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Posttranslational Modification of CENP-A Influences The Posttranslational modification of CENP-A influences the conformation of centromeric chromatin Aaron O. Baileya,1, Tanya Panchenkob,1, Kizhakke M. Sathyanc, Janusz J. Petkowskid, Pei-Jing Paie, Dina L. Baif, David H. Russelle, Ian G. Macarad, Jeffrey Shabanowitzf, Donald F. Huntf,g, Ben E. Blackb,2, and Daniel R. Foltza,c,2 Departments of aCell Biology, cBiochemistry and Molecular Genetics, dMicrobiology, fChemistry, and gPathology, University of Virginia, Charlottesville, VA 22908; bDepartment of Biochemistry and Biophysics, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104-6059; and eDepartment of Chemistry, Texas A&M University, College Station, TX 77842-3012 Edited* by C. David Allis, The Rockefeller University, New York, NY, and approved June 13, 2013 (received for review January 7, 2013) Centromeres are chromosomal loci required for accurate segrega- of these proteins are highly divergent, sharing only 24% identity. tion of sister chromatids during mitosis. The location of the cen- Several lysine residues in the canonical H3 N terminus are highly tromere on the chromosome is not dependent on DNA sequence, conserved targets of acetylation and methylation that mediate but rather it is epigenetically specified by the histone H3 variant epigenetic regulation of local chromatin activity (12, 13). Post- centromere protein A (CENP-A). The N-terminal tail of CENP-A is translational modification (PTM) of histones is usually combi- highly divergent from other H3 variants. Canonical histone N natorial, and the net PTM status of these histones can negatively fl termini are hotspots of conserved posttranslational modification; or positively in uence transcription or direct global condensa- however, no broadly conserved modifications of the vertebrate tion of chromatin. The CENP-A N-terminal tail is enriched in CENP-A tail have been previously observed. Here, we report three arginines and lacks most of the well-characterized lysines that are fi targets for modification in histone H3, so the CENP-A tail simply posttranslational modi cations on human CENP-A N termini using fi high-resolution MS: trimethylation of Gly1 and phosphorylation of cannot share many of the modi cations to which H3 is subjected. In contrast, human CENP-A and histone H3 share similar Ser16 and Ser18. Our results demonstrate that CENP-A is subjected phosphorylation sites (Ser10 in H3 and Ser7 in CENP-A) that to constitutive initiating methionine removal, similar to other H3 are modified by the Aurora B kinase during mitosis (14–16). variants. The nascent N-terminal residue Gly1 becomes trimethy- Phosphorylation by Aurora B does not appear to be a conserved α BIOCHEMISTRY lated on the -amino group. We demonstrate that the N-terminal feature of CENP-A–containing nucleosomes because Ser7 is very RCC1 methyltransferase is capable of modifying the CENP-A N poorly conserved even within mammals. terminus. Methylation occurs in the prenucleosomal form and We provide a comprehensive analysis of the posttranslational marks the majority of CENP-A nucleosomes. Serine 16 and 18 be- modifications of human CENP-A. The initiator methionine come phosphorylated in prenucleosomal CENP-A and are phos- (Metinit) is cleaved from CENP-A, similar to canonical histones. phorylated on asynchronous and mitotic nucleosomal CENP-A The major form of the N-terminal tail of human CENP-A is and are important for chromosome segregation during mitosis. posttranslationally modified by combinations of three mod- The double phosphorylation motif forms a salt-bridged secondary ifications: trimethylation of the α-amino group of Gly1 and structure and causes CENP-A N-terminal tails to form intramolecu- phosphorylation at Ser16 and Ser18 . In vitro data show that lar associations. Analytical ultracentrifugation of phospho-mimetic CENP-A N-terminal methylation can be catalyzed by N-terminal CENP-A nucleosome arrays demonstrates that phosphorylation RCC1 methyltransferase (NRMT). CENP-A phosphorylated on results in greater intranucleosome associations and counteracts Ser16/Ser18 forms a salt-bridged secondary structure within the the hyperoligomerized state exhibited by unmodified CENP-A nu- N terminus that allows for intra- and intermolecular interactions cleosome arrays. Our studies have revealed that the major mod- and influences the conformation of reconstituted CENP-A nu- ifications on the N-terminal tail of CENP-A alter the physical cleosome arrays. Expression of a version of CENP-A that cannot properties of the chromatin fiber at the centromere. be phosphorylated results in mitotic errors. epigenetics | kinetochore | mass spectrometry Results CENP-A Is Trimethylated on the α-N Position of Gly1. Human CENP- fi fi entromeres are epigenetically inherited chromosomal loci that A nucleosomes were af nity