It is fundamentally important to determine how signaling pathways control ES cell pluripotency and differentiation, and how these pathways connect to discrete sets of target genes to affect such states. We found that a terminal component of the Wnt signaling pathway, the transcription factor Tcf3, is physically associated with the same genomic sites as the pluripotency regulators Oct4 and Nanog in mES cells. This result reveals that the Wnt pathway is physically connected to the core regulatory circuitry of these cells. This core circuitry consists of two key features: an interconnected autoregulatory loop, and the set of target genes that are mutually bound by the pluripotency transcription factors and Tcf3.
The genome-wide data sets we report here enhance our knowledge of the targets of Oct4, Nanog, and Tcf3. These new data sets were generated using the same protocols and genome-wide tiling microarrays in ES cells grown under identical conditions, allowing more reliable conclusions about the overlap of these factors throughout the genome; previous data sets for these factors came from different mES cells grown in different settings, using different ChIP analysis platforms, and these data were not always genome-wide (Boyer et al. 2005
; Loh et al. 2006
). The new data reveal, for example, the remarkable extent to which Oct4 and Nanog binding overlap throughout the ES cell genome and the striking association of Tcf3 with those sites (). The new data also provide a revised model for the core regulatory circuitry of mES cells, which incorporates Tcf3 and high-confidence target genes of key ES cell regulators ().
The revised model of core regulatory circuitry extends our knowledge of how extracellular signals from the Wnt pathway contribute to stem cell state. Pereira et al. (2006)
demonstrated that Tcf3 binds the Nanog
promoter and represses its mRNA expression in mES cells. Our data confirm Tcf3 binding and function at Nanog
and extend our knowledge of Tcf3 targets to the other well-characterized pluripotency regulators Oct4 and Sox2, as well as most of their target genes. Pereira et al. (2006)
proposed a model wherein Tcf3-mediated control of Nanog levels allows stem cells to balance the creation of lineage-committed and undifferentiated cells. Our results also support this model, but argue that Tcf3 contributes to the balance through its functions in the core regulatory circuitry described here.
Our results suggest that the Wnt pathway, through Tcf3, influences the balance between pluripotency and differentiation in ES cells, as modeled in . Under standard culture conditions, where there is a low-level of Wnt activation, ES cells are poised between the pluripotent state and any of a number of differentiated states. It is well established that Oct4, Sox2, and Nanog act to promote the pluripotent state, as depicted in the model where the influence of these factors is shown by an arrow. Under standard culture conditions, Tcf3 may exist in an activating or repressive complex, but is predominantly in a repressive complex promoting differentiation. The loss of Tcf3 in Tcf3 knockdown cells, would, in this model, favor pluripotency. Wnt stimulation converts the repressive complex to an activating complex and thus promotes pluripotency. Our results suggest that the Wnt pathway, through Tcf3, influences the balance between pluripotency and differentiation by bringing developmental signals directly to the core regulatory circuitry of ES cells. The observation that the Wnt pathway can be manipulated to affect the balance between pluripotency and differentiation suggests that perturbation of this pathway may impact the efficiency of reprogramming somatic cells into pluripotent stem cells.
Figure 5 Model depicting the influence of Wnt pathway components on pluripotency and differentiation in ES cells. ES cells are poised between the pluripotent state and any of a number of differentiated states. Oct4, Sox2, and Nanog act to promote the pluripotent (more ...)