FIGURE 2.

FoxO1 binds to and is required for activation of CYP7A1 gene promoter. A, chromatin was prepared from primary mouse hepatocytes infected with an adenovirus expressing GFP (lanes 1 and 4) or HA-tagged FoxO1 (lanes 2 and 3 and lanes 5 and 6). Chromatin immunoprecipitation assays were performed with an anti-HA (lanes 1, 3, 4, and 6) or mouse IgG (lanes 2 and 5), and immunoprecipitates were analyzed by q-PCR. Results are expressed as -fold change in comparing the level of DNA amplification specifically precipitated by the HA antibody from the chromatin prepared from cells infected with FoxO1 relative to that infected with GFP. The level of DNA amplification precipitated by a normal mouse IgG fraction with chromatin from the FoxO1-infected cells (lanes 2 and 5) and the level of recruitment of FoxO1 to a non-relevant region of the genome at the YY1 locus (lane 6) are shown as negative controls. The data represent the mean of triplicates for two individual experiments and include error bars. Total FoxO1 expression was monitored by immunoblotting total chromatin-associated proteins with the HA epitope antibody from GFP- or HA-FoxO1-infected cells as indicated (inset). B, mouse primary hepatocytes were cultured in the absence of insulin and infected with adenovirus constructs expressing GFP or FoxO1-Δ256, a dominant negative version of FoxO1, as noted. Cells were harvested 24 h after infection, and total RNA was analyzed by q-PCR for CYP7A1 and FoxO1 and normalized to ribosomal protein L32 RNA levels as described under “Experimental Procedures.” In the comparisons marked by an asterisk, p < 0.0007 (*).