Acetylation of histone H4 lysine 5 and 12 is required for CENP-A deposition into centromeres

Centromeres are specified epigenetically through the deposition of the centromere-specific histone H3 variant CENP-A. However, how additional epigenetic features are involved in centromere specification is unknown. Here, we find that histone H4 Lys5 and Lys12 acetylation (H4K5ac and H4K12ac) primarily occur within the pre-nucleosomal CENP-A–H4–HJURP (CENP-A chaperone) complex, before centromere deposition. We show that H4K5ac and H4K12ac are mediated by the RbAp46/48–Hat1 complex and that RbAp48-deficient DT40 cells fail to recruit HJURP to centromeres and do not incorporate new CENP-A at centromeres. However, C-terminally-truncated HJURP, that does not bind CENP-A, does localize to centromeres in RbAp48-deficient cells. Acetylation-dead H4 mutations cause mis-localization of the CENP-A–H4 complex to non-centromeric chromatin. Crucially, CENP-A with acetylation-mimetic H4 was assembled specifically into centromeres even in RbAp48-deficient DT40 cells. We conclude that H4K5ac and H4K12ac, mediated by RbAp46/48, facilitates efficient CENP-A deposition into centromeres.

(C) The Hat1-RbAp46 complex acetylates CENP-A-H4 tetramers on histone H4. The Hat1 acetylation of histone H2A-H2B dimers (orange/red), H3.1-H4 or CENP-A-H4 tetramers (green/light green), and H3.1 or CENP-A mononucleosomes was assayed in the presence or absence of RbAp46. The top panel is an autoradiogram detecting the 14 C-acetylation of histone substrates and the bottom panel is a coomassie stain of the gel.
(E) RbAp48 binds directly to the CENP-A N-terminal tail.
(F) The xHat1/xRbAp46 complex acetylates xCENP-A-H4 tetramers on H4K5 and H4K12. Acetylation reactions were performed in the presence of Hat1 and RbAp46. Mutation of both H4K5 and H4K12 completely eliminated detectable 14 Cacetylation of H4 by Hat1. (B) Immunofluorescence with anti-CENP-C antibody in RbAp48 ON and OFF cells. Bar, 10 µm.
(C) Immunofluorescence with anti-CENP-T antibody in RbAp48 ON and OFF cells. Bar, 10 µm.
(D) Quantification method for signal intensities of centromeres and non-centromeres for SNAP-CENP-A assay used in Figure 4C. For measurement of centromere signals (SNAP-CENP-A) we followed a method described by Hoffman et al. 1 We took integrated intensities of outer area (72 pixels) and inner area (36 pixels) for each SNAP-CENP-A signal and calculated intensities according to a formula describe here. For non-centromere-region, we took integrated intensities from nucleus as inner area (36 pixels) and inner area plus adjacent non-nucleus area (36 pixels) as outer area (total 72 pixels), and then calculate intensities. Ratio of centromere signal to non-centromere-signal was plotted. When centromere signals were diffused, the ratio would be reduced. CENP-A diffused cell was defined as a cell whose centromere signals ratio to non-centromere signals is less than 1.51. Error bars represent the standard deviation (SD). Asterisk indicates statistically significance (p<0.0001) by Student's t-test. (N=100) (E) Signal intensities of centromeres and non-centromeres for SNAP-CENP-A were measured. CENP-A mis-incorporated cell was defined as a cell whose centromere signals ratio to non-centromere signals is less than 2.23. Error bars represent the standard deviation (SD). Asterisk indicates statistically significance (p<0.0001) by Student's t-test. (N=100) (F) Representative images of SNAP-CENP-A labeled with TMR-Star in RbAp48 ON or OFF cells without quench. CENP-T was used as a centromere marker. CENP-A mis-incorporation was observed in RbAp48 OFF cell. Bar, 10 µm.

(G) Percentages of mitotic cells that displayed CENP-A mis-incorporation. Definition of CENP-A mis-incorporation is in (E).
(H) Increase of sequence reads in non-centromere region in RbAp48 OFF cells based on ChIP-seq analysis with anti-CENP-A antibody.

Supplementary Figure 4. Phenotype of cells expressing Y32H RbAp48 mutant
(A) Multiple alignment for RbAp46/p48 sequences from various species. Conserved Y residue is highlighted. Y32H mutation was made.
(B) Representative images of CENP-A signals with anti-CENP-A antibody in RbAp48 OFF cells expressing wild-type RbAp48 or Y32H mutant RbAp48. Bar, 10 µm. (E) Quantification for centromere and non-centromere signals for Figure 5A. HJURP diffused cell was defined as a cell whose centromere signals ratio to non-centromere signals is less than 1.90. Error bars represent the standard deviation (SD). Asterisk indicates statistically significance (p<0.0001) by Student's t-test. (N=100) (F) Western blot analysis with anti-GFP, anti-HA, anti-CENP-A, and anti-tubulin antibodies in whole cell extracts in RbAp48 ON and OFF cells expressing GFP-HJURP.
(G) Western blot analysis with anti-CENP-A and -HJURP antibodies for IP samples with anti-HJURP in RbAp48 ON and OFF cells.