FIGURE 3.

Activator-dependent p300 acetylation preferentially targets the promoter region of HTLV-1 chromatin. A, shown is a schematic depicting physical separation of 208-bp 5 S-positioned mononucleosomes on the extremities from the internal promoter region of HTLV-1 chromatin. E represents an EcoRI digestion site. B, [¹⁴C]acetyl-CoA labeling and electrophoretic mobility shift assay of p300-acetylated promoter and distal nucleosomes is shown. HTLV-1 arrays (0.08 μm) were incubated with (+; 0.16 μm) and without (−) p300 and [¹⁴C]acetyl-CoA and with (0.46, 0.92, or 1.84 μm) and without (−) activators (pCREB/Tax) for 60 min at 30 °C in HAT buffer. [¹⁴C]Acetyl-CoA was removed, and HAT buffer was exchanged for EcoRI digestion buffer using a Bio-Rad spin 6 column before digestion with EcoRI for 120 min at 30 °C. Reaction products were then resolved on a native mixed 1% agarose, 2% polyacrylamide gel and analyzed by Coomassie staining on the left and phosphorimaging on the right. Migration of undigested HTLV-1 chromatin (*), digested 5 S mono- and di-nucleosomes, HTLV-1 promoter, and HTLV-1 promoter-p300-activator complexes are indicated. Relative ¹⁴C incorporation was determined by measured density (using ImageQuant 5.1) of HTLV-1 promoter and HTLV-1 promoter-p300-activator complexes normalized to 5 S mononucleosome signal in the same reaction.