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Posttranslational modification of CENP-A influences the conformation of centromeric

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 , 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 are highly divergent, sharing only 24% identity. tion of sister during . The location of the cen- Several residues in the canonical H3 N terminus are highly tromere on the is not dependent on DNA sequence, conserved targets of and that mediate but rather it is epigenetically specified by the H3 variant epigenetic regulation of local chromatin activity (12, 13). Post- A (CENP-A). The N-terminal tail of CENP-A is translational modification (PTM) of 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 and lacks most of the well-characterized that are fi targets for modification in , 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 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. 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 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.

| | 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. form atop C stable cell line expressing tagged CENP-A (Fig. S1). Affinity 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– 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 ; 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 that occurs adopted conventional histone nomenclature to refer to positions on the side chains of 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. This predominant trimethylation is consistent z +1 z +2 with NRMT-mediated methylation, which transfers single methyl 3 8 groups in a processive manner (19). 357.3 534.8 To determine whether the CENP-A N terminus was a direct c +2 +2 5 c9 substrate for NRMT in vitro, we engineered a recombinant 341.9 z +1 575.6 CENP-A tail (G1–E10) in which Gly1 was exposed for modifi- x0.6 5 60 600.2 cation by NRMT by Factor X cleavage to recapitulate the +4 CENP-A tail following Metinit removal (Fig. 1E). The wild-type 50 [M+4H] [M+2H]+2 CENP-A amino tail (GPRR) was robustly methylated by NRMT 321.2 3 40 641.2 in vitro, using H-S-adenosyl methionine (SAM) as a substrate, z +1 +2 6 whereas CENP-A mutant sequences, which should be defective 30 c 756.3 α 4 c +1 z +1 substrates for -N methylation (GPQR and GPQQ), showed 20 263.8 5 7 little or no methylation (Fig. 1F) (18, 19). MS analysis of the in +1 682.5 +1 913.6 +1 c2 c z8 vitro NRMT-methylated peptide shows that Gly1 is the only eaieAbundance Relative 10 6 214.2 769.6 1069.7 substrate and is completely trimethylated, consistent with the 0 predominant trimethylation observed from in vivo-purified CENP-A 150 250 350 450 550 650 750 850 950 1050 C D m/z (Fig. S2 and ).

B fragments 1 2 3 4 5 6 7 8 9 C memmee CENP-A N-Terminal Tail Is Doubly Phosphorylated at Ser16 and Ser18. c+2 Purification of CENP-A from interphase cells and subsequent c+1 M GPRRRSRKPE MS analysis identified two phosphorylations of the CENP-A N- 0 1 10 terminal tail. Trypsin digestion produced a CENP-A fragment G(me3) P R R R S R K P E +3 100 that encompassed residues R14–R27. The observed [M + 3H] +1 D m/z z 80 ion species ( 551.5814) was 159.9320 Da larger than the z+2 fi m/z 60 calculated mass of the unmodi ed peptide ( 498.2708), which fragments 9 8 7 6 5 4 3 2 1 is consistent with a doubly phosphorylated peptide (−0.54 ppm). 40 Relative Two large-scale proteomic studies reported detection of a Abundance F 40000 20 similar doubly phosphorylated CENP-A peptide; however, au- 0 tomated MS2 spectra interpretation was unable to distinguish 30000 _ me me me which of the five possible sites within the CENP-A peptide were MP me me 20000 me fi c z –

