In vivo mapping of the human adenine nucleotide translocator-2 (ANT2) promoter provides support for regulation by a pair of proximal Sp1-activating sites and an upstream silencer element

Regulatory factors bound to the human adenine nucleotide translocator-2 (AN T2) promoter have been mapped in HeLa cells by in vivo DNase I protection a nd ligation-mediated PCR amplification. Protein binding was detected at onl y three sites within the extended promoter region (to nt - 703). One, start ing at nt -61 and covering the TATA box and transcription start, most proba bly represents occupation by the transcription-initiation machinery. A repe ated Spl element determined by in vitro studies to be the major activation element for the promoter was also protected in vivo on nucleotides responsi ble for strong binding to the zinc fingers. Occupation of two additional up stream Sp1 elements was not observed. The third site occupied in vivo was i dentified previously by in vitro studies as a unique silencer element. Trea tment of cells with trichostatin A to induce hyperacetylation released the silencer-binding protein after 1 h, but had no effect on the Sp1-activating elements. Prolonged treatment (24 h) displaced Sp1 from the activating ele ments. These findings confirm and extend in vitro studies indicating that r egulation of the ANT2 promoter is most probably exerted through a single pa ir of proximal Sp1-activating elements and an upstream silencer, and that c hromatin organization plays a role in the interaction between the two.