Identification of a rapidly formed nonnucleosomal histone-DNA intermediate that is converted into chromatin by ACF.

Abstract

Chromatin assembly involves the combined action of histone chaperones and ATP-dependent motor proteins. Here, we investigate the mechanism of nucleosome assembly with a purified chromatin assembly system containing the histone chaperone NAP1 and the ATP-dependent motor protein ACF. These studies revealed the rapid formation of a stable nonnucleosomal histone-DNA intermediate that is converted into canonical nucleosomes by ACF. The histone-DNA intermediate does not supercoil DNA like a canonical nucleosome, but has a nucleosome-like appearance by atomic force microscopy. This intermediate contains all four core histones, lacks NAP1, and is formed by the initial deposition of histones H3-H4. Conversion of the intermediate into histone H1-containing chromatin results in increased resistance to micrococcal nuclease digestion. These findings suggest that the histone-DNA intermediate corresponds to nascent nucleosome-like structures, such as those observed at DNA replication forks. Related complexes might be formed during other chromatin-directed processes such as transcription, DNA repair, and histone exchange.

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Molecular Cell
Article
Identification of a Rapidly Formed
Nonnucleosomal Histone-DNA Intermediate
that Is Converted into Chromatin by ACF
Sharon E. Torigoe,1Debra L. Urwin,1Haruhiko Ishii,2Douglas E. Smith,2and James T. Kadonaga1,*
1Section of Molecular Biology, University of California, San Diego, La Jolla, CA 92093-0347, USA
2Department of Physics, University of California, San Diego, La Jolla, CA 92093-0379, USA
*Correspondence: jkadonaga@ucsd.edu
DOI 10.1016/j.molcel.2011.07.017
SUMMARY
Chromatinassemblyinvolves thecombined actionof
histone chaperones and ATP-dependent motor pro-
teins. Here, we investigate the mechanism of nucleo-
some assembly with a purified chromatin assembly
system containing the histone chaperone NAP1
and the ATP-dependent motor protein ACF. These
studies revealed the rapid formation of a stable non-
nucleosomal histone-DNA intermediate that is con-
verted into canonical nucleosomes by ACF. The
histone-DNA intermediate does not supercoil DNA
like a canonical nucleosome, but has a nucleosome-
like appearance by atomic force microscopy. This
intermediate contains all four core histones, lacks
NAP1, and is formed by the initial deposition of his-
tones H3-H4. Conversion of the intermediate into
histone H1-containing chromatin results in increased
resistance to micrococcal nuclease digestion. These
findings suggest that the histone-DNA intermediate
corresponds to nascent nucleosome-like structures,
such as those observed at DNA replication forks.
Related complexes might be formed during other
chromatin-directed processes such as transcription,
DNA repair, and histone exchange.
INTRODUCTION
Nucleosome assembly is required for the duplication of eukary-
otic chromosomes, as well as for the packaging of DNA into
chromatin upon transcription and DNA repair. Nucleosomes
are assembled by the combined action of ATP-driven motor
proteins, such as ACF (ATP-utilizing chromatin assembly and
remodeling factor), RSF (remodeling and spacing factor), and
CHD1 (chromo-ATPase/helicase-DNA-binding protein 1) (for
reviews,seeHaushalterandKadonaga,2003;KadamandEmer-
son, 2002; Lusser and Kadonaga, 2004), and histone chaper-
ones, such as NAP1 (nucleosome assembly protein 1), CAF1
(chromatin assembly factor 1), Asf1 (antisilencing function 1),
FACT (facilitates chromatin transcription), nucleoplasmin, and
HIRA (histone regulatory protein A), that deliver the histones to
the sites of chromatin assembly (for reviews, see Campos and
Reinberg, 2010; Corpet and Almouzni, 2009; Das et al., 2010;
Ransom et al., 2010; Avvakumov et al., 2011).
The assembly of periodic arrays of nucleosomes was
observed to be an ATP-dependent process by Worcel and
colleagues (see, for example, Glikin et al., 1984; Ruberti and
Worcel,1986).Thiseffectwasoriginallystudiedincrudeextracts
derived from Xenopus oocytes (Glikin et al., 1984) or Drosophila
embryos (Becker and Wu, 1992; Kamakaka et al., 1993). Then,
the individual components that mediate the ATP-dependent
assembly of chromatin were fractionated, purified, and cloned,
and it was found that a purified and defined system comprising
ACF, NAP1, core histones, ATP, and relaxed DNA is able to
mediate the assembly of periodic arrays of nucleosomes (Ito
et al., 1997, 1999).
Although the efficient assembly of canonical nucleosomes on
extended relaxed DNA is dependent upon a motor protein such
as ACF (see, for example, Ito et al., 1997, 1999; Loyola et al.,
2001;FyodorovandKadonaga, 2002a;Lusseretal.,2005),there
are conditions in which nucleosomes can be formed at physio-
logical salt concentrations in the absence of an ATPase. First,
with short DNA fragments typically ranging from 146 bp to
207 bp, NAP1 and histones can yield mononucleosomes (see,
for example, Mazurkiewicz et al., 2006; Andrews et al., 2010).
