Figure 3: H4K5 and K12 acetylation in the pre-nucleosomal CENP-A–H4 complex are mediated by the RbAp48 complex.

(a) Comparison of levels for H4K5ac or H4K12ac in the pre-nucleosomal CENP-A–H4 complex in RbAp48 ON cells with those in RbAp48 OFF cells. (b) Comparison of levels for H4K5ac or H4K12ac in the pre-nucleosomal CENP-A–H4 complex in Mis18α ON cells with those in Mis18α OFF cells. (c) Quantification of levels of H4K5ac or H4K12ac in the pre-nucleosomal CENP-A–H4 complex in RbAp48 ON/OFF or Mis18α ON/OFF cells. Band intensities for H4K5ac and H4K12ac in a and b were normalized to CENP-A levels. (d) Comparison of levels for H4K5ac or H4K12ac in total pre-nucleosomal fraction in RbAp48 ON cells with those in RbAp48 OFF cells. (e) Immunoprecipitation with anti-HA or anti-HJURP antibodies in DT40 cells expressing HA-RbAp48, followed by western blot analysis with anti-HJURP, anti-HA, anti-CENP-A, anti-H3 and anti-H4 antibodies. (f) Affinity chromatography with histone H3.1 and CENP-A N-termini. Xenopus RbAp46/48 and xHat1 bind to the xCENP-A N-terminus. (g) xRbAp46 binds directly to the xCENP-A N-terminal tail and xHat1 depends on xRbAp46 for xCENP-A association. Tail fusions to GST and presence of Hat1 or RbAp46 is indicated on the left. Input Hat1 and RbAp46 proteins are indicated. (h) The xHat1–xRbAp48 complex acetylates xCENP-A–H4 tetramers on H4K12. Acetylation reactions were performed in the presence of Hat1 and RbAp48. Mutation of H4K12 eliminated detectable ¹⁴C-acetylation of H4 by Hat1. The top panel is an autoradiogram detecting the ¹⁴C-acetylation of histone substrates and the bottom panel is a coomassie stain of the gel.