C modi ed (20, 21). Our analysis of and ions in the R14 R27 2 CENP-A fi fi 10000 ETD MS spectra was of insuf cient coverage to con dently E G1-E10 GFP assign the phosphates to the correct amino acids due to the high 0 concentration of prolines, which do not produce c and z frag- NRMT + + + + _ GPRR _ . . . IEGR . . . ments using ETD. To identify the sites of phosphorylation, we CENP-A wt QR QQ wt a y -GFP Factor X cleavage site took advantage of and ions, which are produced at prolines using ETD, although these ions are less abundant than c and z Fig. 1. CENP-A is trimethylated on the α-amino position of the N-terminal ions (22). The total ion fragment data definitively demonstrated glycine. (A) CENP-A GluC digestions produced an ETD MS2 spectrum of an N- that Ser16 and Ser18 of CENP-A are the sites of phosphorylation terminal peptide trimethylated on the α-N position of glycine 1. Value above (Fig. 2 A and B). Singly phosphorylated and unmodified species fi B α bracket indicates magni cation. ( ) Sequence coverage of -N trimethylated were observed at much lower frequency, 10% and 1%, re- 2 C CENP-A ETD MS spectrum. ( ) GluC cleavages at E10 to produce an N- spectively, relative to double phosphorylated peptides (Fig. 2C). terminal peptide that had been subjected to proteolytic initiating methionine In the 10% of cases where CENP-A contained only a single removal in vivo. A conventional histone nomenclature is adopted for CENP-A ∼ where the initiator methionine is “Met0.” (D) Integrated chromatographic phosphate, 80% of the CENP-A peptides were phosphorylated peak areas of CENP-A G1–E10 methylated forms. Orange hexagons, α-N only on Ser16, and 20% were phosphorylated on Ser18. methylation. (E) A fusion protein containing amino acids 1–10 of CENP-A was α engineered to reveal Gly1 by Factor X cleavage to produce a substrate for CENP-A Is Combinatorially -N Methylated and Phosphorylated. NRMT modification. (F) CENP-A fusion protein was methylated using Combinatorial patterns of PTMs form complex recognition sig- recombinant NRMT and 3H-SAM. Wild-type CENP-A N termini (GPRR-) and nals that underlie a wide range of epigenetic phenomena. To mutants in which R3 and R4 were replaced by glutamine (GPQR- or GPQQ-) determine what combinations of CENP-A α-N methylation and were tested. P < 0.01, Student’s t test, n = 3 independent experiments ± SD. phosphorylation exist on the same protein, we used LysC to

11828 | www.pnas.org/cgi/doi/10.1073/pnas.1300325110 Bailey et al. Downloaded by guest on September 27, 2021 A x20 x100 x20 x50 x20 x10 conclude that these forms become degraded or are not stably +1 +1 +1 +1 +3 c +2 c +1 z +1 +1 a z incorporated in chromatin. y5 [M+3H] 4 [M+2H] 10 12 y12 13 13 514.8 551.9 594.3 827.3 1254.6 1325.7 1341.8 1364.7 1480.5 Prenucleosomal CENP-A was α-N trimethylated; however, the +1 z +1 +1 a +1 c +1 α a3 6 c6 12 13 level of -N trimethylation was lower in prenucleosomal CENP-A 598.3 858.4 1495.7 100 453.0 y +1 1364.7 compared with asynchronous CENP-A nucleosome populations. 11 +1 1173.5 [M+1H] ∼ 90 +1 1654.7 Unmethylated forms were present in a ratio of 2:1 compared y4 +1 +1 a6 c12 416.4 +1 with trimethylated forms. This ratio is nearly inverse to the de- 80 y +1 812.8 10 1408.6 y 1076.5 13 gree of α-N methylation found in asynchronous nucleosomal 70 z +1 1496.7 4 z +1 +1 CENP-A. Phosphorylation levels of prenucleosomal CENP-A 400.2 8 y9 60 796.3 909.4 +1 +1 were identical to CENP-A within the nucleosome, with a ratio of z10 a z +1 10 fi 50 1 a +1 1060.3 1210.5 1:10:100 for unmodi ed and singly and doubly phosphorylated 159.1 5 716.8 +1 c8 40 1056.5 y +1 y +1 Relative Abundance 1 3 30 174.2 359.3 +1 +1 y7 a8 711.5 1011.9 z +1 100 20 2 A meem me P P 246.1 90 10 y +1 2 * MGPRRRSRKPEAPRRRSPSPTPTPGPSRRGPSLGASSHQHSRRRQGWLK * P 262.1 80 0 1 16 18 48 P 0 me meem 200 400 600 800 1000 1200 1400 1600 70 13C x 3 m/z PP 512.1838 100 60 13C x 3 z = +11 B C 508.3617 1.32 ppm me meem P fragments 1 2 3 4 5 6 7 8 9 10 11 12 13 80 50 z = +11 13C x 3 2.02 ppm a+1 60 P tive Abundance 40 me 504.9135 me em 13 c+1 a C x 3 z = +11 40 unmodified 13C x 3 501.0910 0 ppm Rel 30 RRS(ph) PS(ph) PTPTPGPSR Relative 13C x 3 497.6446 z = +11 CENP-A 14 16 18 27 Abundance 20 493.8220 z = +11 0 ppm residue y +1 20 z = +11 1.63 ppm 0 1.34 ppm z +1 10 fragments 13 12 11 10 9 8 7 6 5 4 3 2 1 _ P P P 0 492 494 496 498 500 502 504 506 508 510 512 BIOCHEMISTRY m/z Fig. 2. The CENP-A N-terminal tail is doubly phosphorylated at Ser16 and Asynchronous Nucleosomal A 2 – Ser18. ( ) ETD MS spectrum of doubly phosphorylated CENP-A R14 R27. B erAkaeP CENP-A G1-K48 C 100 meem Value at bracket indicates magnification level for region of spectrum. (B) me P P Sequence coverage of an MS2 spectrum of a trypsin-generated CENP-A 80 60 peptide R14–R27. Signature a, c, y, and z ions indicate the presence of HUMAN GPRRRSRKPEAPRRRSPSPTP C fi 40 BOVINE MGPRRQKRKPETPRRRPASPAP phosphate at Ser16 and Ser18. ( ) CENP-A is unmodi ed and singly or tive a 20 MOUSE MGPR---RKPQTPRRRPSSPAP doubly phosphorylated in a ratio of ∼1:10:100, respectively. Green ovals la CHICK -MPR---PKPRSPRRRGRPPPA

