Tài liệu Báo cáo khoa học: The sequentiallity of nucleosomes in the 30 nm chromatin fibre pptx

The sequentiallity of nucleosomes in the 30 nm
chromatin fibre
Dontcho Z. Staynov
1
and Yana G. Proykova
2
1 Imperial College London, National Heart and Lung Institute, UK
2 School of Earth and Environmental Sciences, University of Portsmouth, UK
The DNA is packed on several levels as chromatin in
the eukaryotic nucleus. The first level of packing,
the highly conserved nucleosome, allows transcrip-
tion, after remodelling and ⁄ or histone modifications ⁄
replacements. The nucleosome core particles have been
reconstituted and crystallized and their structure solved
in detail at 1.9 A
˚
resolution [1–3]. The second level of
packing is the transcriptionally dormant 30 nm chro-
matin fibre. Understanding its structure, as well as the
processes that determine its folding and unfolding, is a
prerequisite for studying the epigenetic mechanism,
which leads to poised-for-transcription or dormant
chromatin [4]. The fibre consists of the entire chroma-
tin of the nucleated avian erythrocytes and comprises
approximately 85% of the chromatin in other cell
types [5].
The structure of chicken erythrocyte chromatin is
the most widely studied in the whole nucleus, as well
as in solution. Using small angle X-ray and neutron
scattering, it has been shown that all the high mole-
cular weight material that diffuses out of the nuclei

after micrococcal nuclease (MNase) digestion is in the
30 nm fibre conformation. It consists of a regular helix
with a diameter of approximately 33 nm and a variable
mass per unit length, which approaches 0.6 nucleo-
somesÆnm
)1
with an 11 nm pitch at 80 mm salt concen-
trations. This implies that there are seven nucleosomes
per helical turn with their flat surfaces almost parallel
to the fibre axis [6–11]. The unusually small cross-
sectional radius of gyration (9.5 nm at 80 mm salt)
suggests a very compact structure with close nucleo-
some–nucleosome contacts.
There are several basic models for the structure of
the fibre that were proposed in the late 1970s and early
1980s, and some variants have been published subse-
quently [4,5,12]. They all comprise regular helices of
more or less seven nucleosomes per turn and thus
approximately satisfy the results obtained by small
angle X-ray and neutron scattering and low resolution
electron microscopy with respect to the packing of
Keywords
30 nm fibre; chromatin structure;
nucleosome
Correspondence
D. Z. Staynov, Imperial College London,
National Heart and Lung Institute, Guy
Scadding Building, Dovehouse Street,
London SW3 6LY, UK
Tel: +44 207 6223644

E-mail:
(Received 29 March 2008, revised 20 May
2008, accepted 23 May 2008)
doi:10.1111/j.1742-4658.2008.06522.x
The folding of eukaryotic DNA into the 30 nm fibre comprises the first
level of transcriptionally dormant chromatin. Understanding its structure
and the processes of its folding and unfolding is a prerequisite for under-
standing the epigenetic regulation in cell differentiation. Although the
shape of the fibre and its dimensions and mass per unit length have been
described, the path of the internucleosomal linker DNA and the sequential-
lity of the nucleosomes in the fibre are poorly understood. In the present
study, we have chemically crosslinked adjacent nucleosomes along the
helix of chicken erythrocyte oligonucleosome fibres, digested the inter-
nucleosomal linker DNA and then examined the digestion products by
sucrose gradient sedimentation. We found that the digestion products con-
tain considerable amounts of mononucleosomes but less dinucleosomes,
which suggests that there are end-discontinuities in the fibres. This can be
explained by a nonsequential arrangement of the nucleosomes along the
fibre helix.
Abbreviations
as, acid soluble; DSP, dithiobis-(succinimidyl propionate); EDC, 1-ethyl-3(3-dimethylaminopropyl)-carbodiimide; MNase, micrococcal nuclease.
FEBS Journal 275 (2008) 3761–3771 ª 2008 The Authors Journal compilation ª 2008 FEBS 3761
nucleosomes in the fibre. However, they were proposed
before the crystal structure of the nucleosome was
solved and do not take into account the topological
constraints imposed on the relationship with respect to
nucleosome orientation and tilt versus chromatin
repeat length. Thus, they differ with respect to the ori-
entation of the nucleosomes and the path of the linker
DNA within the fibre.

High definition structures have not been achieved
because the native fibres comprise a mixture of differ-
ent repeat lengths and could not be crystallized. To
avoid this problem, several studies recently reconsti-
tuted oligonucleosome arrays on a nucleosome by
positioning DNA sequence-repeats differing by multi-
ples of 10 or 11 bp [13–16]. Dorigo et al. [14] used cys-
teine substituted recombinant core histones with or
without linker histone. The electron micrographs of
their reconstitutes show flat ribbons with approxi-
mately five instead of seven nucleosomes per 11 nm,
which do not fold into helical fibres, although they
refer to them as two-start helices. To study nucleosome
sequentiallity in their reconstitutes, Dorigo et al. [14]
crosslinked the samples and digested the linker DNA
with a restriction enzyme. The resultant oligonucleo-
somes migrate in a ‘native’ gel as a ladder with dimers
up to half the size of the original material and support
a two-start helix. In a subsequent study by Schalch
et al. [16], a reconstituted tetranucleosome with a
20 bp linker DNA was crystallized. It was speculated
that this construct allows a two-stranded helix with
close nucleosome contacts in which the flat surfaces of
the nucleosomes are perpendicular instead of parallel
to the fibre axis.
Very different results were obtained by Robinson
et al. [15] using native chicken erythrocyte core histones
and H5 linker histone. They observed two helical
arrays: one for repeat lengths below 210 bp with a
diameter of 33 nm and another for repeat lengths above

