Nucleosomes of polyploid trophoblast giant cells mostly consist of histone variants and form a loose chromatin structure

Abstract

Trophoblast giant cells (TGCs) are one of the cell types that form the placenta and play multiple essential roles in maintaining pregnancy in rodents. TGCs have large, polyploid nuclei resulting from endoreduplication. While previous studies have shown distinct gene expression profiles of TGCs, their chromatin structure remains largely unknown. An appropriate combination of canonical and non-canonical histones, also known as histone variants, allows each cell to exert its cell type-specific functions. Here, we aimed to reveal the dynamics of histone usage and chromatin structure during the differentiation of trophoblast stem cells (TSCs) into TGCs. Although the expression of most genes encoding canonical histones was downregulated, the expression of a few genes encoding histone variants such as H2AX, H2AZ, and H3.3 was maintained at a relatively high level in TGCs. Both the micrococcal nuclease digestion assay and nucleosome stability assay using a microfluidic device indicated that chromatin became increasingly loose as TSCs differentiated. Combinatorial experiments involving H3.3-knockdown and -overexpression demonstrated that variant H3.3 resulted in the formation of loose nucleosomes in TGCs. In conclusion, our study revealed that TGCs possessed loose nucleosomes owing to alterations in their histone composition during differentiation.

Full text

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SCiEnTifiC REPORTS | (2018) 8:5811 | DOI:10.1038/s41598-018-23832-2
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Nucleosomes of polyploid
trophoblast giant cells mostly
consist of histone variants and
form a loose chromatin structure
Koji Hayakawa
1, Kanae Terada1, Tomohiro Takahashi2, Hidehiro Oana2, Masao Washizu2,3 &
Satoshi Tanaka1
Trophoblast giant cells (TGCs) are one of the cell types that form the placenta and play multiple
essential roles in maintaining pregnancy in rodents. TGCs have large, polyploid nuclei resulting from
endoreduplication. While previous studies have shown distinct gene expression proles of TGCs,
their chromatin structure remains largely unknown. An appropriate combination of canonical and
non-canonical histones, also known as histone variants, allows each cell to exert its cell type-specic
functions. Here, we aimed to reveal the dynamics of histone usage and chromatin structure during
the dierentiation of trophoblast stem cells (TSCs) into TGCs. Although the expression of most genes
encoding canonical histones was downregulated, the expression of a few genes encoding histone
variants such as H2AX, H2AZ, and H3.3 was maintained at a relatively high level in TGCs. Both the
micrococcal nuclease digestion assay and nucleosome stability assay using a microuidic device
indicated that chromatin became increasingly loose as TSCs dierentiated. Combinatorial experiments
involving H3.3-knockdown and -overexpression demonstrated that variant H3.3 resulted in the
formation of loose nucleosomes in TGCs. In conclusion, our study revealed that TGCs possessed loose
nucleosomes owing to alterations in their histone composition during dierentiation.
e rodent placenta consists of a labyrinth zone and junctional zone. e latter is further divided into the spon-
giotrophoblast and trophoblast giant cell (TGC) layers1–3. TGCs secrete various proteins such as extracellular
matrix components, cell adhesion molecules, cytokines, and hormones to promote embryo implantation and
maternal adaptations during pregnancy2. TGCs are characterized by their large cytoplasm and polyploid nuclei
resulting from endoreduplication. In contrast to proliferating diploid cells, TGCs undergo repeated DNA repli-
cation without mitosis and accumulate more than 1,000 copies of genomic DNA4,5. Recently, it was revealed that
there were a few under- and over-replicated regions in the TGC genome, which suggested that their genomic
DNA formed a polytene structure6,7. To organize such an unusual genome, TGCs are expected to possess a unique
chromatin structure that has not been explored in details.
e structural organization of eukaryotic DNA in chromatin is intricately linked to the regulation of many
essential cellular processes, such as genomic DNA replication and transcription8,9. Nucleosomes are the struc-
tural units of chromatin and are dynamically remodeled, assembled, and disassembled by histone chaperones10,11.
Genomic DNA is packaged by the core histones, namely H2A, H2B, H3, and H4, and length of the linker DNA
bound to the linker histone H1 varies12,13.
Histones are classied into two types, canonical histones and histone variants (non-canonical histones), based
on the similarity of amino acid sequences and their cell cycle-dependent or independent expression14–16. Genes
for canonical histones form clusters on certain chromosomes and code for few variations in the amino acid
sequences15,17. In contrast, genes for histone variants are located away from the gene clusters and code for higher
variations in amino acid sequences than canonical histones15,16. e most pronounced sequence divergence
1Laboratory of Cellular Biochemistry, Department of Animal Resource Sciences/Veterinary Medical Sciences, The
University of Tokyo, Tokyo, Japan. 2Department of Mechanical Engineering, The University of Tokyo, Tokyo, Japan.
3Department of Bioengineering, The University of Tokyo, Tokyo, Japan. Koji Hayakawa and Kanae Terada contributed
equally to this work. Correspondence and requests for materials should be addressed to K.H. (email: akojih@mail.
ecc.u-tokyo.ac.jp) or S.T. (email: asatoshi@mail.ecc.u-tokyo.ac.jp)
Received: 11 January 2018
Accepted: 20 March 2018
Published: xx xx xxxx
OPEN
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is found in H2A, leading to the highest number of variants. Both H2B and H3 have a few variations in their
sequences, although there is no identied variant for H2B. In contrast, H4 neither displays sequence divergence
nor has any identied variants14,15. High-order chromatin organization in mammals is modulated by several epi-
genetic mechanisms, including DNA methylation and histone post-translational modications18–20. Since there is
a wide variety of histones, chromatin structure might change by changing the combination of histones.
In the present study, we aimed to reveal the dynamics of chromatin structure and histone usage during TGC
dierentiation. For this, we examined the nucleosomal content, chromatin structure, and histone mobility of
TGCs using a trophoblast stem cell (TSC) dierentiation system21, and observed that TGCs had loose nucleosome
structures incorporated with histone variants, such as H2AX and H3.3.
