Afr2 regulation occurs cell-autonomously in vitro but is not conferred on episomal DNA in transient assays

Oncofetal antigens such as alpha-fetoprotein (AFP) are expressed in regenerating liver. The level of AFP gene expression during liver regeneration is regulated by the unlinked, autosomal gene, Alpha-fetoprotein regulator 2 (Afr2). C3H/HeJ (Afr2A/A) mice express 10-fold higher levels of AFP than C57BL/6J (Afr2B/B) mice. Here we show that primary hepatocytes isolated from C3H/HeJ and C57BL/6J mice exhibit differential expression of the endogenous AFP gene, which was attributed to the Afr2 gene locus and indicative of a cell-autonomous mechanism. We show that the Afr2-Response Element (ARE), between 1010 and 838 base pairs upstream of the AFP transcriptional start site, did not modulate reporter gene expression in transfection assays of Hep G2, Hep 3B, Hepa 1.6, and HeLa cell lines. Reporter gene expression in transiently transfected primary hepatocytes was also ARE-independent. Finally, gene expression from reporter constructs delivered by hydrodynamics-based transfection to the livers of C3H/HeJ and C57BL/6J mice after CCl4-induced liver regeneration was ARE-independent. In conclusion, ARE-dependent transcription was not found in transient assays performed in three different systems, two of which retained regulation of the endogenous AFP gene, suggesting that the ARE may not function as a simple transcription factor recognition site.