) Google Scholar, Tyanova et al., 2016Tyanova S. Temu T. Cox J. The MaxQuant computational platform for mass spectrometry-based shotgun proteomics.Nat. Protoc. 2016; 11: 2301-2319Crossref PubMed Scopus (280) Google Scholar). Our single-run EasyPhos workflow produced excellent phosphopeptide coverage, quantifying over 15,000 phosphopeptides in every run (Figure S2C). This yielded a total of 30,726 distinct phosphopeptides from which we identified 37,619 individual phosphorylation sites (Figure 1D). Phosphosite localization confidence was high, with >80% (26,180) of the quantified phosphosites accurately localized to a single amino acid (mean localization probability for quantified sites: 0.96) (Figure S2D; STAR Methods). A total of 17,866 phosphosites and over half of the class 1 phosphosites (14,103) were quantified across all 12 time points analyzed (Figure 1D; Table S1). From our proteome runs, we identified over 160,000 distinct peptides and quantified a grand total of 10,597 proteins across all samples and 9,250 proteins in every sample (Figure 1D). Quantification coverage at the proteome level was also very high, with 9,250 proteins quantified across all profiled time points (Figure 1D; Table S2). Using paired-end RNA sequencing (RNA-seq), we mapped the transcriptome across 8 time points during the 72-h time course and detected a total of 16,734 transcripts (reads per kilobase of transcript, per million mapped reads [RPKM] > 1) corresponding to 13,600 unique genes (Figures 1D and S2E; Table S3). Chromatin immunoprecipitation sequencing (ChIP-seq) analyses of the chromatin, collected from the same 8 time points, using antibodies against common histone modifications (H3K4me1, H3K4me3, and H3K27ac: associated with the promoters of transcriptionally active genes; H3K27me3 and H3K9me2: associated with the promoters of silent genes) and RNA polymerase II (RNAPII) identified several thousand transcriptionally active and/or poised genes (Figures 1D and S2F; Table S4). Principal-component analysis (PCA) and unsupervised hierarchical clustering of the transcriptome, proteome, phosphoproteome, or epigenome revealed clear time-dependent separation of the data (Figures 2A–2C and S3), with global changes to the phosphoproteome evident as early as 5 min post induction (Figure 2C), suggesting that the clustering is driven largely by differences in the underlying biological signal across various time points. PCA analysis of our transcriptomic data, in conjunction with the recently published RNA-seq data obtained from ESCs transitioning out of naive ground-state pluripotency (0 h, 16 h, 25 h-Rex1high, and 25 h-Rex1low) (Kalkan et al., 2017Kalkan T. Olova N. Roode M. Mulas C. Lee H.J. Nett I. Marks H. Walker R. Stunnenberg H.G. Lilley K.S. et al.Tracking the embryonic stem cell transition from ground state pluripotency.Development. 2017; 144: 1221-1234Crossref PubMed Scopus (54) Google Scholar), revealed temporal concordance of the datasets from the two studies (Figure 2A), suggesting that the biological signal driving these temporal clusters is highly reproducible. The transcriptome at 24 h post EpiLC induction clustered with those from 16-h- and 25-h-Rex1high cells (Figure 2A), with the latter previously shown to be in a reversible phase preceding extinction of the naive state (Kalkan et al., 2017Kalkan T. Olova N. Roode M. Mulas C. Lee H.J. Nett I. Marks H. Walker R. Stunnenberg H.G. Lilley K.S. et al.Tracking the embryonic stem cell transition from ground state pluripotency.Development. 2017; 144: 1221-1234Crossref PubMed Scopus (54) Google Scholar). Consistent with 25-h-Rex1high cells, Rex1 (Zfp42) expression in cells at 24 h post EpiLC-induction remained high at the mRNA and protein level (Tables S2 and S3). In contrast, the transcriptome at the 36-h time point during ESC to EpiLC transition clustered with that of the 25-h-Rex1low cells, the primary products of exit from naive pluripotency (Kalkan et al., 2017Kalkan T. Olova N. Roode M. Mulas C. Lee H.J. Nett I. Marks H. Walker R. Stunnenberg H.G. Lilley K.S. et al.Tracking the embryonic stem cell transition from ground state pluripotency.Development. 2017; 144: 1221-1234Crossref PubMed Scopus (54) Google Scholar). Consistent with 25-h-Rex1low cells that exited the naive ground state, Rex1 expression was downregulated by ∼10-fold at 36 h post EpiLC induction (Table S3). Collectively, these data suggest that by about 36 h post induction, cells had exited the naive pluripotent state. To understand the sequence of molecular events and the temporal kinetics that transform cellular identity, we next examined the timing, scale, and magnitude of changes to the proteome, phosphoproteome, transcriptome, and epigenome as ESCs transition through various phases of pluripotency. Our analyses revealed that phosphoproteome dynamics precede ordered waves of epigenomic, transcriptomic, and proteomic changes (Figures 2D and S4). Notably, abo" @default.
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• W2950402574 date "2019-05-01" @default.
• W2950402574 modified "2023-10-10" @default.
• W2950402574 title "Multi-omic Profiling Reveals Dynamics of the Phased Progression of Pluripotency" @default.
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