Results
The repertoire of histones in TGCs is limited compared to that in undierentiated cells. TSCs
were induced to dierentiate and collected every other day till day 10 of dierentiation. Expression of PL-I pro-
tein, a specic marker for TGCs, was detected aer day 6 of dierentiation using western blotting (Fig.1a), indi-
cating that dierentiated TSCs (dTSCs) could be regarded as TGCs as early as day 6 of dierentiation in terms of
hormone expression.
To reveal the histone composition of TGC nucleus, the expression of histone-coding genes (20, 14, 9, 10, and
8 genes for H2A, H2B, H3, H4, and H1, respectively) was investigated using RT-quantitative PCR (RT-qPCR).
Since mRNAs of some histone-coding genes have a stem–loop structure but no poly-A tail at their 3′ UTR14,22,23,
we used random hexamers and oligo(dT) as primers for cDNA synthesis. The qPCR analysis revealed that
Figure 1. e variation in canonical histone subtypes and histone variants decreases during dierentiation
of trophoblast stem cells (TSCs). (a) Western blot analysis showing the protein level of trophoblast giant cell
(TGC) marker PL-I in TSCs and dierentiated TSCs (dTSCs). Cytoplasmic protein was used. Gapdh was
used as an internal control. (b) e mRNA levels of histone-coding genes were evaluated using RT-qPCR,
normalized to Actb expression, and visualized as a heatmap. Both random hexamers and oligo(dT) were used as
primers for cDNA synthesis. Color scale bars indicate the expression level of each gene relative to that of Actb.
Genes encoding histone variants described in Fig.2 are shown in red.
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mRNAs of ten H2A genes (Hist1h2ab, Hist1h2ad, Hist1h2af, Hist1h2ag, Hist1h2ao, Hist2h2aa2, Hist2h2ac, H2afx,
H2afz, and H2afy), ve H2B genes (Hist1h2bg, Hist1h2bh, Hist1h2bj, Hist1h2bl, and Hist2h2bb), four H3 genes
(Hist1h3b, Hist1h3g, Hist1h3i, and H3f3b), nine H4 genes (Hist1h4a–d, Hist1h4f, Hist1h4h, Hist1h4j, Hist1h4k,
and Hist1h4m), and three H1 genes (Hist1h1a, Hist1h1b, and H1f0) were highly expressed in the undieren-
tiated state (Fig.1b). Interestingly, most genes showing high expression in TSCs were downregulated during
dierentiation. In contrast, expression of three out of ten H2A genes (Hist2h2ac, H2afx, and H2afz), two out
of ve H2B genes (Hist1h2bg and Hist1h2bj), one out of four H3 genes (H3f3b), three out of nine H4 genes
(Hist1h4c, Hist1h4d, and Hist1h4m), and one out of three H1 genes (H1f0) remained relatively high throughout
Figure 2. e histone variants H2AX, H2AZ, and H3.3 are predominantly present in TGC chromatin. (a,b)
Protein levels of the H2A variants H2AX, H2AZ (a), and canonical H3 H3.1/3.2, and the H3 variant H3.3 (b) in
TSCs and dTSCs. Upper panel shows western blotting results for H2AX, H2AZ, pan-H2A (a), H3.1/3.2, H3.3
and pan-H3 (b). Western blotting was performed using total histones equivalent to 0.5 μg of genomic DNA.
Bottom panel shows H2AX, H2AZ, H3.1/3.2, and H3.3 levels in dTSCs, which were normalized to the pan-H2A
(a) or pan-H3 level (b) (Fig. S1). e values were expressed as a ratio relative to the maximum average. e
mean ± standard deviation (S.D.) of three independent experiments are shown. *P < 0.05 versus TSC (Student’s
t-test). (c) Immunouorescence staining of H2AX, H2AZ, H3.1/3.2, and H3.3 in TSCs and dTSCs at day 4, 6,
and 8 of dierentiation. Bars = 10 μm.
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dierentiation. Expression of Hist1h2bc and Hist1h1c was observed at day 6 of dierentiation in dTSCs. us, the
complexity of histones in dTSC nuclei was markedly lower compared to that in TSCs.
Histone variants H2AX and H3.3 predominantly occupy the chromatin of TGCs. Since the
expression of H2afx (encoding H2AX), H2afz (encoding H2AZ), and H3f3b (encoding H3.3) was maintained
at a high level throughout the dierentiation of TSCs (Fig.1b), the protein levels of H2AX, H2AZ, H3.3, and
the canonical histone H3.1/3.2 were estimated using western blot analysis by normalizing the intensity values of
each band to those of pan-H2A or -H3 levels (Fig.S1). e level of H2AX increased from day 6 and plateaued at
day 8 (Fig.2a). For H2AZ, expression increased by day 2 and remained high until day 6; however, it drastically
decreased to less than half of the level observed for TSC aer day 8 (Fig.2a). Notably, the level of H3.3 increased
at day 6, in contrast to that of the canonical type H3.1/3.2 (Fig.2b).
To reveal their subnuclear localization, immunouorescence staining was performed. H2AX and H2AZ were
detected broadly in the nucleoplasm but not in the nucleolus, and H2AZ signals were observed as dotted foci
compared to H2AX signals. e intensity of H2AX increased as the nucleus expanded throughout dierentiation
(Fig.2c). e intensity of the H2AZ signal remained constant until day 6 of dierentiation and reduced remark-
ably at day 8 (Fig.2c). In contrast to TSCs and dTSCs at day 4, H3.3 could be detected in dTSC nuclei at day 6
and 8, while H3.1/3.2 was barely detectable (Fig.2c). e dTSCs with a small nucleus (non-TGCs, most likely
spongiotrophoblast or syncytiotrophoblast cells) at days 6 and 8 of dierentiation exhibited a lower expression of
H2AX and H3.3 than putative TGCs (Fig.S2).
