Prof The Hebrew University of Jerusalem Jerusalem, Yerushalayim, Israel
Abstract: The mechanisms by which the cell-cycle directs embryonic-stem-cells differentiation outcomes are not fully understood. To gain deeper insights, we performed a CRISPR/Cas9 genomic-screen on pre-sorted G1 and G2/M ESCs, identifying hundreds of genes essential for the transition from pluripotency to differentiation. Notably, many of these genes are influenced by the cell-cycle state at the onset of differentiation, even if they are not expressed during pluripotency. By incorporating temporal measurements, we revealed a link between the exit from pluripotency and differentiation decisions, demonstrating that fate selection is already influenced during the pluripotent state. Next, we focused on Jarid2, a subunit of PRC2.2, which emerged as an essential G2/M-dependent differentiation regulator. Interestingly, other PRC2 components, while essential for differentiation, did not exhibit cell cycle sensitivity. The majority of G2/M Jarid2 KO ESCs failed to survive differentiation, while the small fraction that endured exhibited an immature differentiation state, retaining ability to reactivate pluripotency. We showed that Jarid2 selectively recruits RNA polymerase II Ser5 to genes involved in extra-embryonic endoderm-like (XEN-like) differentiation specifically during G2/M explaining the preference of G2/M cells to activate XEN. Using cell-cycle pre-sorted ESCs and single-cell RNA-seq, we further characterized Jarid2's role in ESCs differentiation; While EpiSC-like cells did not require Jarid2—indicating their status as a premature differentiation state. XEN-like cells originated mostly from persisting G2/M ESCs, exhibited a mixed expression with pluripotent markers. Moreover, the activation of more defined states, such as mesendoderm and pre-epithelium were unable to activate without Jarid2. Additionally, chromatin immunoprecipitation for H3K27ac revealed that the loss of Jarid2 disrupts the activation of enhancers critical for differentiation while promoting H3K27me3 deposition, resulting in the repression of pluripotency. In summary, our screen identified a novel set of cell-cycle-sensitive genes, providing a valuable resource for the stem cells research community. Our focus on Jarid2 further elucidated its pivotal role in regulating early steps of differentiation.
