FOXA factors are critical members of the developmental gene regulatory network (GRN) composed of master transcription factors (TF) which regulate murine cell fate and metabolism in the gut and liver. How FOXA factors dictate human liver cell fate, differentiation, and simultaneously regulate metabolic pathways is poorly understood. Here, we aimed to determine the role of FOXA2 (and FOXA1 which is believed to compensate for FOXA2) in controlling hepatic differentiation and cell metabolism in a human hepatic cell line (HepG2). siRNA mediated knockdown of FOXA1/2 in HepG2 cells significantly downregulated albumin (p < .05) and GRN TF gene expression (HNF4α, HEX, HNF1ß, TBX3) (p < .05) and significantly upregulated endoderm/gut/hepatic endoderm markers (goosecoid [GSC], FOXA3, and GATA4), gut TF (CDX2), pluripotent TF (NANOG), and neuroectodermal TF (PAX6) (p < .05), all consistent with partial/transient reprograming. shFOXA1/2 targeting resulted in similar findings and demonstrated evidence of reversibility of phenotype. RNA‐seq followed by bioinformatic analysis of shFOXA1/2 knockdown HepG2 cells demonstrated 235 significant downregulated genes and 448 upregulated genes, including upregulation of markers for alternate germ layers lineages (cardiac, endothelial, muscle) and neurectoderm (eye, neural). We found widespread downregulation of glycolysis, citric acid cycle, mitochondrial genes, and alterations in lipid metabolism, pentose phosphate pathway, and ketogenesis. Functional metabolic analysis agreed with these findings, demonstrating significantly diminished glycolysis and mitochondrial respiration, with concomitant accumulation of lipid droplets. We hypothesized that FOXA1/2 inhibit the initiation of human liver differentiation in vitro. During human pluripotent stem cells (hPSC)‐hepatic differentiation, siRNA knockdown demonstrated de‐differentiation and unexpectedly, activation of pluripotency factors and neuroectoderm. shRNA knockdown demonstrated similar results and activation of SOX9 (hepatobiliary). These results demonstrate that FOXA1/2 controls hepatic and developmental GRN, and their knockdown leads to reprogramming of both differentiation and metabolism, with applications in studies of cancer, differentiation, and organogenesis.
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.
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