To investigate the expression of aforementioned histone variants in TGCs in vivo, we collected parietal TGCs
and embryos at the embryonic day 9.5 (E9.5) (Fig.3a). Western blotting analysis revealed that the expression of
H2AX, H2AZ, and H3.3 was signicantly higher in parietal TGCs than that in embryos, in contrast to that of
canonical H3.1/3.2 (Fig.3b and c). H2AX and H3.3 signals were distributed in the entire nucleoplasm in parietal
TGCs. ese two histone variants were undetectable in diploid cells, most probably in parietal endodermal cells
present in the Reichert’s membrane (Fig.3d). Similar H2AZ signal intensities were detected in parietal TGCs, as
well as diploid cells. e expression status of these histone variants indicates that dTSCs at day 6 of dierentiation
recapitulate the features of TGCs in vivo.
Taken together, our results showed that during the early stage, dTSCs predominantly expressed H2AZ and
H3.1/H3.2; however, dTSCs aer day 6 of dierentiation and parietal TGCs in vivo predominantly expressed the
H2AX and H3.3 isoforms/variants of H2A and H3, respectively. Since these histones are categorized as histone
variants, which have unique functions in the formation of loose nucleosome16, we hypothesized that dierentia-
tion towards TGCs potentially destabilizes the histone–histone and histone–DNA interactions in nucleosomes.
TGCs have loose nucleosome structures. To visualize chromatin dynamics in a single cell, a mouse
TSC line stably expressing GFP-fused H4 (H4-GFP) was established and GFP uorescence was monitored for
96 h during TSC dierentiation (days 2–6 of dierentiation). A majority of the cells underwent cell division
three times within the rst 48 h (days 2–4 of dierentiation), followed by the expansion of nuclear size (Fig.4a
and MovieS1) most likely due to endoreduplication. Aer the last cell division, GFP foci, possibly representing
heterochromatin, became apparent and gradually increased in number with the simultaneous appearance of a
GFP-poor area (i.e., areas with less nucleosomes).
e micrococcal nuclease (MNase) digestion assay using unxed chromatin demonstrated that TSCs and
dTSCs at days 2 and 4 of dierentiation showed typical nucleosomal structures, since bands corresponding to
mono-, di-, and tri-nucleosomal sizes were observed (Fig.4b). A band of barely digested DNA (>3 kb) also
appeared in both TSCs and dTSCs. Remarkably, aer day 6 of dierentiation, digested genomic DNA did not
show obvious bands for di- and tri-nucleosomes, but showed blurred bands instead. e MNase digestion assay
using xed chromatin conrmed that aer day 6 of dierentiation, like TSCs, dTSCs also formed nucleosomal
structures (Fig.4c). Of note, di- and tri-nucleosomal bands were lower in dTSCs aer day 6 than in TSCs and
dTSCs at days 2–4 (Fig.S3), which may reect the dierence of structures of linker DNA regions. ese results
using unxed and xed chromatin indicated that not only the linker DNA regions, which ank the nucleosome
core, but also the DNA surrounding histone core was more easily digested by MNase aer day 6 of dierentiation
in dTSCs than in TSCs.
Results of the MNase digestion assay suggested that dTSCs aer day 6 of dierentiation had a more relaxed
nucleosomal structure than TSCs. To conrm this, we evaluated the nucleosomal stability of dTSCs expressing
H4-GFP in increasing concentrations of NaCl buer by single-cell imaging using a microuidic device (Fig.5a).
In this device, H4-GFP-TSCs or -dTSCs at day 8 of dierentiation were individually placed using optical tweezers
in microscale reaction chambers (hereaer referred to as micropockets), which were located along the sides of
the main channel. Cells were permeabilized by introducing the Triton X-100 buer into the main channel, and
subsequently GFP uorescence was observed aer sequential exposure to 0.5 to 2 M NaCl buer for ~20 min
each (Fig.5a). A notable decrease in the intensity of H4-GFP uorescence in dTSCs aer the exposure to 0.75 M
NaCl was observed, whereas only a slight decrease was observed in TSCs. Aer exposure to 1 M NaCl, H4-GFP
in dTSCs was barely detectable (Fig.5b). Quantication of GFP intensity revealed a more marked decrease in
dTSCs than in TSCs (Fig.5c).
To validate the results of the nucleosomal stability assay, proteins were extracted in 0.5 or 1 M NaCl buer
from the cell pellets of TSCs and dTSCs at days 2–10 of dierentiation and histones were detected using western
blotting (Fig.5d). As expected, dTSCs, particularly aer day 6, showed higher solubility of core histones than
TSCs in a 1 M NaCl solution. In contrast, the solubility of H1 showed an opposite trend (Fig.5d and e).
Taken together, our results showed that as TSCs dierentiated, an increase in the number of speckled foci
of core histones and histone-less area in nucleus was observed. Parallely, loose nucleosome structures became
apparent in dTSCs, which mostly consisted of polyploid TGCs.
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Figure 3. Expression status of histone variants H2AX, H2AZ and H3.3 in TGCs in vivo. (a) Western blotting
analysis of TGC marker PL-1 and decidual marker Desmin in the cytoplasmic protein samples. Bio. Rep.
indicates biological replicates. (b,c) Western blotting analysis of H2AX, H2AZ (b), H3.1/3.2, and H3.3 (c) in the
nuclear protein of parietal TGCs and embryos. e graphs below the blots show H2AX, H2AZ, H3.1/3.2, and
H3.3 levels in parietal TGCs and embryos, which were normalized to the pan-H2A or pan-H3 level. e values
were expressed as a ratio relative to the maximum average. e mean ± S.D. of three independent experiments
are shown. *P < 0.05 versus parietal TGCs (Student’s t-test). (d) Immunouorescence staining of H2AX, H2AZ,
H3.1/3.2, and H3.3. White arrowheads indicate diploid cells in Reichert’s membrane. Bars = 20 μm.
