UC San Diego

During pancreas development, undifferentiated cells must undergo stepwise progression toward particular cell fates. The emergence of the distinct cell types of the pancreas relies on the capacity of lineage intermediates to properly interpret and respond to environmental inductive cues, an ability termed developmental competence. Enhancers have been shown to be critical in spatiotemporal gene regulation during development. However, how enhancers acquire the ability to

translate signals from the extracellular environment into cell-type-specific transcriptional responses during development is poorly understood. Epigenetic priming and activation of enhancers by the pioneer transcription factors FOXA1 and FOXA2 has been proposed to confer developmental competence in various stages of pancreas lineage specification. We have previously shown that enhancers are first recognized by the pioneer transcription factors FOXA1 and FOXA2 when competence is acquired. To elucidate the roles and requirement of FOXA1 and FOXA2 for pancreas differentiation and enhancer priming, human embryonic stem cell (hESC) lines harboring shRNAs targeting FOXA1 and FOXA2 or were generated. Using differentiation of these hESC lines as a model for pancreas development, we employed qRT-PCR, ChIP-qPCR, and protein detection methods to address the effect, if any, the loss of these transcription factors have on pancreas differentiation. . As a follow up, CRIPSR/Cas9-mediated gene editing was also employed to generate FOXA1/2 knockout lines in order to serve as a reliable platform for future experiments.