Re 0 ZFISH -MPRHHKRKPSTPRRRSPPASL represent degree of phosphorylation on peptide. me me me me me me ^TSA G1 methyl ___me me me _ _ XEN TR MRPGSSRRKSRPPRRVSPPLPT ^ P P P S16/S18 P P P TPA phos generate a peptide of the entire N-terminal tail of CENP-A. The PTM combination most abundant of the CENP-A G1-K48 peptide was a combina- fi α Mitotic Mitotic torially modi ed peptide that included -N-trimethylation and D Mito. E F Pre-nucleosomal Pre-nucl. vs. Nucleosomal phosphorylation of Ser16 and Ser18 (Fig. 3 A and B). We con- Pre-nuc. fi Mito. CENP-A M0-K48 CENP-A G1-K48 rmed the identity of this peptide by performing a targeted ETD Nuc. 100 100 2 A–C 1 Pre-nuc MS analysis (Fig. S3 ). CENP-A trimethylated, doubly 3 Asyn. 80 80 phosphorylated peptides were threefold more abundant than the Nuc. 60 Nuc M 60 unmethylated, phosphorylated peptides in three independent 2 G2 G1 40 40 CENP-A nucleosome purifications. Modifications reside in regions S 20 20

C relative to G1-K48

of the CENP-A tail that are well conserved in vertebrates (Fig. 3 ) Relative Peak Area fi 0 Relative Peak Area 0 suggesting that these modi cations may occur in several species. ___ac ac ac me me me M0 acetyl ___me me me G1 methyl me me me _ _ _ _ S16/S18 P P P P P P S16/S18 P P P P CENP-A N-Terminal Tail Is Combinatorially Modified Before Nucleosome P P phos phos Deposition. The CENP-A prenucleosomal complex accumulates PTM combination PTM combination during mitosis and is recruited to centromeres in G1 phase (Fig. Fig. 3. CENP-A N-terminal tails are marked with combinations of stable 3D). To identify the modifications of prenucleosomal CENP-A, phosphorylation and α-N methylation. (A) LysC digestion yields a CENP-A we purified CENP-A from the chromatin-free extract of mitoti- G1–K48 peptide. A full MS spectrum (17 averaged scans) shows combina- cally enriched cells (Fig. S4 A–D). We found high levels of torial α-N trimethylation and phosphorylations of the same peptides in HJURP peptides and did not observe peptides derived from asynchronously cycling cells. Accurate mass and charge state is reported for histones H2A and H2B. These results are consistent with ex- the 13C x 3 isotopes. The most abundant form is trimethylated at Gly1 and clusive purification of the CENP-A prenucleosomal complex (8) doubly phosphorylated at Ser16 and Ser18. Green oval, phosphorylation; (Dataset S1C). orange hexagon, methylation. (B) Integrated chromatographic peak areas of We generated intact N-terminal tail peptides from pre- CENP-A G1–K48 PTM forms. n = 3 independent biological replicates. Error bars C nucleosomal CENP-A using LysC (Dataset S1D). Ten percent of represent SEM. ( ) Comparison of vertebrate CENP-A protein sequences reveals conservation at observed PTM sites: N termini (blue), proline–arginine prenucleosomal CENP-A tails retained the initiating methio- (red), and serine (green). (D) Soluble and chromatin fractions of cells blocked nine, and a small fraction of this group was α-N acetylated on E A A B in mitosis were used to purify populations of (circle 1) prenucleosomal, and Met0 (Fig. 3 and Figs. S5 and S6 and ). The presence of nucleosomal CENP-A from (circle 2) asynchronously-dividing cells and (circle 3) M0 is in contrast to the nucleosomal CENP-A where all of the mitotic cells. Integrated chromatographic peak area quantified CENP-A tail α peptides were subjected to Metinit removal and no -N group PTM forms: (E) prenucleosomal containing Metinit;(F) prenucleosomal and were observed. Because Metinit-containing forms mitotic nucleosomal fractions lacking Metinit. Acetylation (blue square) was were not observed in analysis of nucleosomal CENP-A, we observed only when Metinit was present.