In such cases, the free ends of the short DNA fragments are
able to wrap around the histone octamers to form mononucleo-
somes. Second, with longer DNA substrates, such as plasmid
DNA, the efficiency of spontaneous nucleosome formation in
the absence of a motor protein is significantly enhanced by
negative supercoiling (see, for example, Pfaffle and Jackson,
1990; Nakagawa et al., 2001). In such cases, the formation of
nucleosomes is driven forward by the release of DNA supercoil-
ing energy, which is relieved by the wrapping of the DNA around
thehistoneoctamer.Intheeukaryoticnucleus,however,thebulk
of the DNA is relaxed (Sinden et al., 1980; Giaever and Wang,
1988). Hence, we have focused on the mechanism of ACF-
dependent assembly of extended nucleosome arrays with
relaxed DNA substrates.
In this work, we studied the early steps in the formation of
nucleosomes. We sought, in particular, to address the apparent
paradox that nucleosome-like structures appear to form im-
mediately (within seconds) upon passage of DNA replication
forks (see, for example, McKnight and Miller, 1977; McKnight
et al., 1978; Sogo et al., 1986), whereas canonical ‘‘mature’’
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nucleosomes, as characterized by nuclease digestion and sedi-
mentation properties, are more slowly generated after approxi-
mately 10 to 20 min (see, for example, Seale, 1975, 1976; Levy
and Jakob, 1978; Worcel et al., 1978; Schlaeger and Knippers,
1979; Klempnauer et al., 1980; Jackson and Chalkley, 1981).
To this end, we examined the initial steps in the formation of
chromatin with the purified chromatin assembly system that
comprises ACF, NAP1, histones, ATP, and relaxed DNA. We
investigated the mechanisms by which the histones could be
depositedontotheDNAandconvertedintonucleosomes.These
studiesrevealedanonnucleosomal histone-DNAcomplexthatis
rapidly formed within 15 s in the absence of ACF. These interme-
diate species, which resemble nucleosomes by atomic force
(scanning probe) microscopy, can be slowly converted into
canonical chromatin by ACF. It is likely that this nonnucleosomal
intermediate in the chromatin assembly pathway corresponds to
the rapidly formed nucleosome-like species that was observed
at DNA replication forks.
RESULTS AND DISCUSSION
A Stable Nonnucleosomal Complex Is Formed prior
to Nucleosome Assembly
In our analysis of the initial steps in chromatin assembly, we
examined when the histones become associated with the DNA
template (Figure 1). Specifically, we sought to distinguish
between model A, wherein histone deposition occurs concur-
rently with nucleosome formation, and models B and C, in which
histone deposition precedes the formation of canonical nucleo-
somes (Figure 1A). To this end, we carried out template associ-
ation experiments in which NAP1-histone complexes are first
incubated with DNA template #1 and subsequently challenged
with DNA template #2 prior to the addition of ACF (see reaction
scheme at bottom of Figure 1B). If the histones stably associate
with template #1 (as in models B and C), then there would be
preferential chromatin assembly on template #1 relative to tem-
plate#2.Alternatively,ifthehistonesdonotstablyassociatewith
the DNA template prior to ACF-mediated chromatin assembly
(as in model A), then there would be roughly equivalent levels
of assembly on templates #1 and #2.
We monitored the extent of chromatin assembly by using the
DNA supercoiling assay, which is based on the observation that
the wrapping of DNA around the core histone octamer results in
a change in the linking number of approximately ?1 (Germond
etal.,1975;Simpsonetal.,1985).Thus,inthisassay,theformation
of nucleosomes is detected by the supercoiling of relaxed closed
circular DNA templates in the presence of topoisomerase I. As
controls, we confirmed that the chromatin assembly reactions
are dependent upon NAP1 and ATP (Figure S1A available online).
We performed the template association experiments with
equivalent masses of two DNA templates of approximately
Figure 1. Core Histones Associate with the DNA Template prior to
ACF-Catalyzed Nucleosome Assembly
(A) Three models for the mechanism of chromatin assembly. In model A, core
histone deposition and nucleosome formation occur in a single step, whereas
in models B and C, the histones are initially deposited onto the DNA to give
anon-nucleosomalintermediate that is subsequently converted into canonical
(‘‘mature’’) chromatin by ACF.
(B) Core histones stably associate with DNA prior to chromatin assembly by
ACF. Template association experiments were performed with equivalent
masses of two different circular DNA templates of approximately 3 kbp (3K)
and 8 kbp (8K). Core histones (amount sufficient for complete assembly of one
DNA template) were initially incubated with template 1 (either 3K or 8K). After
5 min, template 2 (either 8K or 3K) was added to the mixture, which was then
allowed to incubate for another 5 min (to allow potential exchange of histones
from template 1 to template 2) prior to the addition of ACF. Purified top-
oisomerase I was included in the reactions, and the formation of nucleosomes
was monitored by the DNA supercoiling assay. The 3K and 8K templates were
resolved by agarose gel electrophoresis. For each reaction, the percent su-
percoiling of template 1DNA [(supercoiledDNA/total DNA)3 100] is indicated.
