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OCR for page 101
Colloquium
Histone H3 variants specify modes of
chromatin assembly
Kami Ahmad and Steven Henikoff*
Fred Hutchinson Cancer Research Center, 1 100 Fairview Avenue North, A1-162, Seattle, WA 98109
Histone variants have been known for 30 years, but their functions
and the mechanism of their deposition are still largely unknown.
Drosophila has three versions of histone H3. H3 packages the bulk
genome, H3.3 marks active chromatin and may be essential for
gene regulation, and Cid is the characteristic structural component
of centromeric chromatin. We have characterized the properties of
these histones by using a Drosophila cell-line system that allows
precise analysis of both DNA replication and histone deposition.
The deposition of H3 is restricted to replicating DNA. In striking
contrast, H3.3 and Cid deposit throughout the cell cycle. Deposition
of H3.3 occurs without any corresponding DNA replication. To
confirm that the deposition of Cid is also replication-independent
(Rl), we examined centromere replication in cultured cells and
neuroblasts. We found that centromeres replicate out of phase
with heterochromatin and display replication patterns that may
limit H3 deposition. This confirms that both variants undergo Rl
deposition, but at different locations in the nucleus. How variant
histones accomplish Rl deposition is unknown, and raises basic
questions about the stability of nucleosomes, the machinery that
accomplishes nucleosome assembly, and the functional organiza-
tion of the nucleus. The different in viva properties of H3, H3.3, and
Cid set the stage for identifying the mechanisms by which they are
differentially targeted. Here we suggest that local effects of
"open" chromatin and broader effects of nuclear organization help
to guide the two different H3 variants to their target sites.
Nucleosomes are the fundamental units of chromatin. con-
sisting of 146 bp of DNA wrapped around an octamer of
four core histones. Histone deposition occurs primarily as DNA
replicates to complete chromatin doubling (1~. During S phase
of the cell cycle, new histones are produced in abundance for
immediate replication-coupled deposition. In most metazoans,
this abundant S-phase synthesis results from the tight regulation
of tens to hundreds of intronless histone genes that have special
3' untranscribed regions instead of poly(A) tails (2~. However,
some histones are produced from orphan genes outside of S
phase. In Drosophila, orphan genes encode two H3 variants: one
encodes Cid, the centromeric histone (3), and two encode H3.3,
the replacement variant (Fin. 1; refs. 4 and 5~. These variants
nave equivalents In many other eukaryotes (6, 7~. The H3.3
histone is nearly identical to H3, differing at only four amino acid
positions. Cid differs profoundly from H3 in sequence, showing
some significant identity only within the histone fold domain.
Surprisingly, these three histones have different deposition
properties. H3 and H3.3 are deposited as DNA replicates, but
coin n~.~ anct Fact can be deposited at sites that are not
undergoing DNA replication (Fig. 2; refs. 8 and 9~. Whereas only
a minor fraction of the bulk genome is packaged into Cid- and
H3.3-containing nucleosomes, each variant is targeted to differ-
ent specialized sites, with Cid localizing to centromeres and H3.3
to transcriptionally active genes. Specific localization of centro-
meric H3-like histones (CenH3s) has been observed in various
animals (10), fungi (11, 12), and plants (13~. Also, an H3.3-like
histone targets the transcriptionally active macronucleus in
~ O
. . . . .
. .. .. ~ . ~ ..
www.pnas.org/cgi/doi/10. 1 073/pnas. 172403699
HFD
~H3
__H3.3
al 's. ~ Ills I l' Ill I Cid
Fig. 1. Drosophila produces three versions of H3 histones. H3 and H3.3 are
nearly identical throughout their N-terminal tail and histone fold domains
(HFD), with only four amino acid residue differences. Cid is much more
diverged, and can only be aligned to H3 in the HFD (49). Black boxes indicate
identities to H3.
ciliates (14~. Thus, the targeting of H3 variants is likely a feature
of every eukaryotic cell, where centromeres and transcribed
regions are the major loci of activity in metaphase and inter-
phase, respectively. Both kinds of loci use a distinct pathway for
nucleosome assembly (8, 9), and here we explore the properties
of this process.
H3 Variants Determine the Nucleosome Assembly Pathway
Studies of histone deposition have generally been done using
crude extracts, purified components or pools of cells from which
bulk chromatin is extracted (15~. These methods reveal the
average properties of chromatin, and have shown that the bulk
of chromatin doubles as DNA replicates. Extensive in vitro work
has demonstrated that the assembly of nucleosomes is a stepwise
process in which deposition of an (H3.H4~2 tetramer is followed
by addition of two H2A H2B dimers (1~. The new histones are
brought to the replication fork in a complex with chromatin
assembly factor 1 (CAFE. CAF1 appears to be recruited to the
replication fork by binding to the ring-shaped proliferating cell
nuclear antigen (PCNA) that encircles the DNA template at
each replication fork (16~. Histones from the parent DNA are
distributively segregated to the two sister chromatics behind the
replication fork, and the gaps in their nucleosomal arrays are
rapidly filled by step-wise assembly of new nucleosomes. These
nucleosomes are then matured by addition of linker histones and
covalent modification of histone tails to complete chromatin.
Nucleosomes containing H3 variants comprise only a small
proportion of bulk chromatin, and thus their properties have
been generally undetectable. However, replacement H3 variants
can become enriched in the chromatin of nonreplicating cells
(17-19). This means that other ways of depositing histones must
exist; but because such variant enrichment was only detectable
in unusual cell types (such as long-lived neurons or spermato-
This paper results from Arthur M. Sackier Colloquium of the National Acaclemy of
Sciences, "Self-Perpetuating Structural States in Biology, Disease, anc] Genetics," heic]
March 22-24, 2002, at the National Academy of Sciences in Washington, DC.
