Transitions in the Human CENP-A
Nucleosome In Vivo
Minh Bui,1Emilios K. Dimitriadis,2,5Christian Hoischen,4,5Eunkyung An,3Delphine Que ´net,1Sindy Giebe,4
Aleksandra Nita-Lazar,3Stephan Diekmann,4and Yamini Dalal1,*
1Laboratory of Receptor Biology and Gene Expression, National Cancer Institute
2Laboratory of Biomedical Engineering and Physical Sciences, National Institute of Biomedical Imaging and Bioengineering
3Laboratory of Systems Biology, National Institute of Allergy and Infectious Diseases
NIH, Bethesda, MD 20892, USA
4Fritz Lipmann Institute, Jena, Germany
5These authors contributed equally to this work
In eukaryotes, DNA is packaged into chromatin by
canonical histone proteins. The specialized histone
H3variantCENP-A provides an epigeneticandstruc-
tural basis for chromosome segregation by replacing
H3 at centromeres. Unlike exclusively octameric
canonical H3 nucleosomes, CENP-A nucleosomes
have been shown to exist as octamers, hexamers,
and tetramers. An intriguing possibility reconciling
these observations is that CENP-A nucleosomes
cycle between octamers and tetramers in vivo. We
tested this hypothesis by tracking CENP-A nucleo-
somal components, structure, chromatin folding,
and covalent modifications across the human cell
cycle. We report that CENP-A nucleosomes alter
from tetramers to octamers before replication and
revert to tetramers after replication. These structural
transitions are accompanied by reversible chap-
erone binding, chromatin fiber folding changes, and
previously undescribed modifications within the
histone fold domains of CENP-A and H4. Our results
reveal a cyclical nature to CENP-A nucleosome
structure and have implications for the maintenance
of epigenetic memory after centromere replication.
Every metaphase chromosome has a centromere, a unique
chromatin structure to which kinetochore complexes and
spindle microtubules attach during mitosis (Bloom and Joglekar,
2010). Centromeric chromatin is comprised of nucleosomes
containing a centromere-specific histone H3 variant, CENP-A,
which is required for establishing the kinetochore prior to every
mitotic event over the replicative life span of eukaryotic cells.
Thus, CENP-A is a key epigenetic determinant of centromere
identity and function.
In contrast to canonical nucleosomes, which organize the
bulk of eukaryotic genomes into octamers composed of H2A,
H2B, H3, and H4, CENP-A nucleosomal structure remains
controversial. Whereas yeast and human CENP-A can assemble
into conventional octameric nucleosomes in vitro (Camahort
bles into rigidified protein tetramers in vitro (Black et al., 2004;
Sekulic et al., 2010). Furthermore, octameric (Camahort et al.,
2009), hexameric (Mizuguchi et al., 2007), and right-handed
(Furuyama and Henikoff, 2009) CENP-A nucleosomes have
been documented in yeast, whereas tetrameric ‘‘hemisomes’’
containing CENP-A, H2A, H2B, and H4 have been identified in
asynchronous Drosophila and human cells (Dalal et al., 2007;
Dimitriadis et al., 2010). In contrast, a recent study using overex-
pressed CENP-A has reported the presence of unstable oc-
tamers in fly cells (Zhang et al., 2012). These studies point to
an inexplicable variability in structure for a nucleosome whose
function is both critical and conserved.
An unexplored possibility to explain such variability in struc-
ture might be that CENP-A nucleosomal organization is dynamic
over the cell cycle, so that CENP-A forms octamers after
completion of assembly at G1, but transits through stable tetra-
meric intermediates (Allshire and Karpen, 2008; Probst et al.,
2009) that are generated after replication (Dalal and Bui, 2010;
Henikoff and Furuyama, 2010; Black and Cleveland, 2011) or
mitosis (Bloom and Joglekar, 2010). To investigate this hypoth-
esis, we tracked CENP-A nucleosomes over the cell cycle in
human cells by using a combination of chromatin biochemistry,
atomic force microscopy (AFM), coimmunoprecipitation (co-IP)
experiments, Fo ¨rster resonance energy transfer (FRET), and
liquid chromatography coupled to tandem mass spectrometry
(LC-MS/MS). We report that native CENP-A nucleosome are
tetrameric at early G1, convert to octamers at the transition
from G1 into S phase, and revert back to tetramers after
replication, a state they assume for the rest of the cell
cycle. These structural changes are accompanied by reversible
Cell 150, 317–326, July 20, 2012 ª2012 Elsevier Inc. 317
binding of the CENP-A chaperone HJURP and changes in chro-
matin fiber folding. Furthermore, we uncover previously unde-
scribed covalent modifications in both CENP-A and H4 histone
fold domains, which occur at the key transition point from G1
into S phase. We discuss implications of our findings for the
inheritance of centromeric domains after replication.
Heterotypic CENP-A Nucleosomes Bind the Chaperone
HJURP at G1 and G2 Phases but Not at S Phase
We first examined whether histone or kinetochore components
in the centromeric fiber change over the cell cycle. To address
this, human cells were synchronized at early G1, G1/S, S,
G2/M, and M phases (Experimental Procedures and Fig-
ure S1A available online). Chromatin arrays were released from
these cells by mild nuclease digestion, followed by chromatin
immunoprecipitation (ChIP) with an anti-CENP-A antibody to
enrich for native CENP-A nucleosomes (Dimitriadis et al., 2010)
Components present within long- and short-length arrays of
bulk chromatin (BC) and CENP-A chromatin were analyzed
on high-sensitivity protein gels (Experimental Procedures). As
expected, BC from these cells depicts the normal equivalence
of canonical histones, within which CENP-A is detectable (Fig-
ure S2A,western blots [WB]). Ourprevious results demonstrated
with H2A, H2B, and H4 on long-, moderate-, and short-length
chromatin arrays even when H3 is depleted, suggesting that
CENP-A nucleosomes are heterotypic (Dimitriadis et al., 2010).
