How does a cell narrow from many possible fates to one? This question is highly relevant to pluripotent stem cells, which generate all tissue types of the adult organism. One answer is that pluripotent cells in vitro and in the peri-implantation mouse embryo undergo global rewiring of their chromatin landscape to prepare for lineage commitment. In this dissertation, I explore molecular mechanisms that regulate chromatin state during early development.
The emergence of permissive chromatin and hypertranscription in pluripotent cells enables rapid proliferation and lineage induction. We found that cellular growth pathways, most prominently translation, perpetuate the euchromatic state and hypertranscription of mouse embryonic stem (ES) cells. Inhibition of translation rapidly depletes euchromatic marks and reduces nascent transcription in ES cells and blastocysts. Our results identify a positive feedback loop between chromatin state and translational output, whereby high translational output sustains levels of unstable euchromatin regulators and may set the pace of proliferation at peri-implantation.
The transition to a permissive chromatin state coincides with widespread loss of facultative heterochromatin. Repressive histone H3 lysine 27 tri-methylation (H3K27me3), deposited by Polycomb Repressive Complex 2 (PRC2), is redistributed from broad distal blankets to mark the promoters of developmental genes. In this thesis, I report a post-translational mechanism for control of PRC2. I found that the deubiquitinase Usp9x regulates PRC2 stability and activity in ES cells, and Usp9x levels capture the molecular transitions at implantation with remarkable fidelity. Transcriptome and chromatin analyses reveal that Usp9x-high cells bear a molecular signature of the pre-implantation embryo, whereas Usp9x-low cells resemble the post-implantation, gastrulating epiblast. These findings indicate that physiologic decline of Usp9x expression destabilizes PRC2 and helps constrict H3K27me3 during lineage induction. Deletion of Usp9x in pluripotent epiblast cells results in delayed repression of early lineage genes and developmental defects by embryonic day 9.5.
Studies in ES cells and early embryos provide insight into the regulatory logic that not only shapes embryonic development, but also underlies cell fate transitions more generally. The mechanistic interdependence of euchromatin, transcription and translation may apply to other fast-proliferating cells. Usp9x recurs as a marker of “stemness”; is essential for fly, mouse, and human development; and is mutated in various neurological disorders and cancers. Thus, the work described in this thesis has implications for stem/progenitor cell compartments, stem cell-based therapeutics, tissue regeneration and engineering, and cancer.