FIGURE 5.

AF17 down-regulates H3 K79 methylation in bulk histones. A, AF17 expression was substantially decreased by transfection of AF17-specific RNAi constructs. 293T cells were stably transfected with pSilencer-2.1-U6-Hygro vector (Vec) or its derivatives bearing AF17-specific siRNA#10 or siRNA#11. Total RNA was isolated and examined by real-time RT-qPCR for AF17, which was normalized to β-actin. n = 3. *, p < 0.05 versus vector. B, AF17 knockdown increases H3 K79 methylation. As in A, acid extracts of 293T cells were analyzed by IB with antibodies recognizing di- and trimethylated H3 K79 (H3me2K79 and H3me3K79), dimethylated H3 K9 (H3 me2K9), or α-tubulin. Coomassie staining was performed with an identical gel. C, AF17 overexpression impairs H3 K79 di- and trimethylation in an LMB-sensitive manner. pGFP-Dot1a was transfected into 293T cells along with pRFP vector or RFP-AF17. 16 h later the cells were treated with vehicle or nuclear export inhibitor LMB (10 nm) for another 16 h followed by IB of the acid extracts as shown in B.