210 bp with a diameter of 45 nm and with the flat
surfaces of the nucleosomes close to parallel to the fibre
axis. The overall shapes of their reconstitutes are very
similar to the fibres observed in ‘native’ chromatin.
Most striking are the two very different structures
presented by the two groups for the 177 bp as well as
the 207 and 208 bp repeat lengths, which differ by the
presence ⁄ absence of the linker histone. Apparently,
the reconstitutes of the two groups cannot represent
the same structure and additional evidence is needed.
Both groups have discussed their results with respect
to discriminating between single-start (sequentially
arranged nucleosomes) and two-start nonsequential
helices. Other possible nonsequential helices were
ignored. Neither group considered the very informa-
tive results obtained by DNase I digestion of native
chromatin, which produces ‘dinucleosome repeat’ pat-
terns. Such patterns in which the even multiples are
strong can be produced only if the adjacent nucleo-
somes are digested at alternating sites and, thus, the
odd multiple fragments are attenuated and the even
multiple fragments dominate the pattern. These results
unambiguously show that there is a common structure
of the fibre in which the consecutive nucleosomes in
samples of several different repeat lengths have alter-
nating orientations, as extensively discussed elsewhere
[5,12,17,18].
The question of the sequentiallity of the nucleo-
somes in the fibre is essential. Because a variety of
higher order structures might be capable of reconstitu-

tion with a repeat-sequence DNA, a key question is
how do the reconstitutes of the two groups compare
with the fibres obtained from natural chromatin?
In the present study, we examined the sequentiallity of
the nucleosomes in 30 nm fibres, which diffuse out of
chicken erythrocyte nuclei after a mild MNase diges-
tion without further manipulations. We used the ratio-
nale of Dorigo et al. [14], which involved crosslinking
and nuclease digestion. To demarcate the adjacent
nucleosomes along the fibre, we used nonspecific
protein–protein crosslinkers with two different spans:
(a) dithiobis-(succinimidyl propionate) (DSP) (also
known as Lomant’s reagent), a cleavable bifunctional
ester with 1.2 nm span and a noncleavable, contact-site
crosslinker and (b) 1-ethyl-3(3-dimethylaminopropyl)-
carbodiimide (EDC). Subsequently, the samples were
redigested with MNase and fractionated by sedimenta-
tion on sucrose gradients. Instead of obtaining half the
size of the original material, we observed only a slight
decrease of its size and a considerable number of
mononucleosomes. These results do not support the
two-start helix arrangement, but a higher order nonse-
quential arrangement of the nucleosomes in the fibre
with end-defects as described below.
Experimental rationale
It has been shown that, at moderate ionic strength dif-
ferent, crosslinkers can covalently crosslink linker- and
core-histones beyond a single nucleosome and thus are
able to demarcate adjacent nucleosomes along the
helix of the 30 nm fibre [5]. In the present study, we

used internucleosome histone crosslinking and subse-
quent nuclease digestion to distinguish between differ-
ent arrangements of the nucleosomes in the fibre. The
rationale is illustrated in Fig. 1. Three topologically
different arrangements of the nucleosomes along the
fibre have been suggested [5].
Nucleosome sequentiallity in the 30 nm fibre D. Z. Staynov and Y. G. Proykova
3762 FEBS Journal 275 (2008) 3761–3771 ª 2008 The Authors Journal compilation ª 2008 FEBS
A sequential arrangement
The order of nucleosomes along the fibre helix follows
their order along the DNA. If a 9-mer fragment of
continuous helix of nucleosomes (Fig. 1Aa) is exten-
sively crosslinked, the adjacent nucleosomes will be
crosslinked and the continuity of the linker DNA will
not be required to keep them together. Figure 1Ab
shows the sedimentation profile of an oligonucleosome
sample comprising 7- to 9-mers and half the quantity
of 6- to 10-mers. After 100% crosslinking and nuclease
digestion to mononucleosome size DNA, the sedimen-
tation profile of the sample remains the same. The
80% crosslinked sample will show a decrease of the
average size of the original oligonucleosome sample
and smaller size oligomers will appear (Fig. 1Ac). If
the nucleosomes are interdigitated, as shown by Rob-
inson et al. [15], there will be more internucleosomal
crosslinks, which will stabilize the fibre structure, and
the sedimentation profiles of digestion products will be
intermediate between those shown in Fig. 1Ab,Ac.
A multi-start helix
Nucleosomes are arranged in a multistrand sequence

