FIG. 9.

Co-occupancy of H3 and H3.3 with histone PTMs. (A) Schematic diagram indicating the experimental protocol used in the sequential ChIP assays. Note that the antibody order was reversed for all experiments and similar results were obtained (see Fig. S6 in the supplemental material). The primers used were located in the promoter region of the CD69 and heparanase genes, as indicated. (B and C) Enrichment of DNA of the CD69 gene promoter seen when chromatin was immunoprecipitated first with either anti-H3 or anti-H3.3 antibody and then the indicated anti-PTM antibody. Data are shown for H3 (B) and H3.3 (C) in Jurkat T cells left NS or stimulated for 4 h with PI (ST). Graphs show the n-fold change in signal relative to the NS sample. (D and E) Sequential ChIP assays were performed as in panel B, except that the heparanase gene promoter was analyzed. Data are depicted as in panels B and C, with H3 data shown in panel D and H3.3 data depicted in panel E. Data pooled from two independent experiments are shown, with the mean ± the standard error plotted. Note that the H3.3-enriched K9me2 ChIPs for CD69 and heparanase have not been plotted, as no enrichments were detected above the background levels in these sequential ChIP experiments.