Abbreviations: Ri, replication-inclepenclent; CenH3, centromeric H3-iike histone; HP1, het-
erochromatin protein 1; H3K9me, H3-methylatec] iysine-9; PCNA, proliferating ceil nuclear
antigen.
*To whom reprint requests shouic] be ac~c~ressecl. E-maik steveh~fhcrc.org.
PNAS 1 December 10, 2002 1 vof. 99 1 supple. 4 1 16477-16484
OCR for page 102
replication
. or us
C-id
H4:
centromeres
am_
1 ~
H3 _ H3.3_ __ _ RC _
H3.3 RI
He
active genes
,8~-_
Fig. 2. The three versions of histone H3 determine the mode of nucleosome
assembly. The deposition of H3 is strictly replication-coupled (RC), and H3 is
recruited to replication forks for chromatin doubling. Deposition of Cid (blue)
is exclusively replication-independent (Rl), and normally occurs only at cen-
tromeres. H3.3 (green) undergoes RC deposition, and Rl deposition at active
loci. Open chromatin at centromeres and at active genes may promote histone
replacement. Transcriptional activity, chromatin remodeling factors, and RNA
polymerases (orange) will unfold the chromatin fiber and disrupt nucleosome
(gray) structure (open chromatin). Transcriptionally inactive regions are not
subjected to these forces and remain in a closed configuration. Flanking
heterochromatin and H3-containing blocks within the centromeric domain
presents the H3K9me epitope, thereby binding HP1 (red) and resulting in a
compacted, closed chromatin structure. Cid-containing nucleosomes cannot
be methylated in this way, and thus remain comparatively open. The special-
ized N-terminal tail of Cid may alter the linker DNA between nucleosomes,
also contributing to the open chromatin configuration. Rl deposition of H3.3
is limited to the open chromatin at active genes and Rl deposition of Cid is
limited to the open chromatin in centromeric domains.
cytes), studies of the phenomenon have been limited. The ability
to tag histones and examine their deposition properties in single
cells has allowed us to gain insight into chromatin assembly
processes.
We developed a cytological assay system for studying repli-
cation and chromatin assembly by using Drosophila Kc cells, a cell
line that displays a regular cell division schedule (Fig. 3A) and
a consistent tetraploid karyotype. Organization of the Drosoph-
ila nucleus is visually simple, because the late-replicating het-
erochromatin typically coalesces into a compartment in the
nucleus, termed the chromocenter (Fig. 3B). This provides both
a temporal and spatial distinction between the early replicating,
gene-rich euchromatin, and the late-replicating heterochroma-
tin. DNA replication can be tracked either by pulse-labeling with
nucleotide analogs or by using anti-PCNA antibody. Further-
more, by introducing histone-GFP fusion constructs and pro-
ducing a pulse of the tagged protein, we can track histone
deposition during the cell cycle. Using this system, we have been
able to quantitatively examine DNA replication and histone
deposition in unsynchronized populations of cells (3, 8, 9~.
GFP-tagged H3 shows exclusively replication-coupled depo-
sition, displaying co-localization with replication markers and
showing no detectable deposition in cells in which replication has
been blocked (9~. The N-terminal tail of H3 is required, sug-
gesting that the H3 tails of tetramer particles interact with
accessory factors at some early step in nucleosome assembly in
ViVO.
In contrast to the properties of GFP-tagged H3 in cells, tagged
H3.3 deposits in a replication-independent manner at actively
transcribing loci (9~. Deposition can occur in any stage of the cell
cycle, and we demonstrated that it is not accompanied by
unscheduled DNA synthesis. Incorporation of H4 also occurs at
these target sites, as expected for deposition of (H3.3.H4~2
tetramers; but how replication-independent (RI) histone depo-
sition occurs is virtually unknown.
Tagged Cid can also deposit throughout the cell cycle (8),
suggesting that its deposition is also replication-independent.
1 6478 1 www.pnas.org/cgi/doi/10. 1 073/pnas. 172403699
\
A M
B
Fig. 3. Drosophila Kc cells. (A) The cell cycle is ~20 h long, and S phase has
two distinct periods: early S phase, when all euchromatin (gray) replicates, and
late S phase, when all heterochromatin (black) replicates. Eighty percent of
cells show ~15 chromosomes, and this karyotype has been stable for >2 years.
(B) The morphology of a Drosophila interphase nucleus. All heterochromatin
typically associates into a chromocenter (black). Centromeres (red) are en-
closed within the chromocenter, with the nucleolus (light gray) next to it. The
active rDNA genes (green) are located within the nucleolus.
However, this conclusion depends on knowing the timing of
centromere replication. We have shown that centromeres rep-
licate within a defined portion of S phase (8~. The evidence for
this conclusion has been challenged (20), and so here we examine
the available data on centromere replication timing. We confirm
that Drosophila centromeres replicate as isolated domains within
later-replicating heterochromatin.
Centromeres Replicate Before Their Surrounding
Heterochromatin
Historically, centromeres have been thought to replicate very
late in the cell cycle. This is because they are embedded within
pericentric heterochromatin, which replicates late. Analysis has
usually relied on visualization at mitosis; but mitotic chromo-
somes have inherently low resolution because they are highly
condensed. Indeed, a recent study showed that Drosophila
centromeres cannot be resolved from heterochromatin in 44% of
spread mitotic chromosomes (21~. Despite this limitation, Sul-
livan and Karpen (20) concluded from the analysis of normal
mitotic chromosomes that Cid-containing chromatin replicates
on the same late schedule as pericentric heterochromatin. How-
ever, this could be late replication in pericentric heterochroma-
tin that was mix-scored as replication of centromeres.