and are not consecutive. Figure 1Ba illustrates a rib-
bon, which can fold into a two-start helix fibre, with
linkers either parallel or perpendicular to its axis.
Fig. 1. Schematic presentation of different nucleosome arrangements in the 30 nm fibre and the expected sucrose gradient sedimentation
profiles after 100%, 90% or 80% chemical crosslinking of nucleosomes and digestion of the DNA to mononucleosome size of samples con-
sisting of a mixture of hepta-, octa- and nonanucleosomes and half the amount of hexa- and decanucleosomes (6- to 10-mer sample). (A) (a)
Nonamer of a sequential single helix. (b, c) Sedimentation profiles after crosslinking and subsequent digestion of all DNA linkers of the 6- to
10-mer sample: (b) 100% crosslinked and (c) 80% crosslinked. (B) (a) Nonamer in a two start nonsequential (ribbon) arrangement. Numbers
indicate the number of consecutive nucleosomes along the DNA chain. (b–d) Sedimentation profiles of the oligonucleosome sample before
digestion (b) and after digestion of 100% crosslinked (c) and 90% crosslinked sample (d). (C) (a) Hexamer in a single helix nonsequential
arrangement. (b–d) Sedimentation profiles of the 6- to 10-mer sample: (b) original, (c) after digestion of 100% crosslinked and (d) 90% cross-
linked sample. The thick red line demarcates adjacent nucleosomes, expected to be crosslinked. Numbers under the horizontal axes of the
sedimentation profiles denote mono- di-, etc. nucleosomes.
D. Z. Staynov and Y. G. Proykova Nucleosome sequentiallity in the 30 nm fibre
FEBS Journal 275 (2008) 3761–3771 ª 2008 The Authors Journal compilation ª 2008 FEBS 3763
Digestion of the same mixture of 6- to 10-mers
(Fig. 1Bb) of a 100% crosslinked sample will produce
half the size of the original sample (Fig. 1Bc). If the
crosslinking is not complete, smaller size oligomers will
appear (Fig. 1Bd) but the maximum of the main peak
will be approximately half the size of the original
sample.
Single-stranded helices with nonsequentially arranged
nucleosomes
Due to nucleosomes’ nonsequentiallity, the fibres have
end-defects (not envisaged in either of the structures
shown in Fig. 1A,B); namely, one or two missing end-
nucleosomes, as well as one or two end-nucleosomes
separated from the rest by longer distances. Thus,
these end-nucleosomes are probably non-interacting

with the continuous helix and may not be crosslinked
to the rest. One such arrangement, the (–3,5) arrange-
ment, is shown in Fig. 1Ca [18]. Thus, nuclease diges-
tion will shorten the size of the original sample on
average by three nucleosomes and will produce a frac-
tion of 3 ⁄ n mononucleosomes, where n is the average
number of nucleosomes per fragment. The expected
sedimentation profiles of the same 6- to 10-mer sam-
ple, before and after nuclease digestion, are shown in
Fig. 1Cb–d. Because the closely interacting nucleo-
somes are always an even number (Fig. 2), digestion
will produce a mononucleosome fraction and a mix-
ture of even multiples. If some of the end-nucleosomes
are crosslinked to the rest via linker–linker or linker–
core histone crosslinks, the mononucleosome fraction
will be less than 3⁄ n and crosslinking will produce
some odd number oligonucleosomes in the digest.
Thus, the sedimentation profile might not be as clear-
cut as shown in Fig. 1Cc,d, but there would be a
mononucleosome fraction and enriched even-multiples
of oligonucleosomes in the main fraction. Incomplete
(90%) crosslinking will also produce some odd number
oligomers.
As shown in Fig. 1, the differences among the
expected sedimentation profiles of the oligonucleosome
samples after crosslinking and MNase digestion are
expected to be considerable and some incomplete
crosslinking, or cross-chain crosslinking, will not
change their characters.
Figure 2 shows that, in the nonsequential (–3,5)

arrangement, the number of nucleosomes making close
contacts is always even [18]. Thus, there are no close
contacts in the di- and trinucleosomes, whereas only
two nucleosomes are close to each other in tetra- and
pentanucleosomes. In hexanucleosomes, there are four
nucleosomes in close proximity.
Results
Chicken erythrocyte nuclei were digested with MNase
and the material that diffused out of the nuclei was
fractionated by sedimentation through sucrose gradi-
ents. All chromatin samples from dinucleosomes to
high molecular weight material contained indistin-
guishable ratios of linker to core histones (see supple-
mentary Fig. S1).
Chromatin crosslinked with DSP
DSP has been used previously for histone crosslinking
to establish the proximity of different histones inside
the nucleosome or of nucleosomes in the fibre [5,19]. It
is a cleavable crosslinker with two succinimidyl groups,
which react with lysines independently. The maximum
span of crosslinking is 1.2 nm.
A sample of oligonucleosomes, consisting mainly of
tetra-, penta-, hexamers, minor tri- and heptamer com-
ponents with an average number of nucleosomes in the
main peak of 4.9, was extensively crosslinked. It was
digested with MNase for different lengths of time and
agarose gel electrophoresis of the DNA exhibited the
characteristic nucleosome repeat (not shown). After
removal of free crosslinker by dialysis, it was fraction-
ated on sucrose gradients. Figure 3A,B shows that the

