Figure 4

BRG1 binds to acetylated Tat. A) CEM G₁/S cell extracts (2 mg) were mixed with 100 μg of biotin-Tat peptides (aa 42–54), incubated for 2 h, and washed. Bound proteins were separated on a 4–20% SDS/PAGE and Western blotted. Lane 1, input; lane 2, unacetylated Tat peptide; and lane 3, acetylated Tat peptide. B) GST-Tat (wild-type and mutant) was purified over a glutathione column and eluted. Acetylated GST-Tat was incubated with p300 and acetyl-CoA (62), washed, and incubated with cyclin T₁/cdk9 (top) or SWI/SNF (bottom). Bound complexes were run on a 4–20% SDS/PAGE and Western blotted for the presence of cyclin T₁ or BRG1. Lane 1, wild-type Tat; lane 2, Tat with a lysine to arginine change at residues 41, 50, and 51; lane 3, wild-type Tat and p300; lane 4, mutated Tat and p300. C) GST proteins and TNT lysates containing ³⁵S-labeled Tat or BRG1 were incubated for 2 h at 4°C. Complexes were centrifuged; bound labeled proteins were denatured, subjected to SDS-PAGE, dried, and autoradiographed. D) Top: Schematic of the biotinylated HIV-1 LTR DNA used in the pull-down assay. Bottom: Immobilized chromatin HIV-1 LTR templates were incubated with CEM extract and wild-type or mutated Tat, then acetylated in the presence of GST-HAT. Samples were incubated with 100 ng SWI/SNF and all four cold nucleotides. Templates were washed and proteins were separated on a 4–20% SDS/PAGE for Western blot analysis. Bottom: remaining histones after transcription. E) CEM cells were treated with hydroxyurea and nocodazole, and samples were processed at 9 h post-release. The extracts were supplemented with purified SWI/SNF (all lanes), plus wild-type Tat (lanes 2 and 6), acetylated Tat (lanes 3 and 7), and Tat mutated at positions 50/51 (lanes 4 and 8). Anti-acetyl Tat 50/51 antibodies were added at time zero (lanes 10–12). Bottom: histone stain from immobilized DNA after transcription.