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

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.

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-

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

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)

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

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-

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

)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

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

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

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

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

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

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
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