Due to the higher resolution of the different supercoiled species with the 3K
plasmid than the 8K plasmid, the quantitation of percent supercoiling is likely
to be more accurate with the 3K plasmid than the 8K plasmid. As references,
supercoiled and relaxed DNAs were also included. The positions of relaxed,
supercoiled, and nicked DNA species are shown. The asterisk indicates
a contaminant in the preparation of the 3K plasmid.
See also Figure S1.
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3 kbp (‘‘3K’’) and 8 kbp (‘‘8K’’) that can be resolved by agarose
gel electrophoresis (Figure 1B). These experiments revealed
essentially exclusive assembly of chromatin onto template #1
relative to template #2 (Figure 1B). Template association by the
histones was observed whether the 3K plasmid or the 8K
plasmid was used as template #1. Thus, prior to the formation
of nucleosomes by ACF, the histones associate with template
#1 as a nonnucleosomal histone-DNA complex that does not
induce DNA supercoiling. This property suggests that DNA in
the nonnucleosomal histone-DNA complex is not fully wrapped
around the core histone octamer.
As observed previously (Ito et al., 1999; Fyodorov and Kado-
naga, 2002a; Lusser et al., 2005), a small amount of DNA super-
coiling is observed with NAP1 and histones in the absence of
a motor protein (Figure 1B). The extent of the ACF-independent
DNA supercoiling is typically about 20%–25% of that obtained
with ACF. Given that NAP1 can spontaneously form nucleo-
somes on supercoiled DNA (see, for example, Nakagawa et al.,
2001), the ACF-independent partial supercoiling of the relaxed
DNA is likely to be due to the spontaneous folding of a fraction
(?20%–25%) of the deposited histones into canonical nucleo-
somes. It is important to note, however, that the amount of
DNA supercoiling in the absence of ACF does not increase sig-
nificantly upon extended incubation forup to 90 min (FigureS1B)
(Ito et al., 1999; Fyodorov and Kadonaga, 2002a; Lusser et al.,
2005). Hence, the efficient assembly of chromatin onto relaxed
DNA with purified recombinant NAP1 requires an ATP-utilizing
motor protein such as ACF or CHD1.
ACF comprises the Acf1 polypeptide along with the ISWI
ATPase. The ISWI polypeptide alone is able to catalyze the
assembly of chromatin, although with a lower efficiency than
ACF (see, for example, Ito et al., 1999; Fyodorov and Kadonaga,
2002b). To assess whether the Acf1 subunit of ACF is required
for conversion of the histone-DNA complex into chromatin, we
performed template association experiments with purified ISWI
protein. As shown in Figure S1C, we observed that ISWI protein
alone is able to convert the nonnucleosomal complex into chro-
matin. Hence, this activity can be carried out by the core ISWI
subunit of ACF.
To complement the DNA supercoiling data, we characterized
the histone-DNA complexes by partial micrococcal nuclease
digestion analysis (Noll and Kornberg, 1977). As shown in Fig-
ure S1D, digestion of the histone-DNA complexes formed in the
absence of ACF yields a diffuse heterogeneous mixture of DNA
fragments, which is in contrast to the distinct bands of DNA
that are obtained upon micrococcal nuclease digestion of
ACF-assembled chromatin. Notably, the digestion products
obtained from the histone-DNA complexes resemble the micro-
coccal nuclease digestion products of nuclei from HTC cells
that were pulse-labeled with [3H]thymidine for 2 min (Smith
et al., 1984). In both cases, there is a broad range of DNA frag-
ments with some detectable bands that may derive from mono-
and dinucleosomes. The absence of distinct bands derived
from periodically spaced oligonucleosomes is unlike the pattern
seen in bulk chromatin or chromatin that is assembled in the
presence of ACF (Figure S1D). Hence, these findings, combined
with the DNA supercoiling data (Figure 1B), suggest that the
nonnucleosomal complexes are distinct from chromatin.
Formation of the Nonnucleosomal Complex and Its
Conversion into Nucleosomes
To understand the properties of the nonnucleosomal complex,
we investigated its stability as well as the rates of its formation
and ACF-catalyzed conversion into canonical nucleosomes. To
analyze its stability, we formed the complex on template #1
and then incubated it with template #2 for time periods ranging
from 5 min to 120 min prior to the addition of ACF (Figure 2A).