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A change from canonical H3 to variant H3.3 in nucleosomes plays an important role in estab-
lishment of the unique TGC chromatin structure. Western blotting and immunouorescence stain-
ing suggested a change of core histone H3 from canonical H3.1/3.2 to variant H3.3 during TGC formation. To
investigate the role of H3.3 in establishment of the unique chromatin structure in dTSC nucleus, we performed
H3.3 knockdown (KD) and overexpression (OE) experiments. e expression vectors for H3.3-KD or -OE were
transfected into dTSCs at day 4 of dierentiation. e cells were cultured for another four days and collected at
day 8 of dierentiation for each assay. KD and OE were conrmed using western blotting (Fig.6a). In the MNase
digestion assay, the nucleosomal bands corresponding to di- and tri-nucleosomal sizes became more conspicuous
in H3.3-KD dTSCs compared to those in the control cells (LacZ-KD + Flag) (Fig .6b and c). Rescue experiments
revealed that OE of H3.3 in H3.3 KD dTSCs reversed the appearance of nucleosomal bands, whereas OE of H3.1
increased the band intensity. Moreover, enlargement of the nucleus was signicantly inhibited by H3.3 KD, which
was rescued by H3.3 OE, but not by H3.1 OE (Fig.6d). H3.3-KD dTSCs also signicantly decreased the number
of cells containing more than 4n DNA content compared to the control cells (Fig.6e).
ese results indicated that H3.3 played an important role in formation of the unique chromatin structure and
nuclear expansion in dTSCs by replacing canonical H3.
Decrease in histone chaperone variation may be one of the key events during alteration of
chromatin state from TSCs to TGCs. e spatiotemporal deposition and eviction of histones by their
dedicated chaperones are required to control the chromatin structure for cell-specic gene expression10,11,24. We
next examined the expression of 17 genes encoding histone chaperones using RT-qPCR. Surprisingly, 12 out of
the 17 genes showed signicantly decreased expression levels upon the induction of dierentiation (Fig.7a). e
expression of Nap1l2 and Hirip3 decreased aer the induction of dierentiation; however, their expression levels
increased at day 10 of dierentiation. e expression of Daxx, Nap1l4, and Nap1l5 decreased at day 2 of dier-
entiation; however, their expression was upregulated and became similar to that in dTSCs at day 4. ese results
suggested that these histone chaperones could aect the mobility of histones in nucleus and organization of the
unique chromatin structures in dTSCs aer day 6 of dierentiation.
We nally examined whether there were any dierences in the mobility of histones in TSCs and polyploid
dTSCs by fluorescence recovery after photobleaching (FRAP) analysis using H4-GFP-TSCs and -dTSCs at
day 8 of dierentiation. One-half of the nucleus was bleached and uorescence intensity was measured in the
presence of cycloheximide to suppress uorescence recovery resulting from de novo protein synthesis (Fig.7b).
Quantitative measurements indicated that H4-GFP was quickly recovered in TSCs (Fig.7c). In contrast, H4-GFP
Figure 4. Nuclei of TGCs containing loose chromatin. (a) Live cell imaging for H4-GFP during dierentiation.
Representative snapshots from a live cell movie (MovieS1), which was recorded for 96 h (tracking the same
eld), are shown. Bars = 5 μm. (b,c) MNase digestion analysis using chromatin from TSCs and dTSCs. Unxed
(b) or xed (c) chromatin was digested with MNase and electrophoresed on an agarose gel with (+) MNase
and without (−) MNase as the negative control. Mono, mono-nucleosome; Di, di-nucleosomes; Tri, tri-
nucleosomes.
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TSC
day 2
day 4
day 6
day 8
day 10
d
ab
H2A
H2B
H4
H3
H1
dTSC
TSC
day 2
day 4
day 6
day 8
day 10
0.5 M
1 M
0.75 M
1.5 M
2 M
TSC
dTSC
at day 8
NaCl Conc.
c
0
0.2
0.4
0.6
0.8
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0.50.75 11.5 2
Relative intensity
NaCl Conc. (M)
Inlet
Outlet
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Set a cell
into micropocket
Flow Flow NaCl Buffer
0.5 ~ 2 M
Flow
Permeabilization
Optical tweezers
Fluorescence imaging
Triton X-100
Buffer
Gapdh
dTSC
0.5 M NaCl 1 M NaCl
TSC
dTSC at day 8
Diffusion Diffusion
240 μm
50 μm
Top view
Main channel
260 μm
e
dTSC
day 2
day 4
day 10
day 8
day 6
TSC
0
2
4
H3
3
1
**
*
0
4
2
1
3
H4
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day 2
day 4
day 10
day 8
day 6
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5
*
*
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10
20
30
H1
dTSC
day 2
day 4
day 10
day 8
day 6
TSC
*
*
*
H2A
0
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6
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day 2
day 4
day 10
day 8
day 6
TSC
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6
8
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day 2
day 4
day 10
day 8
day 6
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*
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Relative intensity
(NaCl Ext. / Total histone)
**
Figure 5. Nuclei of TGCs show decreased nucleosomal stability. (a–c) Nucleosome stability assay using
a permeabilized individual cell in the microuidic device. (a) Overview of the microuidic device and
experimental procedure for the nucleosome stability assay using a single cell. (b) Representative green
uorescence observed in H4-GFP-TSC and -dTSC at day 8 following exposure to NaCl buer at the indicated
concentrations in the micropockets. Bars = 5 μm. (c) Measurement of uorescence intensity of H4-GFP in
TSCs and dTSCs at day 8. Fluorescence intensities were measured using the ImageJ soware, and the nuclear
position in each cell was determined using the genomic DNA image obtained aer GelRed staining. Values
indicate the mean ± S.D. (n = 5) and are relative to the value obtained aer the 0.5 M NaCl treatment, which
is arbitrarily set as 1. (d) Nucleosome stability assay. Each sample was extracted using a buer containing 0.5
or 1 M NaCl. Extracted proteins equivalent to 0.5 μg of genomic DNA were subjected to 15% SDS-PAGE. (e)
Proportion of each histone in the NaCl extract to total histone. Band intensity following western blotting of 1 M
NaCl-extracted histone (d) was calculated and normalized to that of the corresponding band in Fig.S1. e
mean ± S.D. of three independent experiments are shown. *P < 0.05 versus TSC (Student’s t-test).
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in dTSCs at day 8 did not fully recover aer photobleaching for 30 min. us, polyploid dTSCs showed less
potency for histone exchange, even though they had loose nucleosomal structures.
Discussion
In the present study, we found that TGC nuclei had loose nucleosomes. Further, the variation in histone subtypes/
variants decreased in TGCs, and relatively high expression of the histone variants H2AX, H2AZ, and H3.3 was
observed. Previous reports indicated that these variants were required for normal embryogenesis, but their role(s)
in trophoblast cells was unknown25–27. Our study suggested that global histone reorganization from canonical
histones to these variants during TSC dierentiation was associated with the formation of a unique chromatin
structure for TGCs (Fig.8).