We next examined whether CENP-A transits through a homo-
typic state (i.e., H2A/B free; Mizuguchi et al., 2007) during the
human cell cycle. However, whether from G1, G1/S, S, and
G2/M cells, long CENP-A chromatin arrays contain H2A, H2B,
and H4 (Figure 1A). Such arrays are associated with key inner
kinetochore proteins such as CENP-C and CENP-N (Figure 1A,
WB) (Carroll et al., 2010; Screpanti et al., 2011) and contain H3
(Figure 1A,two-color WB), indicative of alternating domains typi-
cally found at centromeres (Sullivan and Karpen, 2004). Centro-
meric immunoprecipitates (IPs) are enriched in CENP-A and
depleted in H3 (Figure S2B) and contain centromere-specific
alpha satellite DNA (Figure S2C), supporting their centromeric
origin. When the arrays are made shorter by prolonging
nuclease treatment, until the input is almost exclusively
mononucleosomes, CENP-A nucleosomes copurify with core
histones H2A, H2B, and H4 in equimolar amounts, even when
Figure 1. CENP-A Nucleosomes Are Heterotypic throughout the Cell Cycle and Bind the Chaperone HJURP at G1, G1/S, and G2 Phases but
Not at S Phase
Denaturing protein gel analysis of (A) native CENP-A IP from long chromatin arrays shows CENP-A contains H2A, H2B, and H4 at all points of the cell cycle and
binds kinetochore proteins CENP-C and CENP-N (WB panel).
(B and C) Native CENP-A IP from short chromatin arrays (B) and mononucleosomal input (C) demonstrate that CENP-A is always associated with H2A, H2B, and
H4, but not with H3.
(D) Western blot of HJURP within the CENP-A IP from (B) demonstrates loss of the HJURP at S phase, and return of HJURP at G2 phase.
(E) Extracted chromatin fibers stained for HJURP (green) and CENP-A as centromeric marker (red) demonstrate that HJURP is lost from centromeric fibers at S
phase but enriched in G2 and G1 phases. Scale bars, 1 mm. See also Figure S2.
318 Cell 150, 317–326, July 20, 2012 ª2012 Elsevier Inc.
H3 is completely depleted within the IP (Figures 1B and 1C).
Thus, these data indicate that H2A and H2B are intrinsic compo-
nents of native CENP-A nucleosomes derived from active
centromeres at all points of the cell cycle examined.
In parallel, we also analyzed the CENP-A histone chaperone
HJURP in CENP-A chromatin arrays. At G1 phase, consistent
with its role in depositing CENP-A (Dunleavy et al., 2009; Foltz
et al., 2009; Shuaib et al., 2010), HJURP is bound to chromatin
(Figure S2D, HJURP IP) and specifically to short CENP-A
chromatin arrays (Figure 1D, CENP-A IP, HJURP WB). However,
in contrast to its yeast homolog Scm3, which is bound to centro-
meres throughout the cell cycle (Xiao et al., 2011), we observed
that HJURP is diminished in CENP-A chromatin from G1/S
through S (Dunleavy et al., 2011) but reappears at G2 phase
(Figure 1D, CENP-A IP, WB). A reciprocal HJURP IP likewise
demonstrates that CENP-A copurifies with HJURP during G1,
is depleted at S, but returns during G2/M phase (Figure S2D,
HJURP IP, CENP-A WB).
To test this observation further, we performed HJURP immu-
nofluorescence on centromeric chromatin fibers (Sullivan and
Karpen, 2004). These data show that HJURP localizes onto
centromeric fibers at G1 and G1/S, is absent at S phase, but re-
turns at G2 phase (Figures 1E and S2E). Thus, HJURP associa-
tion to CENP-A chromatin is cell-cycle-dependent (Figures 1D,
1E, S2D, and S2E).
CENP-A Nucleosomes Cycle between Tetramers and
Octamers In Vivo
HJURP is known to be a CENP-A deposition chaperone (Hu
et al., 2011, Foltz et al., 2009, Dunleavy et al., 2009, Shuaib
et al., 2010). The loss of HJURP binding from CENP-A chromatin
at the G1 into S transition might signal completion of assembly,
heralding a structural shift in CENP-A nucleosomes. To test this
idea, we turned to single-molecule analysis of native CENP-A
chromatin. AFM is a powerful single-molecule imaging method
(Zlatanova and van Holde, 2006) that has been used extensively
to investigate chromatin dynamics (Shukla et al., 2010; Torigoe
et al., 2011, Bintu et al., 2011). Using AFM, we have previously
shown that native CENP-A nucleosomes contain H2A, H2B,
and H4, but not H3, and are tetrameric rather than octameric in
asynchronous fly and human cells (Dalal et al., 2007; Dimitriadis
et al., 2010). To investigate whether CENP-A nucleosomes alter
their structure over the cell cycle, we used AFM to measure
dimensions of native CENP-A chromatin obtained from experi-
ments described above (Figures 1A, 1B, and 1D).
Consistent with previously published results (Dimitriadis et al.,
2010), BC nucleosomes are exclusively octameric, averaging
2.6 nm in height and 268 nm3in volume (Figures 2A, 2B, and
S3A and Table 1). To ensure that IP or AFM conditions do not
nucleosomes remain within the octameric range (Figures 2A
and 2B overlay of BC and H3-IP, Table 1, and Figures S3B
and S3C). In contrast, native CENP-A nucleosomes similarly
purified present remarkably variable structures over the cell
cycle. At early G1, the point at which new CENP-A assembles
at centromeres (Hemmerich et al., 2008; Jansen et al., 2007;
Schuh et al., 2007), CENP-A nucleosomes are tetrameric, aver-
aging 1.5 nm high and 125 nm3in volume (Figures 2A, 2B, and
S3and Table 1). At the G1/S transition, CENP-A nucleosomes
have a broader distribution, with the majority averaging 1.9 nm
high and 168 nm3in volume, and an octameric subfraction,
averaging 2.5 nm high and 251 nm3in volume (Figures 2A, 2B,
and S3 and Table 1). As cells enter S phase, the overwhelming
BC / H3 IP
168 ± 33 nm3
251 ± 80 nm3
273 ± 53 nm3
242 ± 52 nm3
198 ± 72 nm3
190 ± 53 nm3
125 ± 35 nm3
1.9 ± 0.28 nm
2.5 ± 0.77 nm 2.7 ± 0.34 nm1.5 ± 0.5 nm 2.6 ± 0.5 nm1.8 ± 0.27 nm1.8 ± 0.38 nm2.6 ± 0.6 nm
268 ± 92 nm3
5 101502010 4084062010 2001020 0 10 20300 10 20
0510150102020010 2001020300102001020010 20
Figure 2. CENP-A Nucleosomes Alter from Tetramers to Octamers at S Phase
Single-molecule AFM measurements depicting (A) heights and (B) volumes of purified H3 and bulk chromatin (BC) nucleosomes show they have octameric
octameric particle values. BC values are in gray, H3 values are in green, and CENP-A values are in blue. Average values for heights and volumes are indicated
under each graph. Table 1 gives a detailed overview of the data. See also Figures S3, S4A, and S4B.