Bailey et al. PNAS | July 16, 2013 | vol. 110 | no. 29 | 11829 Downloaded by guest on September 27, 2021 forms (Fig. 3F, Fig. S5B). The same pattern of phosphorylation by Ser16 and Ser18 phosphorylation contribute to the protection was also detected for all Metinit-containing forms. of the P1–P1′ and P2–P1 bonds. To determine whether trypsin inhibition was an intrinsic property of the peptide and dependent Fraction of α-N Trimethylated CENP-A Increases with Cell Cycle on phosphorylation, we digested a synthetic CENP-A E10–G24 Progression. Centromere-bound nucleosomal CENP-A in mito- peptide using trypsin. At 19 h, trypsin digestion of the un- sis has existed for a minimum of one cell cycle. For this reason, modified peptide resulted in equal parts zero- and one-missed- mitotic nucleosomal CENP-A represents an “old” population of cleavage forms (Fig. 4C). During the same amount of time, CENP-A protein (Fig. 3D). To determine the modification state trypsin digestion of a doubly phosphorylated peptide produced of CENP-A during mitosis, we isolated CENP-A nucleosomes only the two-missed-cleavage form, R14–G24. from nocodazole-treated cells (Fig. S4 A–D). Analysis of our Arginine–phosphoserine electrostatic salt bridging is highly mitotically enriched nucleosomal CENP-A sample identified stable as both arginine and phosphoserine are ionized at physi- significant amounts of H2A, H2B, H3, H4, and CENP-C, but no ological pH (25). We reasoned that arginine–phosphate salt HJURP peptides (Dataset S1E), consistent with purification of bridges could form intramolecular interactions within CENP-A an exclusively nucleosome population. N-terminal α-N methyl- tails and intermolecular interactions between individual tails. ation and phosphorylations on mitotic CENP-A was observed on Therefore, we analyzed the conformation of doubly phosphory- the same sites as in mitotic prenucleosomal and asynchronous lated E10–G24 synthetic peptide using ion mobility-mass spec- nucleosomal CENP-A populations. Mitotic nucleosomal CENP-A trometry (IM-MS). IM-MS drift time measurements are used to was almost entirely trimethylated; less than 10% of CENP-A calculate the ion-neutral collision cross-section (CCS) of peptide forms were unmethylated (Fig. 3F, Fig. S5C, Dataset S1F). ion conformations (26). Unmodified E10–G24 CENP-A peptides + ([M + 3H] 3 ions) exhibit CCS profiles that deviate from a trend Tandem Phosphorylation Induces Secondary Structure in CENP-A N line of randomly coiled peptides by −7.6% (major peak), which Termini. We noted a correlation between Ser16/Ser18 phos- indicates that E10–G24 exists in an already-compact conforma- phorylation and trypsin missed cleavages within CENP-A pep- tion (Fig. 4 D and E). Phosphorylated E10–G24 exhibits a CCS tides (Fig. 4 A and B). Unphosphorylated CENP-A peptides profile that deviates −11.0% (major peak) from randomly coiled were efficiently cleaved by trypsin at the P1–P1′ cleavage site to peptides (Fig. 4 D and E). Therefore, the conformation of produce the S16–R27 peptide. In contrast, singly phosphorylated phosphorylated E10–G24 peptide is further compacted relative peptides were observed as a mixture of zero-, one-, and two- to the unmodified E10–G24, consistent with intramolecular salt missed-cleavage peptides. Peptides phosphorylated at both Ser16 bridging of phosphorylated CENP-A N termini producing a and Ser18 were almost exclusively detected in the two-missed- more compact structure. cleavage form. The P1–P1′ bond of R15–S16ph should be To assess intermolecular interactions mediated by CENP-A cleaved at a lower rate because it is followed by an acidic residue tail phosphorylation, we analyzed synthetic E10–G24 peptide for in the P1′ position (23, 24), and the P2–P1 bond between should potential dimers by MS. Noncovalent dimeric and monomeric be the preferred site. Instead, we observed that the P3–P2 bond species have identical m/z values but can be distinguished by is the primary site of cleavage. Structural constraints can in- their isotopic peak pattern. Separation of isotopic peaks of a fluence the efficiency of trypsin cleavage (25), and we hypothe- species is dictated by charge state, and calculating the molec- sized that one or more arginine–phosphate salt bridges formed ular weight at a given m/z value reveals stoichiometry. Doubly

