Sp1 and chromatin environment are important contributors to the formation of repressive chromatin structures on the transfected human adenine nucleotide translocase-2 promoter

The influence of chromatin on the human adenine nucleotide translocase isof orm 2 (ANT2) promoter was investigated in transfected cells treated with th e deacetylase inhibitors butyrate and trichostatin A (TSA). Both inhibitors activated the expression of reporter plasmids transfected into HeLa cells, indicating that the promoter was suppressed by hypoacetylated chromatin an d activated by hyperacetylation. Inhibitor-dependent activation was traced to the two Sp1-activation elements within the proximal promoter region, ind icating that the Sp1 elements are repressed by chromatin structure. Repress ive chromatin structures were also formed on the promoter integrated into a stable chromatin environment, as shown by the effects of TSA and butyrate on 14 single-cell-derived NIH3T3 clones bearing the stable integrated ANT2 promoter. Both the basal expression of the luciferase reporter gene and the response to TSA and butyrate varied widely between clones. The range of ba sal expression (4000-fold) was due partially to variation in the formation of repressive chromatin, since clones with low basal expression were induce d by TSA, but those with high basal expression were less effected. These da ta indicate that chromatin environment surrounding the integrated DNA exert s a strong influence on chromatin-dependent repression of the ANT2 promoter , and that the ability of Sp1 to activate ANT2 expression is compromised in the repressed state.