ARTICLE
https://doi.org/10.1038/s42003-023-05750-w

OPEN

The nucleolar shell provides anchoring sites for
DNA untwisting1234567890():,;

Jumpei Fukute1,2, Koichiro Maki

1,2,3,4 ✉ & Taiji Adachi

1,2,3,4

DNA underwinding (untwisting) is a crucial step in transcriptional activation. DNA underwinding occurs between the site where torque is generated by RNA polymerase (RNAP) andthe site where the axial rotation of DNA is constrained. However, what constrains DNA axial
rotation in the nucleus is yet unknown. Here, we show that the anchorage to the nuclear
protein condensates constrains DNA axial rotation for DNA underwinding in the nucleolus. In
situ super-resolution imaging of underwound DNA reveal that underwound DNA accumulates in the nucleolus, a nuclear condensate with a core–shell structure. Speciﬁcally, underwound DNA is distributed in the nucleolar core owing to RNA polymerase I (RNAPI)
activities. Furthermore, underwound DNA in the core decreases when nucleolar shell components are prevented from binding to their recognition structure, G-quadruplex (G4). Taken
together, these results suggest that the nucleolar shell provides anchoring sites that constrain
DNA axial rotation for RNAPI-driven DNA underwinding in the core. Our ﬁndings will contribute to understanding how nuclear protein condensates make up constraints for the sitespeciﬁc regulation of DNA underwinding and transcription.

1 Laboratory of Cellular and Molecular Biomechanics, Department of Mammalian Regulatory Network, Graduate School of Biostudies, Kyoto University, Sakyo,

Kyoto, Japan. 2 Laboratory of Biomechanics, Institute for Life and Medical Sciences, Kyoto University, Sakyo, Kyoto, Japan. 3 Department of MicroEngineering, Graduate School of Engineering, Kyoto University, Sakyo, Kyoto, Japan. 4 Department of Medicine and Medical Science, Graduate School of
Medicine, Kyoto University, Sakyo, Kyoto, Japan. ✉email: maki@infront.kyoto-u.ac.jp
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In a cell nucleus, genomic DNA can be underwound(untwisted)1 or overwound (twisted)2 by mechanobiochemical factors3, regulating the access of polymerases4–6
and nucleases7,8 to the genetic code. In addition, under/overwound DNA can adopt non-B secondary structures9,10 andsupercoiled structures (plectonemes)11, contributing to the multiscale genome conﬁguration12. Thus, DNA under/over-winding
plays a pivotal role in the spatiotemporal regulation of genomic
processes such as transcription, replication, and repair.
According to a model proposed by Liu and Wang13, DNA
under/over-winding occurs between the site where torque isgenerated and the site where the axial rotation of DNA is constrained. During gene transcription, RNA polymerase (RNAP),
whose rotation around the DNA helical axis is constrained,
generates torque to induce axial rotation of the template DNA14;
however, torque-generating RNAP alone is not sufﬁcient for
DNA under/over-winding15,16. By contrast, DNA becomesunder/over-wound when DNA axial rotation is constrained
behind/ahead the RNAP15,16. Therefore, constraints on DNA
axial rotation are critical to determining the under/over-winding
regions along genomic DNA.
DNA looping has been proposed as a major constraint on
DNA axial rotation. DNA loop formation by bridging proteins,
such as LacI in prokaryotic cells17,18 and cohesin in eukaryotic
cells19,20, could constrain the axial rotation of DNA at the bridging sites. However, multiple underwound genomic regions(~100 kb) have been found within a single loop (>1 Mb)21, suggesting the existence of constraints by looping-independent
factors.
In this study, we hypothesized that anchoring to nuclear protein condensates22 constrains the axial rotation of DNA. To test
this hypothesis, we ﬁrst identiﬁed the intranuclear location where
underwound DNA is produced by performing in situ superresolution imaging. Next, we investigated how DNA axial rotation is constrained in the nucleus by focusing on the interactionof DNA with nuclear condensates.
Results
Biotinylated psoralen binds to underwound DNA in the
nucleus. We ﬂuorescently labeled underwound nuclear DNA
with psoralen19,21,23,24, an intercalator that preferentially binds tounderwound DNA rather than relaxed DNA25. An in vitro
crosslinking assay26 veriﬁed that biotinylation of psoralen for
avidin-based ﬂuorescent labeling retains its preferential binding
to underwound DNA (Supplementary Fig. 1), as previously
shown by sequencing23,27–29. Biotinylated psoralen (bio-psoralen) was used to ﬂuorescently label underwound DNA in
MC3T3-E1 mouse osteoblast-like cells (Fig. 1a). First, cell
membranes were gently permeabilized with digitonin to enhance
the incorporation of bio-psoralen into the nucleus23,30. Then, thecells were treated with bio-psoralen and irradiated with UV light,
followed by paraformaldehyde ﬁxation, permeabilization, andﬂuorescent labeling with NeutrAvidin Protein, DyLightTM 488.
To validate each staining step, we performed staining while
omitting each step: digitonin treatment, bio-psoralen incorporation, UV irradiation, or NeutrAvidin Protein, DyLightTM 488
treatment (Fig. 1b). Only samples that had been subjected to all
four staining steps (Fig. 1b, right-most panel) showed prominent
nuclear signals in all cells. Blockade of the biotin–avidin interaction via treatment with free avidin diminished nuclear biopsoralen signals (Supplementary Fig. 2a). In addition, DNAdegradation by DNase I treatment decreased nuclear bio-psoralen
signals (Supplementary Fig. 2b). As bio-psoralen binds to pairwise RNA secondary structures31 and underwound DNA, we also
assessed the possibility that bio-psoralen binds to nuclear RNA.
2

