| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Institute of Pharmacology, Department of Biological Sciences and Biotechnology, State Key Laboratory of Biomembrane and Membrane Biotechnology, Institutes of Biomedicine, School of Medicine, Tsinghua University, Beijing, China
2Correspondence: GIBH, Chinese Academy of Science, Guangzhou 510663, China. E-mail: pei-duanqing{at}gibh.ac.cn
SPECIFIC AIMS
The successful derivation and establishment of human embryonic stem (ES) cell lines have generated strong interest in harnessing the therapeutic potential of these pluripotent cells to treat human diseases. At the present, there are considerable challenges in maintaining these ES cells in a pluripotent state or triggering ES cells to differentiate into a particular cell type. In fact, the precise mechanism that regulates stem cell self-renewal and pluripotency remains largely unknown.
The homeodomain transcription factors Oct4 and Nanog have been proposed as master regulators of stem cell self-renewal and pluripotency. However, it is not clear how ES cells maintain the critical activity of these two and other potential master regulators at the transcription level. We would like to determine the molecular mechanism that governs the regulation of Oct 4 and ES cells.
PRINCIPAL FINDINGS
1. Oct4 induced mouse ES cells differentiation by suppressing Nanog
Our initial aim was to define the relationship between Nanog and Oct4, two transcription factors implicated in stem cell self-renewal and pluripotency. More specifically, we would like to define if Oct4 and Nanog regulate each other in ES cells. To this end, we compared the phenotypes of ES cells that either over-express or underexpress Oct4 or Nanog by their expression constructs or siRNA constructs. To our surprise, ES cells over-expressing Oct4 are morphologically identical to those with Nanog knocked down by siRNA (Fig. 1
A), suggesting that both factors may have opposing function inside ES cells. Indeed, analysis of pluripotent and differentiation markers revealed that these two cell populations expressed identical markers of endoderm origin (Fig. 1B
), suggesting that ES cells over-expressing Oct4 phenocopy those with Nanog knockdown. Interestingly, we also noticed that the expression of Nanog was suppressed in ES cells with elevated Oct4 (Fig. 1B
, top panel), arguing that over-expressed Oct4 mediates its effect via repressing Nanog directly.
|
To test this possibility, we analyzed the effect of Oct4 on the Nanog promoter directly. As shown in Fig 1C
, the Nanog promoter functioned as expected with high activities in F9 and ES cells and low and no activities in P19 and NIH3T3, respectively, paralleling the endogenous levels of Nanog in these cells. Oct4 when coexpressed suppressed the activity of Nanog promoter in a dose-dependent fashion while activating the control reporter bearing Oct4 binding sites (Fig 1D
). There is one Oct4 binding site at –181 position within the Nanog promoter of which we confirmed its occupation by Oct4 by chromatin immunoprecipitation assay (ChiP) as shown in Fig. 1E
. Deletion analysis shown in Fig. 1F
demonstrated that the two constructs bearing this Oct4 binding site were repressed by Oct4 in nonpluripotent NIH3T3 cells. Mutation at the Oct4 binding site rendered the 926bp promoter insensitive to coexpressed Oct4 or Oct4 siRNA, suggesting that Oct4 regulates Nanog promoter specifically (Fig. 1G
). These data strongly suggested a negative role of Oct4 in regulating Nanog. Indeed, the concentration of Nanog in ES cells over-expressing Oct4 is also reduced significantly as monitored by Western blot (Fig. 1H
). Together, these data demonstrate that elevated Oct4 induces ES cell differentiation by suppressing Nanog.
2. Biphasic regulation of Nanog by Oct4 in ES cells
The repressive effect of Oct4 we observed on Nanog promoter appears to be contradictory to the recent report that Oct4 is required to maintain Nanog activity in ES cells by RNAi-mediated knockdown. To reconcile this difference, we monitored Nanog promoter activity in a range of Oct4 siRNA concentrations. Figure 1I
demonstrated an initial up-regulation of Nanog by lower doses of Oct4 siRNA (lanes 3, 4 vs. 2), and higher doses suppressed Nanog activity as reported. Figure 1J
shows that the mutant plasmid bearing Oct4 site mutation has much lower activity than the wild-type promoter (Fig. 1J
, lanes 3 vs. 2), suggesting that Oct4 does play an essential role in maintaining Nanog expression at steady-state concentration. Taken all these observations together, we can conclude that Oct4 regulates Nanog biphasically, i.e., a steady-state concentration maintains its expression, while an elevated concentration suppresses its expression.
