Site-directed mutagenesis studies showed the greatest effect on enhancer function when either site 1 or site 4 was mutated, with activity decreasing to 40% of the wild type. Mutating multiple sites affected activity more dramatically. When sites 1, 2, 4 and 6 were mutated, activity decreased to 10% of the wild type. All combinations of multiple mutants tested displayed approximately a multiplicative reduction in activity compared to the respective single mutants, suggesting that each site is acting independently.