Using co-staining with StrandBriteTM, a speciﬁc RNA marker, weobserved no remarkable co-localization of bio-psoralen andStrandBriteTM signals (Supplementary Fig. 2c), and RNA signal
was signiﬁcantly decreased after RNase A treatment (Supplementary Fig. 2d, e). On the other hand, the nuclear signal of biopsoralen was sustained (Supplementary Fig. 2d, f). These results
conﬁrmed that bio-psoralen-bound DNA were ﬂuorescently
labelled in a nucleus.
To assess whether bio-psoralen predominantly binds to DNA in
the underwound state, we examined changes in its binding after
treating cells with bleomycin (BLM), which induces the transition ofunder/over-wound DNA to relaxed state by introducing both singleand double-strand breaks21,32. Naughton et al. had utilizedbiotinylated trimethyl psoralen (bTMP, not bio-psoralen) in humancells and veriﬁed the binding speciﬁcity of bTMP by showing that
BLM treatment decreased bTMP signal21. Consistent with Naughton’s ﬁndings, BLM treatment decreased the amount of biopsoralen-bound genomic DNA (Fig. 1c) and the ﬂuorescenceintensity of bio-psoralen in the nucleus (Fig. 1d, e). These results
indicate that bio-psoralen predominantly binds to underwound
DNA in the nucleus, verifying the speciﬁcity of our staining method.
Underwound DNA colocalizes with nuclear proteins. We
identiﬁed the intranuclear location of underwound DNA and associated nuclear proteins at super-resolution using lattice-pattern
structured illumination microscopy (Lattice-SIM2) (Fig. 2a). Notably,
bio-psoralen-labeled underwound DNA exhibited a distinct distribution from 4′,6-diamidino-2-phenylindole (DAPI), a minor grove
binding dye. This difference would be explained by the stronger
interaction of DAPI with overwound DNA33, which has a decreased
minor groove width34. Co-staining of underwound DNA and several
marker proteins revealed that underwound DNA was distributed in
the vicinity of RNA polymerase II phosphorylated at serine 2(RNAPIIS2P) (Fig. 2b). This result is consistent with previous reportson the generation of underwound DNA behind RNAPII duringtranscription elongation21,29. The observation of underwound DNA
in proximity with RNAPII, which was in agreement with the fact thatRNAPII generates torque to induce DNA underwinding, encouraged
us to further investigate using the developed methodology. Notably,
several bright foci of underwound DNA existed in nuclei (Fig. 2c,arrows). In these foci, underwound DNA was distributed adjacent toclusters of topoisomerase I (TopI), which is involved in the relaxationof under/over-wound DNA35,36, further supported that our imaging
technique speciﬁcally detects underwound nuclear DNA. It is
important to mention here that the distribution of TopI has beenreported to span nucleoplasm37, suggesting that some portion ofproteins potentially leak to cytoplasm by digitonin treatment38. To
assess the roles of TopI and topoisomerase II (TopII) in the regulation of DNA underwinding, we performed an inhibitor experimentusing camptothecin (for TopI inhibition) and etoposide (for TopII
inhibition) (Supplementary Fig. 3). Although we expected that TopI/
II inhibition would increase underwound DNA and produce astronger bio-psoralen signal, Top I/II inhibition decreased thenuclear bio-psoralen signal, indicating a decrease in the amount ofunderwound DNA. Such responses, as previously reported19,21,
might be caused by secondary effects of TopI/II inhibitor treatment,such as DNA strand break36 and reduced RNAP activity29,39. Taken
together, we successfully elucidated the intranuclear location ofunderwound DNA and associated nuclear proteins.
Underwound DNA accumulates in the nucleolar core. As TopI
is known to distribute in nucleoli37, the site of ribosome biogenesis, we examined whether DNA underwinding occurs inribosome DNA (rDNA). We stained rDNA by ﬂuorescence insitu hybridization (FISH) and found that bio-psoralen signal

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aMild
permeabilization

Bio-psoralen
incorporation

UV
irradiation

Fluorescentlabeling

Digitonin

NeutrAvidinDyLightTM 488→ Fixation andpermeabilization

Digitonin (-)

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DylightTM 488

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Fluorescence intensity ofbio-psoralen in the nucleus (a.u.)

6.0

BLM

Bio-psoralen-bound genomic DNA
in 1000 ng total DNA (ng)

c

20 μm

3000

2000

1000

0

DMSO BLM

Fig. 1 Fluorescent labeling of intracellular underwound DNA. a Procedure for bio-psoralen ﬂuorescent labeling. b Fluorescence images of DAPI controls
and bio-psoralen (DyLightTM 488) in samples subjected to all or only three of the staining steps. c Quantiﬁcation of bio-psoralen-bound genomic DNA
extracted from DMSO control and BLM-treated cells (100 µM, 15 min). BLM introduces single-/double-strand breaks and induces the transition ofunderwound DNA to relaxed DNA. Three independent biological replicate experiments were performed (n = 3). Statistical signiﬁcance was assessed using
the two-sided paired t-test. P* < 0.05 (P = 0.0367). d Fluorescence images of DAPI and bio-psoralen in DMSO- and BLM-treated cells. e Fluorescenceintensity of bio-psoralen in the nucleus. The circle, centerline, box, upper/lower whiskers, and dots represent the mean, median, 75th/25th percentiles, 1.5
times the interquartile range, and outliers respectively. n = 150 cells. Statistical signiﬁcance was assessed using the two-sided Mann–Whitney U-test.
P** < 0.01 (P = 3.05 × 10−50).