Cell 150, 317–326, July 20, 2012 ª2012 Elsevier Inc. 319
majority of CENP-A nucleosomes are indistinguishable from H3
octameric nucleosomes, averaging 2.7 nm high and 273 nm3in
volume (Figures 2A, 2B,and S3 and Table 1). In late S phase,
CENP-A nucleosomes persist as octamers, averaging 2.6 nm
high and 242 nm3in volume (Figures 2A, 2B, and S3 and
Table 1). In contrast, postreplicative CENP-A nucleosomes
from G2/M phase also form nucleosomal arrays (Figure S3) but
are consistent with tetrameric dimensions, averaging 1.8 nm
high and 198 nm3in volume (Figures 2A and 2B and Table 1).
CENP-A nucleosomes from early mitotic cells (Experimental
Procedures) remain tetrameric, averaging 1.8 nm high and
190 nm3in volume (Figures 2A, 2B, and S3 and Table 1). Thus,
CENP-A nucleosomes cycle between tetrameric and octameric
configurations in vivo.
Octameric volumes for S phase CENP-A nucleosomes could
result from an increase not just in height but also in diameter,
perhaps resulting from nonhistone binding. A priori, this possi-
bility seems less likely because histone gels depict an equiva-
lence of CENP-A, H2A, H2B, and H4, but no significant enrich-
ment of other small proteins that might contribute to octameric
dimensions at S phase (Figure 1B). Nevertheless, we also exam-
ined diameters of CENP-A nucleosomes. These data show
that CENP-A nucleosomal diameters are similar over the cell
cycle, and consistent with the observed lateral diameter of H3
nucleosomes (Figure S3). Therefore, volume increase in CENP-A
nucleosomes from late G1 through late S reflects doubling of
nucleosomal heights, supporting the interpretation that they
To confirm that CENP-A nucleosomes transition to octamers
at S phase and back to tetramers at G2/M, we examined their
DNA content. G1/S, S, and G2/M CENP-A short chromatin
arrays were treated with proteinase K to remove histones, and
contour lengths of nucleosomal DNA released were measured
by AFM (Experimental Procedures). At G1/S, CENP-A mononu-
cleosomal DNA ranges from 100–150 bp (Figures S4A and S4B).
In contrast, by S phase, CENP-A nucleosomes yield multiples
of ?150–200 bp of DNA, consistent with classical octameric
organization. By G2/M, CENP-A nucleosomal DNA decreases
to ?115 bp in length, consistent with previous measurements
of the CENP-A mononucleosomal footprint in vivo (Ando et al.,
2002), and with diminutive heights and volumes observed in
our analyses above (Figures 2A and 2B).
We sought an alternative approach to confirm that CENP-
A:CENP-A intranucleosomal interactions are increased at S
phase. In order to perform this experiment, we used a stable
cell line expressing GFP-CENP-A alongside native (untagged)
CENP-A (Dimitriadis et al., 2010) and collected cells at G1/S
and S phase as before. Mononucleosomal input chromatin
was extracted from these G1/S and S phase cells under condi-
tions used previously (Figure 1C) and an anti-GFP antibody
used to perform IP. Previous data have shown that EGFP-
CENP-A in these cells is associated with the core histones
H2A, H2B, and H4 (Dimitriadis et al., 2010). Here, the EGFP-
tagged nucleosomes were analyzed for EGFP-CENP-A relative
to native CENP-A content by using an anti-CENP-A antibody,
followed by quantitative laser scanning. As can be seen, very
little native CENP-A is present in the G1/S phase EGFP-
CENP-A IP. In contrast, native CENP-A is enhanced 7-fold
in the S phase EGFP-CENP-A IP (Figure S4C, CENP-A WB).
These data support the interpretation that CENP-A:CENP-A
intranucleosomal interactions are increased at S phase and
are consistent with octameric dimensions uncovered by AFM
Thus, AFM analyses, DNA measurements, and co-IP analyses
(Figure 2A, 2B, S4A–S4C, and Table 1) indicate that, whereas
canonical H3 nucleosomes have octameric dimensions through-
out the cell cycle, CENP-A nucleosomes are predominantly
tetramers in early G1 phase, alter to octamers at the end of G1
through S phase, and revert to tetramers after replication.
The CENP-A Chromatin Fiber Is Highly Dynamic In Vivo
We were intrigued by the possibility that changes in CENP-A
structure and components observed above might be reflected
in chromatin fiber folding. To testwhether chromatin fiber folding
changes in vivo, we measured interactions between CENP-A
molecules in living cells by using FRET. FRET occurs when two
fluorescently tagged proteins are within 10 nm of each other
in vivo, which is the average distance between nucleosomes
Table 1. AFM Measurements of CENP-A Nucleosomal Dimensions Indicate a Change in Structure over the Cell Cycle
Cell-Cycle PhaseNucleosomeNHeights (nm)Volume (nm3)Diameter (nm)NInferred Organization
G2/MCENP-A 4791.8 ± 0.27198 ± 7214.6 ± 2.20584tetramer
MCENP-A11771.8 ± 0.38190 ± 5314.2 ± 2.03498tetramer
Early G1CENP-A1691.5 ± 0.5125 ± 3513.7 ± 1.93169tetramer
G1/SCENP-A13721.9 ± 0.28
2.5 ± 0.77
168 ± 33
251 ± 80
12.7 ± 0.76288transition to octamer
2.7 ± 0.34273 ± 53
13.3 ± 1.86246octamer
2.6 ± 0.5242 ± 52
13.2 ± 1.36124octamer
2.9 ± 0.5345 ± 56
14.4 ± 1.78394octamer
2.6 ± 0.6268 ± 92
13.6 ± 1.25497octamer
3.2 ± 0.4300 ± 46
13.1 ± 1.3298octamer
3.3 ± 0.16302 ± 65
12.8 ± 1.0126octamer
Bolded numbers denote values within the predicted range for canonical octameric nucleosomes, values rounded up to 1 decimal point. N = number of
nucleosomes measured. BC = bulk chromatin; H3 = H3 IP; CENP-A = CENP-A IP.
