Figure 7.—

Yta7bd interacts with histone H3. (A) Yta7 protein preferentially interacts with histone H3. Yta7bd-GST fusion, or GST alone, was incubated with purified histones and isolated on glutathione resin. Histones associated with Yta7 were visualized by immunoblotting. Input (I) and bound (B) lanes are shown. (B) Yta7bd interacts with the N-terminal tail of histone H3. Recombinant Yta7bd, or GST alone, was incubated with streptavidin Dynabeads coated with biotinylated peptides representing different regions and modification states of histone H3 (methylation and acetylation). Bound Yta7bd was resolved by SDS–PAGE and visualized by Coomassie staining. (C) Quantification of Yta7bd binding from B. Yta7bd binding is shown as a percentage relative to H3 1-20 binding. (D) Full-length Yta7 protein interacts with the N-terminal tail of histone H3. Purified Yta7-PrA was incubated with streptavidin Dynabeads coated with biotinylated peptides representing different regions and modification states of histone H3. Bound Yta7-PrA was resolved by SDS–PAGE and visualized by immunoblotting for the PrA tag. (E) Yta7 associated histone H3 was acetylated at Lys56. Yta7-PrA was immunopurified and coenriching proteins were resolved by SDS–PAGE/Coomassie staining. Copurifying histone H3 was treated in-gel with d6-acetic anhydride to isotopically mark unmodified lysines and then digested with trypsin. Shown is a section of a mass spectrum containing the H3K56 peptide as either acetylated (K56ac) or unacetylated (K56). In accordance to the monoisotopic peak areas, H3K56 was 33% acetylated.