To test the functional role of the putative FOXA sites in the 4E3 region, we mutated each of these sites and tested them in transient transfections in HepG2 cells. Mutation of sites 1 and 4 decreased the activity by 60% and 65%, respectively (Figure 6), whereas mutations at sites 2, 3, and 5 reduced the activity at least by 40%. Disruption of site 6 did not have a significant effect. To test the function of these sites in a combinatorial fashion, we mutated multiple sites. A double mutant of sites 1 and 4 lost most of the activity, exhibiting only 0.2-fold of the wild type enhancer. The activity was further decreased to 0.1-fold when multiple sites were mutated in conjunction with site 1 (Figure 6).