Figure 6. A change from H3.1/H3.2 to H3.3 is important for the establishment of loose chromatin in TGCs.
(a) Western blotting analysis for H3.3-knockdown (KD) and H3.3-overexpressing (OE) dTSCs. e vectors for
H3.3-KD and Flag-H3.3-OE were transfected at day 4 of dierentiation, and all experiments were performed
four days aer transfection (at day 8 of dierentiation). (b,c) MNase digestion analysis using chromatin from
H3.3-KD and -OE dTSCs. (b) Gel electrophoresis image showing MNase-digested or -undigested genomic
DNA. (c) Densitometry graph of electrophoresis image (le half) shown in (b) plotted using the ImageJ
soware. Mono, mono-nucleosome; Di, di-nucleosomes; Tri, tri-nucleosomes; Tetra-, tetra-nucleosomes. (d)
Nuclear sizes of H3.3-KD and -OE dTSCs at day 8 of dierentiation. Nuclear size was calculated from randomly
selected DAPI images using the CellProler soware. *P < 0.05 versus LacZ-KD + Flag (Wilcoxon rank-sum
test). (e) Genomic DNA content of H3.3-KD and -OE dTSCs. DNA content was analyzed using ow cytometry
of cells stained with propidium iodide. e percentages (mean ± S.D.) of nuclei with DNA content >4n are
indicated in each graph. *P < 0.05 versus LacZ-KD + Flag (Student’s t-test).
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Figure 7. Repertoire of histone chaperones and mobility of histones are limited in dTSCs. (a) Expression of
genes encoding histone chaperones. Graphs show the expression level of various histone chaperones determined
using RT-qPCR. Values indicate the mean ± S.D. aer being normalized to Actb expression, and are indicated
relative to the levels in TSCs, which were arbitrarily set as 1. (b,c) FRAP analysis for H4-GFP-TSCs and -dTSCs
at day 8. One-half of the nuclei of living H4-GFP-TSCs and -dTSCs at day 8 was bleached (white squares in b),
and the averages of the relative uorescence intensity (mean ± S.D.) of the bleached area up to 55-min post-
bleaching are plotted (n = 10) (c). Bars = 5 μm.
Figure 8. Proposed model for TGC nucleosome structure.
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Polytene nuclei of Drosophila melanogaster showed similar histone contents as the non-polytene nuclei28. In
addition, HPLC fractionation of histones indicated that TGCs at day 6 of dierentiation had a similar histone
composition prole as that of TSCs29. ese observations explained why there was no dierence in the ratio
of each pan-histone (H2A, H2B, H3, and H4) to total histone between polytene and non-polytene nuclei of
Drosophila melanogaster and between TSC and TGC nuclei at day 6 of dierentiation. In both studies, however,
histone variants were not distinguished from canonical histones and the alteration in the expression level of each
histone subtypes/variants during dierentiation was not monitored. In this study, we evaluated the expression
proles of almost all histone-coding genes in details and found that the repertoire of canonical histones and
histone variants reduced following the dierentiation of diploid TSCs into polyploid TGCs. Although the spe-
cic functions of histone variants have been well studied16, the functional dierences among canonical histone
subtypes have not been studied. However, we recently discovered a novel canonical H2A subtype-specic his-
tone O-GlcNAcylation29. is discovery suggested that not only the histone variants, but also the composition
of the subtypes of canonical histones should be considered while studying the epigenetic organization of a cell.
Expression level of each histone variant and canonical histone subtype may aect the formation of chromatin
architecture and epigenetic regulation in TGCs.
Relaxed and transcriptionally active chromatin regions are thought to be maintained by H2AZ and H3.330.
e presence of H2AZ and H3.3 in the genome potentially correlated with actively transcribed genes, indicating
that H2AZ and H3.3 were necessary for creating relaxed and transcriptionally active chromatin structures31–33.
e present study showed that the level of H3.3 markedly increased at the expense of canonical H3 (H3.1/3.2).
Meanwhile, H2AZ level was maintained until day 6 of dierentiation, followed by upregulation of H2AX level
from day 6 of dierentiation, unlike canonical H2A levels during dierentiation to TGCs even in the endore-
duplication cycle. e genes encoding canonical histones are expressed at the S phase of the cell cycle14–16. In
contrast, histone variant genes, including H2afx, H2afz, and H3f3b, are expressed in a cell cycle-independent
manner. Since dTSCs with expanded nuclei, focused on in this study, appeared to express DNA replication mark-
ers (Fig.S4), the upregulation of H2AZ, H2AX, and H3.3 expression might not be due to cell cycle arrest but is
specic to the dierentiation into TGCs. Moreover, the H3.3-KD and -OE experiments in dTSCs demonstrated
that H3.3 played an important role in creating loose chromatin structures. Based on the present study and previ-
ous reports on the function of H2AX, H2AZ, and H3.3, these H2A and H3 variants were potentially responsible
for the formation of a loose nucleosome structure that was unique to TGCs.
Histone variants provide chromatin with temporally and spatially regulated alterations, and are required for
transcriptional control of specic genes, repair of DNA damage, and DNA replication16. e H3.3-KD and -OE
experiments using dTSCs indicated that expansion of nuclear size and polyploidization were controlled by the
exchange of canonical H3 with variant H3.3. e mechanisms of polyploidization in TGCs were mainly explained
by the role of cell cycle regulators such as p57Kip2 and cyclin E1/E234,35. Factors associated with regulating the
nuclear size of TGCs were also identied36,37. However, the previous studies did not focus on the inuences of
chromatin structures on these unique TGC phenotypes. Here, we demonstrated the importance of nucleosomal
components in determining TGC phenotypes and identied H3.3 as a novel promoter of nuclear expansion and
polyploidization. In the TGC genome, over- and under-replicated regions are present in a locus-dependent man-
ner6,7. Considering that chromatin structures can aect the DNA replication machinery9, H3.3 may also play a
role in establishing this non-uniform polyploid genome. In future, genome-wide analysis of H3.3 distribution in
TGCs will validate this hypothesis.