320 Cell 150, 317–326, July 20, 2012 ª2012 Elsevier Inc.
in arrays, and thus has been used to study chromatin fiber inter-
actions (Hellwig et al., 2008, 2011; Hemmerich et al., 2008).
EGFP-CENP-A localizes accurately and can partially rescue
CENP-A depletion within centromeres (Kalitsis et al., 2003). It
associates with H2A, H2B, H4, and DNA in vivo (Dimitriadis
et al., 2010), is able to interact with CENP-C (Trazzi et al.,
2009), and has been widely used as a surrogate for tracking
native CENP-A behavior (Wan et al., 2009).
We assessed interactions between centromere-localized
C-terminal and N-terminal EGFP- and mCherry- CENP-A pairs
by using synchronized cell populations (Figures S1A and S5A).
Equivalently tagged control H3.1 pairs show significant FRET
between each other at all points of the cell cycle, including at
mitosis, consistent with a globally compacted chromatin fiber
(Figures 3, S5B and Table S1). In contrast, C terminally tagged
CENP-A pairs yield remarkably different results. In early G1, we
observed significant CENP-A-EGFP:CENP-A-mCherry FRET
(p < 0.001; Figure 3 and Table S1). The FRET signal remains
high over G1 and at G1/S (Figure 3 and Table S2). However,
2 hr into S phase, the FRET signal drops to background
levels (Figure 3) and remains insignificant throughout the
subsequent G2 and mitosis phases. Analysis of N terminally
tagged CENP-As similarly yields high FRET during G1 and
G1/S but no appreciable FRET during S, G2, and M phases (Fig-
ure S5C and Table S1).
FRET detection between CENP-As in G1 is consistent with
CENP-A assembly, which peaks at G1 (Hemmerich et al.,
2008; Jansen et al., 2007), increasing CENP-A occupancy on
the centromeric chromatin fiber and possibly resulting in a highly
compacted fiber (Marshall et al.,2008). Conversely, loss of FRET
between N and C terminally tagged CENP-A pairs, from mid S
to the next G1 is indicative of lack of physical proximity between
CENP-A termini, possibly resulting from greater spatial dis-
tances between CENP-A nucleosomes, a more extended
chromatin fiber (Dalal et al., 2007), or from inner kinetochore
proteins binding to CENP-A termini (Hellwig et al., 2011; Carroll
et al., 2010; Trazzi et al., 2009), thus preventing higher-order
In order to confirm our interpretation that the FRET data reflect
chromatin fiber folding, we analyzed FRET before and after
disrupting the chromatin fiber with mild nuclease treatment.
We first confirmed FRET occurs between C terminally tagged
CENP-A in untreated G1 phase nuclei (Figure S5D and Table
S2), which recapitulated our in vivo results above (Figure 3). In
contrast, upon treating G1 phase nuclei with mild nuclease
(MNase), thus disrupting higher-order chromatin fiber folding
in situ, FRET between C terminally tagged CENP-A in late G1
disappears (Figure S5D and Table S2). From these data, we
infer that CENP-A FRET reflects interactions between CENP-A
nucleosomes within a densely packed chromatin fiber during
G1. Conversely, the loss of FRET between CENP-As in S phase
suggests that the centromeric chromatin fiber is altered as cells
transition from G1 into S phase.
We were curious to test whether disappearance of FRET
between CENP-A arrays in early S phase could be due to
disruption by of CENP-A chromatin fiber by DNA polymerases.
We synchronized and released cells into G1/S in the presence
of aphidocolin, an inhibitor of DNA polymerases a and d, thus
blocking replication. Because the cells are stalled at G1/S, we
observe continued CENP-A/CENP-A FRET for 4 hr (Figure S5E).
In contrast, after synchronizing and releasing cells at G1/S, but
inhibiting DNA polymerase only one hour after replication has
initiated, FRET between CENP-As is lost (Figure S5E, Table
S2). This behavior is similar to that of wild-type cells, which
lose FRET by mid S phase (Figure 3). Because centromeres
are late replicating in human cells (Shelby et al., 2000), 1 hr of
DNA polymerase activity is insufficient for the replication
machinery to run through human centromeres. Thus, the loss of
CENP-A FRET signal by mid S phase likely derives from a less
folded centromeric chromatin fiber in advance of replication.
The Histone Fold Domains of CENP-A and H4 Have
Modifications at G1/S
AFM and FRET experiments indicate that CENP-A structural and
dynamic transitions peak during G1/S (Figures 2A, 2B, and 3),
concurrent with the loss of HJURP binding in S phase (Figures
1C and 1D). We were curious to know whether covalent modifi-
cations within CENP-A/H4 could contribute to the changes
noted above. We purified DNA-bound CENP-A/H4 complexes
from G1/S, S, and G2/M cells (Figure S1C), excised them from
gels (Figure S6A), and subjected them to LC-MS/MS (Experi-
Six unique peptides from CENP-A were identified (Experimen-
tal Procedures). Remarkably, in G1/S phase-derived CENP-A,
the mass spectra revealed enrichment of an ion that corre-
sponds to a doubly charged tryptic peptide containing acety-
lated Lysine 124 (m/z 714.90, Figures 4A and S6B). Pep-
tide fragmentation in MS/MS mode and manual validation
allowed unequivocal confirmation of the CENP-A peptide
Figure3. FRETMeasurements RevealstheCENP-AChromatinFiber
Undergoes a Transition at G1/S Phase
FRET measurements between CENP-A C termini and H3 C termini across the
cell cycle. H3 FRET is constant across the cell cycle (red), whereas CENP-A
FRET shows high FRET in G1 (Black bars) but no FRET from S through M
(mitosis) or C (Cytokinesis) phase (Grey bars). Black or red bars, significant
FRET (p < 0.001); gray bars, no FRET. See also Table S1, S2, and Figure S5.
