Introduction of Polycomb‐mediated Compaction and Phase Separation Activity in Stem Cells Impairs Differentiation

Abstract

In multicellular organisms, the Polycomb group (PcG) proteins play a significant role in maintaining the repression of specific target genes across development. There are five proteins in the mammalian Cbx family that can participate in Polycomb Repressive Complex 1 (PRC1) that differ in their levels of in vitro polynucleosome compaction and phase separation activity. Notably, Cbx7, the main Cbx protein expressed in embryonic stem cells (ESCs), lacks these two activities. We hypothesize that the lack of compaction and phase separation activity in Cbx7 is significant in the maintenance of pluripotency and in early differentiation. By not committing to a more inaccessible chromatin structure, it may allow “poised” genes in stem cells to become activated efficiently upon differentiation, while also priming them for more complete repression in alternate lineages. I inserted a region of the Cbx2 protein known to be necessary for compaction and phase separation activity into Cbx7. This chimeric Cbx7 protein is capable of both compaction and phase separation, even with a smaller fragment of this region than we have previously shown to be required for these activities. This demonstrates that the chromatin compaction and phase separation activities of Cbx2 are transferrable to a related protein and are held within a single region. I engineered ESCs to express this compaction-capable Cbx7 in place of the wild-type protein. These cells are impaired in their ability to properly form embryoid bodies and neural progenitor cells. They show modified expression patterns of developmental genes across both of these differentiation time courses, including reduced activation of lineage-specific genes and incomplete repression of alternate lineages. In neural progenitor cells, this is additionally associated with the maintenance of Polycomb binding at neural-specific loci over the course of differentiation. Thus, the addition of chromatin compaction and phase separation activity to Cbx7 results in defects in the redistribution of PcG proteins during differentiation, and a corresponding dysregulation of developmentally regulated target genes. This demonstrates that the addition of phase separation and compaction activity to Cbx7 in stem cells has a biological effect on gene regulation and pluripotency.