The lack of a quantitative framework around the dynamics of gene expression and its determinants represents a major hurdle for capturing transcription regulation into regulatory models. Embryonic Stem Cells (ESC) provide an excellent cellular model to quantitatively define the principles of gene regulation at the biochemical and (epi)genetic level. In SysStemCell, I will focus on two distinct mouse pluripotent cell states. My recent studies have shown that the epigenome and transcriptome of ESCs maintained in serum-free medium complemented with two kinase inhibitors and LIF (‘2i ESC’) are distinct from classical ESCs cultured in the presence of serum and LIF (‘serum ESC’). Importantly, the 2i ESCs reflect pre-implantation stage ICM cells whereas the widely studied serum cells reflect post-implantation ESCs. These two distinct pluripotent states are interconvertible in vitro, providing an unique and accessible model to explore the regulation of the pre- to post-implantation phase of early embryonic development. I will combine state-of-the-art proteomics and chromatin-based methods, including a novel in vivo UV femtosecond laser crosslinking approach, and in depths bioinformatics in an iterative manner to define the full compendium of transcription (co)factors (TF) and TF-modules that define the two cell states. The dynamics of long- and short-range interactions between these modules will be assessed and correlated with the epigenetic state and chromatin structure. Novel and existing factors will be assessed for their role in differentiation from 2i to serum and reverse programming using knockout and forced expression strategies. Collectively, my studies will provide an invaluable resource for the community and a deep insight in the mechanisms and general principles that orchestrate gene expression in particular in the hitherto unexplored transition at implantation during early mouse development.