ABP3 - P2 - P1 - P1’ - P2’ - P3’ CENP-A-LAP 100% P2 P1 P1’ Expected cleavage site 80% 14 15 16 27 % Fig. 4. CENP-A Ser16/Ser18 phosphorylation forms 60 RRSPSPTPTPGPSR C CENP-A E10-G24 a compact secondary structure. (A) Nomenclature P2 P1 P1’ 40% 100 RSPSPTPTPGPSR B 10 14 15 16 24 % for proteolytic sites. ( ) The percentage of trypsin 80 20 SPSPTPTPGPSR EAPRRRSPSPTPTPG cleavage at the P1–P1′,P1–P2, and P2-P3 sites for 60 Relative Abundance 0% 40 RRSPSPTPTPG _ peptides derived from affinity-purified CENP-A. (C) RSPSPTPTPG P P 20 P The percentage of trypsin cleavage at the P1–P1′, 0 SPSPTPTPG – – fi NT 0 0.25 1 419 D CENP-A E10-G24 CENP-A E10-G24 + 2 phos P1 P2, and P2 P3 sites for unmodi ed and phos- Gaussian Fit Gaussian Fit phorylated synthetic CENP-A peptides E10–G24 CENP-A E10-G24 + 2phos P2 P1 P1’ Peak Sum Peak Sum digested with trypsin over time were followed by 100 10 14 15 16 24 Major D 80 100 Major 100 liquid chromatography (LC)-MS. ( ) IM-MS analysis + Relative Abundance EAPRRRS(ph)PS(ph)PTPTPG 60 + 3 fi RRS(ph)PS(ph)PTPTPG of the monomeric [M 3H] ion of unmodi ed 40 Minor – 20 RS(ph)PS(ph)PTPTPG and doubly phosphorylated E10 G24 peptide is % 0 S(ph)PS(ph)PTPTPG % used to calculate the ion-neutral collision cross sec- NT 0 0.25 1 4 19 Minor tion (CCS) for each species. CCS values for the major-

Hours Trypsin Digest Relative Abundance Relative Abundance 0 0 and minor-contributing conformers were calculated 300 320 340 360 300 320 340 360 as Gaussian peaks from the sum of each peptide. (E) 2 E F CCS (Å2) CCS (Å ) Tryptic digested BSA peptides analyzed by IM-MS CENP-A E10-G24 are plotted according to CCS and molecular weight. CENP-A E10-G24 + 2 phos Comparison of phosphorylated (square) and un- [M + 3H]+3 BSA tryptic peptides modified (triangle) E10–G24 peptide with the ran- Random coil linear trend dom coil trend line reveals that phosphorylation

