Probing transcriptional regulation in mammalian cells

The transcriptional regulatory networks that govern developmental cell fate decisions are important components of the process by which multicellular organisms develop from unicellular zygotes. These networks comprise an interconnected set of transcriptional regulatory relationships that allow a cell to change gene expression programs in response to extracellular signals. Understanding how these networks control cell fate decisions requires an understanding of how individual transcriptional regulatory proteins function within the network. To study the functions of these network proteins, methods of specifically modulating their activities are necessary.;Chapter 2 describes the use of a new method for controlling protein activity in which fusion of a protein of interest to an intein-based domain renders the activity of that protein dependent on the small molecule 4-hydroxytamoxifen (4-HT). We show that 4-HT-dependent inteins previously developed in our lab can be used to control the activities of proteins in mammalian cells. We demonstrate the 4-HT-dependent activity of two murine transcription factors fused to evolved inteins. In addition, one of these transcription factor fusions is used to render osteoblast differentiation of a murine embryonic fibroblast cell line dependent on 4-HT. Our results suggest that small-molecule-dependent inteins may represent a general method for controlling transcription factor activity in mammalian cells and that they may therefore be useful tools in the study of transcriptional regulatory networks.;Chapter 3 describes a study of the mechanism by which activation of the Wnt signaling pathway contributes to the maintenance of pluripotency in murine embryonic stem (mES) cells. We present evidence that the pluripotency-associated transcription factor Nanog is a direct transcriptional target of Wnt signaling, and we show that Nanog is required for Wnt-mediated maintenance of pluripotency in mES cells. Our findings suggest that the activation of Wnt signaling promotes mES cell pluripotency by upregulating Nanog expression via both canonical and noncanonical Wnt signaling mechanisms. Furthermore, our conclusions support the proposal that the transcriptional mediators of Wnt signaling may be important components of the transcriptional regulatory network responsible for the maintenance of pluripotency in mES cells.