The goal of the project is to define the role of key regulators of pluripotency and differentiation in human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs). Specifically, this project focused on the regulation and actions of 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 and iPSCs is not known, whether its functions are similar to its functions in mouse ESC. The work performed in the second funding period focused on mapping key protein modifications of TCF-3 including phosphorylation. Phosphorylation is a frequently used method to modify the actions of proteins – to either inhibit protein activity, stimulate activity, or change the ability to interact with other proteins. Human embryonic stem cell lines were modified to express a tagged version of TCF-3 for rapid isolation and purification from cell extracts. Purification was performed rapidly and under specialized conditions to prevent loss of any protein modifications. Mass spectrometry identified multiple phosphorylation sites and other forms of protein modification.
Work during this period also focused on developing strategies to identify key target genes regulated by TCF-3. Methods to remove TCF-3 from hESCs were developed and validated on levels to ensure bona fide removal without artefactual effects on hESCs. Removal of TCF-3, or “knockdown” will be used to investigate how hESCs cope in the absence of TCF-3. Preliminary results suggest that TCF-3 exerts control over a set of gene regulators as well as cellular signals. For these experiments, only early, immediate events that occur upon TCF-3 removal were assessed. A focus on early events is a purposeful effort to identify immediate-reacting, key regulatory steps. 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.