puri ed from micrococcal nuclease (MNase)-digested chromatin from an asynchronously growing direct chromosome segregation. Kinetochores form atop C stable cell line expressing tagged CENP-A (Fig. S1). Affinity centromeres to couple microtubules to sister chromatids. Nucle- fi ∼ fi puri cation yielded 50 pmol of CENP-A nucleosomes in a osomes harboring the centromere-speci c histone-H3 variant, soluble form (Fig. S1 C and D). centromere protein A (CENP-A), are a conserved and es- Purified nucleosomes were digested into peptides using the sential component of the eukaryotic centromere. CENP-A is endoproteinases trypsin, LysC, or GluC, and then analyzed using the best candidate for epigenetically marking the location of the online reversed phase separation coupled to high-resolution MS fl centromere (1, 2). Centromeres in ies and humans are com- (Fig. S1E). We readily detected CENP-C and histones H2A and prised of discontinuous patches of CENP-A nucleosomes, in- H2B, which are known to tightly associate with CENP-A when in terspersed with H3 nucleosomes (3). CENP-A nucleosomes directly recruit CENP-C and CENP-N, which are part of a large constitutive centromere associated network (CCAN) of proteins Author contributions: A.O.B., T.P., I.G.M., D.F.H., B.E.B., and D.R.F. designed research; A.O.B., recruited to mediate the processes of kinetochore formation and T.P., K.M.S., J.J.P., P.-J.P., and D.L.B. performed research; T.P. contributed new reagents/ microtubule attachment necessary for mitosis (4–6). analytic tools; A.O.B., T.P., K.M.S., J.J.P., D.H.R., I.G.M., J.S., D.F.H., B.E.B., and D.R.F. analyzed CENP-A nucleosomes are assembled by the CENP-A–specific data; and A.O.B., B.E.B., and D.R.F. wrote the paper. assembly factor Holliday Junction Recognition Protein (HJURP) The authors declare no conflict of interest. in early G1 phase (7–10). The CENP-A–histone H4 heterodimer *This Direct Submission article had a prearranged editor. is recognized by HJURP via the CENP-A targeting domain, 1A.O.B. and T.P. contributed equally to this work. forming a prenucleosomal complex that exists during G2 and 2To whom correspondence may be addressed. E-mail: [email protected] or blackbe@ mitosis (8, 9, 11). mail.med.upenn.edu. Human CENP-A and histone H3.1 are 56% identical within This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. the histone fold; however, the unstructured N-terminal tail domains 1073/pnas.1300325110/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1300325110 PNAS | July 16, 2013 | vol. 110 | no. 29 | 11827–11832 Downloaded by guest on September 27, 2021 its nucleosomal form (Dataset S1A). Ninety-eight percent of the unmodified to an increased shift of 42 Da. High-resolution MS CENP-A primary sequence was covered by peptides that were data show the Δ42 Da species is due to the addition of three detected at greater than 500:1 signal-to-noise. This level of methyl groups (Δ42.0470 Da, 3.12 ppm mass accuracy). The sensitivity allows detection of modified forms of CENP-A pep- accurate mass measurement is not consistent with the addition of tides present at less than 0.2% of the most abundant peptide a single acetyl group (Δ42.0106 Da, 31.47 ppm mass accuracy) form (Dataset S1B). (Fig. S2 A and B). Fragmentation of the Δ42 Da species using 2 The initiating methionine (Metinit) was removed in all forms election transfer dissociation (ETD) produced an MS spectrum observed in a GluC digest (Fig. 1 A and B). Removal of the ini- that revealed Gly1 as the trimethylated amino acid. Glycine lacks tiating methionine occurs cotranslationally by methionine amino- a side-chain, so the N-terminal primary amine (α-N) is the only A–C peptidase (MetAP) activity. Because the Metinit of CENP-A is possible site of trimethylation (Fig. 1 ). CENP-A N-terminal removed, similar to all other histone H3 variants, we have trimethylation differs from known histone methylation that occurs adopted conventional histone nomenclature to refer to positions on the side chains of arginine and lysine residues. of CENP-A amino acids where the first amino acid after the NRMT directly methylates the α-amino terminus of regulator fi fi cleaved initiating methionine is designated as position 1 (17). of chromosome condensation 1 (RCC1) (18). Af nity puri ed Four different species of the CENP-A G1–E10 peptide were CENP-A shows a pattern of complete N-terminal methylation D – observed with masses differing by ∼14 Da, ranging from (Fig. 1 ). Almost 86% of the observed CENP-A G1 E10 pep- tides are trimethylated on Gly1 and incompletely methylated CENP-A (mono- and dimethylated) peptides constitute only 0.6% of the CENP-A G1–E10 peptides derived from nucleo- c +1 c +2 +3 z +2 c +2 z +1 c +1 somes. The remaining 13% of CENP-A nucleosomes are un- A 3 6 [M+3H] 7 7 4 4 370.3 385.3 427.9 456.8 463.3 513.2 526.2 modified on Gly1.
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