By genetically ablating Tcf3 from mESC, we showed that Tcf3 broadly functioned to counteract the effects of pro-self-renewal factors Nanog and Oct4. The data presented here were consistent with our previously published finding that Tcf3 repressed Nanog expression, and they provide substantial new insights by identifying Nanog-independent effects of Tcf3. As opposed to transient RNAi-based approaches, the use of TCF3−/− mESC lines revealed a stable, steady-state inhibitory effect of Tcf3 on pluripotent cell self renewal. As a result of increased expression of Tcf3/Oct4/Nanog-regulated genes, TCF3−/− mESC were capable of indefinite self renewal in the absence of Lif-Jak-Stat3 stimulation. Taken together, these findings suggest that Tcf3 limits the ability of Nanog and Oct4 to activate target genes that promote self renewal.
While the analysis of gene expression data revealed a strong inverse correlation between the effects of Tcf3 and the effects of both Oct4 and Nanog, the observed effects are inconsistent with a simple hierarchical relationship between Tcf3 and the proposed Nanog-Oct4-Sox2 network of regulators. Since siRNA-mediated knockdown of Oct4 and Nanog each reduced Tcf3 levels (), the majority of effects of Nanog and Oct4 on gene expression cannot occur through regulation of Tcf3 levels. Since Oct4 and Sox2 levels were not significantly different in TCF3−/−
mESC, the effects of Tcf3 cannot occur through regulation of Oct4 or Sox2 levels (). Although Nanog was increased by ablation of Tcf3, the overlap between Tcf3 and Nanog was actually less than that between Tcf3 and Oct4 (). The closer relationship between Tcf3- and Oct4- mediated effects (ρ = −0.78) compared to Tcf3- and Nanog- mediated effects (ρ = −0.61), or Oct4- and Nanog- mediated effects (ρ = 0.70), further supports a model in which Tcf3 and Oct4 directly regulate the same target genes. The repressor activity of Tcf3 in mESC 27
and the activator activity of Oct4 9, 39
are consistent with our observation that Tcf3 and Oct4 caused essentially opposite effects on co-regulated genes. Therefore, these new data suggest that effects of Tcf3 occur in parallel to effects of Nanog and Oct4 and oppose the effects of Nanog and Oct4.
These conclusions are consistent with a recently published report identifying genome-wide chromatin occupancy for transcription factors in mESC. The study by Cole et al used the ChIP-CHIP method to reveal that Tcf3, Oct4, and Nanog bound to a significantly overlapping set of putative promoter regions, including those for one another’s genes13
. They also showed that Tcf3-bound genes were more frequently increased upon shRNA reduction of Tcf3 than unbound genes 13
. The co-occupancy of Tcf3, Oct4 and Nanog on putative target genes provides a good mechanism for our finding of the parallel effects of Tcf3 with Nanog and Oct4 on gene expression in mESC (, ). Thus despite important differences between the two studies (discussed later), our results support the overall conclusion that Tcf3 is an integral component of mESC circuitry.
Differences between our study and the Cole et al study could provide the most interesting insights into Tcf3’s regulation of self renewal. Knockdown of Tcf3 by shRNA elevated Oct4, Sox2 and Nanog after 48 hours 13
whereas constitutive knockout of the TCF3
gene elevated only Nanog levels (, ). Although the reason for the difference in steady state effects versus dynamic effects is not known, it is tempting to speculate that Tcf3 represses a mechanism of inhibiting Oct4 and Sox2 expression. Regardless of the underlying mechanism, the normal levels of Oct4 and Sox2 in TCF3−/−
mESC are important. The increased levels of Oct4 and Sox2 after shRNA reduction of Tcf3 precluded conclusions that gene expression effects on co-occupied genes could be caused by increased Oct4, by increased Sox2, or by decreased Tcf3. In contrast, changes to the steady state levels of gene expression in TCF3−/−
mESC can be confidently concluded to be Oct4-independent and Sox2-independent.
Model depicting steady state effects of gene expression on self renewal
It is interesting to consider that Tcf3 inhibits self renewal yet it is stably expressed in self renewing pluripotent cells. Reconciling this apparent contradiction can be aided by combining classic and new perspectives on the role of pluripotency in embryonic development. Teratoma and lineage tracing experiments using epiblast cells from post-implantation staged mouse embryos showed that the majority of cells in pre-streak and early-streak embryos gave rise to cells in multiple germ layers40, 41,42, 43
. Recently, cells cultures from post-implantation embryos (e5.5–e6.5) were clonally derived and maintained in vitro, and shown to be pluripotent by teratoma and embryoid body assays 44, 45
. These findings demonstrated that the epiblast progeny of the ICM retained pluripotency until lineage commitment during gastrulation, at which time lineage commitment must occur rapidly. Molecular genetic experiments revealed that mESC lineage commitment was blocked by high levels of Nanog (all lineages) 3, 46
and perturbed by ectopic expression of Tcl1 (neurectoderm), Esrrb (meso/endoderm), Tbx3 (mesoderm), and Sox2 (mesoderm) 6
. These observations support a requirement for a mechanism to limit the activities of stem cell self renewal circuitries for timely differentiation of pluripotent cells in embryos. Our data have revealed that Tcf3 provides a fundamental molecular mechanism that could accommodate this need, and Tcf3 protein expression in the epiblast is consistent with this model (unpublished observations). Thus, in contrast to the model proposed by Cole et al in which Tcf3-repressor stimulates differentiation 13
, we propose Tcf3 function is analogous to that of a current limiter in electronic circuits as Tcf3 must prevent over-activation of transcriptional circuits promoting pluripotent cell self renewal ().
The cellular consequences of loss of Tcf3 in mESC have been described here. Effects on embryonic development have been described by us previously 30
; however, our previous studies did not examine effects on pluripotency of cells because the identity of pluripotency factors was largely unknown at the time. It will be interesting to examine the role of Tcf3 inhibition of self renewal circuits for normal embryonic development. In addition it will be important to determine if absence of Tcf3 will provide a benefit to the induction of pluripotent cells by reprogramming cells from adult mammals 16–18
. Given its effects on Nanog and Oct4 downstream target genes, and the Lif-independent self renewal of TCF3−/−
mESC, one would anticipate that absence of Tcf3 could provide a selective advantage to cells undergoing reprogramming. As such, inhibition of Tcf3 could promote more efficient reprogramming by stabilizing an epigenetic program of pluripotent cell proliferation.