FOXO1 cysteine-612 mediates stimulatory effects of the coregulators CBP and PGC1α on FOXO1 basal transcriptional activity

Hepatic production and release of metabolites, nutrients and micronutrient transporters is tightly regulated at the level of gene expression. In this regard, transcription factor FOXO1 modulates the expression of genes such as G6PC and SELENOP, encoding the catalytic subunit of glucose 6-phosphatase and the plasma selenium transporter selenoprotein P, respectively. Here, we analyzed the role of cysteine residues in FOXO1 in controlling its activity with respect to regulation of G6PC and SELENOP in HepG2 human hepatoma cells. None of the seven FOXO1 cysteines affected FOXO1 binding to DNA or its basal subcellular distribution. Whereas overexpression of wildtype FOXO1 caused a strong induction of both G6PC and SELENOP promoter activities and mRNA levels, the induction was lowered by approx. 50% if cysteine-deficient FOXO1 was overexpressed instead. Only the most C-terminal of the seven FOXO1 cysteines, Cys612, was required and sufficient to ensure full FOXO1 transactivation activity. Coexpression of FOXO1 coregulators, CBP or PGC1 alpha, had a strong synergistic effect in stimulating G6PC promoter activity and expression, fully relying on the presence of FOXO1 Cys612. Similarly, a synergistic effect of FOXO1 and CBP was observed for SELENOP. In contrast, stimulation of SELENOP by PGC1 alpha was independent of FOXO1-Cys612, due to the close proximity of a hepatocyte nuclear factor-4 alpha binding site to the FOXO1 binding site within the SELENOP promoter, as demonstrated using mutant SELENOP promoter constructs. In summary, full basal FOXO1 transactivation activity relies on Cys612, which mediates synergistic effects of coregulators, CBP or PGC1 alpha, on FOXO1 transcriptional activity. The extent of Cys612 contribution depends on the promoter context of FOXO1 target genes.