partly overlapped with that of rDNA (Fig. 3a). This result indicated that DNA underwinding occurs in rDNA as well.
Nucleoli have a core–shell structure consisting of three subcompartments40: ﬁbrillar center (FC) and dense ﬁbrillar component (DFC) forming the core, and granular component (GC)
forming the shell (Fig. 3b). This structure enables the successive
steps of ribosome biogenesis, with rRNA transcription taking
place at the FC/DFC interface, processing in the DFC, andassembly with ribosomal proteins in the GC40,41. Although
digitonin potentially affects the integrity of the nucleolar subcompartments38, we conﬁrmed that the characteristic nucleolar
structure where shell component proteins surround corecomponent proteins was retained after digitonin treatment(50 μg/mL, 1 min) (Supplementary Fig. 4). Under this conditionof digitonin treatment, we co-stained bio-psoralen and marker
proteins of each sub-compartment, including RNA polymerase I(RNAPI, an FC marker), ﬁbrillarin (FBL, a DFC marker), andnucleophosmin (NPM, a GC marker). As a result, we found that
underwound DNA was mainly distributed in the FC and DFC(Fig. 3c), indicating that underwound DNA is generated in thevicinity of RNAPI in the FC and extends to the DFC. Comparedto the region inside the nucleolus, underwound DNA was lessabundant outside the nucleolus (Fig. 3d, e). These results
suggested that DNA axial rotation is constrained in the GC(shell), resulting in RNAPI-driven DNA underwinding in the FC
and DFC (core).

DNA axial rotation is constrained by anchoring to the
nucleolar shell via GC protein–G-quadruplex (G4) interaction.
To identify the driving force of DNA underwinding, we performed inhibitor experiment for RNAPI. The prominent biopsoralen foci in nucleoli disappeared after inhibition of RNAPI
transcription initiation using CX-546142 as well as inhibition ofelongation using Actinomycin D (ActD) (Fig. 4a). The intensity
proﬁle also showed that the bio-psoralen signal was signiﬁcantly
decreased inside the nucleolus (Fig. 4b–d). Of note, in theseexperiments, the treatment time was limited to 15 min, to avoid

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DAPI

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Fluorescence intensity of bio-psoralen (×104, a.u.)

a0

Bio-psoralen

RNAPIIS2P

Bio-psoralen
RNAPIIS2P

Bio-psoralen

TopI

Bio-psoralen
TopI

0.5 μmDAPI

Fig. 2 In situ super-resolution imaging of underwound DNA. a Co-staining of DAPI (left) and bio-psoralen (right) displayed in XY (top) and XZ (bottom)
sections along the dashed yellow line in the XY section. Heatmap is shown for bio-psoralen ﬂuorescence intensity. b Co-staining of DNA (DAPI), biopsoralen, and RNAPIIS2P. c Co-staining of DNA (DAPI), bio-psoralen, and TopI. Arrows indicate prominent foci of bio-psoralen.

cell death (Supplementary Fig. 5). These results indicated that
DNA is underwound by RNAPI transcription in nucleoli.
Having identiﬁed the driving force of DNA underwinding in
the nucleolus, we next explored possible factors that constrain
DNA axial rotation. We focused on DNA anchoring to the GC asa factor constraining DNA axial rotation. The GC is a condensate
that consists of rRNA and self-assembling proteins, including
NPM40. NPM is known to interact with the G-quadruplex (G4)
formed in rDNA43, a non-B DNA structure composed of stacked
G-tetrads44. Based on these reports, we examined whether DNA
axial rotation is constrained by DNA anchoring to the GC via theinteraction between NPM and G4. We performed an inhibition
experiment using the G4-selective ligand TMPyP4, which inhibits
the interaction with NPM43. As we expected, chromatin
immunoprecipitation (ChIP) analysis showed that TMPyP4
treatment prevented NPM from binding to putative G4 forming
sequences (PQS) in rDNA (Fig. 5a). Then, cells treated with orwithout TMPyP4 were stained with NPM and bio-psoralen(Fig. 5b). A lower NPM signal was detected in TMPyP4 treatedcells because NPM could not bind to G4, and subsequently left
the nucleoli and diffused into the nucleoplasm43. Importantly,
bio-psoralen signal in the nucleolus was signiﬁcantly decreased by
TMPyP4 treatment (Fig. 5c, d). rDNA still existed in the
nucleolus after TMPyP4 treatment (Fig. 5e), indicating that the
inhibition of NPM-G4 interaction relaxes the underwound rDNA
inside the nucleolus. This result suggested that the release of
4

