Identification of the Post-translational Modifications Present in Centromeric Chromatin

Abstract

The centromere is the locus on the chromosome that acts as the essential connection point between the chromosome and the mitotic spindle. A histone H3 variant, CENP-A, defines the location of the centromere, but centromeric chromatin consists of a mixture of both CENP-A-containing and H3-containing nucleosomes. We report a surprisingly uniform pattern of primarily monomethylation on lysine 20 of histone H4 present in short polynucleosomes mixtures of CENP-A and H3 nucleosomes isolated from functional centromeres. Canonical H3 is not a component of CENP-A-containing nucleosomes at centromeres, so the H3 we copurify from these preparations comes exclusively from adjacent nucleosomes. We find that CENP-A-proximal H3 nucleosomes are not uniformly modified but contain a complex set of PTMs. Dually modified K9me2-K27me2 H3 nucleosomes are observed at the centromere. Side-chain acetylation of both histone H3 and histone H4 is low at the centromere. Prior to assembly at centromeres, newly expressed CENP-A is sequestered for a large portion of the cell cycle (late S-phase, G2, and most of mitosis) in a complex that contains its partner, H4, and its chaperone, HJURP. In contrast to chromatin associated centromeric histone H4, we show that prenucleosomal CENP-A-associated histone H4 lacks K20 methylation and contains side-chain and α-amino acetylation. We show HJURP displays a complex set of serine phosphorylation that may potentially regulate the deposition of CENP-A. Taken together, our findings provide key information regarding some of the key components of functional centromeric chromatin.

Fig. 1.

Copurification of histones H3 and H4 with affinity-tagged CENP-A in nucleosomal and prenucleosomal fractions. A, Affinity purification of CENP-A was used to identify the post-translational modifications present on centromeric histone H3 present in neighboring nucleosomes and H4 present in either neighboring nucleosomes or the CENP-A nucleosome itself by digestion of chromatin with MNase followed by affinity purification. B, Chromatin digested with Micrococcal Nuclease (MNase) to mostly Mono-, Di- and Tri- nucleosomes was used as the input for the anti-GFP affinity purificiation (IP). Ethidium bromide stained gel of purified DNA is shown. C, Coomassie stained gel of the CENP-A LAP IP shows that the CENP-A LAP nucleosomes are isolated as complexes, which include the core histones. Recombinant (CENP-A/H4)₂ is shown for comparison. D, Western blots show quantitative amounts of histones H3 and H4 are present in CENP-A-LAP immunoprecipitated samples when compared with a mixture of recombinant histones.

Fig. 2.

Post-translational modification of CENP-A-associated histone H3. A, Histone H3 amino-terminal tail peptides and post-translational modifications identified by LC/MS (ETD) following LysC digestion of CENP-A purified chromatin. The absolute amounts of recovered peptides were estimated by comparing peptides to internal standard peptides. Signature fragment ions in MS2 spectra were used to measure the degree of methylation at specific sites on combinatorially modified peptides containing multiple methylation sites. B, Histone H3 peptides and posttranslational modifications identified by LC/MS (ETD) following trypsin digestion of CENP-A purified chromatin. Peptide identification and quantitation was as descried in A. C, Full MS spectrum of H3 A1-E50 intact tail peptide PTM forms. Expected and observed m/z ratios for +11 ions used to determine the PTM state of each peptide are shown in supplemental Fig. S3A. A highly abundant form (asterisk, box) corresponding to four methylations was selected for MS2 analysis by data-dependent acquisition and is shown in panel D. D, H3 A1-E50 tail containing four methylations was fragmented using ETD (25 ms) and observed in an averaged 13 MS2 scans. E, Fragment ion data conclusively show dimethylation of K9 and dimethylation of K27.

Fig. 3.

Centromeric chromatin is enriched for hypoacetylated H3 and limited methylation. A, Relative abundances of methylated and acetylated PTM-forms of GluC-generated H3.1 IAT found in the Asynchronous Nucleosomal sample. B, The enrichment of H3 tail PTM forms in centromeric H3 compared with bulk chromatin was compared as the log fold difference between the relative abundance of the modified peptides. Combinations of methylation and acetylation that are enriched in centromeric chromatin are indicated in red. N.D. indicates that the modified form was not detected in the analysis of general chromatin. Absent from centromere indicates that the modified form was observed in the general chromatin but not in the CENP-A associated fraction.

Fig. 4.

Post-translational modification of CENP-A-associated histone H4. A, Histone H4 peptides and posttranslational modifications identified by LC/MS (ETD) following LysC digestion of CENP-A purified chromatin from asynchronously dividing cells. The absolute amounts of recovered peptides were estimated by comparing peptides to internal standard peptides. B, Full MS spectrum showing the parent ions for histone H4 tails marked by 1–2 acetylations and 0–3 methylations. C, D, H4 S1-R23 tail generated by AspN digestion containing one acetylation and two methylations was fragmented using ETD (25 ms) and observed in an averaged 15 MS2 scans. Acetylation was localized to the N terminus and both methylations were localized to K20.

Fig. 5.

Comparison of H4 PTMs between centromeric and general chromatin. A, Relative abundances of methylated and acetylated PTM-forms of AspN-generated H4 S1-R23 amino-terminal tail peptide found in the Asynchronous Nucleosomal sample. B, Comparison of H4 tail PTM forms from bulk chromatin versus centromeric chromatin. Histone H4 tail PTM forms were compared using the log of the fold difference between the relative abundance of centromeric H4 modified forms versus those observed in general chromatin. Centromeric chromatin enriched methylation and acetylation are indicated in red. N.D. indicates that the modified form was not detected in the analysis of general chromatin. Absent from centromere indicates that the modified form was observed in the general chromatin but not in the CENP-A associated fraction.

Fig. 6.

Histone H4 present in the CENP-A prenucleosomal complex is acetylated and hypomethylated. A, Schematic showing the purification approach to identify posttranslational modification on components of the CENP-A prenucleosomal complex from mitotically arrested cells. B, Full MS spectrum of AspN digested samples showing the parent ions for H4 tails marked by 3 acetylations and 0–3 methylations. C, H4 S1-R23 tail containing three acetylations was fragmented using ETD (25 ms) and observed in an averaged 6 MS2 scans. Acetylation was localized to the N terminus, K5, and K12.

Fig. 7.

Prenucleosomal CENP-A-Bound H4 is N-terminally acetylated and lacks H4K20 methylation. A, Relative abundances of methylated and acetylated PTM-forms of AspN-generated H4 S1-R23 amino-terminal tail peptide found in the Mitotic Prenucleosomal sample. B, Comparison of H4 tail PTM forms from Mitotic Prenucleosomal CENP-A-LAP versus Asynchronous Nucleosomal H4 forms observed in this study shows H4 molecules bound to prenucleosomal CENP-A are enriched in forms that lack H4K20 methylation and contain α-N-terminal acetylation as well as acetylations at K5 and K12.

Fig. 8.

Phosphorylation of the CENP-A chaperone HJURP. A, Schematic showing the location of previously known and novel sites of phosphorylation detected in this study. The percentage phosphorylation for each site is shown below. Two peptides that contained phosphorylation simultaneously at two sites were identified. B, The phosphorylated peptides that were identified and the integrated peak area used to determine the relative degree of phosphorylation.