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SMAD2/3 - FOXH1 regulation during cell cycle progression upon differentiation 10.17863/CAM.93362 Perrin, Marion Vallier, Ludovic Throughout development, balance between differentiation and proliferation is key to ensure the proper induction of the initial layout of the future body plan and to later produce the correct amounts of each cell type. While the characteristics of cell cycle regulation in pluripotent and differentiated cells have been well described, the way they coordinate cell fate acquisition is only beginning to be elucidated. Indeed, the activity of TGFβ signalling via its downstream effector SMAD2/3 is directed by cell cycle regulators and especially cyclin Ds. Indeed, cyclin Ds direct cell fate propensity either by activating CDK4/6 activity, which results in blocking SMAD2/3 nuclear entry or by directly binding to developmental genes and co-recruiting epigenetic modifiers. Despite the importance of these interplay, the molecular mechanisms orchestrating transcriptional networks during progression of the cell cycle upon differentiation remain to be fully uncovered. In this dissertation, I combined cell cycle synchronisation mediated by nocodazole with our well-established endoderm differentiation protocol to study the coordination between cell cycle and differentiation. This approach first revealed that the transition from pluripotency to definitive endoderm requires two cell cycles. I then focused on the forkhead transcription factor FOXH1, a well-known SMAD2/3 binding partner during endoderm formation. Attempts to generate a human FOXH1 knockout stem cell line revealed that FOXH1 is essential for selfrenewal. Alternatively, its essential role in endoderm but not mesendoderm formation was confirmed with an inducible knockdown system against FOXH1. Going further, I showed that FOXH1 has a unique role in the first cell cycle which is required to form definitive endoderm in the second cycle. Finally, I investigated whether this unique feature could be explained by FOXH1 chromatin binding pattern by performing ChIP-Seq or by its dynamic interactions with co-partners by performing co-immunoprecipitation. Altogether, this work uncovers that FOXH1 orchestrates different cellular states in coordination with cell cycle progression and thus suggests that its role is modulated by the molecular context. Altogether, these data confirm that studying key factors of differentiation in the context of cell cycle progression can lead to unravelling new mechanisms involved in the temporal acquisition of cell identity. University of Cambridge 2022-03-31 Thesis Doctoral Doctor of Philosophy (PhD) eng https://www.repository.cam.ac.uk/handle/1810/345939 https://apollo8-f-pro.lib.cam.ac.uk/bitstreams/6e9aa609-3915-4b92-98e1-1d53975129d4/download 325626a51f98b9499236df9588c24e33 https://www.rioxx.net/licenses/all-rights-reserved/ FOXH1 Gastrulation Stem cells TGFb signalling