di tri
tetra penta
hexa
1
1
1
1
1
2
2
2
2
2
3
3
3
3
4
4
4
5
5
6
Fig. 2. Schematic presentation of di- to hexanucleosomes in the
(–3,5) arrangement. The thick red lines illustrate closely spaced
nucleosomes. Numbers indicate consecutive nucleosomes along
the DNA chain.
Nucleosome sequentiallity in the 30 nm fibre D. Z. Staynov and Y. G. Proykova
3764 FEBS Journal 275 (2008) 3761–3771 ª 2008 The Authors Journal compilation ª 2008 FEBS
crosslinking resulted in a partial loss of resolution and

a drop in the sedimentation velocities. Digestion with
MNase produced 10% mononucleosomes and as little
as 3.5% dinucleosomes (Fig. 3C). The average number
of nucleosomes per chain in the main peak decreased
to 4.1. One third of the optical density sedimented
slower than the mononucleosome fraction. It com-
prised an acid soluble (as) oligo-nucleotide fraction
(14%) at the top of the gradient, and a well-defined
band, S, of mononucleosome-size naked DNA (17%).
Further MNase digestion (Fig. 3D,E) led to an
increase of band S by up to 50%, but did not change
the overall result; the proportion of mononucleosomes
increased to 15% and dinucleosomes to 7%. The main
peak was centered at tetranucleosomes (approximately
40%) with the even numbers (2-, 4- and 6-mers)
slightly more pronounced than the odd numbers
(3- and 5-mers). At longer times of digestion, more
than 50% of the sample was converted into the frac-
tion S. Breaking the disulfate bond in the middle of
the crosslinker produced only mononucleosomes and
the band S. DNA gel electrophoresis of the fractions
from Fig. 3D,E showed that approximately 90% of
the DNA in the main peak as well as the mononucleo-
somes and band S are all in the 140–160 bp size
interval (not shown). The high percentage of mono-
nucleosomes obtained after MNase digestion with only
a small amount of dinucleosomes, as well as the grad-
ual decrease of the number of nucleosomes in the main
peak, indicates that crosslinking is almost complete in
the middle of the fibre, but some of the end-nucleo-

somes are not crosslinked to the rest and therefore
must originate from end-defects in the fibre. In differ-
ent experiments, the mononucleosome fraction was
always in the range 0.9–1.6 per chain (and often higher
than 1.0).
A sample of eight to 12 nucleosomes was cross-
linked, digested with MNase, and further digested with
trypsin for different lengths of time. The sedimentation
profiles are shown in Fig. 4. It is seen that MNase
(Fig. 4B) produces a similar profile as in Fig. 3B, with
prominent fractions as, S and mononucleosomes, but
that di- to penta-nucleosomes are of negligible
amounts due to the larger size of the starting material.
Absorbance at 254 nm
F
E
D
C
B
A
S123456as
Distance from top of the gradient
Fig. 3. UV absorbance profiles of sucrose gradients of an oligonu-
cleosome sample mainly comprising tetra- penta- and hexamers
and minor tri- and heptamer components. (A) Control (no crosslink-
ing). (B–E) Extensively crosslinked with DSP and digested with
20 unitsÆmL
)1
MNase for 0, 8, 16 and 32 min respectively. (F)
Showing the sample used in (E) but reduced to break the crosslin-

ker. Numerals 1, 2, etc., denote mono-, di-, etc., nucleosomes.
Fig. 4. UV absorbance profiles of sucrose gradients of a sample of
eight to 12 nucleosomes. (A) Control (no crosslinking). (B–F) Exten-
sively crosslinked with DSP, digested with 20 unitsÆmL
)1
MNase
for 20 min and subsequently digested with trypsin for 0, 1.5, 5, 15
and 45 min. Numerals 1, 2, etc., denote mono-, di-, etc., nucleo-
somes.
D. Z. Staynov and Y. G. Proykova Nucleosome sequentiallity in the 30 nm fibre
FEBS Journal 275 (2008) 3761–3771 ª 2008 The Authors Journal compilation ª 2008 FEBS 3765
Short trypsin digestion times caused an increase of the
mononucleosome fraction without appearance of di- to
penta-nucleosomes (Fig. 4C,D,E) and all the oligonu-
cleosome fractions appeared only after a long digestion
(Fig. 4F).
Histone gel electrophoresis of the samples cross-
linked with DSP showed that crosslinking of core
particles produced a histone octamer, whereas cross-
linking of oligonucleosomes produced higher multiples
corresponding to their size, as reported previously [5].
It is not clear why treatment with DSP slows down the
sedimentation of all oligonucleosomes including the
mononucleosome fraction. The final product of MNase
digestion of all the fractions, including S, is mononu-
cleosome size DNA and the ten nucleotide periodicity
is preserved in the DNase I digests (not shown). Tryp-
sin digestion leads to digest products of the same limit
as that observed in the untreated nucleosomes (see
supplementary Fig. S2). This suggests that, in all prob-