NPM from G4 by TMPyP4 treatment allowed DNA axial
rotation, resulting in the transition of underwound DNA torelaxed DNA. These ﬁndings suggest that DNA axial rotation isconstrained by anchoring to the nucleolar shell via GC
protein–G4 interaction, contributing to RNAPI-driven DNA
underwinding in the core (Fig. 5f).
Discussion
The in situ imaging analysis of underwound DNA demonstrated
that anchoring to nuclear condensates constrains the axial rotation of DNA, facilitating RNAP-driven DNA underwinding. We
showed that nucleoli contain torque-generating RNAP in theircores and anchoring sites in their shells, enabling the accumulation of underwound DNA. In the nucleolar shell (GC), proteins
with self-assembling and DNA-binding domains may contributeto the constraint of DNA axial rotation. NPM, a major protein in
the GC, forms pentamers via its N-terminal domain45 andassembles with nucleolar proteins, rRNA, and itself via an
intrinsically disordered region to form huge condensates46,47 thatbind to rDNA via G4-binding C-terminal domains43,48. This
exerts high rotational drag to constrain DNA axial rotation at the
NPM-bound region. This mechanism can be generally applied to
RNAPII-mediated transcription, in which DNA-binding transcription factors assemble with coactivators to form
condensates49. The assembly of transcription factors is

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Merge

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0.5 μm

4.5

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rDNA (×103)

5 μm

rDNA

Bio-psoralen (×104)

a

Nucleolus

12Distance (μm)

Fibrillar center (FC)Marker: RNAPI
Dense fibrillar component (DFC)
Marker: FBL
Granular component (GC)
Marker: NPM

Core

Shell

Merge

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01.4Fluorescence intensity of bio-psoralen(×104, a.u.)

3.5

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Fluorescence intensity of NPM (×104, a.u.)

Fluorescence intensity of bio-psoralen(×103, a.u.)

d

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GC

Bio-psoralen

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Merge

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DFC

5 μm

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FC

Bio-psoralen

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c

1.2
1.0
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Inside Outside

d (Normalized distance)
Fig. 3 Accumulation of underwound DNA in the nucleolar core. a Co-staining of bio-psoralen and ribosome DNA (rDNA). b Diagram of nucleolar subcompartments; FC and DFC form the core and GC forms the shell. c Co-staining of bio-psoralen and RNAPI (top), ﬁbrillarin (middle), and NPM (bottom).
d Intensity proﬁle of bio-psoralen and NPM. Intensity proﬁle in each nucleolus was measured along the radial axis from the centroid of the nucleolar
outline, which was identiﬁed with NPM. The normalized distance d was calculated based on the distance between the centroid and the intersection of radial
axis and the nucleolar outline. n = 68 nucleoli/11 cells. The intensity proﬁle was smoothed by moving-average. The standard deviation is shown as a band.
e Mean intensity of bio-psoralen inside (0 ≤ d ≤ 0.5) and outside (1.5 ≤ d ≤ 2.0) the nucleolus. n = 68 nucleoli/11 cells. Statistical signiﬁcance was assessedusing the two-sided Mann–Whitney U-test. P** < 0.01 (P = 1.58 × 10−25).

dynamically regulated by phosphorylation50 and ligand
binding51. These dynamic processes alter the degree to which
DNA axial rotation is constrained and possibly contribute to thespatiotemporal regulation of DNA underwinding.
We found that G4 is a key DNA structure for anchoring to the
GC. As G4 is enriched in promoter regions44, underwound DNA

is expected to accumulate in promoter regions of rDNA tandem
repeats. Furthermore, DNA underwinding promotes G4 formation by facilitating DNA strand separation9,10. Taken together
with our ﬁndings, this information suggests a positive feedback
mechanism of DNA underwinding where G4-mediated constraints facilitate DNA underwinding, which, in turn, promotes

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NPM

Bio-psoralen

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NPM

Fluorescnece intensity of bio-psoralen (×103, a.u.)

0.5 μmMerge

CX-5461

5 μm

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ActD

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Fluorescnece intensity of bio-psoralen(×104, a.u.)

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Fluorescence intensity of NPM (×104, a.u.)

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Merge

InsideOutsideFig. 4 DNA underwinding in the nucleolus is driven by RNAP I transcription. a Co-staining of bio-psoralen and NPM in DMSO-, 3 µM CX-5461- and
50 nM Actinomycin D (ActD)-treated cells. The treatment time is 15 min. CX-5461 and Act. D inhibit the RNAPI activities in the initiation and elongation
steps, respectively. b Intensity proﬁle of bio-psoralen and NPM in CX-5461-treated cells (n = 135 nucleoli/19 cells). The dotted line shows the intensity
proﬁle in DMSO-treated cells (n = 89 nucleoli/13 cells). c Intensity proﬁle of bio-psoralen and NPM in ActD-treated cells (n = 120 nucleoli/17 cells).
d Mean intensity of bio-psoralen inside (0 ≤ d ≤ 0.5) and outside (1.5 ≤ d ≤ 2.0) the nucleolus. n = 89 nucleoli/13 cells for DMSO-treated cells, n = 135
nucleoli/19 cells for CX-5461-treated cells and n = 120 nucleoli/17 cells for ActD-treated cells. Statistical signiﬁcance was assessed using the two-sided
Steel-Dwass test. P** < 0.01 (P = 0.00118 for DMSO vs CX-5461, P = 0.001 for DMSO vs ActD). n.s., not signiﬁcant (P = 0.9 for DMSO vs CX-5461,
P = 0.353 for DMSO vs ActD).

