Abstract
Cell cycle and differentiation decisions are tightly linked; however, the underlying principles that drive these decisions are not fully understood. Here, we combined cell-cycle reporter system and single-cell RNA-seq profiling to study the transcriptomes of mouse embryonic stem cells (ESCs) in the context of cell cycle states and differentiation. By applying retinoic acid, a multi-linage differentiation assay, on G1 and G2/M pre-sorted ESCs, we show that only G2/M ESCs were capable of differentiating into extraembryonic endoderm cells (XENs), whereas cells in the G1 phase predominantly produce Epiblast Stem Cells. We identified ESRRB, a key pluripotency factor that is upregulated during G2/M phase, as a central driver of XEN differentiation. Furthermore, enhancer chromatin states based on WT and Esrrb-KO ESCs show association of ESRRB with XEN poised enhancers. Cells engineered to overexpress Esrrb during G1 allow ESCs to produce XENs, while ESRRB-KO ESCs lost their potential to differentiate into XEN. In addition, Embryonic bodies (EBs) are not affected by deletion of ESRRB but trigger apoptosis upon attempts to apply direct XEN differentiation. Taken together, this study reveals an important functional link between Esrrb and cell-cycle states during the exit from pluripotency. Finally, the experimental scheme of single cell RNA-seq in the context of cell cycle can be further expanded into other cellular systems to better understand differentiation decisions and cancer models.
Competing Interest Statement
The authors have declared no competing interest.
Footnotes
In this manuscript, using specific cell-cycle stage sorting via FUCCI, together with single-cell RNA-sequencing, we establish Esrrb, an estrogen related receptor, as a key player in balancing between pluripotency maintenance and differentiation priming. We demonstrate that Esrrb upregulates during G2/M and binds to enhancers associated with differentiation programs. Using retinoic acid protocol as a representative differentiation assay that mimics exit from pluripotency, we demonstrate that only G2/M ESCs are able to differentiate into Extraembryonic Endoderm cells (XEN), whereas G1 ESCs contribute only to the epiblast stem cells. Confirming this, G1 ESCs engineered to overexpress Esrrb were able to become XEN cells while Esrrb KO cells completely lost their potential to become XEN cells. Furthermore, we show that cell-cycle dependency occurs only when the signal to differentiate meets the cells when they are still pluripotent and not after differentiation has initiated. Our data support a model in which Esrrb upregulation during ESCs G2/M phase, and its binding to XEN-poised enhancers are key in promoting XEN differentiation.