Cell 150, 317–326, July 20, 2012 ª2012 Elsevier Inc. 321
as ‘‘119VTLFPK(acetyl)DVQLAR130’’ (Figures 4A and S6B). Simi-
larly, LC-MS/MS analysis of CENP-A-bound H4 from G1/S
phase cells also revealed an acetylation site on H4’s Lysine 79
residue in the tryptic peptide ‘‘79K(acetyl)TVTAMDVVYALK91,’’
showing the sequence of fragment ions that confirm the
presence of the acetyl group on K79 (m/z 748.90, Figures 4B
Whereas histone fold domain (HFD) modifications in histone
H3 have been identified that impact the pseudo-dyad of
the canonical octamer in vitro (Neumann et al., 2009; Simon
et al., 2011), there have been no such reports for histone vari-
ants such as CENP-A. The structural consequence of CENP-A
K124 and H4 K79 acetylation, buried in the interface of the
HFD and DNA in octamers, is likely to be significant (Figures
4C and 4D). CENP-A K124 is located within the a3-helix
domain and is within 7A˚ of the DNA double helix at the
pseudo-dyad of the octamer (Figure 4C). Adding an acetyl
group to K124 could have the structural consequence of
neutralizing the positively charged lysine surface, which might
loosen histone-DNA contacts. Similarly, H4 K79 is located
within the Loop 2 domain juxtaposed near the DNA double helix
(Figure 4D), and an acetylation on this residue could loosen
the DNA-histone interface, thereby increasing accessibility of
the CENP-A nucleosomal interior to nonhistone proteins or to
Our data indicate that heterotypic CENP-A nucleosomes
(Figures 1A–1C) undergo significant changes in binding of
the chaperone HJURP (Figures 1D, 1E and S2), nucleosomal
structure (Figures 2A, 2B, S3, and S4), chromatin fiber folding
(Figures 3 and S5), and covalent modifications (Figures 4A, 4B,
S6B, and S6C), during the transition from G1 into S phase
(summarized in Figure S7). These findings are significant
because they reconcile previous contradictory observations
of CENP-A octamers and tetramers in diverse organisms,
which likely reflect cycling of the CENP-A nucleosomes.
Although we did not directly observe CENP-A hexameric or
homotypic tetramer intermediates during the transition from
Figure 4. CENP-A Lys124 and H4 Lys79 Are Acetylated at G1/S Phase
(A) MS/MS spectrum showing CENP-A K124 is acetylated in the peptide ‘‘VTLFPK(acetyl)DVQLAR’’. Location of the parent peptide ion prior to fragmentation is
indicated withablue diamond. Peptide fragmentation ions identified are indicated inthespectraand on thepeptide sequence.Themasses of ionsb9,y8,y9, y10
are diagnostic of K124 acetylation. See also Figure S6B.
(B) MS/MS spectrum showing fragmentation of H4 ‘‘K(acetyl)TVTAM(oxi)DVVYALK’’ as a doubly charged, monoisotopic ion, m/z 748.90 (?0.83 ppm difference
from the theoretical m/z). Location of the parent ions prior to fragmentation is indicated with a green diamond. Peptide fragmentation ions identified are indicated
in the spectra and on the peptide sequence. All fragment ions of the b series have masses diagnostic of K79 acetylation. See also Figure S6C.
(C) Model of the CENP-A octamer showing two CENP-As (red), DNA (blue), core histones (gray), CENP-A K124 (yellow), and CENP-A H115, R118, and D125
(green). Dotted lines represent distances (A˚) between residues.
(D) Distance (A˚) between H4K79 and DNA and H4K79 and CENP-A F84. Models were generated in PyMol by using published crystallographic coordinates
(Tachiwana et al., 2011). See also Figure S6.
322 Cell 150, 317–326, July 20, 2012 ª2012 Elsevier Inc.
G1 into S phase, it is plausible that such intermediates exist
during interconversion between heterotypic tetramers and
Our findings implicate novel mechanisms in the assembly-
disassembly kinetics of the centromeric fiber. One speculative
mechanism whereby CENP-A nucleosomes could be converted
from one form to another in vivo is through the action of
chromatin remodelers (Perpelescu et al., 2009; Torigoe et al.,
2011). The chromatin remodeler RSF, which binds to centro-
meres at late G1 and is required for CENP-A inheritance (Perpe-
lescu et al., 2009), could initiate reorganization of the CENP-A
chromatin fiber and recruit histone acetyltransferases that
catalyze acetylation of CENP-A and H4, delaying the formation
of stable octamers (Figure 5). At the end of G1, loss of such
modifications, accompanied by chromatin remodeling, could
influence the release of the chaperone HJURP, thus resulting
in the stabilization of the four helix bundle in the CENP-A
octamer (Hu et al., 2011). A plausible alternative scenario is
that changes in intrinsic stability (Conde e Silva et al.,
2007), or processes such as transcription (Bintu et al., 2011),
may alter CENP-A nucleosomal structure directly at various
points of the cell cycle. CENP-A mononucleosomes have
been demonstrated to be inherently unstable when subjected
to unwinding stress in vitro (Dechassa et al., 2011), and, ectop-
ically expressed CENP-A can be detected in the dimeric form in
fly cells, suggesting that unstable CENP-A octameric nucleo-
somes might also exist in vivo (Zhang et al., 2012). Such
unstable octameric CENP-A nucleosomes could disassemble
during passage of DNA replication machinery. Consequently,
our observation of the return of the chaperone HJURP concur-
rent with CENP-A tetrameric nucleosomes at G2 phase
might reflect HJURP-dependent recycling of old CENP-A into
tetrameric nucleosomal intermediates, after late replication of
centromeric DNA in human cells (Dunleavy et al., 2011; Shelby
et al., 2000). This state would persist until a burst of new
CENP-A protein at the next G1 promotes completion of
octamers prior to replication (Figure 5). New avenues that arise
from this work include elucidating how HJURP is released and
rebound over the cell cycle, identifying histone modifiers and
chromatin remodelers that target CENP-A, and the mechanism
by which CENP-A chromatin is reorganized throughout the cell
In the absence of new CENP-A assembly during replication,
random segregation of old CENP-A would be expected to result
in unequal centromere domains at sister centromeres, unequal
attachment to the mitotic spindle, and subsequently aneuploidy.