G4 formation via DNA strand separation. As G4 is prevalent ingenomic DNA44, G4-mediated positive feedback may play apivotal role in regulating DNA underwinding.
It should be noted that factors other than anchoring to the
nucleolar shell could constrain DNA axis rotation in nucleoli. For
example, upstream binding factor (UBF) binds to rRNA coding
region while forming a nucleoprotein structure calledenhancesome52. DNA looping by enhancesome formation might
constrain DNA axial rotation, in a similar way to that of LacI17,18.
6

G4 formation itself has also been suggested to constrain DNA axis
rotation. In vitro experiments previously showed that G4 formation hinders the transmission of negative supercoil (underwound DNA) due to the formation of a more rigid structure53.
Besides anchoring to the nucleolar shell, these factors could beconstraints and determine the genomic landscape of under/overwound DNA.
In this study, we identiﬁed RNAPI transcription as the driving
force of DNA underwinding in the nucleolus. RNAPI transcribes

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PQS-1(-31813)

PQS-3(-18092)

Non-PQS(-2155)

PQS-4(-1918)Intergenic spacer

18S

1.2
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n.s.

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PQS-2

at

PQS-1

Water

b

PQS-2
(-24321)

Bio-psoralen intensity (×104, a.u.)

Normalized percent input(relative to water)afReeling of rDNA

Shell (GC)

NPM

Constraint
DNA
motionUnderwound DNA
DNAmotionG4Core (DFC)Core (FC)faster and pauses less often than RNAPII owing to the intrinsic
backtracking recovery activities54, suggesting that RNAPI exhibits
stronger torque capacity than RNAPII55. This efﬁcient torquegeneration by RNAPI would help the accumulation of underwound DNA in the nucleolus. In the future, single molecule
experiments such as direct torque measurements14,56, would

TorqueRNAP I

reveal how RNAPI generates torque and how it differs fromRNAPII/III. Besides the type of RNAP, transcriptional activities
in each genomic region also affect DNA underwinding. Mouse
rDNA consists of more than 100 tandem repeats and 30–50% ofthem are transcriptionally active57. The variation in transcriptional activities of each repeat would generate the non-uniform

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Fig. 5 GC protein–G4 interaction locally constrains axial rotation to induce nucleolar DNA underwinding. a qPCR analysis for chromatin
immunoprecipitation (ChIP) DNA samples using NPM antibody for cells without (water) and with TMPyP4 treatment (100 µM, 15 min). TMPyP4 is a G4selective ligand that inhibits the interaction with NPM. For the negative control, ChIP samples with normal rabbit IgG was used. Each target region for qPCR
is shown in the upper panel with the relative position from the transcription start site. Four putative quadruplex sequences (PQS) and one non-PQS weretargeted. The percent input (%) was normalized by the control (water) sample. Three independent biological replicate experiments were performed(n = 3). Statistical signiﬁcance was assessed using the one-sample t-test. P*1 < 0.05 (P = 0.0132), P*2 < 0.05 (P = 0.0177), P**3 < 0.01 (P = 0.000997),
P**4 < 0.05 (P = 0.00175). n.s., not signiﬁcant (P = 0.185). All bar graphs show mean ± s.d. b Fluorescence images of bio-psoralen and NPM in cells without(water) and with TMPyP4 treatment (100 µM, 15 min). The right panel is a line proﬁle of the ﬂuorescence intensity of bio-psoralen and NPM along thedashed lines in the merged images. c Intensity proﬁle of bio-psoralen and NPM in TMPyP4-treated cells (n = 84 nucleoli/16 cells). The dotted line shows
the intensity proﬁle in water-treated cells (n = 108 nucleoli/15 cells). d Mean intensity of bio-psoralen inside (0 ≤ d ≤ 0.5) and outside (1.5 ≤ d ≤ 2) the
nucleolus. n = 108 nucleoli/15 cells for water-treated cells and n = 84 nucleoli/16 cells for TMPyP4-treated cells. Statistical signiﬁcance was assessedusing the two-sided Mann–Whitney U-test. P** < 0.01 (P = 3.35 × 10-26), P* < 0.05 (P = 0.0148). e Immuno-FISH staining for bio-psoralen, FBL and rDNA
for water-treated cells and TMPyP4-treated cells (100 µM, 15 min). f Proposed mechanism of DNA underwinding in nucleoli. During transcription, rDNA
is reeled through RNAPI condensates (FC/DFC interface). Anchoring of the rDNA to the GC (shell) via NPM–G4 interaction constrains the axial rotationof DNA, facilitating DNA underwinding between RNAPI and the anchoring sites. This leads to the accumulation of underwound DNA in the FC and
DFC (core).

