Genome binding/occupancy profiling by high throughput sequencing
Summary
The CBX family of proteins is central to proper mammalian development via key roles in Polycomb-mediated maintenance of repression. CBX proteins in differentiated lineages have chromatin compaction and phase separation activities that might contribute to maintaining repressed chromatin. The predominant CBX protein in pluripotent cells, CBX7, lacks the domain required for these activities. We inserted this functional domain into CBX7 in embryonic stem cells to test the hypothesis that it contributes a key epigenetic function. ESCs expressing this chimeric CBX7 were impaired in their ability to properly form embryoid bodies and neural progenitor cells and showed reduced activation of lineage-specific genes across differentiation. Neural progenitors exhibited a corresponding inappropriate maintenance of Polycomb binding at neural-specific loci over the course of differentiation. We propose that a switch in the ability to compact and phase separate is a central aspect of Polycomb group function during the transition from pluripotency to differentiated lineages.
Overall design
Embryonic stem cells expressing different versions of CBX7 (WT, CaPS, KO) were differentiated into embryoid bodies and neural progenitor cells. Localization of PRC1 and H3K27me3 was determined in ESCs and differentiated cells by ChIP-Seq and by CUT&RUN. ChIP-Seq peaks were called over input values, and CUT&RUN peaks were called over IgG control values. Replicates were averaged where present. All comparisons were made between experiments that were performed at the same time.
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