It is plausible that an evolutionary conserved mechanism exists
to ensure distribution of CENP-A nucleosomes after centromeric
DNA replication. Thus, it is likely that cycling of CENP-A nucleo-
somal structure occurs in other organisms and that such cycling
will track closely with assembly by CENP-A chaperones. Our
observations that histone modifications, chromatin fiber folding,
and chaperone binding changes accompany structural transi-
tions in the centromere provide insight into a fundamental
problem arising from replication-independent assembly of
histone variants. These findings reveal a cyclical nature for
Human cell lines HeLa and HEK293 were synchronized at various stages of
the cell cycle by using a double-thymidine block (see Cell-Cycle Synchroniza-
tion), harvested, washed twice with PBS + 0.1% Tween, and nuclei were ex-
tracted with TM2 Buffer (20 mM Tris [pH 8.0], 2 mM MgCl2) supplemented
with 0.5% Nonidet P40 Substitute (Sigma Cat number 74385). For mitotic
cells, early prophase cells were used which retain nuclear membranes, along
with an antibody specific to phosphorylation of CENP-A Ser 7. To release
chromatin, MNase (Sigma Cat number N3755) digestion was performed at
0.2–0.4 units/ml for 30 s, 2.5 min, and 4 min at 37?C to generate long,
moderate, and short chromatin arrays, and the reaction stopped with 10 mM
EGTA. An aliquot of nuclei was used for extracting and analyzing DNA to
Figure 5. Cyclical Oscillations of HJURP and CENP-A Drive
Model depicting how cyclical oscillations in CENP-A nucleosomal structure
could derive from changes in HJURP chaperone binding mediated by chro-
matin remodeling, histone modifications, and cell-cycle events such as
Cell 150, 317–326, July 20, 2012 ª2012 Elsevier Inc. 323
confirm bulk chromatin array length. Remaining nuclei were extracted over-
night at 4?C in 103 volume of low-salt buffer (0.53 PBS, 5 mM EGTA,
0.5 mM PMSF). Chromatin IP was performed by using Dynabeads Protein G
(Invitrogen catalog number 100-07D) or prehydrated sepharose-protein G,
with primary antibodies as listed below. Three biological replicates were per-
formed for each experiment.
Antibodies Used for ChIP and Western Blot
CENP-A, rabbit CENP-A (Santa Cruz catalog number sc-22787) and rabbit
CENP-A (Millipore catalog number 07-574); Mitotic CENP-A, rabbit phos-
pho-serine 7 CENP-A (Millipore catalog number 07-232) (early prophase cells
still have nuclear membranes); HJURP, goat HJURP (Santa Cruz catalog
number sc-168091); CENP-C, goat CENP-C (Santa Cruz catalog number sc-
11285); and CENP-N, goat CENP-N (Santa Cruz catalog number sc-69152).
Chromatin Fiber Analysis by Immunofluorescence
PBS-washed HeLa cells were counted and diluted to 300,000 cells/ml in
hypotonic buffer (75 mM KCl, 13 PBS). After 10 min of incubation at room
temperature, 200 mL of cells were cytospunned for 10 min at 4,000 rpm, lysed
for 15 min in lysis buffer (2.5 mM Tris HCl [pH 7.5], 0.5 mM NaCl, 1% Triton
X-100, 0.4 M urea), fixed for 10 min in fixation buffer (4% PFA [paraformalde-
hyde], 13 PBS), and permeabilized for 7 min in permeabilization buffer (0.1%
Triton X-100, 13 PBS). After blocking for 30 min in 13 PBS supplemented with
0.5% BSA and 0.01% Triton X-100, extracted fibers were incubated overnight
at 4?C with mouse anti-CENP-A (1:200, Abcam) or rabbit anti-HJURP (1:50,
Santa Cruz) antibodies diluted in blocking buffer. After three washes with 13
PBS supplemented with 0.05% Tween, slides were incubated for 3 hr at
room temperature with Alexa Fluor 488 goat anti-rabbit IgG (1:300, Invitrogen)
or Alexa Fluor 568 goat anti-mouse IgG (1:300, Invitrogen). DNA was counter-
stained with DAPI (50 ng/ml in 13 PBS). Extracted fibers were observed with
a DeltaVision RT system (Applied Precision) controlling an interline charge-
coupled device camera (Coolsnap; Roper) mounted on an inverted micro-
scope (IX-70; Olympus). Images were captured by using a 1003 objective at
0.1 mm z sections, deconvolved, and projected by using softWoRx. Two
biological replicates and three technical replicates each were performed.
AFM Imaging of Chromatin and DNA
Imaging of CENP-A, bulk chromatin and DNA was performed as described
(Dimitriadis et al., 2010) with the following modifications. Imaging was per-
formed by using standard AFM equipment (Multimode AFM and the Bio-
scope Catalyst, Bruker-Nano, Inc., Santa Barbara, CA) with silicon cantilevers
(OTESPA and TESP-SS with nominal resonances of ?300 kHz, stiffness of
?42 N/m, and tip radii of 3–7 nm and FESP with ?75 kHz, 2.8 N/m and
7 nm, respectively, Bruker-Nano) in noncontact tapping mode. Usually, 5 ml
stock solution of CENP-A chromatin or 1,0003 diluted solution of bulk chro-
matin fraction was deposited on APS-mica pretreated with magnesium2+.
