Sp1 activates and inhibits transcription from separate elements in the proximal promoter of the human adenine nucleotide translocase 2 (ANT2) gene

Expression of the adenine nucleotide translocator 2 (ANT2) gene is growth regulated, We report a feature of the ANT2 promoter that involves a novel regulatory function for the Spl transfactor. We show that expression from the ANT2 proximal promoter is modulated through three Spl elements, two of which activate and one of which partially inhibits transcription, The inhibitor site, box C, is juxtaposed to transcription start (nucleotides -7 to -2), Spl bound to box C decreases transcription initiation, This was demonstrated by introducing mutations in box C which (a) increased chloramphenicol acetyltransferase expression in the transient transfection assay and (b) inhibited binding of both purified Spl and Spl in crude nuclear extracts, The activating elements (A and B boxes) are located at adjacent sites in the distal region of the proximal promoter, Mutation of either box inhibits transfection by 90%, indicating that they act in a synergistic manner, Supershift experiments with crude nuclear extracts showed that only Spl was bound to the three GC boxes, The finding that Spl acts as an activator/inhibitor within the same promoter region was verified in NIH3T3, HeLa, JEG3, and COS-1, indicating that this dual effect of Spl is widely preserved, These data suggest a unique role for Spl and raise the possibility that growth activation of the ANT2 gene is regulated by the interaction of Spl on the A, B, and C boxes.