3. FoxD3 as an activator of Nanog
The second aim was to identify additional factors that may positively regulate the expression of Nanog, thus, counteracting the repressive effects of Oct4 observed in the previous section. FoxD3 is expressed among pluripotent cells such as P19, F9, and ES cells but not in nonpluripotent NIH3T3 cells. We thus hypothesized that FoxD3 may play an important role in maintaining ES cell pluripotency in a similar fashion as Nanog. We then performed cotransfection of Nanog promoter reporter with FoxD3 expression vector in F9 EC cells and demonstrated that FoxD3 is a very potent activator of Nanog in a pluripotency specific manner, apparently through a FoxD3 binding site between –269 and –544 in Nanog promoter.
4. Oct4 activity is maintained at the steady-state concentration by a negative feedback loop in ES cells
The third aim was to establish a regulatory circuit among FoxD3, Nanog, and Oct4 in ES cells. The apparent biphasic regulation of Nanog by Oct4 observed in Fig. 1
prompted us to investigate the regulation of Oct4 by FoxD3 and Nanog. To this end, we cloned the promoter region of Oct4 and analyzed its expression in the context of Nanog, FoxD3, and Oct4 itself. First, we evaluated the activity of this promoter in pluripotent P19 and F9 cells and nonpluripotent NIH3T3 cells. As expected, the Oct4 promoter has robust activity in pluripotent P19 and F9 cells but not in NIH3T3 cells. When analyzed with Nanog and FoxD3, the Oct4 promoter was activated strongly by both factors in a dose-dependent fashion. Interestingly, when cotransfected with Oct4, we found that Oct4 protein appeared to be a repressor of its own promoter. These data suggested that Oct4 had a feedback loop to limit its own quantity. We then performed siRNA knockdown experiment to down-regulate endogenous Oct4 and confirmed the inhibitory effect of Oct4 even at the endogenous concentration. Thus, we concluded that Oct4 behaves as a persistent repressor of its own promoter, in contrast to its biphasic regulation of Nanog (see Fig. 1
).
The negative autoregulation of Oct4 promoter by Oct4 protein raises the possibility that Oct4 works as a negative feedback mechanism to ensure its own expression set at a steady state in ES cells. Indeed, our data demonstrate a feedback inhibitory loop that counterbalances the activation potential of FoxD3 and Nanog on Oct4. The suppression of Oct4 by Oct4 itself is sufficient to achieve the critical concentration of Oct4 at equilibrium, a hallmark function of Oct4 in regulating stem cell pluripotency. Taken together, our results demonstrate a regulatory circuitry among FoxD3, Nanog, and Oct4, which ensures the proper expression of Oct4 in ES cells.
5. Neither Oct4 nor Nanog is dispensable in sustaining the self-renewal of mouse ES cells
The interdependence between Nanog and Oct4 to maintain their transcription activity suggests that both master regulators may cooperate to sustain stem cell pluripotency. We cotransfected the RNAi vectors for Oct4 or Nanog either alone or with Nanog or Oct4 expression vectors, respectively, and then analyzed the self-renewal activity of these transfected cells. Stem cells expressing siRNAs for Oct4 or Nanog failed to undergo self-renewal. The overexpressed Nanog also failed to compensate the loss of Oct4 in sustaining self-renewal. Conversely, overexpression of Oct4 failed to rescue the loss of Nanog. These data demonstrate that two master regulators, Oct4 and Nanog, need to function in parallel in maintaining ES cell self-renewal and that neither is dispensable and nor capable of compensating the role of the another.
CONCLUSIONS AND SIGNIFICANCE
We conclude that Oct4, Nanog, and FoxD3 form an interactive and interdependent circuit for the regulation of stem cell pluripotency. Nanog is a direct target of Oct4, and the suppression of Nanog by Oct4 can help explain the previous observation that overexpression of Oct4 leads to endoderm-like differentiation in ES cells. Furthermore, we demonstrated that FoxD3 also participates in the regulation of stem cell self-renewal and pluripotency by activating the expression of the two known master regulators, Nanog and Oct4, in ES cells. We propose that this FoxD3-Nanog-Oct4 circuit may anchor a much larger network of transcription factors dedicated to stem cell self-renewal and pluripotency.