We have addressed this uncertainty by analyzing mitotic
chromosome replication patterns, providing brief 15-min pulses
to Kc cells and examining mitotic figures after a chase. This
provides a "snapshot" of replication at single points in the cell
cycle. We observed examples of heterochromatin replication
patterns similar to those previously reported (20), where labeling
overlaps Cid spots (Fig. 4A). However, we also observed unam-
biguous examples of chromosomes that were intensely labeled
throughout the euchromatic arms, with foci directly coinciding
with centromeres (Fig. 4B). These centromeric foci are sur-
rounded by heterochromatin that did not replicate during the
labeling pulse. We attribute the inability of Sullivan and Karpen
to observe early centromeric replication foci to the continuous
labeling protocol they used, where all surrounding heterochro-
matin will always be labeled when earlier-replicating sites ac-
quire label. When late-replicating heterochromatin is labeled,
this will often overlap Cid-containing chromatin. If overlapping
heterochromatin accounts for the apparent late replication of
centromeres when analyzed on mitotic chromosomes, then its
removal should improve the visualization of earlier-replicating
Ahmacl ancl Henikoff
OCR for page 103
Fig. 4. Centromeres replicate with euchromatin in tetraploid Kc cells and in
larval diploid neuroblasts. (A) Mitotic X chromosomes from cells pulsed with
dig-dUTP nucleotide analog (green) and then chased for 4 h show heavy
labeling in the heterochromatin surrounding centromeres (Cid, red), as ex-
pected for incorporation during late S phase. (B) Mitotic X chromosomes from
cells pulsed with dig-dUTP and then chased for 10 h were in early S phase at
the time of the pulse, because they show heavy labeling in the euchromatic
arms. There are also foci of incorporation corresponding to both sister cen-
tromeres. (C) Pulse-labeling and imaging of interphase Kc cells shows that
centromeres replicate in the early S-phase period when euchromatin is also
replicating. Wetracked cell survival and S-phase progression overa 5-h period,
and mitotic index over a 25-h period in all labeling experiments. These
parameters were indistinguishable from control, untreated cultures. In la-
beled cultures after 7 h we observed ~98% labeling of mitotic figures,
indicatingthatvirtuallyallcellsinSphaseatthetimeofthepulsereceivedthe
nucleotide analog. (D) Neuroblast centromeres are contained within one to
three heterochromatic chromocenters (H3K9me, blue). Pulse-labeling with
dig-dUTP reveals foci of DNA replication in two centromeric spots and in
euchromatin. Cultured cells and dissected larval brains were labeled and
prepared as described (8).
centromeres. Indeed, this has been observed for minichromo-
somes deficient in flanking heterochromatin (20~.
Our previous experiments using interphase Kc cells revealed
that ~90% of centromere replication occurs when euchromatin
is replicating. The remaining 105to may have been late replication
in centromeric regions, but is more likely the result of nearby
heterochromatic replication foci that could not be resolved from
sites with Cid. Such early replication of centromeres is not
limited to tetraploid Kc cells (Fig. 4C) we have observed
similar replication patterns in diploid larval neuroblasts (Fig.
4D)- although the much shorter cell cycle time and the more
irregular chromocenter limits quantitative analysis. Therefore,
this early timing of centromere replication appears to be general
for Drosophila cells.
This feature of centromeres extends to other eukaryotes. It has
long been known that budding yeast centromeres replicate early
in the cell cycle (224. In mammals and plants, centromeres
appear to replicate conspicuously earlier than similarly repetitive
heterochromatic DNA (23-25~. Thus, whereas the absolute
timing of centromere replication in the cell cycle appears
variable (26), the relative timing of replication in euchromatin,
centromeres, and heterochromatin is consistent.
High-Resolution Mapping of Centromere Replicons
A series of progressively more direct experiments have provided
insight into the fine structure in the centromere region. A model
Ahmad and Henikoff
A
flanking centromeric domain flanking
het. ~ - ~ het.
_
Cid H3
1
2 ^
Fig. 5. Replication within centromeric domains. (A) Cid and H3 appearto be
interspersed as alternating blocks in the centromeric domain. Replication
within the domain occurs before replication of the flanking heterochromatin.
We consider three possible arrangements of replication origins within this
domain. 1: Multiple origins coincide precisely with each block of Cid-
containing chromatin. Firing of these origins would replicate every Cid block
first, after which replication forks proceed into H3-containing chromatin.
2: Multiple origins are distributed throughout the domain, without regard to
the kind of chromatin. At any one time, some Cid-containing and some
H3-containing chromatin would be replicating. 3: A single origin lies in the
centromeric domain, and bidirectional replication duplicates the entire re-
gion. Labeling from short nucleotide analog pulses can distinguish these
arrangements. (B) A pulse-labeled centromeric domain fiber. Kc cells were
labeled with dig-dUTP (8) and fibers prepared according to (30), except that
a high salt buffer described in (60) was used. The stretched fiber shows an array
of Cid spots (red). In this case, the nucleotide analog (green) has incorporated
in the intervening gaps between Cid chromatin. These replication tracts are
scattered throughout the centromeric domain, and this pattern demonstrates
that multiple origins are scattered throughout the domain. The statistically
significant association of replication tracts with non-Cid chromatin across the
domain suggests that origins have a fixed relationship to the chromatin
blocks, and that H3-containing blocks replicate out of phase from Cid-
containing blocks.
for the centromeric constriction has suggested that loops of DNA
coil through the constriction, with centromeric nucleosomes
lying in the outward parts of these coils, and conventional
nucleosomes in the interior portions (21, 27, 28). This would
account for the polar structure of the entire centromere if
centromeric nucleosomes nucleate kinetochore formation (and
thus microtubule capture) and conventional nucleosomes recruit
cohesins (and thus centromeric cohesion). The linear arrange-
ment of nucleosomes along centromeric DNA would then be
alternating blocks of centromeric and conventional nucleosomes
within the centromeric domain (Fig. SA). A recent study using
stretched chromatin fibers has demonstrated that Cid and H3 are
interspersed in Drosophila, although these are not included in the
same nucleosome (21~. Apparently, blocks packaged in one kind
of nucleosome alternate with blocks packaged in the other. How
could the duplication of such regular but discontinuous arrays of
nucleosomes occur?