We found that the histones remain stably associated with
template #1 for at least 120 min, at which time there is no detect-
able exchange of histones from template #1 to template #2. A
slight decrease in the amount of supercoiled DNA can be seen
at the 60 min and 120 min time points, but this effect appears
to be due to increased nicking of the DNA rather than instability
of the complexes. Then, to examine the rate of formation of the
histone-DNA complexes, we combined NAP1-histones with
template #1 and subsequently added template #2 at time points
ranging from 15 s to 5 min (Figure 2B). These experiments
revealed that formation of the template-associated complexes
is complete within 15 s. Hence, the nonnucleosomal histone-
DNA complexes are fully formed within 15 s and remain associ-
ated with the template for at least 120 min.
To test whether the nonnucleosomal complex has the kinetic
properties of an intermediate in the assembly of chromatin, we
measured the rate of conversion of the histone-DNA complexes
into chromatin. If the nonnucleosomal complex is an interme-
diate in the chromatin assembly process, then the rate at which
it is converted into chromatin would be greater than or equal to
the rate of the overall chromatin assembly reaction, because
an intermediate in a multistep reaction must be converted into
the product at least as rapidly as the overall reaction. By carrying
out standard one-template chromatin assembly reactions, we
compared the rate of the overall chromatin assembly reaction
(in which ACF was combined with NAP1-histones prior to the
addition of DNA) with the rate of conversion of histone-DNA
complexes into chromatin by ACF (Figure 2C). These experi-
ments revealed that the rate of conversion of the histone-DNA
complexes into chromatin is comparable to that of the overall
reaction.
Therefore, the nonnucleosomal histone-DNA complex sat-
isfies the two fundamental criteria for an intermediate in the
chromatin assembly process—first, the intermediate can be
converted into chromatin, and second, the rate of conversion
of the intermediate into chromatin is comparable to the rate of
the overall chromatin assembly reaction. Combined with the
observation that the intermediate is formed within 15 s, the
data lead to a model for chromatin assembly in which there is
rapid ACF-independent formation of the non-nucleosomal
histone-DNA intermediate followed by a rate-limiting ACF-medi-
atedconversion oftheintermediate intocanonical nucleosomes.
The Nonnucleosomal Intermediate Lacks NAP1
Next, we sought to determine whether the nonnucleosomal
intermediate contains NAP1, which would enable us to distin-
guish between models B and C in Figure 1A. To address this
issue, we formed the intermediate with NAP1-histones and
templateDNA,anddepletedtheHis6-taggedNAP1fromsolution
by incubation with Ni-NTA agarose followed by pelleting of the
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agarosebeads(Figure3A).Asareference,wecarriedoutparallel
depletions with NAP1-histones in the absence of DNA. The re-
sulting supernatant and pellet fractions were analyzed by
western blot for NAP1 and histones (H2A-H2B) and by agarose
gel electrophoresis and ethidium bromide staining for DNA. In
the absence of DNA, the NAP1 and histones are in the pellet,
and are not detectable in the supernatant. However, in the pres-
ence of DNA, most of the histone-DNA complex is in the super-
natant,whereasessentiallyalloftheNAP1andasmallamountof
histones and DNA are in the pellet (Figure 3A). In addition, the
Figure 2. Dynamics of the Formation of the Nonnucleosomal Histone-DNA Intermediate and Its Conversion to Canonical Nucleosomes
by ACF
(A) The nonnucleosomal histone-DNA complexes remain associated with the DNA template for at least 120 min. Template association experiments were per-
formed as in Figure 1B, except that the time of incubation of the template 1-histone complex with template 2 (to allow potential exchange of histones from
template 1 to template 2 prior to the addition of ACF) was varied as indicated.
(B)The nonnucleosomal histone-DNA complexes are formed within 15 s. Template association experiments were performed as in Figure 1B, except that the time
of addition of template 2 to the template 1 + NAP1-histone mixture was varied as specified.
(C) The nonnucleosomal histone-DNA complexes can be converted into chromatin by ACF at a rate that is comparable to that of the overall ACF-catalyzed
chromatin assembly reaction. Standard (one-template) chromatin assembly reactions were performed in parallel under conditions that compared the rate of
overall chromatin assembly with the rate of conversion of the non-nucleosomal histone-DNA complexes into chromatin by ACF. Purified topoisomerase I was
included in the reactions, and the formation of nucleosomes was monitored by the DNA supercoiling assay.
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relative amounts of the four core histones in the histone-DNA
complex in the supernatant (Figure 3A, lane 4) are the same as
those in the input sample (Figure 3A, lane 1), as assessed by
SDS-polyacrylamide gel electrophoresis and silver staining (Fig-
ure S2A). These results show that most of the non-nucleosomal
histone-DNA complex is not associated with NAP1.
We then tested whether the NAP1-depleted histone-DNA
complexes can be assembled into chromatin by ACF. These
experiments revealed that ACF can catalyze the conversion of
the NAP1-deficient histone-DNA complexes into chromatin at
a rate that is comparable to that seen with mock-depleted
complexes (Figure 3B and Figure S2B) as well as the overall
chromatin assembly reaction (Figure 2C). Moreover, the addition
of purified NAP1 to the NAP1-depleted histone-DNA complexes
does not affect the rate or efficiency of chromatin assembly by
ACF (Figure 3B and Figure S2B). These findings indicate that
the non-nucleosomal intermediate lacks NAP1, and are there-
fore consistent with model C in Figure 1A.