|
FOOTNOTES
1 These authors contributed equally to this work. 
To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.05-5543fje
This article has been cited by other articles:
|
|
 |
|
  J. Wang, D. N. Levasseur, and S. H. Orkin Requirement of Nanog dimerization for stem cell self-renewal and pluripotency PNAS, April 29, 2008; 105(17): 6326 - 6331. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 Z. Wang, T. Ma, X. Chi, and D. Pei Aromatic Residues in the C-terminal Domain 2 Are Required for Nanog to Mediate LIF-independent Self-renewal of Mouse Embryonic Stem Cells J. Biol. Chem., February 22, 2008; 283(8): 4480 - 4489. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 H. J. Kang, D. H. Adams, A. Simen, B. B. Simen, G. Rajkowska, C. A. Stockmeier, J. C. Overholser, H. Y. Meltzer, G. J. Jurjus, L. C. Konick, et al. Gene Expression Profiling in Postmortem Prefrontal Cortex of Major Depressive Disorder J. Neurosci., November 28, 2007; 27(48): 13329 - 13340. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. Xu, S. D. Pope, A. R. Jazirehi, J. L. Attema, P. Papathanasiou, J. A. Watts, K. S. Zaret, I. L. Weissman, and S. T. Smale Pioneer factor interactions and unmethylated CpG dinucleotides mark silent tissue-specific enhancers in embryonic stem cells PNAS, July 24, 2007; 104(30): 12377 - 12382. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. Li, G. Pan, K. Cui, Y. Liu, S. Xu, and D. Pei A Dominant-negative Form of Mouse SOX2 Induces Trophectoderm Differentiation and Progressive Polyploidy in Mouse Embryonic Stem Cells J. Biol. Chem., July 6, 2007; 282(27): 19481 - 19492. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 B. Boer, J. Kopp, S. Mallanna, M. Desler, H. Chakravarthy, P. J. Wilder, C. Bernadt, and A. Rizzino Elevating the levels of Sox2 in embryonal carcinoma cells and embryonic stem cells inhibits the expression of Sox2:Oct-3/4 target genes Nucleic Acids Res., March 27, 2007; (2007) gkm059v2. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 H. Niwa How is pluripotency determined and maintained? Development, February 15, 2007; 134(4): 635 - 646. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
1 kb) recapitulates the endogenous expression of Nanog. Endogenous Nanog transcripts in NIH3T3, P19, F9, and ES cells were analyzed by reverse transcription (RT)-polymerase chain reaction (PCR). Activity of Nanog promoter was also analyzed in these cell lines to show its specificity in pluripotent cells as indicated as described (16). D) Oct4 suppresses activity of Nanog promoter in F9 cells. Nanog promoter construct (NanP, lanes 2–5) and Oct4 reporter (6w-lu, lanes 6–9) were cotransfected with Oct4 expression constructs with increasing doses into F9 EC cells, and luciferase activities were measured and analyzed as described previously (16). E) Binding of Nanog promoter by Oct4 in ES cells. Chromatin immuno-precipitation analysis was performed to demonstrate in vivo binding of Nanog promoter by Oct4. F) Oct4 suppresses Nanog promoter constructs in NIH3T3 cells. Cotransfecton of Oct4 with reporters carrying deletions in the Nanog promoter was performed and analyzed as described in D. G) A mutation in the Oct4 binding site abolished regulation of Nanog promoter by Oct4. Reporters carrying wild-type and mutant promoters were cotransfected with Oct4 or Oct4 siRNA vectors and analyzed as in D. H) Overexpressed Oct4 reduces the expression of Nanog in ES cells. ES or Oct4 transfected ES cells were lysed and fractioned by 10% SDS-PAGE. Endogenous Nanog or transfected Oct4 protein was detected by anti-Nanog or anti-FLAG (Oct4 is FLAG tagged) antibodies, respectively. Levels of actin in the cell lysate were detected by antiactin antibody as a loading control. I) Regulation of Nanog by Oct4 siRNA in F9 cells. Nanog promoter construct (NanP, lane2–6) was cotransfected with increasing doses of Oct4 siRNA construct (lane 3–6) into F9 cells, and activities were analyzed by luciferase assay. J) Oct4 binding site is required for activity of Nanog promoter in F9 cells. Wide-type and mutant Nanog promoter constructs (lanes 2 and 3) were transfected to F9 cells, and their activities were evaluated by luciferase assay as in D.