The alternating pattern of nucleosomes on stretched chroma-
tin fibers is reminiscent of replication patterns on fibers from
normal chromatin (29~. Replication origins within a chromatin
domain often appear to be regularly spaced with an interval of
50-100 kb, and these origins fire synchronously. Perhaps the
PNAS | December ~o, 2002 | vof. 99 | suppl. 4 | 16479
OCR for page 104
nucleosome blocks in the centromeric regions correspond to an
underlying regular arrangement of replication origins through-
out the entire centromeric domain. If Cid-containing blocks
include the origins for these domains, and if replication initiates
at a time when H3 is not available, ultimately only the RI
deposition of Cid will package these blocks. The later replicating
stretches would incorporate H3 as it becomes available. In this
way, the fine pattern of replication would maintain the discon-
tinuous Cid arrays over an extended region.
Our model for maintaining the higher-order chromatin struc-
ture of the entire centromere has precise requirements for
replication patterns in this region: a discontinuously spaced
arrangement of origins must correspond to the blocks of Cid-
containing chromatin (Fig. SO, pattern 1~. At least two other
patterns of replication in this region can be imagined. Firstly, all
Cid- and H3-containing blocks might replicate simultaneously
(pattern 2~. Secondly, a single origin might replicate the entire
domain (pattern 3~.
In a study of stretched centromeric fibers (21), Blower et al.
showed that centromeric domains replicate out of phase with
surrounding heterochromatin. This result is consistent with our
previous demonstration that centromere replication precedes
that of surrounding heterochromatin (8~. Within centromeric
domains, it was reported that H3- and CenH3-containing chro-
matin replicate concurrently (21~. However, any fine structure to
the replication patterns in these domains might have been
obscured by the 2-2.5-h labeling period this study used, because
this is enough time for even just two bidirectional replication
forks from a single replicon to transit the entire domain. Despite
this qualification, a data trace in this work (figure SC in ref. 21)
was intriguing. This trace appears to show that edges of CenpA
signal significantly coincided with edges of nucleotide incorpo-
ration, with gaps between the CenpA blocks. If confirmed, this
might be an example of discontinuous replication tracks corre-
sponding to blocks of centromeric chromatin.
We investigated this possibility by pulse-labeling cells for only
15 min. To prepare stretched chromatin fibers, we disrupted
nuclei spread on a glass slide in a high-salt buffer. As the buffer
runs off the slide, it pulls chromatin fibers behind it. We
identified stretched centromeres and examined those fibers in
which nucleotide incorporation was unambiguous. In each of
these cases it was clear that replication was occurring in discrete
patches scattered throughout the centromeric domain (Fig. SB).
These replication tracks must arise from multiple origins, and
thus we can rule out the two possibilities that the entire domain
replicates from a single origin, or that the whole domain
replicates simultaneously.
These patches corresponded significantly with the segments
between Cid-containing chromatin. Thus, from published exper-
iments (21) and the experiments described here it appears that
replication occurs in two discrete phases: all CenH3-containing
chromatin within a domain replicates, and at a different time all
H3-containing chromatin replicates. Therefore, replication
within this domain is discontinuous and initiates from multiple
. .
Orlglns.
Identifying the precise location of origins within the centro-
meric domain is problematic, because of three difficulties in-
herent to interpreting stretched chromatin fibers. Firstly, al-
though these fibers are stretched to about 50-100 times their
interphase size (21), the radius of H3 or CenH3 spots is
substantially less than the resolution of light microscopes. Sec-
ondly, the intensity of spots along a fiber is variable, implying that
the fiber is unevenly stretched. Thirdly, fibers are inherently
spotted even DNA in situ hybridization with probes that should
uniformly label an extended region always appear spotty (30~.
These effects mean that a lack of overlap between different
signals could result from artifacts in fiber preparation, and
conversely that overlap can result from unstretched segments. It
16480 1 www.pnas.org/cgi/doi/10.1073/pnas.172403699
is apparent from fiber preparations that artifacts are occurring,
because some sites on fibers do not appear to be packaged with
any histories (21~. These concerns can only be addressed with
improvements in fiber technology.
Identifying the position of origins within centromeric domains
is a critical issue to address, because the deposition of H3 must
occur as its DNA substrate replicates. The maintenance of this
interspersed arrangement of chromatin must involve the differ-
ential regulation of the replication-coupled deposition of H3 and
the RI deposition of Cid.
Inferring the Rl Machinery
Given that deposition of any H3 must occur in the form of
(H3.H4~2 tetramers, there must be discrimination of H3-
containing tetramers from tetramers containing variants. Our
analysis of RI assembly initiated the mapping of discriminating
sites within the histone variants (9~. We found that one type of
discrimination is a cluster of three residues within the histone
fold domain (HFD) of H3 that limits it to replication-coupled
deposition. Furthermore, because both Cid and H3.3 undergo
RI deposition but have mutually exclusive targets, there must be
additional discrimination between these variants.