We further examined whether the NAP1-depleted histone-
DNA complexes yield periodic nucleosome arrays upon in-
cubation with ACF. To clarify this point, we incubated the
NAP1-depleted complexes with ACF under standard chromatin
assembly conditions and then subjected the reaction products
to micrococcal nuclease digestion analysis. These experiments
revealed that the NAP1-depleted complexes are converted into
canonical periodic nucleosome arrays by ACF (Figure 3C).
Hence, the NAP1-depleted complexes satisfy the criteria for an
intermediate, astheyyield chromatin ata rate thatis comparable
to the overall reaction.
Analysis of Chromatin Assembly with Histone H1
Chromatininmetazoanscontainsapproximatelyonemoleculeof
histoneH1pernucleosome(see,forexample,BatesandThomas,
1981; Happel and Doenecke, 2009). We therefore analyzed the
assembly of chromatin in the presence of purified histone H1.
To examine the effect of H1 on the formation of the histone-
DNA intermediate, we carried out template association experi-
ments in the presence of one molecule of histone H1 per core
histone octamer. As shown in Figure 4A, we observed template
association during chromatin assembly in the presence of H1.
Thus, the histone-DNA intermediate is formed and converted
into chromatin by ACF in the presence or absence of histone H1.
Then, we compared the properties of naked DNA, the histone-
DNAintermediate(DNA,corehistones,NAP1),chromatinlacking
Figure 3. The Nonnucleosomal Histone-DNA Intermediate Does Not Contain NAP1
(A) NAP1 is not associated with the nonnucleosomal histone-DNA complexes. Nonnucleosomal histone-DNA complexes were formed by incubation of NAP1-
histonesandrelaxedtemplateDNA,andthenincubatedwithNi-NTAagarosebeads(QIAGEN)towhichtheHis6-taggedNAP1canbind(lanes4and5).Thebeads
were pelleted, and the resulting supernatant (S) and pellet (P) fractions were analyzed by western blot with antibodies against NAP1 and histones H2A-H2B. In
addition, the DNA content of the supernatant and pellet was analyzed by agarose gel electrophoresis and staining with ethidium bromide. As a reference, NAP1-
histone complexes in the absence of DNA were analyzed in parallel (lanes 2 and 3). Samples of the input (I) NAP1, histones, and DNA were also included (lane 1).
(B)TheNAP1-depletedintermediate canbeconvertedintochromatinbyACFataratethatiscomparabletothatoftheoverallchromatinassembly reaction.Upon
depletion of NAP1 in (A), the non-nucleosomal histone-DNA complexes in the supernatant (S) fraction were incubated with ACF for the indicated reaction times.
ChromatinassemblywasmonitoredbyDNAsupercoilinganalysisinthepresenceofpurifiedtopoisomeraseI.Assemblyreactionswereperformedinparallelwith
NAP1-depleted samples (middle), NAP1-depleted samples that were subsequently supplemented with purified NAP1 (equivalent amount of NAP1 as depleted;
bottom),andmock-depletedsamples(top).The%supercoiling[(supercoiledDNA/totalDNA)x100]versustimeforeachseriesofreactionsisshowninFigureS3.
The positions of relaxed (Rel), supercoiled (SC), and nicked (N) DNA are indicated.
(C) The NAP1-depleted histone-DNA complexes can be converted into periodic nucleosome arrays by ACF. NAP1-depleted histone-DNA complexes
were incubated with ACF and ATP under standard chromatin assembly conditions. Samples that were obtained either before or after incubation with ACF were
subjected to partial micrococcal nuclease digestion analysis. As a reference, mock-depleted histone-DNA complexes that were incubated with ACF were
analyzed in parallel.
See also Figure S2.
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Figure 4. Analysis of Chromatin Assembled with Histone H1
(A) Template association during chromatin assembly occurs in the presence of histone H1. Chromatin assembly reactions were performed as in Figure 1B, except
thathistoneH1wasincludedatapproximatelyonemoleculeofH1percorehistoneoctamer.Theasteriskdenotesacontaminantinthepreparationofthe3Kplasmid.
(B) Partial micrococcal nuclease digestion analysis of chromatin at different stages of assembly. Naked DNA (DNA only), core histone-DNA complexes (DNA,
NAP1, core histones), chromatin lacking H1 (DNA, NAP1, core histones, ACF, ATP), and histone H1-containing chromatin (DNA, NAP1, core histones, H1, ACF,
ATP) were subjected to micrococcal nuclease digestion analysis. Each sample was treated with three different concentrations (increasing from left to right) of
micrococcal nuclease. The three concentrations of micrococcal nuclease used with the different samples were identical. The DNA size markers are the 123 bp
ladder (Invitrogen). The white dots denote positions of DNA fragments derived from mono-, di-, and trinucleosomes.
