Most IFN-γ-induced histone modifications deposited at the HLA-DRA gene are a consequence of active transcription. (A) The diagram depicts the protocol used to document the effect of DRB on IFN-γ-induced HLA-DRA gene activation. (B) The effect of DRB on the phosphorylation of Pol II was assessed by western blotting (left), whereas its effect on HLA-DRA promoter occupation by RFX (right) and CIITA (middle) was measured by ChIP. (C) HLA-DRA mRNA accumulation, the recruitment of Pol II at the promoter, and the levels of the indicated modifications were measured after 0, 6 and 12 h of induction with IFN-γ, and after 12 h of induction in the presence of DRB for the last 6 h. Results are shown for measurements made at the promoter (Pol II and R17Me2), at +300 (H4Ac, H3Ac, H3K4Me3, H3K4Me2 and H3K9Me3) and in exon 5 (H3K36Me3). Similar results were obtained at other positions in the modified regions (data not shown). The mean and SD are shown for two experiments. (D) Protein levels of the indicated histone modifying factors were measured by western blotting in cells treated as in panel C.