Replication-coupled nucleosome assembly is aided by acces-
sory factors that are recruited to the replication fork by binding
to PCNA. However, the process of RI deposition must be
different, because RI deposition of H3.3 does not require
portions of the histone that are required for replication-coupled
deposition (9~. Furthermore, the lack of PCNA during gap phase
deposition raises the question of what is recruiting histones to the
sites. The phenomenon of CenH3 targeting has raised expecta-
tions that a specific, localized chromatin assembly factor or
histone modification will be involved in the targeting of CenH3s
(26, 31, 32~. Indeed, a chromatin remodeler of the RSC family,
P/BAF, localizes to kinetochores during mitosis of mammalian
cells (33~. Furthermore, RSC mutations in budding yeast have
been previously described to have altered chromatin structure
specifically around centromeres (34), and perhaps RSC activity
is involved in assembly of centromeric nucleosomes. Mutations
in CAF and Hir genes also give centromere defects, and it has
been suggested that these factors are involved in loading the
yeast CenH3 Cse4p (35~. However, a role for any of these factors
does little to explain the specific targeting of CenH3s, because
these factors are all widely distributed in the nucleus (33, 36~.
The best candidate for a uniquely centromere-localized chro-
matin assembly factor is the Mis6 protein in fission yeast (12~.
This protein is required for centromeric localization of the
CenH3 SpCENP-A, but Mis6 homologs in budding yeast (Ctf3;
ref. 37) and in mammals (CENP-I; ref. 38) localize to centro-
meres but are not required for targeting CenH3s. Thus, Mis6
proteins appear to be structural components of centromeres, not
histone assembly factors.
An alternative model is that some feature of centromeric
chromatin facilitates the targeting of its specialized histones. An
obvious candidate for this feature is that centromeric nucleo-
somes themselves bind to and thereby recruit new CenH3
tetramers for future deposition. Such an interaction is a possible
molecular mechanism for direct templating of centromere du-
plication (39~. Regardless of whether CenH3 targeting involves
specialized co-factors, templating, or both, the question remains
as to why it should use an RI pathway.
The targeted deposition of H3.3 to active genes is likewise
replication-independent, although transcription-coupled assem-
bly may facilitate (H3.3-H4~2 deposition. Perhaps H3.3 targeting
is mediated by a component of RNA polymerase complexes.
Because RNA polymerases move processively along the DNA
during transcription, a contiguous transcribed segment of DNA
might incorporate the H3.3 variant. Alternatively, RI deposition
of H3.3 may be facilitated by any of a number of ATP-dependent
Ahmacl ancl Henikoff
OCR for page 105
chromatin remodeling complexes to target specific sites near
transcription units. Any candidate factor might be expected to
preferentially use H3.3 instead of H3, but whether there is any
such discriminating factor is unknown, because all in vitro studies
of higher eukaryotic chromatin assembly have been performed
with H3. We anticipate that this will soon be addressed. How-
ever, the prospects for identifying a unique remodeler that is
required for RI deposition are uncertain, because budding yeast
mutants that eliminate any known chromatin assembly factors do
not eliminate chromatin assembly (1~. Thus, we need to consider
the possibility that RI deposition at active genes and at centro-
meres uses generic remodeling activities, and that components or
structural aspects common to both centromeres and actively
transcribed genes may result in RI histone deposition at both
kinds of sites.
Opening a Space for Histone Replacement
The deposition of histories throughout the cell cycle by a
replication-independent process implies that previously existing
nucleosomes are unraveled, and their histones released. It is
known that the process of transcription results in a local unfold-
ing of the chromatin fiber and an "open" chromatin configura-
tion (Fig. 2; ref. 15~. Although transcription of nucleosomal
templates with bacterial polymerases can occur in vitro without
displacing histone octamers from DNA (40), in vivo assays
demonstrated that a measurable amount of transcription-
dependent histone displacement does occur in eukaryotic nuclei
(41~. In fact, recent experiments revealed that, even in vitro,
RNA polymerase II is virtually unable to transcribe nucleosomal
DNA under physiological conditions (42~. Transcription re-
quires that histone-DNA contacts be broken for polymerase to
transit the nucleosomal DNA. Although transcription can occur
without histone displacement if the histone octamer releases
some contacts with DNA and maintains others (40), at some
frequency all contacts might be released. The histone octamer
would then simply fall off. Additionally, localized remodeling
factors will disrupt nucleosome structure as they act. The in vitro
and in vivo observations can be reconciled if histone displace-
ment occurs occasionally as nucleosomes are disrupted.
Constraints on nucleosomes in a compacted chromatin fiber
(i.e., "closed" chromatin) would limit histone displacement.
Although internucleosome forces within inactive chromatin are
uncharacterized, they have been inferred from numerous exper-
iments, including the tendency of nucleosomes within hetero-
chromatin to form extremely regular and fixed arrays (43~. A
likely constraint in heterochromatin arises from the multimeric
associations that occur between heterochromatin-specific non-
histone chromatin proteins. Attention has focused on the het-
erochromatin protein-1 (HP1~. HP1 is recruited to heterochro-
matic DNA by binding, through its chromodomain, to the H3 tail
when it is methylated at lysine-9 (H3-K9me; ref. 44~. The chrome
shadow domain of HP1 mediates associations between HP1
molecules, and multimers of HP1 bound to methylated histone
tails provides one basis for constraining arrays of nucleosomes.
Although the state of chromatin in heterochromatin and in
actively transcribed regions is well known, less is known about
the chromatin fiber packaged by centromeric nucleosomes.