(C) Extensive micrococcal nuclease digestion analysis of chromatin at different stages of assembly. Samples that are similar to those in (B) were extensively
digested with micrococcal nuclease for 1 min. The relative concentrations of micrococcal nuclease are indicated. The DNA size markers are the 123 bp ladder.
The positions of DNA fragments derived from core particles (‘‘Core’’) and chromatosomes (‘‘Chr’’) are denoted. The relative amounts of total mononucleosome
species (core particles and chromatosomes) are indicated.
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H1 (DNA, core histones, NAP1, ACF), and H1-containing chro-
matin (DNA, core histones, NAP1, ACF, H1), which represent
key stages in the assembly of chromatin. To examine the struc-
ture of mono- and oligonucleosomal species, we carried out
partial micrococcal nuclease digestion analysis (Figure 4B). As
noted above, digestion of the histone-DNA intermediate yields
a diffuse heterogeneous mixture of DNA fragments, which are
similar to those seen upon micrococcal nuclease digestion of
newly synthesized DNA (Smith et al., 1984). Upon the addition
of ACF, periodic arrays of nucleosomes are generated. We also
found that the H1-containing chromatin is more nuclease resis-
tant than the H1-deficient chromatin as well as the histone-
DNA intermediate, which are, in turn, more resistant than naked
DNA. In addition, we observed an increase in the nucleosome
repeat length upon addition of histone H1. The increased repeat
length and decreased micrococcal nuclease sensitivity of H1-
containing chromatin relative to H1-deficient chromatin are
similar to the properties of chromatin associated with bulk DNA
relative to newly synthesized DNA in nuclei (see, for example,
Levy and Jakob, 1978; Schlaeger and Knippers, 1979).
Next, to investigate the nature and amount of mononucleo-
somespecies,weperformedanextensivemicrococcalnuclease
‘‘limit’’ digestion of DNA, histone-DNA complexes, and chro-
matin lacking or containing H1 (Figure 4C). First, with the chro-
matin samples, we saw ?147 bp DNA species (derived from
core particles) in the absence of H1 and a mixture of ?166 bp
and ?147 bp DNA fragments (derived from chromatosomes
[H1-containing mononucleosomes; see Simpson (1978)] and
core particles) in the presence of H1. Second, with the histone-
DNA complexes, we observed only about 15%–20% of the
amountofthe?147bpDNAspecies(whichroughlycorresponds
to the low level of ACF-independent nucleosome formation
[see, for example, Figure 1B]) that was obtained with the corre-
sponding H1-deficient chromatin. Third, with the naked DNA,
the DNA was extensively digested and ?147 bp DNA species
were not seen. Hence, the results of the ‘‘limit’’ digests reveal
that the histone-DNA complexes are much more sensitive to
micrococcal nuclease digestion than nucleosomes.
Template Association Involves the Initial Deposition
of Histones H3 and H4
Chromatin assembly in cells appears to involve the initial depo-
sitionofhistonesH3andH4prior totheincorporation ofhistones
H2A and H2B (see, for example, Worcel et al., 1978; Cre ´misi and
Yaniv, 1980). We therefore examined template association by
the histones with equimolar amounts of purified preparations
of histones H3-H4 and H2A-H2B (Figure S3). First, we carried
out standard chromatin assembly reactions with H2A-H2B
alone, H3-H4 alone, and all four core histones (Figure 5A). With
the DNA supercoiling assay, there was no detectable supercoil-
ing with H2A-H2B alone and a small amount of negative DNA
supercoiling with H3-H4 alone. (The negative supercoiling by
Figure 5. The Deposition of Histones H3-H4, but Not H2A-H2B, Is Sufficient for Template Association
(A) Chromatin assembly reactions with H2A-H2B alone, H3-H4 alone, or all four core histones. Chromatin assembly reactions were performed, as outlined in the
schemeat thebottomof the figure, inthe absence or presence of ACF with equimolar amounts of theindicated histones. The reaction products wereanalyzed by
using the DNA supercoiling assay.
(B) Order-of-addition reactions reveal that H3-H4, but not H2A-H2B, is sufficient for template association. Template association reactions were carried out, as
outlined in the scheme at the bottom of the figure, by modification of the basic procedure used in Figure 1B. Histones (H2A-H2B, H3-H4) were added either at
0 min (prior to the addition of template 2) or at 2 min (after the addition of template 2). For each reaction, the order of addition of H3-H4 and H2A-H2B is indicated.
The percent supercoiling (%SC) is given for both the 3K and 8K templates.
See also Figure S3.