As for the H2A variants, the level of both H2AZ and H2AX increased during the dierentiation of TSCs.
H2AX has the potential to form destabilized nucleosome structures38, suggesting that it plays a role in establish-
ing and maintaining loose chromatin regions in TGCs. Phosphorylated H2AX, also called γH2AX, is well known
for its role in mediating DNA damage response39,40. Since TGCs showed more chromatin instability than TSCs,
genomic DNA might have more opportunities to become “naked” in TGCs than in TSCs, which increases the
possibility of DNA damage. Nevertheless, genomic DNA must be protected to some degree for maintaining its
cellular functions under any circumstances. e marked increase in H2AX level in TGCs might be explained by
its role in genome protection against various environmental stresses.
In general, it is known that heterochromatin regions increase during dierentiation of stem cells into ter-
minally dierentiated cells41. Indeed, an increase in the number of heterochromatin foci was observed aer
the induction of dierentiation in mouse embryonic stem cells (ESCs) using several dierent methods, such
as immunostaining of the heterochromatin protein HP1, DAPI staining42, and monitoring the GFP-fused
methyl-CpG-binding domain43. In the present study, the increase in heterochromatin regions in a TGC was
shown using single-cell imaging analysis. Importantly, despite an increase in heterochromatin, results of the
MNase digestion assay and nucleosome stability assay indicated that the TGC had a relaxed, loose nucleosomal
structure. us, a TGC nucleus can be regarded as having increased heterochromatin regions and regions with
highly relaxed chromatin in the same genome. Isoforms of the linker histone H1 may be responsible for this
unique constitution of the TGC genome. H1 is generally thought to condense chromatin structures, leading to
the repression of gene expression44,45. In this study, the nucleosome stability assay showed a marked decrease in
H1 solubility in NaCl solution aer day 8 of dierentiation, in contrast to the solubility of core histones. In the ter-
minally dierentiated dTSCs, the expression levels of two H1 genes, namely Hist1h1c (encoding H1.2) and H1f0
(encoding H1.0), increased. erefore, we hypothesize that the heterochromatin regions observed in TGC nuclei
were formed and maintained mainly by the linker histones H1.2 and H1.0. Genome-wide distribution analysis
and KD of these H1 isoforms can help in validating this hypothesis.
Deposition and replacement of histones are important to maintain and alter the overall chromatin structure,
which is mainly controlled by histone chaperones10,11. In TGCs, the diversity of histone chaperones as well as the
histone variety decreased during dierentiation. is decrease in diversity may be related to the hindered mobil-
ity of histones in TGCs than in TSCs, as shown by FRAP analysis. Our results also indicated that the expression of
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Daxx, a H3.3-specic chaperone46, increased and remained high during the dierentiation process, along with the
existence of H3.3 in the nucleus of TGCs. Gene expression of H2AX- and H2AZ-specic chaperones, members of
the FACT complex (Ssrp1 and Supt16h) and Hirip347,48, respectively, signicantly decreased aer the induction of
dierentiation. us, it seemed unlikely that these H2A variants were dependent on these dedicated chaperones
for being incorporated into the TGC nucleosomes at least by day 8 of dierentiation. Developmental process and
cell fate decisions are coupled to chromatin remodeling and concomitant replacement of histone molecules by
specic histone chaperones24. e limited usage of histone chaperones in TGCs may support not only the estab-
lishment/maintenance of the unique chromatin structures but also TGC dierentiation.
An increase in the amount of genomic DNA is associated with the enlargement of nucleus and cell body dur-
ing dierentiation, which is a common event in polyploid cells49. However, an increase in the DNA content is even
more intense than the expansion of nuclear size in some highly polyploid cells, including TGCs. Furthermore,
it is known that unlike diploid cells, transcript levels are not proportional to the amount of DNA or gene copy
number in polyploid cells50. e present study, which analyzed nucleosomal contents and chromatin structures in
TGCs, indicated that a unique combination of histone variants in TGCs establishes the characteristic chromatin
structure, which enables an enormous amount of DNA to be precisely packaged in the nucleus and results in a
unique gene expression system.
Methods
Reagents. All reagents were purchased from Wako Pure Chemical, unless otherwise mentioned. Antibodies
used in this study are listed in TableS1. Primer sequences are shown in TableS2.
Cell culture. Mouse trophoblast stem cells (B6TS4 line) derived from a C57BL/6 N blastocyst in our labo-
ratory were cultured and maintained in an undierentiated state in the 70CM + F4H medium51. Dierentiation
was induced by withdrawal of FGF4 and heparin. e cells were harvested using 0.05% trypsin–1 mM EDTA in
phosphate buered saline (PBS) without divalent cations [PBS(−)], washed with PBS(−), collected in 1.5 or 2 mL
tubes, snap frozen in liquid nitrogen, and stored at −80 °C until further use (DNA/RNA/protein extraction).
Collection of parietal TGCs. e parietal TGCs on Reichert’s membrane were harvested from the con-
cepti of ICR mice (Charles River, Japan) on day 9 of pregnancy, according to the method described in a previous
report6. e embryos and decidual cells were harvested and used as control diploid cells. e tissues were rinsed
in PBS(−) to remove blood, collected in 1.5 mL tubes, snap frozen in liquid nitrogen, and stored at −80 °C until
use. Immunouorescence staining was performed by placing the Reichert’s membrane samples, which contain
parietal TGCs, on amino silane (APS)-coated glass slides (Matsunami, Japan) using ne tweezers.
RT-qPCR using the BioMark system. Total RNA was isolated from cells using the TRIzol reagent
(Invitrogen), according to the manufacturer’s instructions. cDNA was synthesized from 1 μg total RNA using
either of the two primers, namely random hexamers or oligo(dT)20, and the SuperScript III First-Strand Synthesis
System (Invitrogen). RT-qPCR was performed using a high-throughput gene expression platform based on
microuidic dynamic arrays (BioMark, Fluidigm)52. Data were processed by automatic global threshold setting
with the same threshold value for all assays and linear baseline correction using the BioMark Real-Time PCR
Analysis soware (Fluidigm). Results of RT-qPCR for histone-coding genes by BioMark were visualized as a
heatmap using the MeV soware53.
