Life Science: Medical Biology

Irregular organization in the human chromosomes
revealed by X-ray scattering
How is a long strand of DNA organized in the cell?
In molecular biology textbooks, we often find a typicalfigure (Fig. 1(a)). To begin with, DNA is wrappedaround histones and forms a "nucleosome" (10-nmfiber) structure. This nucleosome has been assumed
to be folded into the regular "30-nm chromatin fiber."In one of the famous models, the "hierarchical helicalfolding model," it is assumed that the 30-nm chromatinfiber is folded progressively into larger fibers such as~100-nm and then ~200-nm fibers, to form the final
mitotic chromosomes (Fig. 1(b)) or large chromatinfiber (chromonema fiber) in interphase cell nuclei.
What do mitotic chromosomes actually look likein cells? To see chromosomes as intact as possible,
we performed cryo-electron microscopy (cryo-EM) [1].
Surprisingly, cryo-EM and subsequent computational
image processing did not reveal any 30-nm chromatinfibers in the mitotic chromosomes, but merely auniform disordered structure, which strongly argues
against the "established view" (Figs. 1(a) and 1(b)).

(a)

(b)

DNA

2 nm

Nucleosome

30 nm fiber
100 nm fiber
200-250 nm fiber

11 nm

500-750 nm fiber

30 nm chromatin fiber
30 nm

Mitotic chromosome

700 nmHierarchical helical structure?

Chromosomes
X-ray

2θ

Scattering Intensitylog ( I×S2 ) (arb. units)

(c)

0~30
30 nm

(d)
~6nm
nm
~6~11 nm0.05 0.10 0.15 0.20

Scattering Vector S (nm–1)

Fig. 1. (a) Textbook image of chromatin, (b) hierarchicalhelical folding model, (c) experimental setting of smallangle X-ray scattering, and (d) a typical scattering
pattern of human mitotic chromosomes.

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On the other hand, cryo-EM observation can examine
only a limited portion of a chromosome, not the whole,because the section thickness is only around 50 nm.
It was thus difficult to observe possible hierarchicalregular structures in the chromosomes, if any.
To investigate the bulk structure of the mitotic
chromosomes in solution, we performed small-angle
X-ray scattering (SAXS) [2,3]. Generally, when X-rays
are irradiated onto non-crystal materials, scattering atsmall angles can generally reveal periodic structures
within samples (Fig. 1(c)). At beamline BL45XU,
the synchrotron X-ray beam was irradiated onto
isolated human chromosomes at the bottom of a
glass capillary, and scattering patterns were recoded(Fig. 1(c)). A typical scattering pattern of mitotic
chromosomes showed three peaks: 30-nm, weak 11nm, and 6-nm peaks (Figs. 1(d) and 2(a)). This result
is completely consistent with those of experiments
conducted by Langmore & Paulson [4]. While the11 nm and 6 nm peaks are believed to originate fromthe spatial arrangement of nucleosomes, but the 30nm peak was assumed to represent the side-by-side
packing of the 30-nm chromatin fibers, and has long
been regarded as strong evidence for the existenceof these fibers in chromosomes. However, why could
we not observe such a 30-nm structure in the mitotic
chromosomes by cryo-EM?
To elucidate the nature of the 30-nm peakobserved by SAXS, the isolated chromosomes wereexamined by cryo-EM. Again, no 30-nm chromatinstructures were observed inside chromosomes.
However, the cryo-EM images unexpectedly
showed that the chromosome surface was coatedwith electron-dense granules of the similar size asribosomes (Fig. 2(a), bottom). Biochemical analyses
confirmed that they are ribosomes contaminating thechromosome surface. The ribosomes were regularly
stacked with a ~30 nm spacing. We removed theribosomes from the chromosome surface by washingwith an isotonic buffer, with maintaining the size andshape of chromosomes (Fig. 2(b), bottom). Strikingly,
in the chromosomes, no 30-nm peaks were detectedby SAXS (Fig. 2(b), top). However, other peaks
originating from the internal structure of nucleosomes
remained (Figs. 2(a) and 2(b)). We then concluded
that regularly folded 30-nm chromatin fibers are
essentially not present in human mitotic chromosomes.
Next, we investigated an entire region of mitotic
chromosomes at beamline BL29XUL using a newly
developed apparatus for ultra-small-angle X-ray

