Mechanisms of pluripotent cell state transitions

Abstract

Cell state transitions enable the differentiation of stem and progenitor cells into more mature and specialized cell types and are, thus, fundamental to the formation of multicellular organisms. Developmental progression is largely a unidirectional process. However, expression of reprogramming factors is sufficient to de-differentiate mature somatic cells, suggesting that cellular plasticity persists even in terminally differentiated cell types. Multiple signaling pathways, epigenetic regulators, metabolic sensing cascades and transcription factors (TFs) contribute to differentiation and de differentiation. However, if reprogramming requires the reversion of naturally occurring developmental mechanisms remains unknown. A suitable model system to study cell state transitions in vitro are lineage-related mouse embryonic stem cells (ESCs) and epiblast stem cells (EpiSCs) which are derivatives of the pre implantation blastocyst and the post-implantation epiblast, respectively. Interconvertibility of ESCs and EpiSCs provides an experimental model to explore to which extent lineage progression and reprogramming overlap mechanistically. In a collaborative project, I contributed to the characterization of a novel ESC differentiation pathway: in a genome-wide clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated 9 (Cas9) screen we identified multiple components of a conserved amino acid signaling pathway as crucial drivers of ESC progression. Mechanistically, the lysosome activity, the Ragulator protein complex, and the tumor-suppressor Folliculin (Flcn) enable the Rag GTPases C and D to bind and seclude the TF Tfe3 in the cytoplasm. Ectopic nuclear Tfe3 represses specific developmental and activates metabolic transcriptional programs which are associated with in vivo development. In collaboration with geneticists, we identified point mutations in a Tfe3 domain required for cytoplasmic inactivation as a potential cause of a human developmental disorder. This work reveals an instructive and biomedically relevant role for metabolic signaling in licensing embryonic cell fate transitions. In my main PhD project, we aimed to identify cell state transition regulators which both are required for exit from the ESC state and inhibit acquisition of the induced pluripotent cell (iPSC) identity upon reprogramming of EpiSCs. We therefore performed a large-scale loss-of-function reprogramming screen in sensitized EpiSCs. Comparison with ESC differentiation screens revealed the constitutively expressed TF Zfp281 as a unique bidirectional regulator of cell state interconversion. We identified the histone methyltransferase Ehmt1 and the zinc finger TF Zic2 as differentiation-specific protein interaction partners of Zfp281 and showed that subtle chromatin binding changes of Zfp281 during ESC progression translate into activation of Ehmt1 and stabilization of Zic2 on promoters and enhancers. Genetic gain- and loss-of function experiments confirmed a critical role of Ehmt1 and Zic2 downstream of Zfp281 both in driving exit from the ESC state, and in restricting reprogramming of EpiSCs. This study reveals that the cell type invariant chromatin association of Zfp281 provides an interaction platform for remodeling the cis-regulatory network underlying cellular plasticity.