However, these regions appear to be open. Centromeric DNA is
sensitive to micrococcal nuclease digestion both in budding yeast
(45) and in the central core region of fission yeast centromeres
where SpCCENP-A-containing nucleosomes reside (12), and
plant meiotic centromeres appear decondensed (46~. In addi-
tion, early replication is a feature of open chromatin, and
centromeric chromatin replicates before surrounding hetero-
chromatin. An open configuration may arise from at least three
sources. First, all CenH3s lack a canonical H3 tail (3~. Because
methyl-modification of lysine-9 appears to be the key epitope to
maintain heterochromatin, the lack of this site in centromeric
, .
Ahmad and Henikoff
nucleosomes means that such regions cannot become hetero-
chromatic. Indeed, the heterochromatin protein HP1 is not
associated with chromatin packaged by CenH3s (47, 48~. Second,
our recent study of Cid homologs in drosophilids has uncovered
DNA minor-groove binding motifs in the Cid tail outside of the
nucleosome core (49~. Extension of the Cid tail along linker
~ , v
DNA between nucleosomes may inhibit compaction of the
nucleosome strand, thus maintaining these regions in an open
configuration (Fig. 2~. Third, chromatin remodeling factors that
destabilize nucleosomes are found both at active genes and
centromeres, and their activity will promote histone replace-
ment. We suggest that an open chromatin configuration is the
common basis for RI deposition at centromeres and at actively
transcribed genes.
The Rl Target Sites for H3 Variants Are in Distinct Nuclear
Compartments
If open chromatin were the sole basis for RI deposition, then we
would expect that active genes and centromeres would incorpo-
rate both H3.3 and CenH3s. However, their deposition is
mutually exclusive. This exclusivity is likely to rely on multiple
mechanisms that act on all steps in nucleosome assembly.
Factors that discriminate between H3.3 and Cid would be the
best candidates for directing these variants to their targets.
However, the organization of the nucleus provides a clue as to
another way in which exclusive targeting may be accomplished.
Centromeric DNA in Drosophila is flanked by repeated se-
quences that are packaged into heterochromatin, and this forms
a compartment at interphase in which centromeres are embed-
ded in heterochromatin (Fig. 3B). The active rDNA genes are the
primary sites of H3.3 deposition and they are also found in a
distinct nuclear compartment, the nucleolus, next to the chro-
mocenter. This functional nuclear organization is very simple to
see in Drosophila, where all heterochromatin typically associates
into one large chromocenter, and the active rDNA arrays also
often associate to present one large nucleolus. In fact, this
general compartmentalization is almost invariant in eukaryotes,
and has led to the idea that heterochromatin somehow protects
centromeres and NORs (504. Although both Cid and H3.3
undergo RI deposition, their exclusive targeting could in part be
accomplished by restricting one or both variants within the
nucleus. For example, unincorporated (Cid H4~2 tetramers
might be sequestered within the heterochromatic chromocenter.
Cid deposition would then appear targeted to the centromere,
because this is the only site within the chromocenter with open
chromatin.
Whether (Cid H4~2 tetramers are actually sequestered in this
way is unknown. Indeed, whether sequestering substrates can
have any effect on reactions within the nucleus has become a
pressing issue (51~. Many nuclear components remain mobile,
but functional experiments argue that certain effects in the
nucleus actually only occur when components are sequestered
(52~. It is likely that some reactions in the nucleus are relatively
independent of localization because they associate efficiently
with their partners and their reactions proceed quickly. Con-
versely, reactions that involve weak interactions or multiple steps
may require raising the effective concentration of their sub-
strates by nuclear sequestration.
We have previously suggested that the heterochromatic com-
partment is involved in histone traffic within the nucleus (8~. The
basis of this hypothesis was our realization that Cid-containing
chromatin behaves unusually during S phase. Generally, the
deposition of H3 quickly follows DNA replication. However, the
replication of Cid-containing centromeric DNA occurs without
H3 deposition (8), implying that the normal coupling between
replication components and nucleosome assembly components
must be broken. Because this coupling is thought to result from
an interaction between chromatin assembly factor 1 (CAF1~-
PNAS | December 10, 2002 | vo~. 99 | suppl. 4 | 16481
OCR for page 106
Fig. 6. Overexpression of Cid mix-localizes to euchromatin by Rl deposition.
Kc cells were transfected with a HS-CidGFP construct (3) with a modified
translational start sequence (61). Cells were induced to produce high levels of
Cid-GFP (red), and then immediately pulse-labeled with nucleotide analog
(green) to identify cell cycle stages. The heterochromatic compartment is
labeled with an anti-HP1 antibody (blue; ref. 61). (A and B) Overexpressed Cid
incorporates at centromeres but also mix-incorporates throughout euchro-
matin. Mis-incorporation in euchromatin occurs by a replication-independent
process, because it occurs both in early S phase (A) and in late S phase (B) cells.
(C) After a chase of 6 h, mitotic chromosomes that were induced during S
phase show labeling at both sister centromericfoci (arrowhead), and through-
out the euchromatin arms.
histone complexes and PCNA, the simplest explanation for
uncoupling the two processes would be to sequester replicative
nucleosome assembly factors away from centromeres. We imag-
ined that unincorporated H3-containing tetramers might be
sequestered in euchromatin in the first half of S phase, and would
thus never (productively) see the replication forks at centro-
meres within the heterochromatic compartment. This uncou-
pling might be necessary to prevent dilution of centromeric
nucleosomes by conventional nucleosomes that would assemble
after replication-coupled deposition. Genetic experiments in
budding yeast and Drosophila suggest that CenH3s and H3 do
compete for assembly (21, 53~.