ababilty, DSP changes the buoyant density of the
samples without changing the structure of the nucleo-
somes. Bearing in mind that the two-succinimidyl
groups react independently, some lysines that normally
interact with DNA are bound by one of these groups.
Therefore, several of their positive charges are neutral-
ized and the histone-DNA interactions are weakened,
with or without the establishment of covalent bonds
with other lysines. Apart from the as oligonucleotides
at the top of the gradients, the remainder of the DNA,
including the naked DNA in fraction S, comes from
digested nucleosomes. Thus, after crosslinking, the
nucleosomes must have been intact. However, these
nucleosomes become less stable and some of them do
not survive the subsequent dialysis.
Chromatin crosslinked with EDC
The water-soluble carbodiimide EDC has been used to
crosslink H1-histone to itself and to core histones.
Although it is noncleavable and does not allow easy
identification of the crosslinked products, it offers
some important advantages over the cleavable crosslin-
kers. First, it is a contact-site (zero length) crosslinker
and thus it excludes long-range bridges between non-
interacting amino acids. Second, it binds to an acidic
aminogroup first, and only subsequently makes a pep-
tide bond with an adjacent lysine [20]. Thus, it does
not interfere with the majority of the lysines that inter-
act with DNA and the chromatin structure is less
likely to be damaged.
In a repetition of the experiments shown in Figs 3

and 4, a sample comprising 6–18 nucleosomes per
chain was crosslinked with EDC and sedimented in a
sucrose gradient (Fig. 5A). Extensive digestion with
MNase, which broke more than 90% of the DNA
linkers according to DNA electrophoresis, produced
13.5% mononucleosomes (approximately two per
chain), 0.5% dinucleosomes and a negligible amount
of tri- and tetranucleosomes (Fig. 5B). The average
size of the oligonucleosome fraction decreased from
approximately 14 to 12 nucleosomes per chain. This
result is principally as that obtained by DSP cross-
linking, although crosslinking does not change the
sedimentation velocity of the samples and does not
produce the free DNA fraction S. It is interesting that
MNase digestion produces less than three mononucleo-
somes per chain, even after crosslinking with this zero
length crosslinker. Most probably, some of the end-
nucleosomes are crosslinked to the rest via linker–
linker or linker–core histone links. Digestion of this
material with trypsin initially caused a gradual
decrease of the number of nucleosomes per chain in
the main peak, with a corresponding increase in the
Fig. 5. UV absorbance profiles of sucrose gradients of an oligonu-
cleosome sample of six to 18 nucleosomes per chain crosslinked
with EDC. (A) Crosslinked but undigested. The profile of the un-
crosslinked sample is identical to (A) (not shown). (B–D) Digested
with 20 units
ÆmL)1
MNase at 37 °C for 40 min and with trypsin for
(B) 0 min; (C) 0.5 lgÆmL

)1
trypsin for 6 min; and (D) 2 lgÆmL
)1
tryp-
sin for 30 min.
Nucleosome sequentiallity in the 30 nm fibre D. Z. Staynov and Y. G. Proykova
3766 FEBS Journal 275 (2008) 3761–3771 ª 2008 The Authors Journal compilation ª 2008 FEBS
percentage of mononucleosomes by up to 25%;
approximately 2.7 nucleosomes per chain (Fig. 5C).
The oligonucleosome fraction has a maximum at ten
nucleosomes and two shoulders around six and eight
nucleosome sizes. There are other shoulders beyond
ten nucleosomes but their sizes cannot be estimated
accurately (Fig. 5C). As with the samples that were
crosslinked with DSP, after extensive digestion with
trypsin, almost all of the material was converted into
mononucleosomes (Fig. 5D). This experiment was
repeated several times with preparations consisting of
a different number of nucleosomes per chain and
crosslinked for different lengths of time (from 30 min
up to 5 h) in a 30–80 mm Na
+
ion concentration. The
proportion of mononucleosomes in the sucrose gradi-
ents after MNase digestion was always more than two
per chain. Most probably, the histones were cross-
linked mainly via their N- and C-terminal tails and
extensive digestion with trypsin separated them from
each other (Fig. 5D).
Because the EDC crosslink is not cleavable, how

far the histones are digested by trypsin in the oligo-
nucleosome fraction cannot be determined but trypsin
digestion of crosslinked mononucleosomes shows very
similar digest limits to the noncrosslinked mono-
nucleosomes (see supplementary Fig. S3). Crosslinking
with EDC does not change the sedimentation veloci-
ties, nor does it cause sliding of the nucleosomes
along the DNA. The repeat length is preserved and
the background is low (see supplementary Fig. S4A).
It only slows down the digestion rate of the MNase
to approximately one quarter of the original rate (see
supplementary Fig. S4B). Prior trypsin digestion of
this material increases the rate of digestion by MNase
several fold without causing nucleosome sliding (see
supplementary Fig. S4C). There is no indication that
DNA is crosslinked to histones and thereby protected
from nuclease digestion. Some crosslinked samples
were dialyzed against distilled water and dried. Poly-
acrylamide gels of this material were stained first for
DNA and subsequently for proteins. It was seen that
the DNA and histones moved independently. Further-
more, when this material was first dissolved in 0.25 m
HCl and the supernatant was precipitated with 20%
trichloroacetic acid, no DNA was observed in the gels
(results not shown). Thus, the histones are not cross-
linked to DNA and the reduction in digestion rate
and its recovery after partial trypsin treatment
suggests that the histone–histone crosslinking has
introduced some steric hindrance to nuclease around
the DNA linkers (i.e. the linkers are buried inside the