APS-mica was prepared as previously described (Dimitriadis et al., 2010).
The samples were incubated for 10 min, rinsed gently to remove salts, and
dried in a stream of inert Argon before imaging. Images were acquired at
high resolution and preprocessed on the NanoScope instrument software.
AFM Image Analysis
Automated image analysis was performed as described (Dimitriadis et al.,
2010) by using NIH ImageJ software (NIH) and Nanoscope Software (Veeco/
Bruker AFM). An algorithm was developed to first localize the nucleosomes
under investigation and then perform automated statistical analysis of their
height and volume distributions. To achieve this, images were thresh-holded
to remove probe convolution that causes objects to appear dilated in AFM
imaging. Using particle analysis routines, the base area (at half-height) and
total height of all nucleosomes in each image were automatically measured.
Filters were employed to reject particles that were not circular or elliptical in
shape, thereby ensuring that measurements were made of nucleosomes at-
taching to the mica in the same orientation, and therefore projecting a uniform
shape. For the particles segmented this way, the radii of the equivalent circles
were calculated and their statistical distributions plotted and fitted with
Gaussian functions. The histograms always displayed a peak of ?12–14 nm,
slightly larger diameter than the known nucleosomal radius of ?11 nm, which
represents residual dilation from the finite AFM tip size of 2–3 nm. Sub-popu-
further statistics of heights and volumes. Volumes were computed as the sum
ofpixel valueswithin thesegmented baseofeachparticle. Inadditionto arrays
ranging from 5-15 CENP-A nucleosomes, various large protein complexes
(diameter > 25 nm, height > 10 nm) were observed associated with CENP-A
tetrameric nucleosomes. These likely reflect kinetochore proteins, remodeling
logical replicates and 2 technical replicates were performed for each experi-
ment. BC from the same preparation was imaged in parallel to get the baseline
Contour Length Measurement of CENP-A Nucleosomal DNA by AFM
Aliquots of CENP-Aand controlBCnucleosomes weretreated withproteinase
K (0.2 mg/ml, Sigma) for 30 min, equilibrated with 1% SDS in 100 mM Tris pH
8.0, phenol:chloroform extracted (Sigma), ethanol precipitated and dissolved
in 13 PBS for imaging by AFM as above (see AFM imaging of chromatin).
Images were collected and exported to Image J (NIH). DNA populations
were thresholded at roughly half-height. The perimeter and area of each
DNA chain were automatically measured by using particle analysis. Mean
width and length were computed in OriginPro (OriginLab Corp., Nothampton,
MA). For each molecule we measure both its projected area and its perimeter.
These two quantities uniquely determine both the width and the length of the
molecule by solving the two equations A = L*w+p*w2, p = 2*L+2*p*w for the
length (L) and the width (w) of the molecule, where A is the projected area
and P the perimeter. The second terms in the right hand sides of the two equa-
tions apply a small correction to the length at the two ends of the DNA chains
for the AFM tip convolution. Particles were filtered to exclude contaminants by
using low circularity criteria to restrict measurements to linear particles.
Cell-Cycle Synchronization of Hep-2 and HeLa Cells
For cell-cycle-dependent analysis in late G1, S phase, G2, and mitosis, cells
S phase. Cells were blocked with a final concentration of 5 mM thymidine for
18 hr. Cells were released and grown in fresh media for 9 hr, followed by
a second cell-cycle block with 5 mM thymidine for 18 hr. Synchronized cells
were released into S phase by washing and cultivation in fresh media. For
FRET analysis cells were harvested and fixed with 4% paraformaldehyde in
PBS immediately before release and for 8 - 10 hr every 2 hr. Most cells were
in late S phase 6 hr after release and were in G2 or mitosis 8 hr after release.
For analysis of HEp-2 cells in G1 transfected cells were synchronized by
a thymidine block followed by a release for six hours and a subsequent
12 hr treatment with the CDK1 inhibitor RO-3306 (Calbiochem) at a final
concentration of 9 mM. RO-3306 reversibly arrests the cells in late G2 at the
initiation of mitosis (Vassilev et al., 2006). Synchronized cells were washed
and then released into mitosis in fresh media, to enter in G1, 1.5 - 2 hr after
release. To identify the cell-cycle stages, cells in parallel experiments were
transfected with EGFP-CENP-A and mCherry-PCNA and analyzed for the
localization pattern of CENP-F and PCNA at each time point. G1 and G2 cells
were identified by immunostaining of CENP-F. This protein is diffusely located
in the nucleus during G2, forms distinct foci at the kinetochores during mitosis
and is not detectable during G1 (Figure S6). Early, mid, and late S phase were
identified via the localization pattern of PCNA (Figure S6). Mitotic phases were
identified by DAPI staining of DNA, which was stimulated with 405 Diode laser
at low intensity and detected via a 420 nm long path filter.
Imaging and Acceptor Bleaching FRET Measurements and Plasmids
for FRET Study
Please refer to Supplemental Experimental Procedures for FRET analysis.
Large-Scale Purification of Endogenous CENP-A for LC-MS/MS
For each experiment, 10 large flasks of confluent HeLa and HEK cell lines were
synchronized and released at G1/S, mid S, and G2/M phase, by performing
double 5 mM thymidine blocks. Human CENP-A proteins were purified simi-
larly to (Wang et al., 2008), with the following modifications. Cells were lysed
and nuclear slurries prepared with 10% hydroxylapatite (HAP, Acros Organics
Catalog number 37126-1000) in 13 PBS supplemented with 0.35 M NaCl,
324 Cell 150, 317–326, July 20, 2012 ª2012 Elsevier Inc.
0.2 mM EDTA, 0.5 mM PMSF for several hours. The same buffer was used to
gently remove nonhistone proteins away from the HAP-Chromatin matrix, 3
times for 15 min each. The HAP-chromatin matrix was then incubated with 1
3 PBS supplemented with 2 M NaCl to release histones from the DNA-bound
HAP matrix overnight. Under these conditions, histone H3/H4 dimers and
H2A/H2B dimers are released from DNA (Dalal et al., 2005). The 2 M NaCl/
PBS eluted histone preparation was concentrated ?10 fold by using Amicon
centrifugation, dialyzed down to 0.35 M NaCl/PBS. This input was tested
was sequentially immunoprecipitated with anti-H3 antibody (Santa Cruz
Catalog number sc-8654) to preclear excess H3, followed by anti-CENP-A
antibody (Santa Cruz Catalog number sc-22787). Eluted CENP-A IP com-
plexes were analyzed on preparative SDS PAGE gels, an aliquot of which
was confirmed by western blot analysis. The remaining was used for gel exci-
sion and LC-MS/MS analysis.