Histone purication, nuclear and cytoplasmic protein extraction. Histone fractions were col-
lected from two 100-mm dishes containing conuent cells using the Histone Purication Mini kit (Active Motif ),
according to the manufacturer’s instructions. Genomic DNA was also puried from an aliquot of cell pellets used
for histone purication. For western blotting, the amount of total histone was normalized by genomic DNA,
namely total histone equivalent to 0.5 μg of genomic DNA were loaded into each well of SDS-PAGE.
Nuclear and cytoplasmic proteins were extracted using the LysoPure Nuclear and Cytoplasmic Extractor kit,
according to the manufacturer’s instructions. e amount of cytoplasmic and nuclearprotein was estimated using
the BCA assay.
Western blotting. Proteins were fractionated using 15% SDS-PAGE, blotted onto Millipore transfer mem-
branes(Merck Millipore), and incubated at 4 °C with 1 μg/mL of primary antibodies. Protein bands were detected
using a secondary antibody (1:4,000) conjugated to horseradish peroxidase (Jackson ImmunoResearch), and
ImmunoStar basic or ImmunoStar LD as the substrate. Amount of the histone fraction in each sample was nor-
malized to the amount of genomic DNA. All uncropped images are presented in Fig.S5.
Immunouorescence staining. Cells cultured in 4-well plates and parietal TGCs on APS–coated slide
glass were xed using 4% paraformaldehyde for 20 min at room temperature, permeabilized using 0.2% Triton
X-100 for 30 min at room temperature, followed by blocking with 5% BSA (Rockland) and 0.1% Tween 20 in
PBS(−) overnight at 4 °C, and incubation with 1 μg/mL of the primary antibody overnight at 4 °C. e secondary
antibody (1:1,000) was added and incubated for 1 h at room temperature. Nuclei were stained using DAPI (1 μg/
mL; Dojindo). Images were captured using the LSM 700 laser scanning confocal microscope (Zeiss).
Construction of expression vectors. Mouse full-length Hist1h4i (encoding H4) was obtained from
TSC cDNA using PCR amplication. e cDNA was cloned into the pGEM-T Easy vector (Promega), and the
resulting constructs were conrmed using BigDye sequencing (Invitrogen). e cloned cDNA was inserted into
pEGFP-N3 (Clontech) at the NheI and EcoRI sites to generate the expression vectors for H4-GFP.
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Mouse full-length Hist1h3a (encoding H3.1) and H3f3a (encoding H3.3) were obtained using cDNA from
mouse ESCs (with a 3× Flag-tag inserted) by two PCR amplications and subsequently ligated into the pEN-
TR/D-TOPO vector (Life Technologies). e appropriate inserts were conrmed using BigDye sequencing.
3× Flag-fused genes were subcloned into the pCAG-DEST-PGK-Puromycin-IRES-VENUS-pA vector54 using
Gateway LR Clonase (Life Technologies).
For H3.3 knockdown, two specic miRNA sequences targeting the respective UTR regions of H3f3a and
H3f3b were cloned into the pcDNA 6.2-GW/EmGFP-miR vector (ermo Fisher Scientic). Similarly, a sequence
targeting the LacZ-encoding mRNA was cloned and denoted control-miR.
Plasmids were purified using the Quantum Prep Plasmid Midi Prep kit (BIO-RAD), followed by phe-
nol:chloroform:isoamyl alcohol (PCI) extraction and ethanol precipitation. For knockdown and overexpression
experiments using dTSCs, 10 μg plasmids were transfected into dTSCs placed in a 100-mm dish at day 4 of dif-
ferentiation using 20 μL jetPRIME reagents (Polyplus)51. About 24–96-h post-transfection, dTSCs were selected
using 2 µg/mL blasticidin and 1.5 µg/mL puromycin.
Live cell imaging for H4-GFP. To establish a TSC line stably expressing GFP-fused histone H4
(H4-GFP-TSC), TSCs were cultured in 6-well plates to ~50% conuence and subsequently transfected with 3 μg
plasmid and 9 μL jetPRIME reagent per well. Twenty-four hours aer transfection, cells were replated and cul-
tured for one week in a 100-mm dish in the presence of 1 mg/mL G418. Individual G418-resistant colonies were
picked and transferred to each well of a 96-well plate. Cells that expressed the fusion proteins were expanded,
collected, suspended in Cell Culture Freezing Media (CellBanker 1; Takara), frozen in liquid nitrogen, and stored
at −80 °C until further use.
Imaging for GFP was performed using a confocal microscope (CV1000; Yokogawa), equipped with a
heated stage and cover lled with humidied 5% CO2 and 95% air. H4-GFP-TSCs were seeded onto a 35-mm
glass-bottom dish and cultured in TS medium without FGF4/heparin to induce dierentiation. Two days aer
the induction of dierentiation, confocal images were obtained at 30-min intervals for 96 h using a 100× objective
lens.
MNase digestion analysis. MNase digestion analysis for unfixed cells was performed using the EZ
Nucleosomal DNA Prep kit (Zymo Research). Briey, cytoplasm was removed using the Nuclei Prep buer and
the remaining chromatin was washed twice with the Atlantis Digestion buer. Chromatin (equivalent to 1 μg
DNA) was digested using 0.0014 U MNase in the Atlantis MN Digestion buer.
MNase digestion analysis for xed cells was performed using the ChIP IT Express Enzymatic kit (Active
Motif). Cells were xed using 1% formalin for 15 min at room temperature, and cytoplasm was removed using
the 1× lysis buer supplemented with 1 mM phenylmethylsulfonyl uoride (PMSF) and 1× protease inhibi-
tor cocktail. Chromatin (equivalent to 1 μg DNA) was digested using 0.0020 U MNase in the digestion buer.
Cross-linking of DNA was reversed by incubation in 5 M NaCl/lysis buer at 65 °C overnight.
Digested DNA was puried using PCI extraction and ethanol precipitation, and electrophoresed on 1.2%
agarose gel.