distribution of underwound DNA along rDNA, as shown in
Fig. 3a.
This study has potential limitations. First, the bio-psoralen
ﬂuorescence imaging method used in this study involves digitonin treatment, which potentially causes the leakage of DNA andsoluble proteins from the cell nucleus38. An attenuation in the
nucleoplasmic distribution of TopI (Fig. 2c) and NPM (Fig. 3c) indigitonin-treated cells could be caused by the leakage of thesoluble fraction of each protein. Second, the bio-psoralen-based
approach cannot directly detect overwound DNA. Therefore, itremains unknown whether overwound DNA also accumulates in
the nucleolus, and if so, how DNA overwinding occurs. It isnecessary to develop new probes for overwound DNA by utilizing
its binding protein such as GapR, which is helpful for genomewide mapping of overwound DNA2.
DNA underwinding facilitates RNAP recruitment to promoter
regions4,5, whereas excess DNA underwinding is known to promote the release of RNAP from the promoter region30. In the
nucleus, under/over-wound DNA is relaxed by topoisomerases21.
In the present study, TopI was found adjacent to underwound
DNA foci in nucleoli (Fig. 2c). Topoisomerase II (TopII), which
relaxes supercoiled DNA35, is also predominantly localized innucleoli58. Therefore, nucleolar RNAPI-mediated generation ofunderwound DNA could be coupled to TopI/II-mediatedrelaxation, enabling subsequent rRNA transcription. The regulatory mechanisms of underwound DNA generation andrelaxation need to be studied using simultaneous live imaging ofunderwound DNA, RNAP, and topoisomerases.
Methods
In vitro biotinylated (bio-)psoralen crosslinking assay. Theintercalation of bio-psoralen into underwound DNA was examined using an in vitro crosslinking assay26. DNA was crosslinked
by bio-psoralen under UV irradiation, linearized, and denatured
by heat. As bio-psoralen crosslinking prevents DNA heat denaturation, the intercalation of bio-psoralen can be evaluated basedon the amount of non-denatured DNA after heat treatment.
Relaxed and underwound DNA were prepared by incubatingplasmid pBR322 (Takara Bio) with topoisomerase I (TopI, TakaraBio) and gyrase (TopoGEN), respectively, at 37 °C for 1 h, andpuriﬁed using NucleoSpin® Gel and a PCR Clean-up kit(Macherey-Nagel). Bio-psoralen (200 µM; EZ-Link® PsoralenPEG3-Biotin, Thermo Fisher Scientiﬁc) was added to DNA andexposed to 3.6 kJ/m2 of long-wave (365 nm) UV (LUV-4; ASONE) on ice for 30 min. For linearization, DNA was cleaved with
EcoRI (Toyobo) at 37 °C for 1 h. For heat denaturation, one halfof each sample was boiled for 5 min and immediately chilled in8

ice-cold water. Agarose gel electrophoresis was run at 50 V for
1 h, and the gel was stained with 0.1 µg/mL ethidium bromide
solution (Nacalai Tesque) for 30 min. Images were acquired using
ImageQuantTM LAS500 (Cytiva) and DNA bands were quantiﬁedusing Fiji59. The fraction of bio-psoralen-crosslinked DNA wascalculated by dividing the amount of non-denatured DNA by the
total amount of DNA.
Cell culture. MC3T3-E1 mouse osteoblast-like cells (ATCC) were
cultured in minimum essential medium α (Gibco) containing
10% fetal bovine serum (Gibco) and 1% antibiotic-antimycotic(Nacalai Tesque) in a humidiﬁed incubator at 37 °C under a 5%
CO2 atmosphere. For ﬂuorescence imaging, 1 × 104 cells wereseeded on a 7-mm-diameter glass bottom dish (MatTek) for 24 hprior to imaging. For genomic DNA extraction, 2 × 106 cells were
cultured on a 100-mm-diameter Nunc™ EasYDish (Thermo
Fisher Scientiﬁc). For chromatin immunoprecipitation, 4 × 106
cells were cultured on a 150-mm-diameter cell culture dish(Corning).
To verify the speciﬁcity of bio-psoralen for underwound DNA,a perturbation experiment was conducted. Cells were treated with
100 µM bleomycin (Cayman Chemical), which introduces DNA
strand breaks and induces the transition of underwound DNA torelaxed DNA21,32, in complete medium at 37 °C for 15 min. To
examine the roles of TopI/II in the regulation of DNA
underwinding, cells were treated with 10 μM camptothecin (forTopI inhibition) and 10 μM etoposide (for TopII inhibition) incomplete medium at 37 °C for 15 min. To inhibit RNAPI
transcription initiation and elongation, cells were treated with
3 µM CX-5461 (ChemScene LLC)42 and 50 nM Actinomycin D(Nacalai tesque), respectively, in complete medium at 37 °C for
15 min. To inhibit the interaction between nucleolar proteins andG-quadruplex (G4), cells were treated with 100 µM TMPyP4(Abcam)43, a selective G4 ligand, in complete medium at 37 °C
for 15 min.
In situ ﬂuorescence staining of underwound DNA in the cell
nucleus. Fluorescence staining using bio-psoralen was conductedaccording to previous reports19,21,23,24, with modiﬁcation. To
avoid cell detachment from the growth dishes, digitonin and biopsoralen were dissolved in phosphate-buffered saline (PBS) (+),
which was prepared by adding calcium and magnesium solution(Nacalai Tesque) to PBS (–) (Gibco) at a 1:100 dilution. Cells
were treated with 50 µg/mL digitonin (Cayman Chemical) in PBS(+) at 37 °C for 1 min in the dark. After rinsing with PBS ( + ),20 µM bio-psoralen in PBS (+) was added and the cells were
further incubated at 37 °C for 20 min in the dark. Then, the cells

