Fig. 5.
Sin3p-dependent hypoacetylation of histone H4 lysine 16. (A) Level of acetylation on the histone H4 lysine residue after induction of a HO-induced DSB. Using an antibody specific to the acetylated form of histone H4 lysine 16 (Ac-K16), ChIP was performed with extracts from cells with or without HO-induced DSB in the presence or absence of Sin3p (WT or sin3Δ). PCR was done with primers specific to a region ≈2 kb upstream of the HO recognition site. For internal control, primers specific to a region ≈0.5 kb away from the end of chromosome VI-R (Tel) were used. Input represents 10% of the total extract used for immunoprecipitation. (B) Quantification of the histone H4 lysine 16 acetylation after HO DSB induction. For quantification, the ratio of the control signal to the HO signal in the immunoprecipitation samples was calculated, then this ratio was normalized to the same ratio derived from the input samples. Signals from the samples before HO endonuclease induction were assigned as one, and the change in the signal after HO induction was calculated for each experiment. The graph represents the average of at least three experiments; error bars represent the SD, and the P value was calculated by using Student's t test. Quantification was done by using the phosphoimager system and macbas 2.5 software (Fujifilm). (C) Levels of the H2A around the site of HO-induced DSB. ChIP was done with an antibody against histone H2A from cells with or without the HO-induced DSB. PCR was performed with primers described above.