Mutation of constrained GATA1 binding site motifs reduces enhancement more than alteration of lineage-specific ones. The left panel shows the positions of WGATAR motifs that are present in mouse plus multiple mammalian lineages (constrained, gray boxes) or present only in rodents (nonconstrained, white boxes) in six wild-type (wt) DNA segments occupied by GATA1. Mutations in each motif (labeled “ma” for mutation in motif a, etc.) are indicated by an X in the box. The enhancer activity of each wild-type DNA fragment was normalized to 1, and the difference in normalized activity (mutant − wild type) is presented in the graph. The bar (shaded to match the constraint status of the mutated motif) gives the mean of four independent transfection experiments, each assayed in duplicate to give eight measurements, with error bars showing the standard deviation.
