Pancreatic and duodenal homeobox gene 1 (Pdx1) down-regulates hepatic transcription factor 1 alpha (HNF1α) expression during reprogramming of human hepatic cells into insulin-producing cells

Ectopic expression of Pdx1 triggers rapid hepatocyte dedifferentiation by down-regulating liver-enriched transcription factors and liver-specific functional genes such as hepatic nuclear factor-1 alpha (HNF1 alpha), albumin, and AAT. However, the links between Pdx1 over-expression and hepatic gene down-regulation are incompletely understood. HNF1 alpha and HNF4 alpha are important transcription factors that establish and maintain the hepatocyte phenotype. The human HNF4 alpha gene contains two promoters (P1 and P2) that drive expression of P1-(HNF4 alpha 1-6) or P2-(HNF4 alpha 7-9)-derived isoforms, which are used in different tissues and at different times during development. We hypothesized that the relative expression of HNF1 alpha and HNF4 alpha following ectopic Pdx1 expression may promote hepatic cell dedifferentiation and transdifferentiation toward pancreatic beta-cells. We produced lentiviruses expressing Pdx1, Pdx1-VP16, and Ngn3, along with dual-color reporter genes to indicate hepatic and pancreatic beta-cell phenotype changes. Using these PTF alone or in combinations, we demonstrated that Pdx1 not only activates specific beta-cell genes but down-regulates HNF1 alpha. Pdx1-mediated reduction of HNF1 alpha is accompanied by altered expression of its major activator, HNF4 alpha isoforms, down-regulating hepatic genes ALB and AAT. Pdx1 up-regulates HNF4 alpha via the P2 promoter. These P2-driven isoforms compete with P1-driven isoforms to suppress target gene transcription. In Huh7 cells, the AF-1 activation domain is more important for transactivation, whereas in INS1 cells, the F inhibitory domain is more important. The loss and gain of functional activity strongly suggests that Pdx1 plays a central role in reprogramming hepatocytes into beta-cells by suppressing the hepatic phenotype.