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Abstract

Mammalian cell-fate transitions are fundamentally important processes shaping evolution and development. In vitro differentiation and reprogramming of pluripotent stem cells are valuable models to study these processes. Until recently, interrogating the protein regulatory networks involved in cell-fate transitions was hampered by technological limitations. Using the latest mass-spectrometry-based technologies available to date, combined with innovative biochemistry, this thesis presents three projects exploring the proteome rearrangements which occur during neuronal differentiation and reprogramming.

First, we characterized the global proteome dynamics during neuronal differentiation of embryonic stem cells (ESCs) and identified a co-expression protein cluster with high functional enrichment for neurogenic processes. A predictive bioinformatic analysis pointed out Sox2 as a top regulator of this protein group. Like most transcription factors, Sox2 forms complexes with other proteins which influence its target selection. We interrogated the chromatin-associated protein interactome of Sox2 at the beginning and the end of differentiation and found that it undergoes a remarkable stem cell- to neuronal transition. Integrative multi-omic analysis of our interactome data with transcriptomic and chromatin accessibility assays suggest that the joint genome association of Sox2 and selected interactors has a regulatory effect on hundreds of genes involved in embryonic development. Interestingly, this effect can be activating or repressing dependent on the differentiation stage.

The second study explores the potential of epigenetic memory and its proteomic manifestation in the context of induced pluripotency. We demonstrate that unlike primary neurons, neuronal cultures derived from induced pluripotent stem cells (iPSCs) retain the capacity to reprogram back to a pluripotent state. To interrogate the potential proteomic manifestation of epigenetic memory, we compared the initial iPSCs to the neuron-derived iPSCs and found that while their overall proteome composition is highly similar, the neuron-derived iPSCs retain distinct neuronal signatures in addition to the pluripotent ones. We further investigated the spatio-temporal progression of neuronal differentiation within embryoid bodies. As expected, we found that many more proteins change in the embryoid body rim, which is exposed to differentiation-inducing signals compared to the core, which is protected and retains pluripotent proteomic characteristics until the late differentiation stages. Surprisingly, however, we found that key epigenetic and developmental switches involved in pluripotency exit are initiated very early in the embryoid body core, suggesting very fast and efficient cross-communication between the cells layers in these spheroid structures.

Finally, we examined the protein regulatory network associated with the promoter of c-Myc, a gene critically involved with differentiation, development, pluripotency establishment and maintenance. Using a novel technique developed in our lab, we successfully isolated the c-Myc promoter on a single-locus level and were able to characterize the proteins associated with it. These include known c-Myc regulators, as well as many novel candidates. Our study provides a unique and valuable foundation for functional analysis of potential new c-Myc regulators.

In sum, we employed novel biochemical and mass-spectrometry based techniques in different cellular context and successfully expanded the protein regulatory networks driving differentiation and reprogramming.