The goal of the project is to define the role of key regulators of pluripotency and differentiation in human embryonic stem cells (hESCs). Specifically, this project focused on the regulation and actions of the gene expression regulator TCF-3. The role of this factor in mouse embryonic stem cell cultures is known to protect the pluripotent state and cellular responsiveness in a very important way: loss of TCF-3 protein results in a permanent state of pluripotency and inability to differentiate. However, the molecular and cellular characteristics of TCF-3 and its function in hESCs is not known. It is not known whether TCF-3 functions are similar to its functions in mouse ESC. The work performed in the third funding period focused on defining the genes that are modulated by TCF-3 expression. Genome-wide microarray analysis was performed in the presence and absence of TCF-3 and in the presence and absence of differentiation conditions.
Removal of TCF-3, or “knockdown” revealed a specific, small set of genes that are dynamically dependent on TCF-3 for expression. Signals for differentiation that mimic the environment in the developing embryo were used to understand how TCF-3 functions in this setting. Our data suggests that TCF-3 exerts a strong, repressive function within the new signaling context. Microarray analyses, validation and biochemical confirmation have been used to better define the precise role of TCF-3 action. This past year utilized ChIP-seq approached to precisely define the pattern of genome occupancy that TCF-3 has in hESC cultures prior to differentiation. The results suggest that TCF-3 expression is dynamic, constitutively required, and that much of its function is to provide repressive actions. While this has general similarity to the role ascribed to TCF-3 in mouse embryonic stem cells, it appears that the types of genes and processes repressed by TCF-3 are distinct in hESC.