FOXA1 and FOXA2 together control developmental gene regulatory networks, differentiation genes, and metabolism in a human liver cell lines and differentiation genes human stem cell-derived liver progenitors.

Foxa factors (FOXA1/2/3) are master, regulatory transcription factors that are widely known to effect mouse liver organogenesis with stage-dependent effects on cell phenotypes, and play a critical role in the initiating and maintaining developmental gene regulatory networks (GRN) in murine endoderm, gut, and liver. Notably, Foxa factors and these GRN link activation of both differentiation gene and cell metabolism genes. However, despite numerous studies of Foxa factors in murine systems, it remains to be determined how both FOXA2 together with FOXA1 (which is known to compensate for FOXA2 loss) affect differentiation and metabolism in human liver cells. Here, we take an RNAi and transcriptomics approach to evaluate the effects of FOXA1/2 in human liver cells. While the siFOXA1/2 phenotype demonstrated knockdown of FOXA1/2, effects on some members of the liver transcription factors, and albumin (ALB) expression, we observed a much stronger phenotype in the case of lentiviral shFOXA1/2 knockdown, including significant downregulation of both ALB, and liver GRN (HNF4A, HEX, HNF1B, and Tbx3), consistent with reversal of differentiation state. Surprisingly, this was accompanied by accompanied by an unexpected and significant upregulation of stem cell/ germ layer markers like Nanog (pluripotency), Pax6 (neurectoderm), GATA4 (endoderm/mesoderm), and Cdx2 (intestine), as analyzed by qRT-PCR. To determine genome-wide effects of shFOXA1/2 knockdown, we performed RNA-seq of the stable shFOXA1/2 knockdown cell lines, followed by bioinformatics analysis. The data demonstrates global downregulation of liver-specific genes and unexpected upregulation of epithelial differentiation, mesodermal (cardiac), and neural genes (and confirmation of Pax6 upregulation). Interestingly, our bioinformatics analysis together demonstrated widespread changes in genes associated with mitochondrial metabolism and respiration. Our functional analysis of cellular metabolism (seahorse) demonstrated significant changes in glycolysis, including significant decreases in glycolysis, glycolytic capacity, glycolytic reserve. Furthermore, our functional analysis of mitochondrial metabolism in the shFOXA1/2 cells demonstrated significant reductions in basal respiration, maximal respiration, ATP production, and proton leak. Finally, we also demonstrate that FOXA1/2 knockdown downregulates ALB in a model of spontaneous model of liver differentiation.