Dimethyl Arg3 at histone H4 tails is bound by HATs and potentiates H3 acetylation in vitro. (A) Outline of the PCAF in vitro binding experimental procedure. (B) Biotin-conjugated histone H4 tails were incubated with purified PRMT1 in the presence or absence of S-adenosyl methionine (SAM) and PRMT1 was subsequently removed from the reaction. After methylation, histone tails were incubated with 35S-labeled PCAF. The reaction mixtures were then resolved on SDS–polyacrylamide gel electrophoresis and visualized by autoradiography. (C) A total of 4 independent experiments were quantitated by densitometry. Shown are the mean ± SDM of 4 independent experiments. Significant difference by Student t test: *P < .05. (D) Outline of the experimental procedure for analyzing cross talk between dimethyl H4R3 and acH3K9/K14. (E) Purified oligonucleosomes from the PRMT1 KD MEL cells were incubated with PRMT1 in the presence or absence of SAM. After arginine methylation, PRMT1 was removed and then purified p300 and PCAF were added to the reaction mixture for 1 hour at 30°C as shown. The enrichment of dimethyl H4R3 and acH3K9/K14 was analyzed by Western blot analysis using antibodies against dimethyl H4R3, acH3K9/K14, and H3.
