Project description:Mouse embryonic stem (ES) cells are locked into self-renewal by shielding from inductive cues. Release from this ground state in minimal conditions offers a system for delineating developmental progression from naive pluripotency. Here we examined the initial transition process. The ES cell population behaves asynchronously. We therefore exploited a short-half-life Rex1::GFP reporter to isolate cells either side of exit from naive status. Differentiation of Rex1-GFPd2 ES cells was initiated by withdrawing 2i (Kalkan et al., 2016). Undifferentiated 2i-cells and post-2i withdrawal differentiating populations (16h, 25h-Rex1-High, 25h-Rex1-Low) were subjected to proteomic analysis by Mass Spectrometry.
Project description:Pluripotency is highly dynamic and progresses through a continuum of pluripotent stem-cell states. The two states that bookend the pluripotency continuum, naïve and primed, are well characterized, but our understanding of the intermediate states and transitions between them remain 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 naïve 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 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 the multi-layered control of the phased progression of pluripotency and a foundation for modeling mechanisms regulating pluripotent state transitions (www.stemcellatlasorg).
Project description:Translational control plays a central role in regulation of gene expression and can lead to significant divergence between mRNA- and protein-abundance. The translational landscape of early mammalian development and its impact on cellular proteome, however, remains largely un-explored. Here we used genome-wide approaches combined with time-course analysis to measure the mRNA-abundance, mRNA-translation rate and protein expression during the transition of naïve into primed embryonic stem cells (ESCs). We found that the ground state ESCs cultured with GSK3- and MEK-inhibitors and LIF (2iL) display higher ribosome density on a selective set of mRNAs. These mRNAs show reduced translation during the exit from ground state pluripotency and transition to serum/LIF (SL) culture or upon commitment to primed epiblast-like stem cells (EpiLSCs). Strikingly, integrative analysis with cellular proteome indicate a strong translational buffering of this set of mRNAs in 2iL-ESCs leading to stable protein expression levels. Our data reveal that the global alteration of cellular proteome is largely accompanied by transcriptional rewiring. Furthermore, we identified a set of genes (including UHRF1 and KRAS) that undergo selective post-translational regulation during the transition of naïve into primed pluripotency and linked the observed changes to upstream GSK- and MEK/MAPK-signaling pathways using single inhibitor treated ESCs. Thus, we provide a comprehensive and detailed overview of the global changes in gene expression during the transition of naïve to primed pluripotency and dissect the relative contributions of RNA-transcription, translation and regulation of protein stability in controlling protein abundance.