fibre).
Discussion
When compared with the expected results from the
three topologically different arrangements in the
Experimental rationale, our results are incompatible
with the single-helix sequential and the two-start helix
arrangements (Fig. 1A,B) because neither would yield
end-of-fibre discontinuities. The results are consistent,
however, with the (–3,5) nonsequential arrangement
shown in Fig. 1C [18] or some other unenvisaged non-
sequential nucleosome arrangement. The sedimentation
profiles of the chromatin fragments digested with
MNase after crosslinking with two very different cross-
linkers show remarkable similarities and must reflect
the actual proximities of the nucleosomes in the fibre.
There is a considerable amount of mononucleosomes
and much less di- ⁄ trinucleosomes in the products.
The mononucleosomes evidently come from the ends
of the fibre because of the corresponding decrease of
the average number of nucleosomes per chain in the
main peak. Digestion of the EDC crosslinked samples
with MNase produced less than the three mononucleo-
somes per chain expected for the (–3,5) arrangement,
but partial trypsin digestion, which cuts the linker his-
tone tails first, increased their number to approxi-
mately three per chain, with a negligible increase of
di- and trinucleosomes. In other similar experiments
with EDC, there were between two and three mono-
nucleosomes per chain. Thus, some of the end-nucleo-
somes are crosslinked to the rest, even by the

zero-length crosslinker, perhaps via linker histones H1
and H5. Indeed, there is evidence that the tails of these
histones follow the path of the linker DNA [5]. They
are crosslinked first to each other first and subse-
quently to the core histones [19]. The even number
nucleosome fragments (six, eight and ten) is more
prominent in the main peak, as expected from Fig. 2.
The rest of the nucleosomes must interact either
directly or via histone tails and are also crosslinked by
the zero-length crosslinker. Early in trypsin digestion,
when the tails of all H1 and H5 and some H3 histones
were digested, only end-nucleosomes become separated
(Fig. 5C), whereas, after breaking the core histone
tails, all oligonucleosome sizes appeared and, when the
trypsin limit digest was approached, the whole sample
was converted to mononucleosomes (Fig. 5D). Two
alternative explanations of these results were consid-
ered but both appear very unlikely.
In the first explanation, oligonucleosomes smaller
than 6-mers have different hydrodynamic behaviour
compared to longer oligomers and such short oligo-
mers might not fold into a fibre [21]. Such oligomers
D. Z. Staynov and Y. G. Proykova Nucleosome sequentiallity in the 30 nm fibre
FEBS Journal 275 (2008) 3761–3771 ª 2008 The Authors Journal compilation ª 2008 FEBS 3767
would not be crosslinked and, after MNase digestion,
they will produce only mononucleosomes, whereas, in
the longer oligomers, adjacent nucleosomes will be
crosslinked to each other. If this were the case, the
sucrose gradient profiles would be very different from
those observed. Because the mononucleosomes would

be produced from the shorter oligomers, the average
number of nucleosomes in the main peak would
increase, and not decrease as actually observed. More-
over, the gradients shown in Fig. 3C–E show that con-
siderable amounts of nucleosomes are crosslinked,
even in a mixture of tetra- to hexanucleosomes, and
suggest that there are closely interacting nucleosomes
in such short oligonucleosomes.
For the second explanation, it was reported that
oligomers tend to lose some H1 and H5 histones and
the loss is approximately inversely proportional to the
size of the fragments [22]. It can be speculated that, in
a sequentially folded fibre, the lost H1 and H5 histones
come from the ends of the fibre and the end-nucleo-
somes therefore no longer interact with the remainder.
This explanation is highly unlikely for the two reasons.
First, early during trypsin digestion when it is mainly
the linker histones that are cut, more end-nucleosomes
are converted into mononucleosomes. These nucleo-
somes must therefore have been crosslinked to the rest
via the linker histones (i.e. linker histones were present
at the ends of the fibre). Second, the loss of H1 and
H5 depends on the procedure of chromatin extraction.
We used the same protocol of mild digestion with
MNase to footprint H1 ⁄ H5 histones on the chromato-
some and found that only the mononucleosome frac-
tion loses some of the linker histones. The ratio of H5
to H4 histones (which have similar abundances and
can be assessed quantitatively) in the dinucleosome
sample is indistinguishable from that of high-molecular

