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A dual reporter system identifies an intermediate state and sequential regulators of 2-cell-like-to-pluripotent state transition

Zhang, C.; Hao, J.; Shi, M.; Li, Y.-X.; Yao, W.; Wang, Y.

2021-12-17 cell biology
10.1101/2021.12.17.473100 bioRxiv
Show abstract

Mouse embryonic stem cells (ESCs) cycle in and out of 2-cell-like (2C-like) state in culture. The molecular mechanism governing the exit of 2C-like state remains obscure, partly due to the lack of a reporter system that can genetically mark intermediate states during exiting process. Here, we identify an intermediate state that is marked by the co-expression of MERVL::tdTomato and OCT4-GFP (MERLOT) during 2C-like-to-pluripotent state transition (2CLPT). Transcriptome and epigenome analyses demonstrate that MERLOT cells cluster closely with 8-16 cell stage mouse embryos, suggesting that 2CLPT partly mimics early preimplantation development. Through a CRISPRa screen, we identify an ARRDC3-NEDD4-OCT4 regulatory axis that plays an essential role in controlling 2CLPT. Furthermore, re-evaluating previously reported 2C-like state regulators reveals dual function of Chaf1a in regulating the entry and exit of 2C-like state. Finally, ATAC-Seq footprinting analysis uncovers Klf3 as an essential transcription factor required for efficient 2CLPT. Together, our study identifies a genetically traceable intermediate state during 2CLPT and provides a valuable tool to study molecular mechanisms regulating this process.

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