Fig. 4. EGF induces H3S28ph occupancy in Brf1 and TBP promoters and enhances their expression.

(A) EGF enhances occupancy of H3S28ph in the Brf1 promoter. Schematic of the mouse Brf1 promoter and primers used for ChIP assays are designated relative to putative transcription start sites (TSS) and upstream of TSS (top). JB6 cells were treated with EGF and ChIP assays were performed using antibodies to H3S28ph and H3 and qPCR was used to quantify the amplified DNA. The relative occupancy of the proteins was calculated based on the control (no EGF treatment). (B and C) Inhibiting H3S28ph abrogates EGF-enhanced Brf1 and TBP expression. JB6 cells were transfected with WT H3 or mutant H3S28A expression plasmids for 48 hours and then treated with EGF. H3S28ph, H3, c-Myc, Brf1, TBP, TFIIIC₆₃ and β-actin were determined by immunoblot analysis. A representative blot is shown (B). RT-qPCR was performed on RNA isolated from these cells to measure Brf1, TBP, TFIIIC₆₃ and GAPDH transcripts (C). (D) Expression of mutant H3S28A reduces H3S28ph occupancy in Brf1 and TBP promoters. These cells were treated as indicated in B and C. Chromatin was extracted from these cells to perform ChIP assay with H3S28ph antibody. All values shown are the means ± SEM of at least three independent chromatin preparations.