Understanding naïve pluripotency using quantitative mass spectrometry

Zusammenfassung

During embryonic development stem cells progress through different degrees of developmental potential, spanning from naïve at the early stages to primed pluripotency after implantation. Whilst primed stem cells are prone to differentiation, naïve stem cells show higher self-renewal capacity and are considered as the representation of ground state of pluripotency. Dual inhibition of GSK3 and MEK, a combination of drugs known as 2i, can capture in vitro that ground state of pluripotency. Most recently, inhibition of CDK8 (CDK8i), which acts as negative regulator of the Mediator complex in enhancers, also stabilizes this condition. 2i and CDK8i-treated cells form dome-shape colonies, show a homogeneous expression of pluripotency markers and contribute very efficiently to the formation of chimeras. Both treatments activate a similar transcriptional program that resembles the transcriptional profile of pluripotent cells from the preimplantation epiblast. However, the mechanisms responsible for these effects are not fully characterized. Here, we used quantitative mass spectrometry to explore these two transitions from four complementary angles: proteome, metabolome, phosphoproteome and interactome. First, we profiled proteome dynamics comprehensively across seven time points in four different cell lines. These analyses revealed that 2i and CDK8i induce a similar and synchronized proteome response characteristic of the pre-implantation epiblast. Among many others, we found several proteins involved in mitochondrial metabolism consistently up-regulated. To further investigate the implication of this finding, we analyzed the metabolomes of long-term adapted mESCs to 2i and CDK8i and found how their metabolic signatures explain key differences between both treatments, such as epigenetic status. Moreover, given that these events are initiated by kinases inhibition, we sought to delineate the phosphorylation cascades triggered in the early phases of the process and monitored ~14,000 phosphosites within the first 6 hours of treatment. We found that GSK3i/MEKi and CDK8i induce a rapid alteration of phosphorylation networks, mainly affecting pluripotency transcription factors and the transcriptional machinery. Finally, we studied the interactome of key proteins involved in transcriptional control (i.e., Pol II, Mediator and CDK8). As a result, we obtained a comprehensive map of the protein network that interacts with these transcriptional complexes. Our results demonstrate that the stabilization of naïve pluripotency by stimulation of two different routes, proliferation/self-renewal (2i) and enhancer function (CDK8i) undergo similar mechanisms, suggesting that these pathways are highly interconnected in pluripotent cells.