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H3-H4 alone was confirmed by agarose gel electrophoresis of
the DNA species in the absence versus the presence of chloro-
quine [data not shown].) These findings are consistent with
results obtained in previous studies on the deposition of H3-H4
onto DNA (see, for example, Camerini-Otero and Felsenfeld,
1977; Bina-Stein and Simpson, 1977; Peterson et al., 2007). In
addition, upon addition of ACF to H3-H4-DNA complexes, we
observed a reproducible decrease in DNA supercoiling, which
may be due to some disruption or reorganization of H3-H4-
DNAspecies by ACF. With regard to the present study, however,
the main point of this experiment is that chromatin assembly can
be achieved with the purified preparations of H2A-H2B and
H3-H4 and that the properties of H2A-H2B alone and H3-H4
alone are in accord with those observed in previous work.
We then examined the specific roles of H2A-H2B and H3-H4
in template association. To this end, we carried out template
association experiments in which H2A-H2B were added prior
to H3-H4, and vice versa (Figure 5B). When histones H3-H4
were combined with template #1 and then template #2 and
histones H2A-H2B were added sequentially, we observed chro-
matin assembly specifically on template #1 relative to template
#2. In contrast, when H2A-H2B were combined with template
#1 and then template #2 and H3-H4 were added sequentially,
chromatin assembly occurred on both template #1 and template
#2. Therefore, H3-H4, but not H2A-H2B, are sufficient for the
formation of a template associated complex. These findings
indicate that the histone-DNA intermediate is formed by the
initial deposition of histones H3 and H4 onto the DNA, and are
consistent with studies in cells in which the deposition of
H3-H4 was seen to precede the incorporation of H2A-H2B
into chromatin.
Visualization of the Nonnucleosomal Intermediate
To gain a better understanding of the nature of the nonnucleoso-
mal complex, we imaged the intermediate and chromatin by
atomic force microscopy. In these experiments, we performed
template association reactions with the 3K plasmid as template
#1andthe8Kplasmidastemplate#2intheabsenceorpresence
of ACF. As shown in Figure 6A, both the nonnucleosomal inter-
mediate and chromatin exhibit the characteristic ‘‘beads-on-a-
string’’ morphology that has been observed in the imaging of
chromatin by electron microscopy and atomic force microscopy
(see, for example, McKnight and Miller, 1977; McKnight et al.,
1978; Bustamante et al., 1997). Notably, the histones in both
the nonnucleosomal intermediate and chromatin samples are
associated only with the smaller ?3 kbp template #1. Figure 6B
shows magnified images of the histone-DNA complexes and
nucleosomes. The apparent areas (mean ± standard deviation)
of individual histone-DNA particles and nucleosomes were
observed to be 113 ± 69 nm2(n = 338) and 99 ± 55 nm2
(n = 558), respectively. The rather large values for the standard
deviation are partly attributable to heterogeneity in orientations
of individual particles deposited on the surface, overlapping of
some particles, and variations in AFM probe sharpness (see,
for example, Bustamante et al., 1997). The distributions are
shown in Figure 6C. Additional images of the histone-DNA
complexes and chromatin are shown in Figure S4. To within
the obtained resolution, the morphology and size of the non-
nucleosomal histone-DNA particles are similar to those of
canonical nucleosomes.
Histone Deposition and Chromatin Maturation
We have identified a non-nucleosomal histone-DNA interme-
diate that is formed during the assembly of chromatin by ACF.
These histone-DNA complexes do not supercoil DNA like
a canonical nucleosome (Figures 1B), lack NAP1 (Figure 3),
are more sensitive to micrococcal nuclease digestion than nu-
cleosomes (Figure 4), can be formed in the presence of histone
H1 (Figure 4), are formed by the initial deposition of H3-H4 (Fig-
ure 5), resemble nucleosomes by atomic force microscopy anal-
ysis (Figure 6), and can be converted into nucleosomes by ACF
(Figure 3). The rapid rate of formation of the nonnucleosomal
intermediate corresponds to the timing of the appearance of
similar bead-like structures that were observed on newly syn-
thesized DNA at replication forks in Drosophila embryos
(McKnight and Miller, 1977; McKnight et al., 1978), as well as
of the nucleosome-like species that were deduced by the inhibi-
tion of psoralen crosslinking of DNA at SV40 DNA replication
forks (Sogo et al., 1986). In addition, the rate of the ACF-
catalyzed conversion of the nonnucleosomal intermediate into
canonical chromatin correlates with the timing of chromatin
‘‘maturation’’ (see, for example, Seale, 1975, 1976; Levy and
Jakob, 1978; Worcel et al., 1978; Schlaeger and Knippers,
1979; Klempnauer et al., 1980; Jackson and Chalkley, 1981).
Hence, these results, combined with the studies of the proces-
sivity of chromatin assembly by ACF and CHD1 (Fyodorov and
Kadonaga, 2002a; Lusser et al., 2005), lead to a model for
nucleosome assembly in which there is rapid formation of
nonnucleosomal histone-DNA complexes and subsequent
conversion of this ‘‘nascent’’ chromatin into ‘‘mature’’ canon-
ical chromatin by the processive action of a motor protein
(Figure 7).
