Fig. 3: Yta7 exclusively localizes to chromatin.

a Reaction scheme of the Yta7 recruitment assay. b Chromatin was assembled onto 2.8 kb linear DNA coupled to paramagnetic beads. For H3 acetylation, pSAGA and acetyl-CoA were added after nucleosome assembly. H2A, H3, acetylated H3, and Yta7 were analyzed by immunoblotting. For DNA controls, chromatin templates were digested with StuI, which cuts efficiently in the nucleosome-free region (NFR) of the template⁵. Proteins were removed and DNA was analyzed by agarose gel electrophoresis and ethidium bromide (EtBr) staining. For this and all subsequent experiments involving histone immunoblots, H2A and H3 levels were quantified as described in “Methods” section. c Assay of Yta7 recruitment to naked DNA. The experiment was performed as in b except without chromatin assembly. d Reaction scheme of modified Yta7 assay determining nucleosome positioning and chromatin compositing. e Chromatin was assembled as in b and included H3 acetylation by pSAGA and acetyl-CoA. After Yta7 incubation, reactions were washed with high salt (0.6 M KCl). (i) Immunoblot of H2A and H3 after treatment with high concentrations of MNase or DNA digested with StuI were analyzed as in b. (ii) Nucleosomal positioning was determined by partial MNase digestion. After digestion, proteins were removed and the nucleosomal DNA ladder was resolved through 1.3% agarose and stained with ethidium bromide. Shown are representative experiments, which have been biologically replicated three times.