weight chromatin. Furthermore, in the DNase I foot-
print of the dinucleosome, the band resulting from a
cut on the dyad axis (Band S0 at 70 bp) becomes
undetectable as a result of protection by linker histone
[23].
How then do the present results compare with those
from the reconstituted fibres of Dorigo et al. [14] and
Robinson et al. [15]? Both groups show real regular
structures, as indicated by electon micrographs. They
both considered two cases: a continuous helix and a
two-strand fibre and they neglected any possible non-
sequential arrangement of the nucleosomes (Fig. 1C).
Nor did they consider the topological constraints
imposed by the length of the linker DNA on the tilts
of the nucleosomes, as discussed elsewhere [24].
Dorigo et al. [14] studied the sequentiallity of
the nucleosomes in the fibre using crosslinking and
digestion rationale but with cysteine-substituted recom-
binant histone mutants. They obtained unambiguous
proof that their reconstitutes are two-start ribbons.
The most probable explanation for the discrepancy
between the results obtained by Dorigo et al. [14] and
those of the present study is that we examined differ-
ent structures and the reconstitutes used do not repre-
sent the native 30 nm fibre of higher eukaryotes. In
the selected micrographs of Dorigo et al. [14] (see
Figs 4 and S2 therein), the samples appear less con-
densed and look very different from previously pub-
lished micrographs of 30 nm fibres [25], as well as their
Fig. 1. The reconstituted decamers, which are large

enough to be in the fibre conformation [21], are flat
ribbons with approximately five instead of seven
nucleosomes per 11 nm.
The reconstituted oligonucleosomes of Dorigo et al.
[14] can be crosslinked in the absence of H1 histone,
suggesting that H1 is not required for the close nucleo-
some–nucleosome contacts. However, H1 histone is
essential for the fibre stability of chicken erythrocyte
chromatin [5,26]. The loss of linker histone causes
exposure of the linker DNA to DNase I [27] and leads
to shortening of the chromatin repeat length in the
mouse [28]. It can be speculated that the particular
repeat lengths used by Dorigo et al. [14] bring the
nucleosomes into contact. Other causes for the differ-
ences between reconstituted and native fibres might be
the type of the crosslinking used, which, in the study
by Dorigo et al. [14], comprised selective crosslinking
using cysteine modified core histones that may facili-
tate direct nucleosome interactions. The crystallized
tetranucleosome [16] from the same laboratory is out-
side the repeat length interval of higher eukaryotic
chromatin and might have relevance to viral, telomeric
or yeast chromatin in which the presence of linker his-
tone is questionable. Furthermore, the presence of all
oligomers, with dimers up to the half the size of the
original samples after digestion of the linkers, was sug-
gested by Dorigo et al. [14] to indicate incomplete
crosslinking. However, incomplete but random cross-
linking should produce a Poisson distribution of all
sizes with a single maximum. In their gels, there are

two maxima. As shown in Fig. 3A,B of Dorigo et al.
[14], both the even multiples (tetra- and hexamers) are
more abundant than the pentamers. This suggests that
either the samples comprised a mixture of different
structures, the crosslinking is not random, or the
nucleosomes are nonsequentially arranged (not dis-
cussed in their study).
The two structures reported by Robinson et al. [15]
are regular helices and conform to the parameters of
the natural fibres with corresponding repeat length
Nucleosome sequentiallity in the 30 nm fibre D. Z. Staynov and Y. G. Proykova
3768 FEBS Journal 275 (2008) 3761–3771 ª 2008 The Authors Journal compilation ª 2008 FEBS
intervals. Robinson et al. [15] did not examine the
sequentiallity of the nucleosomes, nor did they identify
the path of the linker DNA or the location of the
linker histones. They have assumed that the nucleo-
somes follow the fibre helix and that the linker
histones define the different paths of the linker DNA
for different repeat lengths. It is unclear, however,
if this would be the case because they used the same
linker histone for all repeat lengths [24]. Accordingly,
their results do not contradict the results provided in
the present study and, thus, further experiments are
required to check for compatibility. Nevertheless, the
reconstitutes of Robinson et al. [15] are a good start
for further high definition structural studies.
Conclusions
The present study demonstrates that, after crosslinking
of oligonucleosomes from native nuclei with two
different crosslinkers followed by nuclease digestion,

there is a gradual decrease of the size of the main frac-
tion, and mainly mononucleosomes are liberated.
These mononucleosomes evidently come from end-
discontinuities in the fibre, which can be explained
only by nonsequential arrangements of the nucleo-
somes along the fibre helix. The shoulders in the
digests that represent even-numbered nucleosome frag-
ments (Fig. 5), as well as the stronger tetra- and hexa-
nucleosome bands shown in Fig. 3A,B in the study by
Dorigo et al. [14], suggest a nonsequential arrangement
of the nucleosomes; whether this is the (–3,5) arrange-
ment, or some other as yet unenvisaged structure with
end-defects, remains to be seen.
Experimental procedures
Preparation of chromatin samples
To avoid irreversible damage of the fibre and to minimize
the redistribution of linker histones, all crosslinking and
sucrose gradient fractionation experiments were carried out
at Na
+
ion concentrations in the range 25–60 mm. Chicken
erythrocyte nuclei, freshly prepared or frozen at )70 °Cin
40% glycerol, 10 mm Tris–HCl (pH 7.6), 6 mm MgCl
2
,
25 mm KCl, 35 mm NaCl, 0.2 mm phenylmethanesulfonyl
fluoride, were washed and suspended at 6 mgÆmL
)1
DNA
in digestion buffer [0.25 m sucrose, 1 mm CaCl

