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Mouse Embryonic Stem Cell Study Highlights the Factors Controlling the Totipotent State

Review of “Myc and Dnmt1 impede the pluripotent to totipotent state transition in embryonic stem cells” from Nature Cell Biology by Stuart P. Atkinson

Previous studies of late 1-cell and 2-cell embryos have revealed the transient activation of a group of genes and repeats at this time point [1, 2], suggesting their importance to the establishment of the totipotent state [3]. While mammalian embryos of this stage in development remain scarce for research purposes, studies have provided evidence for the existence of a reversible 2-cell-like state in mouse embryonic stem cell (mESC) cultures [2]. Encouragingly, 2C-like mESCs can also be readily isolated, thereby providing a relevant model system that will allow for a deeper understanding of the mechanisms involved in establishing and maintaining totipotency. 

Now, researchers from the laboratory of Yi Zhang (Howard Hughes Medical Institute/Boston Children’s Hospital/Harvard Medical School, Boston, MA, USA) have combined the forced expression of the Dux transcription factor to drive the efficient formation of 2C-like totipotent ESCs [4, 5] with CRISPR/Cas9 technology to identify transcriptional regulators linked to the totipotent state [6].

Using single-cell RNA-sequencing analysis of mESCs following Dux overexpression, Fu et al. discovered that reprogramming from pluripotency to a 2C-like state first requires the downregulation of pluripotency-associated genes, such as Sox2, Klf4 and Rest, and the entry of cells into an intermediate state. In a second stage, the upregulation of 2-cell-embryo-specific elements, such as MERVL repeats, Zscan4 genes, Spz1 and Zfp352, drives the transition to the 2C-like cell state.

The authors then employed a CRISPR/Cas9 screen to identify those factors crucial to the pluripotency to 2C transition; this approach reproducibly identified Dnmt1, Uhrf1, Ptpn11, Dicer1, Smad7, Myc and Tsc2 as negative regulators and Eif3h, Eif5b and Eif4e2 as positive regulators. Subsequent protein interaction analysis identified multiple networks for negative regulators, including Dnmt1/Uhrf1 for DNA methylation, Grb2/Ptpn11/Sos1 of the MAPK signaling pathway, and Tsc1/Tsc2 of the TOR signaling pathway. 

Given the aim of understanding the transcriptional regulation of the totipotent state, the study focused on how Myc and Dnmt1 prevent the transition process. Interestingly, analyses suggested that Myc acts to prevent the downregulation of pluripotent gene expression key to the first stage of the transition, while Dnmt1 acts to prevent the 2-cell-embryo-specific gene activation required in the second step of the transition.

Overall, the authors anticipate that their determination of Myc- and Dnmt1-associated mechanisms controlling the transition from pluripotency to totipotency will prompt the further exploration of the factors and pathways controlling the establishment of the totipotent state.

For more on how studies in mESCs can help to delineate the factors controlling the totipotent state, stay tuned to the Stem Cells Portal.

References

  1. Falco G, Lee S-L, Stanghellini I, et al., Zscan4: A novel gene expressed exclusively in late 2-cell embryos and embryonic stem cells. Developmental Biology 2007;307:539-550.
  2. Macfarlan TS, Gifford WD, Driscoll S, et al., Embryonic stem cell potency fluctuates with endogenous retrovirus activity. Nature 2012;487:57.
  3. Lu F and Zhang Y, Cell totipotency: molecular features, induction, and maintenance. National Science Review 2015;2:217-225.
  4. Hendrickson PG, Doráis JA, Grow EJ, et al., Conserved roles of mouse DUX and human DUX4 in activating cleavage-stage genes and MERVL/HERVL retrotransposons. Nature Genetics 2017;49:925.
  5. De Iaco A, Planet E, Coluccio A, et al., DUX-family transcription factors regulate zygotic genome activation in placental mammals. Nature Genetics 2017;49:941.
  6. Fu X, Wu X, Djekidel MN, et al., Myc and Dnmt1 impede the pluripotent to totipotent state transition in embryonic stem cells. Nature Cell Biology 2019;21:835-844.