Figure 7: Acetylation-mimetic H4Q5Q12 is incorporated into centromeres even in RbAp48-deficient cells.

(a) Representative images of SNAP-CENP-A labelled with TMR-Star in RbAp48 OFF cells expressing H4_K5K12 or H4_Q5Q12. CENP-T was used as a centromere marker. While CENP-A mis-incorporation was observed in RbAp48 OFF cells expressing H4_ K5K12 (top), the mis-incorporation was not observed in RbAp48 OFF cells expressing H4_Q5Q12. Bar, 10 μm. (b) Fluorescent intensity of non-centromere signals for SNAP-CENP-A (left) and ratio of centromeric CENP-A to non-centromeric CENP-A (right), when acetyl mimetic H4_Q5Q12 was expressed. Error bars represent s.d. Asterisk indicates statistically significance (P<0.0001) by Student's t-test. (N=500). (c) Schematic of experimental design to test xCENP-A assembly in the presence of FLAG-tagged histone H4 mutants in Xenopus egg extract. Proteins, chromatin beads, RNA or calcium were added to frog egg extract at the concentrations and times indicated in the table. CENP-A chromatin beads are prepared with human CENP-A nucleosomes. After xCENP-A assembly, the chromatin was recovered and assayed for histone assembly using FLAG intensities. (d) Fluorescence images of chromatin beads after recovery and immunostaining for human CENP-A to label the input chromatin and FLAG-H4 to label newly incorporated histone H4. (e) Levels of histone H4 assembled into chromatin in the presence of CENP-A. (f) A proposed role for acetylation of H4 tail mediated by the RbAp46/48–Hat1 complex in the process of CENP-A deposition. The CENP-A–H4 complex is acetylated by the RbAp46/48–Hat1 complex before centromere deposition. The CENP-A–H4 binds the N-terminus of HJURP and middle region of HJURP recognize centromeres for centromere deposition. H4 tail acetylations facilitate this process. However, if the acetylation does not occur properly, non-acetylated tail interferes the centromere recognition for HJURP. So that, CENP-A causes mis-localization into non-centromere regions.