Mass Spectrometry Analysis
The components of histone IPs were processed for preparative SDS-PAGE
(Invitrogen Novex Midi Gel System), and stained with Coomassie Brilliant
Blue (Bio-Rad), bands around 11k and 13k were sliced for histone H4 and
CENPA respectively, and each band was in-gel digested with sequencing
grade modified trypsin (Promega, Madison, WI) (An et al., 2006). The resulting
peptides were analyzed by LC-MS/MS at two different settings.
Liquid Chromatography-Tandem Mass Spectrometry
The first set of samples (HeLa and HEK293 cells) was run at the NIAID proteo-
mics core facility. LC-MS/MS was performed on nano-HPLC system (Proxeon
EASY; ThermoElectron, Bremen, Germany) connected to a hybrid mass spec-
trometer (Velos/Orbitrap; ThermoElectron, Bremen, Germany). Each sample
was injected via an auto-sampler and directly loaded on to packed analytical
column at the flow rate of 1.2ul/min and the sample was subsequently sepa-
rated at the flow rate of 400 nl/min. The mobile phases consisted of water
with 0.1% formic acid (A) and 100% acetonitrile with 0.1% formic acid (B). A
linear gradient from 5 to 65% of solvent B was employed over a period of
60 min to separate peptide mixture. Eluted peptides were introduced into
the mass spectrometer through a nano-electrospray source (ThermoElectron,
Bremen, Germany). The spray voltage was set at 1.7 kV and the heated capil-
lary at 250?C. Orbitrap was operated in the data-dependent mode in which
eachcycle consistedof one full-MS survey (m/z,380–2,000)and subsequently
10thMS/MS scans. The targeted ions count in the mass spectrometer trap
was 500,000 for full-MS scans and 10,000 for MS/MS scans. Peptides were
fragmented in the linear ion trap by using collision-induced dissociation with
helium and the normalized collision energy value set at 35%.
The rest of the samples were analyzed at the LSB Cellular Networks
Proteomics Group, and nano-HPLC system (NanoLC 2D; Eksigent, Dublin,
CA) connected to a hybrid mass spectrometer (LTQ Velos with ETD; Thermo-
Electron, Bremen, Germany). Each sample was injected via an auto-sampler
andloaded ontoaC8captrap(0.22mm,2mlbedvolumn;Michrom bioresour-
ces, Auburn, CA) and the sample was subsequently separated by in house
packed column with Magic C18 AQ beads (200 A, 5 mm, 50 mm 3 30 cm,
Michrom Bioresources, Auburn, CA) at a flow rate of 200 nl/min. The mobile
phases consisted of water with 0.1% formic acid (A) and 100% acetonitrile
with 0.1% formic acid (B). A linear gradient from 5 to 65% of solvent B was
employed over a period of 60 min to separate peptides. Eluted peptides
were introduced into the mass spectrometer through a nano-electrospray
source (ThermoElectron, Bremen, Germany). The spray voltage was set at
2 kV and the heated capillary at 250?C. LTQ was operated in the data-depen-
dent mode in which each cycle consisted of one full-MS survey and subse-
quently 10thorder double play with CID and ETD decision tree. The targeted
ions count was same as described previously. MS measurements were per-
formed with the Orbitrap mass spectrometer at 60,000 resolution with accu-
racy better than 10 ppm. Peptides were fragmented in the linear ion trap by
using CID set at 35% and electron transfer dissociation with reaction time
150 ms by using enabled supplemental activation.
Six different peptides (CENP-A amino acid residues: 17–28, 30–42, 57–63,
100–107, 119–124, and 119–130) of CENP-A were identified in this study
by using trypsin (cleaving C-terminal to lysine, K or arginine, R). Sequence
coverage was37.14% (52identifiedaminoacids/140totalaminoacids 3100).
Protein identification was performed against the human UniProt database and
database including CENPA, histone H4, and ubiquitin by using a software
Thermo Fisher Scientific, San Jose, CA; and IP2 equipped with the Sequest
and ProLuCID; Integrated Proteomics Application, San Diego, CA). For Pro-
teome Discoverer, both databases were indexed with assumptions for fully
enzymatic tryptic cleavage with two missed cleavages and the combination
of following possible protein modifications: Met oxidation, Ser, Thr, Tyr phos-
phorylation, Lys acetylation, Lys ubiquitination, and Arg and Lys mono-, di-,
The search result from Proteome Discoverer was filtered with high peptide
confidence value and false discovery rate targeting 0.01. For IP2, MS spectra
ing CENPA, histone4, and ubiquitin was used for the search. Previously
described PTMs were used and allow a maximum of three on each peptide.
The search result from IP2 was filtered with DTASelect 2.0 (Tabb et al.,
2002). The peptides passing the criteria were manually validated.
Supplemental Information includes Extended Experimental Procedures,
seven figures, and two tables and can be found with this article online at
We thank Drs. Shiv Grewal, Tom Misteli, Gordon Hager, Sam John, and
anonymous reviewers for insightful comments and suggestions, and P. Don-
lin-Asp for preliminary FRET experiments. The NIH/NCI Intramural Research
Programs (Y.D., A.N.-L., and E.K.D.), and the Deutsche Forschungsgemein-
schaft (S.D.)supported thiswork. WethankJenniferGertonfor sharing unpub-
lished work complementary to our findings in this manuscript.
Received: October 3, 2011
Revised: December 19, 2011
Accepted: May 18, 2012
Published: July 19, 2012
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