Fig. 9
Swi1 interacts with Vid21, a regulatory subunit or the NuA4 complex, and is involved in efficient acetylation of histone H4. a Two-hybrid interaction between Swi1 and Vid21. Swi1 fused to Gal4-DBD (DNA-binding domain) was tested in the Y190 reporter strain for interaction when combined with Vid21 (C-terminal regions: amino acids 593-985) fused to Gal4-AD (activation domain). Y190 cells harboring Swi1-DBD and Vid21-AD were able to grow in the presence of 3-aminotrizole (3AT). Swi3-AD and Sbp6-AD (Swi1-binding protein 6) were used as positive controls that interact with Swi1. b The indicated GST-fusion proteins were expressed in and purified from S. pombe cells. Glutathione Sepharose beads bound to the indicated GST-fusion proteins were incubated with recombinant Vid21593-985-6xHis. The beads were washed and analyzed by Western blotting using the anti-6xHis antibody. Asterisk shows non-specific bands. c Exponentially growing cells of the indicated genotypes were treated with 100 µg/ml of CHX for the indicated times. Whole-cell extracts were prepared at the indicated times and probed with the anti-FLAG M2 antibody. Western blotting of tubulin was performed as a loading control. Representative images of repeat experiments are shown. d Western blotting of whole-cell extracts was performed using the anti-H4 and anti-acetyl (K5/8/12/16) histone H4 antibody. Quantification of H4 acetylation was performed using Image J software. The level of H4 acetylation was normalized to the total H4 level in each cell extract. Error bars correspond to standard errors of mean (SEM) obtained from three independent experiments