The Roles of Transcription Factors TFAP2a and P63 in Guiding Non-Neural Ectoderm Cell Commitment

Treating embryonic stem cells with bone morphogenetic protein-4 (BMP4) and retinoic acid (RA) upregulate TFAP2 family members and P63, transcription factors that are important for non-neural ectoderm differentiation. Previous research in the lab indicates that TFAP2a and P63 act jointly to regulate early-stage non-neural ectoderm commitment. Our goal was to further understand the roles of these transcription factors in non-neural ectoderm differentiation.
We engineered an inducible plasmid system that overexpressed P63 in TFAP2a-knockout cells (2AKO P63). Differentiation of the TFAP2a knockout cells (2AKO) does not result in non-neural ectoderm formation and these cells differ transcriptionally and epigenetically from wildtype cells. Once transfected with a P63-overexpression plasmid, immunofluorescent staining (IF) and western blotting were used to confirm homogeneity of P63 expression and to optimize the tool for inducer concentration. We then analyzed transcriptional and epigenetic changes in the cells. RNA- seq suggested that 2AKO P63 cells were transcriptionally situated between wildtype and 2AKO cells. ATAC-seq highlighted that there were substantial differences in chromatin accessibility between 2AKO and 2AKO P63 cells (over 15,000 sites changing), while wildtype cells relative to 2AKO P63 cells had approximately 50 sites that differed. We then explored the effect that overexpression of P63 had at later stages of differentiation in the TFAP2a knockout line. The staining and protein data suggested that 2AKO cells did not survive well during keratinocyte maturation, while the 2AKO P63 cells appeared to have committed properly albeit at a slower rate relative to wildtype.
Overall, our study has provided further insights into the step-by-step mechanism by which non- neural ectoderm forms and the importance of the interaction between different transcription factors during differentiation. Deeper understanding of this pathway will help answer fundamental questions about human development that we hope to apply to tissue engineering optimization in a clinical setting in the future.