Given the existence of multiple histone chaperones, there are
probably multiple mechanisms by which the nonnucleosomal
intermediate (or analogous species) can be generated. For
example, in studies of DNA replication-coupled chromatin
assembly by CAF1, Smith and Stillman (1991) observed a chro-
matin assembly intermediate that, like the histone-DNA complex
described in this study, does not induce DNA supercoiling.
Although the CAF1-H3-H4-DNA intermediate differs in content
from the non-nucleosomal complex described in this work,
both species may share a related function and interaction of
H3-H4 with the DNA.
We envision the rapidly formed histone-DNA intermediate to
be a species that resembles an ‘‘open’’ nucleosome in which
the DNA is not fully wound around the histones. Based on the
observation that H3 and H4 are sufficient for template associa-
tion (Figure 5B), the primary contacts are likely to be with
histones H3 and H4. Then, the complete wrapping of the DNA
around the histone octamer to give ‘‘mature’’ chromatin would
be achieved by the ATP-dependent motor activity of ACF. It is
also useful to consider that species that are related to the inter-
mediate thatwedescribein thisstudy could begenerated during
processes such as transcription, DNA repair, and histone
exchange. Hence, these findings may be relevant to a broad
range of phenomena in the nucleus.
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EXPERIMENTAL PROCEDURES
Chromatin Assembly
Chromatin assembly reactions were performed as described by Fyodorov and
Kadonaga (2003). All reactions contained core histones (0.35 mg), NAP1
(1.4 mg), ACF (6 nM), ATP (3 mM), topoisomerase I (1 nM), and an ATP regen-
erationsystem(3mMphosphoenolpyruvate,20U/mlpyruvatekinase)inafinal
volume of 70 ml. The buffer composition of the final reaction mixture was as
follows: 15 mM K-HEPES (pH 7.6), 3 mM Tris, 100 mM KCl, 5 mM NaCl,
5.5 mM MgCl2, 0.1 mM EDTA, 6.6% (v/v) glycerol, 1% (w/v) polyvinyl alcohol
(average MW 10,000), 1% (w/v) polyethylene glycol 8000, 20 mg/ml bovine
serumalbumin,and20mg/mlhumaninsulin.Standard(onetemplate)reactions
contained one relaxed circular DNA plasmid (0.294 mg, pGIE-0, which is the
‘‘3K’’ template), whereas template association (two template) reactions con-
tained two different relaxed circular DNA plasmids (0.294 mg each; pGIE-0
and pJH187, which is the ‘‘8K’’ template). The reaction products were
analyzed by DNA supercoiling and micrococcal nuclease digestion assays
(Fyodorov and Kadonaga, 2003). The percent supercoiling [(amount of super-
coiled DNA/amount of total DNA species) 3 100] in the DNA supercoiling
assays was quantitated with ImageQuantTL (GE Healthcare). It is important
to note that nicked DNA that is packaged into chromatin is included in the
‘‘amount of total DNA species’’ but not in the ‘‘amount of supercoiling DNA.’’
Therefore, ‘‘percent supercoiling’’ reflects the amount of closed circular
plasmid DNA that is packaged into chromatin and does not include the nicked
DNA that is packaged into chromatin. In addition, due to the higher resolution
of the different supercoiled species with the 3K plasmid than the 8K plasmid,
the quantitation of percent supercoiling is likely to be more accurate with the
3K plasmid than the 8K plasmid.
SUPPLEMENTAL INFORMATION
Supplemental Information includes Supplemental Experimental Procedures
and four figures and can be found with this article online at doi:10.1016/j.
molcel.2011.07.017.
Figure 6. Visualization of the Nonnucleosomal Histone-DNA Complexes by Atomic Force Microscopy
(A) Template association experiments were performed, as depicted in the diagram, in the absence or presence of ACF. The resulting samples were immobilized
on mica and then analyzed by atomic force microscopy. Representative images are shown. The sample field for each image is 1 mm 3 1 mm, and the height scale
is from 0 to 4 nm, as shown on the right. The scale bars represent 200 nm.
(B) Magnified images of histone-DNA complexes (?ACF) and nucleosomes (+ACF).
(C) Distribution of apparent areas of individual histone-DNA intermediates (?ACF; n = 338) and nucleosomes (+ACF; n = 558).
See also Figure S4.
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ACKNOWLEDGMENTS
We thank George Kassavetis, Timur Yusufzai, Barbara Rattner, Alexandra
Lusser, Yuan-Liang Wang, Mai Khuong, and Sascha Duttke for critical reading
of the manuscript. We are grateful to Timur Yusufzai for suggesting the NAP1
depletion experiment.S.E.T. was partially supportedby theChancellor’s Inter-
disciplinary Collaboratories Program at University of California, San Diego.
This work was supported by grants from the National Institute of General
Medical Sciences/National Institutes of Health to D.E.S. (GM071552, suba-
ward) and J.T.K. (GM058272).
Received: February 14, 2011
Revised: May 27, 2011
Accepted: July 15, 2011
Published: August 18, 2011
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