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Emi1 for a Spin? – Cell cycle adaptations of embryonic stem cells

From PNAS
By Stuart P. Atkinson

Mouse embryonic stem cells (mESCs) have a remarkably short G1 and G2 phase, which in somatic cells is determined by Cdk activity alongside other cell-cycle related proteins which fluctuate during the cell cycle due to APC/C mediated degradation. APC/C is a large multi-subunit E3 ubiquitin ligase, which can be activated by Cdc20 and Cdh1 interaction at the end of mitosis and inactivated by Emi1 (or Fbxo5) and degradation of Cdh1 just before S-phase. Inhibition of Cdk activity in G1 phase allows the replication factors Cdt1 and Cdc6 to recruit Mcm proteins onto chromatin, form pre-replicative complexes (pre-RCs) and license DNA for replication. Previous studies found that in mESCs APC/C substrates were constant and Cdk activity high throughout the mESC cell cycle (White et alFujii-Yamamoto et al and Yang et al). However, the careful re-appraisal of protein levels and activity in mESC by researchers from the laboratory of Marc W. Kirschner have uncovered oscillations in APC/C substrate levels and Cdk activity which, alongside other key findings, promote the abbreviated cell cycle of mESCs (Ballabeni et al).

Analysis of well-defined APC/C substrates at different phases of the cell cycle using an effective M-phase synchronization protocol demonstrated that levels of these substrates fell after mitotic exit, although not to levels observed in somatic cells. Cell cycle analysis found that APC/C activity in mESCs was high in mitosis, intermediate in the G1 phase, and low in the S phase and Cyclin A and Cdk2 fluctuates in mESC with the Cdk kinase activity necessary throughout cell-cycle progression. The reduced levels of APC/C substrate oscillation as compared to somatic cells were found to be in part due to high levels of Emi1, an APC/C inhibitor. Indeed, it was found that high levels of Emi1 protein and mRNA in self-renewing mESCs were reduced upon mESC differentiation; APC/C-Emi1 complexes were at higher levels in self-renewing mESCs than in differentiating mESCs and depletion of Emi1 in mESCs led to an increase in substrate degradation. Further analysis found that APC/C subunit levels did not alter during differentiation or through the cell cycle, while Emi1 depletion also caused cells to partially accumulate in S-G2 phase and present some re-replication.

Mcm proteins are loaded onto chromatin after mitosis when Cdk activity drops, but are also loaded during G1 phase even though Cdk activity is high, perhaps due to very high levels of the Cdt1 and Cdc6 licensing proteins. While a previous report has found high levels of Cdc6 protein in mESCs (Fujii-Yamamoto et al), Cdt1 levels have been studied less, and this study found that Cdt1 protein and mRNA levels at mitosis (correlated to licensing activity) were higher in self-renewing mESCs as compared to differentiating mESCs. Using an inducible p21 to inhibit Cdk activity allows mitotic entry and cell cycle progression and leads to significantly lower levels of Cdt1. Depletion of the Cdk2 partners Cyclin A and Cyclin E during late S and G2 phase before synchronization in mitosis and release, also led to a decrease in Cdt1 levels. To analyse chromatin licensing, Cyclin A was depleted in mESCs, which were then synchronised and collected at different times during the cell cycle demonstrating that Cdt1 levels were decreased concomitant with reduced Mcm2 chromatin loading. Depletion of Cdt1 and Cdc6 during the S-G2 phase after release from thymidine arrest and synchronisation in mitosis led to an elongated G1 phase confirming that they regulate S phase entry in mESCs.

Overall, this study suggests that in mESCs, APC/C is functional although levels of the inhibitor Emi1 are high leading to an attenuation of APC/C activity, while Cdk1 allows for high levels of Cdt1 allowing chromatin licensing. This adds to a burgeoning field and to an increasing amount of studies which imply a key role for the cell cycle in ESC biology, suggesting that the basic regulatory mechanisms in mammalian somatic cells are present and operating in ESCs but operate in modified ways and are inherently linked to aspects of stem cell function.

 

References

Ballabeni A, Park IH, Zhao R et al.
Cell cycle adaptations of embryonic stem cells.
Proc Natl Acad Sci U S A. 2011 Nov 29;108(48):19252-7.

Fujii-Yamamoto H, Kim JM, Arai K, Masai H.
Cell cycle and developmental regulations of replication factors in mouse embryonic stem cells.
J Biol Chem. 2005 Apr 1;280(13):12976-87.

White J, Stead E, Faast R et al.
Developmental activation of the Rb-E2F pathway and establishment of cell cycle-regulated cyclin-dependent kinase activity during embryonic stem cell differentiation.
Mol Biol Cell. 2005 Apr;16(4):2018-27.

Yang VS, Carter SA, Hyland SJ et al.
Geminin escapes degradation in G1 of mouse pluripotent cells and mediates the expression of Oct4, Sox2, and Nanog.
Curr Biol. 2011 Apr 26;21(8):692-9.