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were exposed to 3.6 kJ/m2 of 365 nm UV on ice for 30 min to
crosslink bio-psoralen with DNA. The cells were rinsed threetimes with PBS (+) and ﬁxed in 4% paraformaldehyde (PFA) in
PBS (–) (Nacalai Tesque) for 15 min. The cells were permeabilized with 0.5% TritonX-100 (MP Biomedicals) in PBS (–) for
30 min, followed by blocking with 1% bovine serum albumin(BSA; Sigma-Aldrich) in PBS (–) for 30 min. After rinsing with
PBS (–), bio-psoralen was ﬂuorescently labeled by incubating thecells with 2.5 µg/mL NeutrAvidin Protein, DyLightTM 488 or
NeutrAvidin Protein, DyLightTM 650 (Thermo Fisher Scientiﬁc)
in 1% BSA, 0.03% Triton X-100 in PBS (–) at 4 °C overnight. For
bio-psoralen staining in RNA- and DNA-degraded cells, cells
were ﬁxed by methanol for 5 min, permeabilized by 0.5%
TritonX-100 in PBS (–) for 15 min, then treated with 1 mg/mL
RNase A (Nippon Gene) or 50 U/mL DNase I (Takara) at 37 °C
for 1 h. After RNA staining was performed as described below,
bio-psoralen was labeled with NeutrAvidin Protein, DyLightTM
650. For bio-psoralen staining upon inhibition of biotin–avidin
interaction, cells were incubated with free avidin (Avidin/Biotin
Blocking Kit, Vector Laboratories) at room temperature for 1 hprior to incubation with NeutrAvidin Protein, DyLightTM 488.
Immunoﬂuorescence and chemical staining. For immunoﬂuorescence staining of nuclear proteins, cells were ﬁxed, permeabilized, and blocked with BSA as described above. The cells
were incubated with the following primary antibodies in 1% BSA
and 0.03% Triton X-100 in PBS (–) at 4 °C overnight: anti-RNA
polymerase II CTD repeat YSPTSPS (phospho S2) (5095, Abcam;
1:500), anti-topoisomerase I (85038, Abcam; 1:500), anti-RPA194
monoclonal (48385, Santa Cruz Biotechnology; 1:100), antiﬁbrillarin monoclonal (2639, Cell Signaling Technology; 1:500),
and anti-NPM 1 monoclonal (32–5200, Invitrogen; 1:500) antibodies. After washing with PBS (–), the cells were incubated withthe following secondary antibodies in 1% BSA and 0.03% Triton
X-100 in PBS (–) at room temperature for 1 h: Goat Anti-Rabbit
IgG H&L-Alexa Fluor® 568 (175471, Abcam; 1:500) and Goat
Anti-Mouse IgG H&L-Alexa Fluor® 568 (175473, Abcam; 1:500).
For nuclear staining, cells were incubated with 4′,6-diamidino-2phenylindole (DAPI; Invitrogen) at 1:500 at room temperaturefor 1 h. For RNA staining, cells were incubated with
StrandBriteTM (AAT Bioquest) at 1:4000 at room temperature for
30 min. For cell viability assays, cells were incubated with 1 μM
SYTOX OrangeTM (Thermo Fisher Scientiﬁc) and 20 μg/mL
bisBenzimide H 33342 trihydrochloride (Hoechst33342; SigmaAldrich) in complete medium at 37 °C for 30 min prior to theobservation.
Immuno-FISH. For the simultaneous imaging of underwound
DNA, nucleolar proteins, and ribosome DNA, Immuno-FISH
was performed as previously described60. In brief, bio-psoralen
and nucleolar proteins (ﬁbrillarin) were ﬂuorescently stained as
described above, except that PFA ﬁxation time was changed from15 min to 20 min. Then, cells were again treated with 4% PFA for
10 min to crosslink antibodies. After washing with 0.05%
TritonX-100 in PBS (–) three times, cells were treated with 1 mg/
mL RNase A at 37 °C for 30 min. Then cells were treated with
0.1 N HCl at room temperature for 2 min, re-ﬁxed with 1% PFA
for 5 min each, and dehydrated using 70% and 100% ethanol for
5 min. Cells were air-dried for 5 min, and incubated with Cy3labeld probes for mouse ribosome DNA (BAC clone RP23225M6, Chromosome Science Laboratory) at 40 °C for 2 h. After
heat denaturation at 70 °C for 5 min, cells were incubated at 37 °C
overnight and washed with 0.01% TritonX-100 in 2× SSC buffer(Nacalai tesque).

