Anti-silencing function 1 (Asf1) binds to H3/H4G94Pand H3/H4 lacking the C-terminal tail in vitro and in vivo. (A) H3/H4G94P (○), H3/H4∆94 (□), and H3/H4 (▼) binding to 1 nM yAsf1*532 observed by fluorescence-quenching titration. The data were fitted with a ligand-depleted binding model (Equation 1). (B) H4G94P coimmunoprecipitates with Asf1 more effectively than wild-type (WT) H4. Strains SNY091 (WT with Asf1-Myc), SNY092 (G94A with Asf1-Myc), SNY095 (G94P with Asf1-Myc), and RMY102 (WT without Asf1-Myc) were subject to anti-Myc immunoprecipitation, followed by western blotting for H3 or H4 (using antibody against H4 acetylated on lysine 12 (H4 K12Ac)). (C) Reduced amounts of H3 K56Ac in the H4G94P mutant is due to reduced overall histone levels, not reduced Rtt109 levels. Equivalent amounts of total protein extracted from strains MYC073 (WT with Rtt109-FLAG), MCY074 (G94A with Rtt109-FLAG), MCY075 (G94P with Rtt109-FLAG), RMY102 (WT) and SNY093 (WT asf1∆) were analyzed by western blotting for the indicated proteins. (D) The loss of total H3 is apparent upon repression of WT histone expression in strains carrying an integrated copy of the gene encoding H4G94P. Total protein extracts of strains W1588-4a (WT), MCY043 (asf1∆), MCY091 (H4), MCY094 (H4G94A), and MYC097 (H4G94P) were made at the indicated times after addition of glucose and western blotted for the indicated proteins. The same DNA equivalents (10 μg DNA) were loaded in each lane.