Foxa1/2 downregulates liver differentiation markers and the endoderm and liver gene regulatory network in human stem cells and in a human stable liver cell line

Foxa2 has garnered considerable interest in its pioneer functions and its role endoderm gut, and liver differentiation, and development, and initiator of gene regulatory networks (GRN) in these tissues. Although Foxa2 has been investigated in these systems, Foxa1 also compensates for its function, and thus may also have compensatory effects in studies of Foxa2 regulation. In this study, we focus on the role for both Foxa1 and Foxa2 in controlling endoderm GRN, liver activation in gut tube, and liver GRN. We first compare endoderm induction protocols, and develop a novel model of liver induction under hypoxic conditions that relies on minimal growth factors and enhanced morphology. We employ an RNAi (siRNA and shRNA) approach to demonstrate the effects of Foxa1/2 on endoderm induction, gut tube activation of the albumin gene. Our data demonstrates widespread regulation of the endoderm and mesendoderm GRN, and albumin, which is significant for activation of the liver differentiation program. We then analyze the Foxa1/2 phenotype in stable liver cell lines, and engineer stable cell lines that demonstrate potential reversibility of cell state. Finally, we perform RNA-seq and bioinformatics analysis that demonstrates global GRN changes and transcription changes due to Foxa1/2 perturbation, including expansive changes in cellular differentiation and metabolism. These data suggest that Foxa1/2 phenotype has time-dependent effects on GRN and widespread effects on GRN, the liver transcriptome, and liver metabolism.