(a)log (I×S2 ) (arb. units)

~30
30 nm
~6 nm

Scattering Intensitylog (I×S2 ) (arb. units)

Scattering Intensity

scattering (USAXS) (Fig. 2(c)) [2,5]. Again, we foundno regular periodic structures, including the 100 –150
or 200 – 250 nm structure, between ~ 50 and 1000
nm in mitotic chromosomes (Fig. 2(d)), contradicting
the hierarchical helical folding model (Fig. 1(b)). The
cryo-EM, SAXS, and USAXS data collectively indicatethat the irregularly folded nucleosome fibers make up
the bulk structure of human mitotic chromosomes.
How is the nucleosome fiber organized intoa mitotic chromosome? Since condensin andtopoisomeraseIIα, which are essential for chromosome
condensation, form an axis in the chromosome,
we assume that they globally hold the nucleosome
fibers around the chromosomal center (Fig. 3(a)) [2].
Locally, nucleosome fiber is folded in an irregularmanner toward the chromosome center (Fig. 3(a)).
Furthermore, SAXS and USAXS also revealed that
there are no periodic structures in the chromatinin the interphase cells [3], suggesting that the bulk

~1111 nm

00.2

0.4

0.6

0.4

Scattering Vector S (nm–1)

X-ray

X-ray(c)No 30-nm
peak

Beamstop
Sample Capillaries

log (I×S2 ) (arb. units)

Scattering Intensity

PinholeGuard Slits(d)NPCNENPC

Fig. 3. (a) Model structure of mitotic chromosomesand (b) interphase chromatin. NE, nuclear envelop;
NPC, nuclear pore complex.

~300~300 nm
nm

Kazuhiro Maeshima a,b,*, Yoshinori Nishino b,c andYasumasa Joti d
Structural Biology Center, National Institute of Genetics
SPring-8 /RIKEN
c
Research Institute for Electronic Science, Hokkaido Universityd
SPring-8 /JASRIab*E-mail: kmaeshim@nig.ac.jp~1000~11000 nm

0.001

(b)

CCD
Detector

1.95 myIrregular folding

Condensin
Nucleosome
A single nucleosome
fiber loop0.6Scattering Vector S (nm–1)~30-nmpeak(a)(b)~30300 nm
n~66 nmn~11 nm00.2

interphase chromatin also consists of irregularly
folded nucleosome fibers (Fig. 3(b)).
In conclusion, X-ray scattering analysis revealed
that interphase chromatin and mitotic chromosomes
have similar local organizations: compact, irregularfolding of nucleosome fibers without the 30-nm
chromatin fiber (Figs. 3(a) and 3(b)). Although
the term "irregular" or "disordered" might give theimpression that the organizations are likely functionallyirrelevant, the irregular folding implies little physicalconstraint and a highly dynamic property, leading
to a high degree of DNA accessibility. The irregularorganization may thus have several advantages intemplate-directed biological processes in interphasenuclei, including RNA transcription and DNA
replication and repair/recombination.

~50 nmm0.01

Scattering Vector S (nm–1)

Fig. 2. (a) SAXS profile of mitotic chromosome
fraction with ribosomes (top) and scheme (bottom),(b) SAXS profile without ribosomes (top) and scheme(bottom), (c) experimental setting for USAXS [5], and(d) USAXS profile of human mitotic chromosomes.

References[1] M. Eltsov et al.: Proc. Natl. Acad. Sci. USA 105 (2008)
19732.
[2] Y. Nishino, M. Eltsov, Y. Joti, K. Ito, H. Takata, Y.
Takahashi, S. Hihara, A.S. Frangakis, N. Imamoto, T.
Ishikawa, K. Maeshima: Embo J. 31 (2012) 1644.
[3] Y. Joti et al.: Nucleus 3 (2012) 404.
[4] J.P. Langmore and J.R. Paulson: J. Cell Biol. 96 (1983) 1120.
[5] Y. Nishino et al.: Phys. Rev. Lett. 102 (2009) 18101.
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