2
,5mm
Tris–HCl (pH 7.6), 60 mm NaCl]. They were digested with
33 unitsÆmL
)1
MNase at 37 °C for 10 min, terminated with
5mm EDTA at final concentration and pelleted by centri-
fugation at 2300 g for 1 min in a microcentrifuge.
The supernatant contained approximately 10–15% of the
DNA and consisted mainly of acid-soluble material and
mononucleosomes with a lower amount of H1 and H5 hi-
stones. The pellet was resuspended in 1 mm EDTA, 5 mm
Tris–HCl (pH 7.6) and 25 mm NaCl, dialysed against the
same buffer overnight and pelleted again. The supernatant
usually contained 60–70% of the total DNA and all the
histones. It was fractionated according to size in 6–40%
isokinetic sucrose gradients in the same buffer on an SW27
Beckman rotor (Beckman Coulter, Fullerton, CA, USA) at
5 °C, 29 500 g. for different times. The material consisted
of a mixture of mono- to 30–40 nucleosomes size frag-
ments, with the most abundant comprising 10–20 nucleo-
somes. Dialysis of the fractionated samples against higher
salt concentration buffers (15–40 mm NaH
2
PO
4
, pH 8.0)
was performed when the sucrose was dialysed out. In this
way, no apparent aggregation was observed. We have pre-
viously shown that, when using this protocol for the isola-

tion of chromatin, only mononucleosomes lost some of the
linker histones [23].
All samples that were used contained equal ratios of
linker to core histones. Histone gel electrophoresis of
samples used in Figs 3–5 are shown in the supplementary
Fig. S1. Although the mean sizes of the three samples are
approximately 5, 10 and 14 nucleosomes per chain, respec-
tively, the ratio of intensities of the bands of H5 to H4
remains the same.
In some experiments, the nuclei were digested with
60 unitsÆmL
)1
MNase and oligonucleosomes were extracted
directly with 60 mm NaCl, 5 mm Tris–HCl (pH 7.6) and
5mm EDTA. The supernatant contained 40–50% of the
total DNA up to 15–20 nucleosomes long. This material
did not show any difference in histone content compared to
the samples shown in Fig. S1.
Crosslinking with DSP
Crosslinking with DSP (Pierce, Rockford, IL, USA) was
carried out in 15–40 mm NaH
2
PO
4
(pH 8), 1 mm EDTA
(31–59 mm Na
+
ions) with 3 mgÆmL
)1
DSP at room

temperature for different times and terminated with 15 mm
glycine and the crosslinker dialysed out. We started with
5 h of crosslinking, but subsequently found out that 10 min
was sufficient.
Crosslinking with EDC
Crosslinking with EDC (Pierce) was carried out in
22–40 mm NaH
2
PO
4
(pH 6.8), 1 mm EDTA (31–62 mm
Na
+
ions) with 5 mgÆmL
)1
EDC at room temperature for
30 min to 12 h and terminated with 1% 2-mercaptoethanol.
No difference was found for the results obtained using
different times for crosslinking.
Redigestion of crosslinked oligonucleosome samples with
MNase was carried out in 30 mm NaH
2
PO
4
(pH 7.6),
1.2 mm CaCl
2
, either at 37 °Corat6°C and terminated
D. Z. Staynov and Y. G. Proykova Nucleosome sequentiallity in the 30 nm fibre
FEBS Journal 275 (2008) 3761–3771 ª 2008 The Authors Journal compilation ª 2008 FEBS 3769

with 5 mm EDTA. Digestion with trypsin (type II; Sigma,
St Louis, MO, USA) was carried out at room temperature
and terminated with soybean trypsin inhibitor (Sigma).
Isokinetic sucrose gradients (6–40%) of crosslinked and
(or) redigested material were run in 30 mm NaH
2
PO
4
(pH 6.8), 5 mm EDTA (46 mm Na
+
ions) in a SW41 rotor
(Beckman) at 5 °C (200 000 g for 12–18 h).
Agarose gel electrophoresis of DNA was carried out in
2% gels in 30 mm NaH
2
PO
4
(pH 6.8), 0.5 mgÆL
)1
ethidium
bromide.
Additional results on linker histone abundance and
MNase and trypsin digestions are presented in the supple-
mentary Figs S1–S4.
Acknowledgements
We are grateful to Drs Daniela Rhodes and Venki
Ramakrishnan for useful discussions. Funding by
Wellcome Trust grant no. 037008 to D. Z. S. is grate-
fully acknowledged.
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Supplementary material
The following supplementary material is available
online:
Fig. S1. Linker to core histones ratios.
Fig. S2. Trypsin digestion of samples crosslinked with
DSP.
Fig. S3. Trypsin digestion of samples crosslinked with
EDC.
Fig. S4. MNase digests of samples crosslinked with
EDC.
This material is available as part of the online article
from
Please note: Blackwell Publishing are not responsible
for the content or functionality of any supplementary

materials supplied by the authors. Any queries (other
than missing material) should be directed to the corre-
sponding author for the article.
D. Z. Staynov and Y. G. Proykova Nucleosome sequentiallity in the 30 nm fibre
FEBS Journal 275 (2008) 3761–3771 ª 2008 The Authors Journal compilation ª 2008 FEBS 3771