One way that a competition between CenH3 and H3 histones
can be probed is to change their relative concentration. We have
previously reported that a tagged Cid protein exclusively depos-
its at centromeres when it was ectopically expressed at low levels
from a heat-shock-inducible promoter (3~. However, it was
apparent that expression from this construct remained low.
Re-engineering the transcriptional start region of the construct
to include a translational initiation consensus site now allows
overproduction of Cid in cells.
To analyze the behavior of excess quantities of Cid protein, we
introduced an overexpression construct into Drosophila Kc cells
(Fig. 6~. Cells receive varying amounts of transfected DNA, and
thus express Cid over a wide range of levels. In cells that express
low amounts of the ectopic protein, Cid localizes to centromeres,
as expected. However, a new localization pattern for Cid is seen
at high expression levels: the tagged protein localizes to centro-
meres and throughout euchromatin. The incorporation pattern
of ectopic Cid is especially clear on mitotic chromosomes from
these transfections, where the tagged protein is incorporated
throughout the euchromatic arms as well as at centromeres (Fig.
6C). We conclude from this result that excess Cid can be
deposited at sites other than centromeres. Normal cells must
have mechanisms to prevent euchromatic deposition, but over-
expression is sufficient, by itself, to overcome this restriction.
16482 1 www.pnas.org/cgi/doi/10.1073/pnas.172403699
The mix-incorporation pattern of Cid shows an interesting
specificity: Cid can deposit at centromeres and euchromatin but
not in heterochromatin (Fig. 6~. Therefore, heterochromatin
must lack the feature that tolerates mix-incorporation, or must
actively exclude Cid. As we have argued above, centromeres and
euchromatin share the feature of open chromatin, which we have
proposed is the first prerequisite for RI deposition of histone
variants. Indeed, the mix-incorporation of Cid into euchromatin
is replication-independent, because it occurs both when euchro-
matin is replicating in early S phase (Fig. 6A), and in late S phase
when euchromatic replication is complete (Fig. 6B). We suggest
that Cid is contaminating open chromatin in the euchromatic
compartment when it is overexpressed.
What normally prevents the deposition of Cid into euchro-
matin? Endogenous Cid is present only at low levels, and
mix-incorporation could be avoided if Cid were sequestered
away from euchromatin in the nucleus. If unincorporated Cid
were sequestered in the heterochromatic chromocenter, it would
be unable to deposit in the closed chromatin of this compart-
ment. Thus, sequestration might serve two purposes: deposition
in euchromatin would be prevented and deposition at centro-
meres would be promoted. Overexpression of CenpA in
mammalian cells also mix-incorporates into euchromatin (544.
Although it has not been examined whether CenpA mis-
incorporation is replication-independent, we expect this to be the
case, because this is how CenpA deposits at centromeres (244.
The idea that histone variants may respect nuclear compart-
ments was first raised by our experiments expressing heterolo-
gous CenH3s in Drosophila Kc and human HeLa cells (3~. These
extremely diverged heterologous histones did not localize to
centromeres in these cells, implying that there is some kind of
specificity for depositing the correct CenH3 at centromeres.
Surprisingly, heterologous histones were preferentially enriched
in the heterochromatic blocks. We suggested that it is a default
ability of cells to enrich diverged H3 variants in the heterochro-
matic compartment. Perhaps heterochromatic enrichment is a
normal first step in the deposition of the endogenous CenH3s.
Those experiments and our overexpression results encourage the
view that nuclear compartments may guide histone variants to
the correct subset of their potential deposition sites. Compart-
ment effects may also affect the RI deposition of H3.3 in an
inverse way to Cid: i.e., sequestering to promote H3.3 deposition
at active genes, and preventing its deposition at centromeres.
Because H3.3 is largely identical to H3, the hypothetical element
that is recognized in H3 and results in its exclusion from
chromocenters during centromere replication may also be
present in H3.3. Perhaps this discrimination against canonical
H3 histones also serves to prevent the RI deposition of H3.3 at
centromeres.
Why Rl Assembly?
RI assembly permits immediate chromatin repair. The unfolding
of chromatin during transcription may be damaging, in that the
forces RNA polymerases apply to their template DNA should at
least occasionally displace histone octamers from DNA (554.
Additionally, histone octamers may sometimes be displaced by
chromatin remodeling factors associated with transcriptional
activity. In either case, these regions must be repackaged into
nucleosomes. Similarly, replacement of CenH3s may be required
to maintain the nucleosomal configuration of centromeres after
mitosis. Bundles of microtubules drag a chromosome to the pole
during anaphase, and the forces they apply (56) may be sufficient
to occasionally pull off histone octamers. Chromatin would then
be stripped of some CenH3 histone octamers. RI deposition
allows repair of this damage. In fact, the RI deposition of CenpA
in mammalian cells seems to occur around the time of mitosis
(244. The deposition of Cid in Drosophila cells occurs throughout
the cell cycle, but may only be required at two points: as
Ahmacl and Henikoff
OCR for page 107
RI
H39
i' t H3 3 ~ anti
RC H3
Suvar3-9
Suvar3-9
1~
silent
Fig. 7. Rl deposition allows switching of heritable chromatin states. Nucleo-
somes in silent heterochromatic are distinctively modified by methylation,
and thereby recruit the HMT Suvar3-9. The silencing epitope can be perpet-
uated by Suvar3-9 through the cell cycle by the methylation of H3 after
replication-coupled deposition (vertical arrow). A gene can be activated
(rightward arrow) at any time in the cell cycle, and the unraveling of meth-
ylated nucleosomes and Rl deposition of H3.3 will remove the silencing
epitope. This abolishes Suvar3-9 recruitment and allows stable activation. Rl
deposition of H3.3 will continue as long as the gene is transcribed. Switching
from an active to a silent state (leftward arrow) can occur by repressing
transcription and methylating the N-terminal tail of H3.3 at Lys-9, once again
recruiting the Suvar3-9 complex.
centromeric DNA replicates to double its chromatin, and after
mitosis to repair stripped chromatin.