380 causes greater compactness relative to the increase y = 0.1022x + 183.11 F fi R² = 0.9584 in molecular weight. ( ) AUC pro les of in vitro as- 360 sembled chromatin arrays showing differential MgCl2- ) 2 dependent folding between H3, CENP-A, and phos- 340 -11.0% -7.6% Major Major pho-mimetic (S16D/S18D) CENP-A arrays. All array

CCS (Å types (minus MgCl ) have overlapping sedimentation 320 -13.9% 2 -9.8% Minor fi Minor pro les, indicating that they are saturated to the same 300 extent. Therefore, the observed differences upon ar- 1200 1400 1600 1800 2000 ray folding are exclusively due to the contributions of MW (Da) different proteins.

11830 | www.pnas.org/cgi/doi/10.1073/pnas.1300325110 Bailey et al. Downloaded by guest on September 27, 2021 phosphorylated E10–G24 peptide was present as highly abun- Phosphorylation of CENP-A Is Required for Proper Chromosome + + dant monomeric species ([M + 2H] 2 and [M + 3H] 3) and lower Segregation. We expressed a CENP-A S16/S18A phospho-mutant + + abundance dimeric species ([2M + 4H] 4 and [2M + 5H] 5)(Fig. in HCT116 cells to determine the effect of CENP-A Ser16/Ser18 S7A). In contrast, no dimeric species were observed for the un- phosphorylation on its function in vivo. Transiently transfected modified peptide (Fig. S7 A and B). Similarly, phosphorylated cells were synchronized in S-phase, released, and fixed after 10 h R14–R27 dimers were observed in analysis of trypsin-digested to examine the progression of cells through mitosis. Expression purified tagged CENP-A (Fig. S7C). Collisional activation of the of CENP-A S16/S18A mutants caused an increase in the number + unique dimer m/z value [2M + 3H] 3 (m/z = 1102.1549, −1.09 of metaphase cells with unaligned relative to ex- ppm) produced an MS2 spectrum corresponding to monomeric pression of wild-type CENP-A (Fig. 5A) consistent with a defect + + peptides of [M + 1H] 1 (m/z = 1652.7) and [M + 2H] 2 (m/z = in chromosome congression. We observed increases in lagging 827.0). These results demonstrate that phosphorylated CENP-A chromosomes as cells undergo chromosome segregation, sug- N-terminal tails can form dimers. gesting that defects in centromere function persist throughout mitosis. The single mutation of either Ser16 or Ser18 shows Phospho-Mimetic CENP-A Nucleosome Arrays Resist Oligomerization. limited effect. Only cells with CENP-A restricted to centromeres Higher-order chromatin condensation is driven in part by histone were analyzed for mitotic defects, suggesting that defects arise tails. We have used in vitro-reconstituted nucleosome arrays to from filling centromere sites with an unphosphorylated CENP-A. examine the behavior of CENP-A nucleosomes within chroma- Taken together, our data suggest a model where individual tin. Here, polynucleosome arrays were assembled using 12 tan- phosphorylated CENP-A tails are normally engaged in phospho- demly repeated 601-nucleosome positioning sequences and dependent intramolecular salt bridging that restricts the ten- recombinant core histones, including either H3.1, CENP-A, or dency to hypercondense chromatin arrays when unphosphory- phospho-mimetic CENP-A S16D/S18D protein along with the lated (Fig. 4F), and that weak oligomerization through CENP-A other core histones (H4, H2A, and H2B). Assembled arrays were tails may occur when CENP-A nucleosomes are densely packed. subjected to analytical ultracentrifugation (AUC) to assess the The inability of these tails to undergo phosphorylation results in + folding characteristics. In the absence of Mg2 , nucleosomes do errors in chromosome segregation (Fig. 5B). not contact each other (i.e., beads-on-a-string conformation). + Folding behavior upon Mg2 addition can be observed at 50% Discussion boundary fraction as the average sedimentation value (Fig. 4F). The CENP-A N terminus is phosphorylated on Ser16 and Ser18 2+ H3-containing arrays become folded upon adding Mg , indi- and α-N trimethylated on Gly1 following Metinit excision. Our in – fi cated by shifting 10S higher. As reported previously, CENP-A vitro experiments demonstrate that NRMT can ef ciently cata- BIOCHEMISTRY + containing arrays subjected to Mg2 also display efficient folding lyze the modification of the N terminus of CENP-A. The human at 50% boundary fraction (27). Additionally, above 50% bound- CENP-A N-terminal Gly–Pro–Arg sequence is well conserved in ary fraction, unmodified CENP-A arrays undergo extensive mammals (Fig. 3C). In addition, Met–Pro–Arg, which is present in oligomerization, which is defined as sedimenting at >60 S (28). CENP-A of both birds and fish, was reported to be a human The sedimentation profile of the phospho-mimetic CENP-A NRMT target in vitro (19). Both CENP-A N-terminal motifs are + arrays in the presence of Mg2 lands between the unmodified H3 highly conserved in Tetrapoda, suggesting a conserved role for and CENP-A profiles. The extent of phospho-mimetic CENP-A α-N methylation modification of CENP-A. array folding as well as oligomerization is more similar to the α-N methylation was shown to mediate stable chromatin lo- H3 array. We conclude that secondary structure generated by calization of RCC1 (29). At centromeres, α-N–methylated CENP-A CENP-A phosphorylation greatly reduces the oligomeric state of may play a similar role in DNA interactions. The addition of the CENP-A arrays. three methyl groups changes the N-terminal glycine so that it

