Functional Dissection of the Mechanism that Lead to Loss of Somatic Cell Identity

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Abstract Adult cells can be forced to change their cell identity and reprogrammed to an embryonic stem cell (ESC)-like induced pluripotent stem cells (iPSCs) state. Reprogramming to iPSCs can be achieved by overexpression of the four transcription factors (TFs) Oct4, Sox2, Klf4, and cMyc (OSKM). Reprogramming takes 2 weeks and the iPSCs are generally derived from less than 1% of the starting cell population. However, the reprogramming is more efficient when we modulate the activity of signaling pathways with small molecule activators or inhibitors. In this regard, we have recently demonstrated that the stepwise-modulation of the Wnt signaling pathway and its downstream transcriptional factors (Tcf1, Tcf3, Tcf4 and Lef1) during different stages of reprogramming promotes the establishment of iPSCs. Our studies also highlight that reprogramming of somatic cells provides a powerful in vitro model system for studying the signaling mechanisms underlying cell fate changes. Importantly, we still do not know how Wnt pathway and other signaling pathways act in somatic cells and limit the various stages of reprogramming. Current understanding of cell fates emphasize that not only the proper set of genes has to be expressed, but also the correct gene regulatory regions have to be engaged by cell type specific TFs and epigenetic markings. Signaling pathway TFs that are often expressed in a cell type specific manner must be targeted to their proper locations in order to maintain cell identity or to create new cell fates upon signal stimulation, however their targeting mechanisms are largely unknown. Our preliminary data show that the Wnt signaling TF Tcf3 dynamically changes its genome-wide binding during reprogramming. By studying this stage-specific behavior of Tcf3, we revealed a previously unknown early barrier of reprogramming, which controls the erasure of somatic Tcf3 sites and the generation of its new reprogramming sites. In this proposal, we will study the role of our newly identified reprogramming-inhibiting TFs as regulators of Tcf3 binding. Furthermore, we propose to identify signaling pathways, which maintain the somatic cell identity and control reprogramming in a stage specific manner. Using our new single cell level immunostaining method, we will determine how these signaling pathways regulate the distinct cell fates during reprogramming. We believe that our work will ultimately lead to (i) a better understanding of how specific cell fates are maintained by signaling pathways, and (ii) also help identifying critical signaling pathways that act during specific cell fate changes. Signaling pathways control many cell fate choices during development and their deregulation can cause diseases. Therefore, understanding the role of signaling pathways in maintaining somatic state and regulating the loss of somatic identity is crucial so that we can modulate these signaling pathways to create or maintain desired cell fates.