Jaager, K. | Search bioRxiv

bioRxiv2019

Cell cycle-balanced expression of pluripotency regulators via cyclin-dependent kinase 1 activity

Embryonic stem cells (ESCs) have a unique ability to remain pluripotent while undergoing rapid rounds of cell division required for self-renewal. However, it is not known how cell cycle and pluripotency regulatory networks co-operate in ESCs. Here, we used stable isotope labeling with amino acids in cell culture (SILAC) combined with mass spectrometry to determine pluripotency proteome dynamics during cell cycle in mouse ESCs. We found the S/G2M-fluctuating pluripotency transcription factors (ESRRB, REST), chromatin regulators (JARID2, TRIM24) and proteins with E3 ligase activity (NEDD4L, PIAS2) to peak in S phase. This expression balance was disrupted upon inhibition of cyclin-dependent kinase 1 (CDK1) activity resulting in the shift of the expression peak from S to G2M. Our results demonstrate that mouse ESCs require CDK1 activity to maintain high S to G2M ratio of pluripotency regulators revealing critical role of cell cycle dynamics in balancing ESC identity.

cell biology↗

bioRxiv2019

G2M splits mouse embryonic stem cells into naïve and formative pluripotency states

Embryonic stem cells (ESCs) express heterogeneous levels of pluripotency and developmental transcription factors (TFs) and their cell cycle is unsynchronised when grown in the presence of serum. Here, we asked whether the cell cycle and developmental heterogeneities of ESCs are coordinated by determining the state identities of G1- and G2M-enriched mouse ESCs (mESCs) at single cell resolution. We found that G2M cells were not all the same and demonstrate their split into the naive and formative (intermediate) pluripotency states marked by high or low Esrrb expression, respectively. The naive G2M sub-state resembles ground state pluripotency of the LIF/2i cultured mESCs. The naive and formative G2M sub-states exist in the pre- and post-implantation stages of the mouse embryo, respectively, verifying developmental distinction. Moreover, the G2M sub-states partially match between the mouse and human ESCs, suggesting higher similarity of transcriptional control between these species in G2M. Our findings propose a model whereby G2M separates mESCs into naive and formative pluripotency states. This concept of G2M-diverted pluripotency states provides new framework for understanding the mechanisms of pluripotency maintenance and lineage specification in vitro and in vivo, and the development of more efficient and clinically relevant reprogramming strategies.

developmental biology↗