MetAP GFP H2B-RFP GFP H2B-RFP mee unmethyl A B memmee mem Kinase * 1 trimethyl 3 NRMT me0 me3 me3 P P

me0 P memmee trimethyl WT HJURP 2 : 1 1 : 10 Mitosis, Nucleosomal Mitosis, Pre-nucleosomal

P phos CENP-A M WT % unaligned chromosomes S16A S18A H3 G2 me memmee nuc S16/S18A G1 meme 2 S PP P me0 me3 ** PP P CENP-A nuc 1 : 3 CENP-A

Asyn., Nucleosomal pre-nuc S16/S8A

C memmee P P CENP-A 1 ----GPRRRSRKPEAPRRRSPSPTP 21

CENP-A H3 1 ARTKQTARKSTGGKAPRKQ----LA 21

% lagging chromosomes WT S16A S18A CENP-A 22 TPGPSRRGPSLGASSHQHSRRRQG 45 S16/S18A H3 22 TKAARKSAPATGGVKKPH-RYRPG 44

Fig. 5. CENP-A Ser16/Ser18 phosphorylation is required for normal mitosis. (A) HCT116 cells transiently expressing -mRFP and GFP-tagged wild- type or S16/S18A CENP-A mutants. The percentage of cells with unaligned metaphase (arrowhead) and lagging anaphase (arrow) chromosomes is higher in the S16/S18A mutant. *P = 0.018, **P = 0.002, Student’s t test,n= 3 independent experiments ± SD. (B) CENP-A PTM begins at translation and persists through the cell cycle. CENP-A α-N trimethylation occurs on a minority of the new prenucleosomal CENP-A and increases with cell cycle progression, con- stituting greater than 90% of the mitotic nucleosomes. (C) Human CENP-A PTM sites Gly1 α-N methylation and Ser16/Ser18 phosphorylation are especially divergent compared with H3.1. Conserved residues are in red.

