Fig. 5

Co-localization and physical interaction of Brpf1 and Moz. (A-D) Brpf1 co-localizes with Moz. Immunofluorescent staining of interphase HEK 293 cells after co-transfection with the indicated versions of GFP-Brpf1 (left panels; green) and FLAG-Moz (middle panels; red), counterstained with DAPI (for DNA; blue); merged images are shown in right-hand panels. Full-length Brpf1 co-localizes with wild-type Moz (A) and with HAT-negative Moz-G675E (B) in a punctate pattern on interphase nuclei. Co-localization is abolished when Brpf1 is N-terminally truncated (C). N-terminal fragment of Brpf1 co-localizes with Moz, but displays a more diffuse distribution (D). (E,F) Schematic structures and co-localization/immunoprecipitation properties of full-length Brpf1, full-length Moz (E), and the various truncations used (F). G-I) Brpf1 physically associates with Moz. (G) Co-IP of full-length Brpf1 and wild-type or HAT-negative Moz(G657E) from co-transfected cells with anti-FLAG (Moz) antibody (left) or anti-HA (Brpf1) antibody (right), analyzed in western blots (upper panels) with the specified antibodies, or assayed for HAT activity on core histones (lower panels). (H) Co-IP of full-length FLAG-Moz and various GFP-Brpf1 deletion constructs with anti-FLAG or anti-GFP antibodies, followed by analysis of complex formation (upper panel) and control for Brpf1 expression levels (lower panel) via anti-GFP western blotting. (I) Co-IP of full-length Moz or C-terminally truncated MozN and various HA-tagged versions of the N-terminal fragments of Brpf1 using anti-FLAG antibody, analyzed in anti-HA western blots. Lower panel shows input control. Brpf1 aa 1-245 fragments that have histidine or cysteine mutations in the zinc-finger domain can still co-precipitate with Moz (lanes 5, 6), whereas the aa 1-149 fragment with an intact zinc finger cannot (lane 4). Scale bars: 5 μm in B; 2.5 μm in D.