Mutational analysis of the γ-globin promoter. (A) Sequence of the γ-globin promoter between −149 and −55. The 10-bp substitution mutations are indicated below. In addition to the 10-bp mutation, M1 has one base changed upstream of −149 in a region that does not appear to be important for butyrate induction (see Fig 3). (B) Activity of mutated γ-globin promoter constructs in K562 cells treated with butyrate, trapoxin, or trichostatin. After transfection, cells were cultured for 36 hours with no addition (Untreated) or with 1 mmol/L arginine-butyrate, 2.5 nmol/L trapoxin, or 100 nmol/L trichostatin. After harvesting, CAT activity was determined in the cell extracts. The CAT activity for each mutant is expressed relative to the activity of the unmutated −198 plasmid (WT) stimulated with butyrate, whose activity is set to 100%. Each construct was tested a minimum of three times with butyrate and the height of the bars represents the mean of all experiments, with the error bars indicating the standard deviation. Each construct was tested two or three times with trapoxin or trichostatin, and a representative experiment is shown.