Bailey et al. PNAS | July 16, 2013 | vol. 110 | no. 29 | 11831 Downloaded by guest on September 27, 2021 resembles a fixed positive charge buried within a hydrophobic Materials and Methods shell. Such a structure might be capable of binding to nucleic Stable Cell Line Generation. CENP-A was C-terminally tagged with two tan- acids through electrostatic interaction with negatively charged dem PreScission protease cleavage sites, a 6xHis, HA tag and GFP. Stable lines phosphates or polarizable π electrons on the aromatic nucleotide were made by retroviral infection as described (4). Polyclonal cell lines were α bases. -N trimethylation could mediate an additional point of FACS sorted for CENP-A expression. Spinner flask cultures were grown at binding directly to underlying or neighboring DNA, perhaps con- a density of 3–8 × 105 cells per mL in Joklik media (Lonza) supplemented tributing to the exceptionally high degree of phasing that CENP-A with 5% (vol/vol) newborn calf serum (HyClone), 1 mM Hepes, 1% penicillin α nucleosomes exhibit on centromeric -satellite DNA (30). streptomycin, 1% L-glutamine (Gibco, Life Technologies). Additional in- We observed that CENP-A, which had been present for longer formation is located in SI Materials and Methods. periods of time in the cell, showed higher degrees of α-N meth- ylation; prenucleosomal CENP-A had the lowest degree of Affinity Purification and Liquid Chromatography-MS Analysis. HeLaS3 cells methylation whereas the CENP-A nucleosomes present in mi- stably expressing tagged CENP-A were harvested and homogenized to tosis, which have been present in the chromatin for at least one generate a chromatin pellet for MNase digestion. CENP-A-LAP (Fig. S1) cell cycle, were almost completely methylated. The increased α α nucleosomes were recovered using GFP beads and subsequently eluted by proportion of CENP-A -N methylation with cell cycle pro- PreScission protease digestion. Eluted proteins were digested using trypsin, gression may be due to accessibility of CENP-A to NRMT activity LysC, or GluC (Roche). Protein digests were HPLC-resolved and analyzed on- or regulation of NRMT activity. The selective destabilization of line using high-resolution MS. Spectra were analyzed as described in SI unmethylated forms of CENP-A perhaps at particular points in Materials and Methods. the cell cycle such as DNA replication could also explain the observed changes in CENP-A methylation. Although it is possible Nucleosomal Array Reconstitution and Analytical Ultracentrifugation. Nucleo- that the unmodified form of CENP-A may represent protein that somal arrays were reconstituted from the indicated histones and DNA using has undergone demethylation, no N-terminal demethylase has yet salt dialysis. The 601 12 × 200-bp DNA template was purified by anion been identified. exchange chromatography. The histones were expressed and purified as Although the N-terminal tail of H3 is considered to be largely unstructured, amino acids 22–28havebeenshowntoform described (27). Reconstituted nucleosomal arrays were characterized by sed- a short α-helix (31). Phosphorylation of human CENP-A Ser16 imentation velocity to obtain the integral distribution of sedimentation co- efficients. Additional information is located in SI Materials and Methods. and Ser18 occurs in the context of an unusual proline-rich se- SI Materials and Methods quence. Moradi and colleagues calculated that Pro-X-Pro Additional experimental details are available in . = sequences (where X Arg, Ser, or Thr) show high propensity to fi adopt beta sheet conformation, rather than polyproline or α-helix ACKNOWLEDGMENTS. We thank the Paci c Northwest National Laborato- fi ries for development of the Isotope Pattern Calculator software. We thank con guration (32). This short sequence of the CENP-A tail is K. Luger (Colorado State), D. Cleveland (UCSD), and A. Straight (Stanford) for completely divergent from H3 and appears to contain up to 4 . This work was supported by National Institutes of Health (NIH) additional residues (Fig. 5C). We find that salt-bridging generates Grant T32 GM08715 (to A.O.B.), Robert A. Welch Foundation Grant A-1176 a local secondary structure as well intermolecular CENP-A tail (to D.H.R.), the American Cancer Society (D.R.F.), NIH Grant R01 GM082989 multimerization. We hypothesize that within highly condensed (to B.E.B.), a Career Award in the Biomedical Sciences from the Burroughs fi Wellcome Fund (to B.E.B.), a Rita Allen Foundation Scholar Award (to B.E.B.), chromatin even low af nity interactions between CENP-A tails and NIH Grant GM037537 (to D.F.H.). T.P. acknowledges support from US could influence chromatin organization and that CCAN proteins National Institutes of Health Grant GM08275 (UPenn Structural Biology may further facilitate this process. Training Grant).

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11832 | www.pnas.org/cgi/doi/10.1073/pnas.1300325110 Bailey et al. Downloaded by guest on September 27, 2021