The mechanisms by which the ES cell cycle is controlled and sustained remain obscure. It is well known that Oct-4 is an essential transcriptional regulator of genes involved in maintaining the undifferentiated pluripotent state of ES cells, but little has been established as to whether the ES cell-cycle fate is regulated by this protein. In the present study, we have characterized the effect of Oct-4 on ES cell-cycle control using ZHBTc4 ES cells. Previous studies have shown that the length of G1
-phase in ES cells is much shorter than in somatic cells [41
], and in the present study we found that Oct-4 down-regulation in ES cells inhibits cellular proliferation by blocking cell-cycle progression in G0
. Deletion analysis of the functional domains of Oct-4 indicated that both the N-terminal and C-terminal transactivation domains of Oct-4 are important for the stimulation of S-phase entry. In addition, we also showed in the present study that the p21
gene is a target for repression by Oct-4 in ZHBTc4 ES cells. Our findings demonstrate that the down-regulation of p21
by Oct-4 may contribute to the maintenance of ES cell proliferation.
Oct-4 is a central mediator of the undifferentiated pluripotent state of ES cells. It was previously reported that maintaining Oct-4 activity within a certain range appears to be critical for stem cell self-renewal, with any increase or decrease triggering differentiation to endoderm/mesoderm or trophectoderm respectively [12
]. Although Oct-4 possesses two separate transactivation domains, Oct-4 deletion mutants lacking either the N-terminal domain or the C-terminal domain are able to maintain the stem cell phenotype [25
]. Consistent with this result, ZHBTc4 ES cells expressing FLAG-tagged Oct-4(ΔC)–EGFP or FLAG-tagged Oct-4(ΔN)–EGFP proteins were positive for undifferentiated stem cell markers, such as AP, SSEA-1, Nanog, Sox2, Fgf-4 and Rex-1 (). However, these ES cells showed smaller colony sizes and were also positive for the expression of markers of differentiated cells, indicating that neither the N-terminal nor the C-terminal domain deletion mutant of the Oct-4 protein is sufficient to maintain the stem cell phenotype. ES cells expressing Oct-4 deletion mutants lacking either the N-terminus or the C-terminus were positive both for the undifferentiated stem cell markers and for markers of differentiated cells (). It would be interesting, therefore, to see whether such ES cells are capable of long-term self-renewal and are pluripotent in vitro
and in vivo
Cell-cycle progression is controlled by the CDKs, and p21 is a CDKI that inhibits G1
CDK–cyclin complexes [44
]. In the present study, the down-regulation of Oct-4 in ES cells induced p21
to a remarkable degree (), in a time-dependent manner, both at the mRNA and protein levels. It is well known that p21 expression is regulated largely at the transcriptional level by a p53-dependent mechanism at the transcriptional level [46
], which suggests that Oct-4 may regulate p21
expression through the p53
tumour suppressor gene. However, although ZHBTc4 ES cells did express p53, the down-regulation of Oct-4 did not significantly change p53 protein levels (B). Having demonstrated that p21
expression is down-regulated by Oct-4 in ES cells, we attempted to determine whether this resulted from a direct effect of Oct-4 with regulatory element(s) in p21
. As shown in , the down-regulation of Oct-4 in ES cells increased p21
promoter activity (D) and the introduction of increasing amounts of Oct-4 led to a reduction in p21
promoter activity (up to 40% of promoter activity) in differentiated cells (HEK-293T cell line; E), suggesting that Oct-4 directly represses p21
promoter activity. However, p21
expression can also be regulated post-translationally by both ubiquitin-dependent and -independent proteasome-mediated degradation [47
]. Thus it would be interesting to determine whether Oct-4 is able to control p21 stability in ES cells and induce pluripotent stem (iPS) cells.
The expression of Oct-4 in doxycyclin-treated ZHBTc4 ES cells led to a consistent increase in the number of cells in S-phase, an effect that was not observed following the expression of EGFP (). Consistent with these results, the down-regulation of Oct-4 in ES cells also led to an increase in the number of cells in G0/G1-phase (D). To clarify the contribution of the different functional domains of the Oct-4 protein to ES cell-cycle progression, we generated ES cell lines carrying functional domain deletion mutants of Oct-4 (A). Deletion of the N-terminal or C-terminal domain of Oct-4 reduced the number of cells in S-phase (), indicating that the overall integrity of Oct-4 is required for its ability to stimulate cell-cycle progression in ES cells.
In conclusion, it is likely that Oct-4 contributes towards maintaining cells in an undifferentiated, pluripotent or totipotent state in two ways: (i) by activating certain key genes and (ii) by silencing others. The findings of the present study provide additional evidence that Oct-4 plays a key role in regulating the ES cell cycle positively, as well as maintaining ES cell pluripotency. Therefore Oct-4-mediated p21 gene down-regulation may be a key mechanism for the stimulation of cell-cycle progression of the ICM.