Fig.3. Tam reduces Brf1 expression and lowers the occupancy of Brf1 in the promoters of Pol III genes.

(A-B) Tam decreased Brf1 expression. MCF-7 cells were starved in DMEM-F12 for 3 h and treated with Tam as indicated in Fig. 2A. The total RNA and cell lysates from these cells were extracted to determine mRNA and protein of Brf1 (A and B) and TFIIIC63 (S1) by RT-qPCT and immunoblot analysis as described previously [22]. (C-F) Tam-lowered Brf1 binding to the promoters. MCF-7 cells were treated with Tam as described above. ChIP assays were performed using Brf1 (C and E) and histone H3 (D and F) antibodies and qPCR to quantify the amplified DNA as described previous [22]. The relative occupancy of the proteins was calculated based on the control (no Tam treatment). All values shown are the means ± SE of at least three independent chromatin preparations.