ARTICLE

Microscopic observation and image analysis. Stained cells wereobserved using an epiﬂuorescence microscope (IX-83, EVIDENT)
and a CCD camera. Lattice-pattern structured illumination
microscopy (Lattice-SIM2, Zeiss) observation was performed atroom temperature using an Elyra7 super-resolution microscope(Zeiss) equipped with a Plan-Apochromat 40×/1.4 oil objective
lens, four diode laser beams (405, 488, 561, and 642 nm), and an
sCMOS camera, using sequential scanning of frames with a step
size of 0.2–0.4 μm. Images were analyzed using Fiji59. To quantify
the ﬂuorescence intensity of bio-psoralen in the nucleus, nuclear
outlines were generated by thresholding for DAPI staining, andmean ﬂuorescence intensities were quantiﬁed within nuclear
outlines. In addition, 3D suite61 was utilized to identify thecentroid of the nucleolus.
Quantiﬁcation of bio-psoralen-bound DNA extracted from thecell nucleus. Bio-psoralen-bound DNA was extracted from thecell nucleus and quantiﬁed. After bio-psoralen incorporation as
described above, genomic DNA was extracted by ethanol precipitation using a NucleoSpin® Tissue kit (Macherey-Nagel) anddissolved in 100 µL of Tris-EDTA buffer (pH 8.0). After treatment with 1 mg/mL RNase A (Nippon Gene) for 1 h, the genomic
DNA was fragmented by applying 30 cycles of 30 sec sonicationand 30 sec pauses using Bioruptor® II (BM Equipment) in icecold water. For biotin–avidin interaction-based puriﬁcation,
100 µL MagnosphereTM MS300/Streptavidin (JSR Life Sciences
LLC) was washed according to the manufacturer’s instructions.
Fragmented DNA was mixed with 2× binding buffer (20 mM
Tris-HCl, 1 mM EDTA, 2 M NaCl, and 0.1% Tween 20), added tothe magnetic beads, and rotated at room temperature in the darkfor 1 h, followed by overnight rotation incubation at 4 °C in thedark. Unbound DNA was removed by washing the magnetic
beads according the manufacturer’s instructions. To release DNA
from the magnetic beads, the samples were incubated in 50 µL
formamide with 10 mM EDTA at 95 °C for 10 min. Finally,
single-stranded DNA was quantiﬁed using QubitTM (Invitrogen).
Chromatin immunoprecipitation (ChIP) and quantitative
PCR (qPCR). For analyzing the interaction between NPM and
G4, ChIP-qPCR was performed. ChIP samples were preparedusing simple SimpleChIP Enzymatic Chromatin IP Kit (Cell
Signaling), according to the manufacturer’s instructions. Brieﬂy,
cells were treated with formaldehyde to crosslink proteins toDNA, followed by nuclei extraction. Nuclei were treated with
0.25 µL of Micrococcal Nuclease (2000 gel units/µL) and sonicated using Bioruptor® II (BM Equipment) in ice-cold water byapplying three cycles of 30 sec sonication with 30 sec pauses
between each cycle. Then, 2% volume of digested chromatin wastransferred to another tube for input samples. The remaining
chromatin was incubated at 4 °C overnight either with anti-NPM
1 monoclonal (32–5200, Invitrogen; 10 µg), anti-Histone H3
monoclonal (4620, Cell signaling; 10 µg), and Normal Rabbit IgG(2729, Cell Signaling; 10 µg). Antibody-bound chromatin fragments were collected using Protein G Magnetic Beads. All samples including input samples were treated with NaCl andProteinase K to revere cross-links, followed by DNA puriﬁcation.
Real-time PCR was performed using TB Green Premix Ex Taq
GC (Takara) on a StepOne Real-Time PCR System (Thermo
Fisher Scientiﬁc). Putative G4 forming sequences were predicted
by G4 hunter web62 and were used for primer design for qPCR.
Primer sequences are shown in Table 1.
Statistics and reproducibility. All experiments performed in the
manuscript were repeated at least two times as independent
experiments/biological replicates. No statistical methods were

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Table 1 Primer sequences for ChIP-qPCR targeting ribosome DNA.
Target region

Forward

Reverse

PQS-1
PQS-2
PQS-3
PQS-4
Non-PQSACCAGTACTCCGGGCGACACTTAGGAACATTTGCAGTCAGTCAGTTGGACTGACTGGCTGCCTTCCTTGGCCAAAGCAGACCGAGTTGC
AACGCTCCAGGCCTCTCAGGTT

AAAAGAGTCCAGAGTGAGCCCGC
TCTGCCTCCGAAGTGCTGGGATTGGTGCCCTCGTCTAGTGTGTCTGACGGGGACAAGAGAGGGCTTACGACACCATCTCCGAGACGCT

used to predetermine the sample sizes. No datapoints were
excluded from the analyses. Statistical analyses were performed
using SciPy63 software. Statistical signiﬁcance was determined by
the speciﬁc tests indicated in the corresponding ﬁgure legends.
Two-sided paired t-test was performed for the comparison of theamount of bio-psoralen-bound genomic DNA. Two-sided
Mann–Whitney U-test was performed for the comparison of
ﬂuorescence intensity between two groups. Two-sided SteelDwass test was performed for the comparison of ﬂuorescenceintensity between three groups. One sample t-test was performed
for the normalized percent input in ChIP-qPCR with the
expected value of 1 for the null hypothesis.
Reporting summary. Further information on research design isavailable in the Nature Portfolio Reporting Summary linked tothis article.

10.
11.

12.
13.
14.
15.
16.

17.

18.

Data availability
The datasets generated and/or analyzed during the current study are available from thecorresponding author on reasonable request. The source data behind the graphs in thepaper can be found in Supplementary Data 1.

19.
20.

Code availability
All codes were performed by publicly available functions of Fiji (ImageJ, version 1.53t)
and SciPy (version 1.11.1). No custom functions were written for the analysis.

21.

Received: 18 August 2023; Accepted: 28 December 2023;

22.
23.
24.

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Acknowledgements
This work was supported by JST, CREST, Grant Number JPMJCR2023, Japan (J.F.,
K.M.), JST, CREST, Grant Number JPMJCR22L5, Japan (T.A.), JST, SPRING, Grant
Number JPMJSP2110, Japan (J.F.), MEXT/JSPS, KAKENHI, Grant Number JP23KJ1255,
Japan (J.F.), and MEXT/JSPS, KAKENHI, Grant Number JP20K20180 and JP23K17196,
Japan (K.M.). We thank Dr. Fumiyoshi Ishidate in ZEISS-iCeMS Innovation Core
Imaging Unit, Kyoto University, Japan, for supporting Lattice-SIM2 imaging anddiscussions.

Author contributions
Conceptualization: J.F., K.M. and T.A.; Methodology: J.F. and K.M.; Investigation: J.F.;
Visualization: J.F., K.M. and T.A.; Funding acquisition: J.F., K.M. and T.A.; Project
administration: K.M.; Supervision: T.A.; Writing—original draft: J.F.; Writing—review &editing: K.M. and T.A.

Competing interests
The authors declare no competing interests.

Additional information
Supplementary information The online version contains supplementary material
available at https://doi.org/10.1038/s42003-023-05750-w.
Correspondence and requests for materials should be addressed to Koichiro Maki.
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