Original article from STEM CELLS

p53-p21-mediated regulation of the G1/S checkpoint pathway of the human cell cycle is a vital control point of proliferation, and the molecular pathways governing the G1/S transition are also known to play a key role in the DNA damage response (DDR).   Human embryonic stem cells (hESCs) appear to have a unique cell cycle, with a shortened G1 phase (Becker et al)which only becomes lengthened upon differentiation (Filipczyk et al).   Further, a lack of p21 protein but, importantly, not of mRNA in hESCs has suggested that p21 plays no role in the G1/S transition (Bárta et al) and therefore the “canonical” p53-p21 axis of the G1/S checkpoint pathway is described as non-functional in hESCs, becoming activated only upon differentiation (Neganova and Lako).   Indeed, it has been proposed that the lack of an active p53-p21 pathway is required to prevent premature differentiation of hESCs (Maimets et al).   Now, a study from the laboratory of Ales Hampl has confirmed that while p53 is active in hESCs following DNA damage, p21 protein is not produced as part of the DNA damage response (DDR).   Furthermore they provide data suggesting that this is due to the actions of multiple microRNA (miRNA) families on p21 mRNA also suggesting that this mode of regulation is important in governing the G1/S transition and cell cycle checkpoint in undifferentiated hESCs (Dolezalova and Mraz et al).

Initial experiments studied the parameters of DNA damage in three hESC lines before and after ultraviolet-C (UVC) irradiation.   Phosphorylation of histone H2AX at Serine 139 (γ-H2AX), used as a measure of DNA damage, peaked at 4 hours while cleavage of PARP and Caspase 3, used as a measure of entry into apoptosis, peaked at 8 hours after UVC irradiation.   Apoptosis levels of hESCs after UVC irradiation was approximately 16% at 4 hours and rose to 42% after 16 hours and was associated with distinct morphological changes.   However hESC-derived NPCs treated with the same levels of UVC irradiation displayed a moderate accumulation of γ-H2AX only and while both cell types accumulated activated p53, this occurred faster in hESCs.   p53-induced proteins (p21, and pro-apoptosis genes GADD45, Bax, and EGR1) were upregulated in hESCs after UVC irradiation with the exception of p21, while in NPCs p21 protein was upregulated but the pro-apoptotic proteins were not, overall suggesting that p21-mediated cell cycle regulation is not operative in hESCs.   However further analysis demonstrated that p21 mRNA was highly upregulated after UVC irradiation in hESCs, indeed bypassing the level observed in NPCs after UVC irradiation.

This suggested that miRNAs may be involved in the regulation of p21 mRNA translation, and may be a major reason for the lack of p21 protein.   miRNAs can induce gene silencing via mRNA degradation or preventing mRNA from being translated through binding to untranslated regions (UTRs) of target mRNAs.   miRNA microarray comparisons of hESCs and NPCs identified 31 miRNAs that differed in their expression, with 26 upregulated and 5 downregulated overall.   The hESC-enriched miRNAs included four members of the miR-302 family and miR-367.   Interestingly members of the miR-302 family have a similar seed sequence to the miR-17-106 family which are known to regulate p21 levels in somatic cells (Ivanovska et al).   Further miRNA analysis after UVC irradiation in hESCs found that 22% of the total miRNAs upregulated were putative regulators of p21 mRNA, having altogether 29 binding sites in the 3’ UTR of p21 mRNA, while members of 4 miRNA families associated with pluripotency were also upregulated (miR-302, miR-371-373, miR-106b and C19MC clusters).   Closer analysis of the pluripotency-associated miR-302 cluster members which could be regulating p21 mRNA found that all miRNAs associated with this cluster (miR-302a, miR-302b, miR-302c, miR-302d, and miR-367) were upregulated greatly in response to UVC irradiation in hESCs.

The direct involvement of the miRNA pathway in p21 regulation in hESCs was examined through shRNA-mediated downregulation of Argonaute (Ago2) and Dicer (Dic1), both of which play key roles in the synthesis of miRNAs.   Transfection of the shRNAs led to the detection of p21 protein after 72 hours in hESCs, and this level increased to higher levels after UVC irradiation.   Co-transfection of the shRNAs with miRNA mimics of all four members of the miR-302 family led to the downregulation of p21 protein and mRNA.   Functional proof of miR-302 cluster members interacting with the p21 mRNA was sought using primary human fibroblasts transfected with a luciferase reporter gene with 3′UTR region of p21 mRNA, the target sequence for miRNAs.   Co-transfection of the miRNA-302 mimics led to a 33% downregulation of luciferase expression compared to controls proving the direct inhibitory role of the miR-302 miRNAs on p21 mRNA.   Finally, to exclude the role of the proteasome in p21 degradation, hESCs and NPCs were exposed to the proteasome inhibitor MG132 which led to an increase in p21 in NPCs only, with p21 protein still undetectable in hESCs.

This study overall suggests that while p53 upregulates its target genes mRNA in response to DDR, p21 is not translated into protein, and that this translation inhibition is mediated by hESC-enriched miRNAs.   This mechanism also appears to be specific to hESCs as this type of regulation did not appear in NPCs in which p21 protein was readily found.   The authors suggest that the functionality of p53 and the lack of p21 protein may prepare the cells to respond to differentiation signals, further demonstrating the importance of understanding the modified cell cycle of hESCs.

References

Bárta T et al. Human embryonic stem cells are capable of executing G1/S checkpoint activation. Stem Cells 2010; 28: 1143–1152.

Becker KA et al. Self-renewal of human embryonic stem cells is supported by a shortened G1 cell cycle phase. J Cell Physiol 2006; 209: 883–893.

Dolezalova D, Mraz M et al. MicroRNAs Regulate p21 (Waf1/Cip1) Protein Expression and the DNA Damage Response in Human Embryonic Stem Cells. Stem Cells. 2012; 30: 1362-72.

Filipczyk AA et al. Differentiation is coupled to changes in the cell cycle regulatory apparatus of human embryonic stem cells. Stem Cell Res 2007; 1: 45–60.

Ivanovska I et al. MicroRNAs in the miR-106b family regulate p21/CDKN1A and promote cell cycle progression. Mol Cell Biol 2008; 28: 2167–2174.

Maimets T et al. Activation of p53 by nutlin leads to rapid differentiation of human embryonic stem cells. Oncogene 2008; 27: 5277–5287.

Neganova I, Lako M. G1 to S phase cell cycle transition in somatic and embryonic stem cells. J Anat 2008; 213: 30–44.

STEM CELLS correspondent Stuart Atkinson reports on those studies appearing in current journals that are destined to make an impact on stem cell research and clinical studies.