Figure 4.

Methylation of TLS could restore the inhibition of pncRNA-D–mediated histone acetyltransferase activity. A, the FLAG bead–purified, baculovirus-expressed p300 was detected by SimplyBlue^TM SafeStain for analysis. Mouse IgG served as a negative control. Star, heavy chains. B, the interaction between p300 and TLS was examined by a precipitation assay as in Fig. 3A, followed by Western blot analysis using an anti-TLS antibody. C, the interaction between p300 and TLS mutants (R476A and R481A) was examined by a precipitation assay as in Fig. 3B, followed by Western blot analysis using an anti-TLS antibody. D, p300 HAT activity was measured by incubation with WT or methylated TLS, histones, and [³H]acetyl-CoA. WT and methylated TLS were sequentially treated with MNase and then incubated with pncRNA-D for comparison of HAT activity. HAT activity was calculated as counts/min (cpm). Background reaction controls were included containing histones and [³H]acetyl-CoA excluding the p300 to measure background labeling radioactive counts. Data are presented in a box plot. n = 3. *, p < 0.01.