The process of RI assembly at active genes provides a novel
level of control over histone modifications. Replacement of
nucleosomes in one modification state by new histones could
switch chromatin to an active state. Initiation of transcription
would start this process, and successive transits of RNA poly-
merases would promote RI assembly. The replacement H3
histone in alfalfa is hyperacetylated (57), and RI assembly with
acetylated histones could enrich such modifications in active
chromatin. However, histone modification by methylation has
appeared more problematic (43~. A number of histone methyl-
transferases (HMTs) have been characterized (44), but no
histone demethylase is known. Methylated lysine-9 in the H3 tail
(H3K9me) is a critical epitope for recruiting heterochromatic
chromatin proteins, because this is the binding site for HP1. HP1
recruits additional heterochromatic proteins including the Su-
var3-9 HMT. Therefore, it is straightforward to imagine how
these recruited proteins could perpetuate a heterochromatic
state through replication-coupled nucleosome assembly and cell
division (Fig. 7~.
Because an irreversible methyl modification appears to specify
the heterochromatic state, it has been unknown how a hetero-
chromatic site could switch to an active state. One route for
switching might be to prevent the methylation of nucleosomes
assembled during replication. Successive cell cycles could then
dilute methylated nucleosomes, allowing eventual activation.
However, more rapid mechanisms for activating silenced chro-
matin must exist. Induction of silenced genes can occur within a
single cell cycle; for example, X chromosomes become reacti-
vated and lose H3K9me during diplotene in the Caenorhabd~t~s
ovary (58~. In addition, our work using a reporter for hetero-
chromatic gene silencing suggests that switching to an active
state can occur in somatic cells without cell division (59~. Thus,
H3K9me can be removed without replication-coupled nucleo-
some assembly.
RI deposition implies that the entire heterochromatic nucleo-
some may be unraveled and replaced (9~. The process of
transcriptional activation may force the disassembly of H3K9me-
containing nucleosomes, followed by RI assembly of an un-
marked nucleosome. Although we do not know the fate of the
Ahmad and Henikoff
Fungi
S_VM
A IG
Cryptococcus S_VM
S IG
_Saccharomvces
,
Neurospora
Sch izosaccharomyces
S_IG
S_IG
S IG
Fig. 8. The relationship of the canonical H3 histones in animals and fungi.
Three residues in the HFD differ between H3 and the replacement H3.3 histone
in metazoans. Basidiomycete fungi (Cryptococcus) also have two canonical H3
histones that can be classed as an H3 and a replacement histone. Ascomycetes
(Saccharomyces, Neurospora, and Schizosaccharomyces) have only one type of
canonical H3, resembling the replacement histone in basidiomycetes. This
phylogeny identifies that ascomycetes have lost their H3 histone, and retain
only a replacement variant.
displaced methylated H3, we do know that RI deposition can
occur at any time in the cell cycle, and thus should be able to
rapidly derepress silencing (Fig. 7~. Conversely, an active gene
could be silenced by methylating the tail of H3.3, which presents
the same lysine-9 epitope. The stability of histone methylation
gives it a distinct advantage over other histone modifications for
heritable effects on chromatin. The possibility of RI deposition
circumvents the irreversible nature of methylation, thus retain-
ing the potential to switch the heritable chromatin state at a later
time.
Conclusions
H3 variants are used to package functionally specialized chro-
matin, where they play vital functional roles. Localizing these
variants to centromeres and to transcriptionally active regions
utilizes an RI process that is distinct from the nonspecific,
replication-coupled method of packaging the bulk genome. We
have argued that RI deposition is the consequence of the
activities that impinge on these sites in the genome and create an
open chromatin structure. This flexibility in histone deposition
may be necessary to maintain the nucleosomal structure of these
regions. In higher eukaryotes, the RI deposition process allows
specialized chromatin to be distinguished at the most basic level,
where histone variants are incorporated into chromatin. The
differences between the generic H3 which packages the bulk of
the genome and the H3 variants may contribute to the physical
properties of specialized regions and recruit particular non-
histone chromatin proteins. Because histones remain associated
with DNA through mitosis, these variants establish heritable
distinctions in chromatin.
Centromeres are a defining feature of eukaryotes, and all are
likely to have a CenH3. However, the utilization of two con-
served versions like H3 and H3.3 is not universal. For example,
budding yeast has only one canonical H3 histone, which under-
goes both replication-coupled and RI deposition (1~. Surpris-
ingly, this is H3.3: phylogenetic analysis reveals that ascomycetes
have lost H3, whereas their sister clade basidiomycetes have both
H3 and H3.3, as do animals (Fig. 8; ref. 9~. Therefore, an H3.3
gene performs all general functions in some organisms. The
extraordinary conservation of H3.3, which is identical from
mollusks to mammals, speaks to its fundamental role in the
eukaryotic nucleus.
We thank Hillary Hayden for help with stretched fiber imaging, Jim
Smothers for plasmid constructs, and Harmit Malik, Paul Talbert, and
Danielle Vermaak for helpful discussions. This work was supported by
the Howard Hughes Medical Institute.
PNAS | December 10, 2002 | vol. 99 | suppl. 4 | 16483
OCR for page 108
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Ahmad and Henikoff
Representative terms from entire chapter:
nucleosome assembly