Nucleosome stability assay using a microuidic device. e microuidic device was fabricated as
described previously55 and placed on an XY motorized stage (Sigma Koki) mounted on an inverted optical micro-
scope (IX-71; Olympus). H4-GFP-TSCs or H4-GFP-dTSCs at day 8 were resuspended in isotonic buer (300 mM
sorbitol and 5 μM Hoechst 33342), introduced into the main channel from the inlet and transported and placed
in the micropockets one-by-one using optical tweezers (Fig.5a). e optical tweezers were operated as described
previously55. Cells were exposed to the Triton X-100 buer [1% Triton X-100, 10 mM Tris-HCl (pH 8.0), 1 mM
EDTA, 10 mM DTT, and GelRed (1:50,000)] in the micropockets for 20 min to permeabilize them. e solution
conditions in micropockets could be rapidly altered by diusion of solutes, when isotonic buer in the main
channel was replaced with the Triton X-100 buer and the newly introduced solution was kept owing55. Aer
permeabilization, the cells were exposed to NaCl buer [0.5/0.75/1/1.5/2 M NaCl, 10 mM Tris-HCl (pH 8.0),
10 mM DTT, and GelRed (1:50,000)] by replacing solutions in the main channel with increasing NaCl concentra-
tion in a stepwise manner. Here, free histone proteins which dissociate from chromatin would diuse out to the
main channel, while nuclei containing chromatin/DNA will remain inside the micropockets due to their small
diusion coecients. Aer the cells were exposed to each concentration of NaCl buer for approximately 20 min,
uorescence images of GFP (histone H4) and GelRed (genomic DNA) were obtained using a high-sensitivity
video camera (EM-CCD camera, C9100-13; Hamamatsu Photonics), and uorescent intensity was quantied
using the ImageJ soware (https://imagej.nih.gov/ij/).
Nucleosome stability assay using frozen cell pellets. Frozen cell pellets were thawed on ice, resus-
pended in buer A [20 mM HEPES (pH 7.9), 0.5 mM DTT, 1 mM PMSF, 1.5 mM MgCl2, and 0.1% Triton X-100]
containing 0.5 or 1 mM NaCl, and incubated for 40 min at 4 °C with constant agitation. Cell lysates were centri-
fuged at 20,000 × g at 4 °C for 30 min. e supernatant was transferred into Amicon Columns (Millipore) and
concentrated by centrifugation at 14,000 × g at 4 °C for 10 min. e extracted liquid was collected in Protein
LoBind tubes by centrifugation at 1,000 × g at 4 °C for 2 min with reverted columns and subjected to western
blotting.
Genomic DNA was also puried from an aliquot of cell pellets used for protein extraction. Cells were dis-
solved in lysis solution [10 mM Tris-HCl (pH 8.0), 5 mM EDTA, 200 mM NaCl, 0.2% SDS, and 200 μg/mL pro-
teinase K] at 55 °C for 30 min. DNA was extracted with PCI, incubated with 5 μg/mL RNase A for 30 min, and
re-extracted with PCI. DNA was precipitated using ethanol and dissolved in Tris-EDTA (TE) buer (pH 8.0).
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Measurement of DNA content. Cells were xed in 70% ethanol and stained using 20 μg/mL propidium
iodide and 100 μg/mL RNase (Sigma). Fluorescence intensity of each cell was analyzed using the BD FACSVerse
ow cytometer (BD Biosciences).
Measurement of nuclear size. Cells cultured in 4-well plates were xed using 4% paraformaldehyde for
20 min at room temperature and the nuclei were stained with DAPI. For measurement of the size of the nucleus
in dTSCs, uorescence images aer DAPI staining were processed using the CellProler soware as described
previously56.
FRAP analysis. FRAP was performed using a confocal microscope (FV-3000; Olympus), equipped with
a heated stage and cover lled with humidied 5% CO2 and 95% air. A confocal image of a eld containing
approximately ve nuclei was obtained using a 60 × lens (512 × 512 pixels, 2 μs/pixel scan speed, 2 AU pinhole,
500–600 nm variable lter, and 0.33% transmission of 488-nm Ar laser). ereaer, one-half of each nucleus
was photobleached using 10% transmission of the 488-nm laser and images were collected using the original
setting at 1-min intervals for 60 min. Fluorescence intensities of the bleached, unbleached, and background areas
were measured using the FV31S-SW soware (Olympus). Aer background subtraction, relative intensity of the
bleached area to the unbleached area was determined and normalized to the initial value before bleaching.
Statistical analyses. Student’s t-test was performed for comparison of western blotting (Figs2a,b, 3b,c and 5e)
and FACS analysis (Fig.6e), and the Wilcoxon rank-sum test was used for comparing nuclear sizes (Fig.6d). A
P-value < 0.05 was considered to be statistically signicant.
Ethics statement. All experiments using mice were carried out according to the institutional guidelines for
the care and use of laboratory animals. e procedures used were approved by the Committee for Life Science
Research Ethics and Safety, Graduate School of Agriculture and Life Sciences, e University of Tokyo. e mice
were humanely euthanized by cervical dislocation to minimize suering.
Data availability. All data generated or analyzed during this study are included in this published article and
its Supplementary Information les.
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Acknowledgements
We are grateful to Kenta Nishitani (e University of Tokyo) and Akiyumi Tashiro (e University of Tokyo) for
providing technical assistance. We thank Miyuki Komura and Hiroshi Toriyama from the Olympus Corporation
for helping with FRAP analysis. is research was supported in part by Grant-in-Aid for Young Scientists B,
KAKENHI (to K.H., Research Project Number: 26850230), the Lotte Shigemitsu Prize (to K.H. and S.T.), and
SICORP, the Japan Agency for Medical Research and Development (AMED) (to S.T.).
Author Contributions
e author contributions are as follows: K.H. and S.T. designed this study; T.T., H.O., and M.W. performed the
nucleosome stability assay using microuidic devices; K.H. and K.T. performed all other experiments; K.H. and
K.T. prepared the manuscript; S.T. nalized the manuscript and supervised the study.
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Additional Information
Supplementary information accompanies this paper at https://doi.org/10.1038/s41598-018-23832-2.
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