FIG.6.

Analysis of acetylation of histones around Py ori core. (A) Schematic representation of TSPy5 with location and orientation of the Py ori core and adjacent Gal4-binding sites. Fragments used to generate the Py ori probe and SV40 ori probe for ChIP assays are indicated by broken lines. (B) Example of ChIP assays from TSA-treated or untreated cells. TSPy5 chromatin fragments immunoprecipitated with anti-acetylated H3 and H4, and DNA input (INPUT) and DNA immunoprecipitated (BOUND) analyzed by Py ori and SV40 ori probes as indicated above the lanes. Antibodies used for ChIP assays and TSA treatment are indicated to the left to the corresponding rows. (C) Average from three independent experiments represented in panel B. Immunoprecipitation ratio for each Gal4 chimera protein was represented as the ratio of bound DNA intensity to input DNA intensity quantified from slot blots. Relative immunoprecipitation ratio was measured by comparing each immunoprecipitation ratio relative to that (set as 1) for cells without TSA treatment. (D) Example of ChIP assays with acetyltransferases using Py ori probe. TSPy5 minichromosome fragments for ChIP assays from Cos7 cells infected with TSPy5 virus and transfected with expression vectors of Gal4 fusion acetyltransferases. DNA input (INPUT) and coprecipitated chromatin DNA with anti-acetyl H3 or H4 antibody (BOUND) analyzed by slot blot using Py ori probe are indicated above the lanes, and Gal4 fusion proteins are indicated at the left. (E) Average from three independent experiments represented in panel D. Relative immunoprecipitation ratio was measured by comparing each immunoprecipitation ratio relative to that (set as 1) for GalDB. IgG, immunoglobulin G.