Original Article

BMAL1-TTK-H2Bub1 loop deficiency

contributes to impaired BM-MSC-mediated
bone formation in senile osteoporosis
Li Jinteng,1,4 Xu Peitao,1,4 Yu Wenhui,1,4 Ye Guiwen,1 Ye Feng,1 Xu Xiaojun,1 Su Zepeng,1 Lin Jiajie,1 Che Yunshu,1
Zhang Zhaoqiang,1 Zeng Yipeng,1 Li Zhikun,1 Feng Pei,2 Cao Qian,2 Li Dateng,3 Xie Zhongyu,1 Wu Yanfeng,2
and Shen Huiyong1
1Department of Orthopedics, The Eighth Affiliated Hospital of Sun Yat-sen University, Shenzhen 518003, P.R. China; 2Center for Biotherapy, The Eighth Affiliated Hospital
of Sun Yat-sen University, Shenzhen 518003, P.R. China; 3Department of Statistical Science, Southern Methodist University, Dallas, TX, USA

During the aging process, the reduced osteogenic differentiation The “osteogenesis-adipogenesis” balance of bone marrow mesen-
of bone marrow mesenchymal stem cells (BM-MSCs) results in chymal stem cells (BM-MSCs) is essential for the maintenance of
decreased bone formation, which contributes to senile osteopo- healthy bone homeostasis. However, during the aging process, differen-
rosis. Previous studies have confirmed that interrupted circa- tiation of BM-MSCs into osteoblasts decreases and differentiation into
dian rhythm plays an indispensable role in age-related disease. adipocytes increases.5,6 This shift in the cell fate of BM-MSCs results in
However, the mechanism underlying the impaired osteogenic reduced bone formation, which contributes to senile osteoporosis.7 The
differentiation of BM-MSCs during aging and its relationship mechanism underlying this abnormal trend of differentiation during
with interrupted circadian rhythm remains unclear. In this aging is still unclear. Therefore, elucidation of the mechanism of oste-
study, we confirmed that the circadian rhythm was interrupted ogenic impairment of aging BM-MSCs and improving osteogenesis of
in aging mouse skeletal systems. The level of the core rhythm BM-MSCs in vivo are important for antiaging bone loss.
component BMAL1 but not that of CLOCK in the osteoblast
lineage was decreased in senile osteoporotic specimens from Circadian rhythms in behavior and physiology are hallmarks of life
both human and mouse. BMAL1 targeted TTK as a circadian- on earth. The endogenous timekeeping mechanism known as the
controlled gene to phosphorylate MDM2 and regulate H2Bub1 circadian clock allows organisms to anticipate the periodic fluctua-
level, while H2Bub1 in turn regulated the expression of tions brought on by the 24 h solar cycle, aligning biological functions
BMAL1. The osteogenic capacity of BM-MSCs was maintained with external changes.8 Brain and muscle Arnt-like protein 1
by a positive loop formed by BMAL1-TTK-MDM2-H2Bub1. (BMAL1) and circadian locomotor output cycles kaput (CLOCK)
Furthermore, we demonstrated that using bone-targeting re- heterodimers drive daily changes in the transcription of circadian-
combinant adeno-associated virus 9 (rAAV9) to enhance controlled genes to control cellular molecular oscillations.9 Previous
Bmal1 or Ttk might have a therapeutic effect on senile osteopo- studies have confirmed that BMAL1/CLOCK heterodimers play an
rosis and delays bone repair in aging mice. In summary, our indispensable role in physiological BM-MSC-mediated bone forma-
study indicated that targeting the BMAL1-TTK-MDM2- tion both in vitro and in vivo.10,11 Moreover, reduced levels of
H2Bub1 axis via bone-targeting rAAV9 might be a promising BMAL1 impair the osteogenic differentiation of BM-MSCs. However,
strategy for the treatment of senile osteoporosis. the role of BMAL1 in impaired senile osteoporosis-related bone for-
mation remains unclear and needs further exploration.

INTRODUCTION
Senile osteoporosis is characterized by age-related bone loss and spe-
cific biological aging in the skeletal system and generally refers to Received 5 August 2022; accepted 13 February 2023;
osteoporosis after the age of 70 years.1 By 2050, the proportion of peo- https://doi.org/10.1016/j.omtn.2023.02.014.
4
ple aged 60 years or older in the total population will reach 22% These authors contributed equally
worldwide, as estimated by the World Health Organization. With Correspondence: Xie Zhongyu, Department of Orthopedics, The Eighth Affiliated
Hospital of Sun Yat-sen University, Shenzhen 518003, P.R. China.
the increase in the aging population, senile osteoporosis and related E-mail: [email protected]
fractures not only enhance the morbidity and mortality of elderly Correspondence: Wu Yanfeng, Center for Biotherapy, The Eighth Affiliated
individuals but also dramatically increase the financial burden on Hospital of Sun Yat-sen University, Shenzhen 518003, P.R. China.
E-mail: [email protected]
public health.2 Therefore, many attempts have been made to discover
Correspondence: Shen Huiyong, Department of Orthopedics, The Eighth Affili-
novel therapeutic medicines, such as teriparatide, risedronate3 and ated Hospital of Sun Yat-sen University, Shenzhen 518003, P.R. China.
herbal medicines.4 E-mail: [email protected]

568 Molecular Therapy: Nucleic Acids Vol. 31 March 2023 ª 2023 The Author(s).
This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
www.moleculartherapy.org

Histone modifications and their associated enzymes can affect chro- RESULTS
matin compaction, nucleosome dynamics, and gene transcription. Disruption of the circadian rhythm and BMAL1 expression in the
Dysregulation of these processes disrupts gene expression homeosta- skeletal system in senile osteoporosis
sis and is frequently observed during the aging process in different To identify the disruption of the circadian rhythm of bone formation
systems, including the skeletal system.12–14 Histone H2B is monoubi- and metabolism in the aging skeletal system, we monitored 18F-labeled
quitylated on Lys120 in mammals (H2Bub1), and H2Bub1 is primar- sodium fluoride (18F-NaF) uptake in the femur in 2-month-old and
ily associated with the transcribed regions of active genes, where it 20-month-old C57BL/6 male mice by a micro-positron emission to-
facilitates transcriptional elongation.15 A stable global level of mography (PET) computed tomographic scanner. During a 24 h
H2Bub1 is necessary for the osteogenic capacity of BM-MSCs.16 dark-light cycle, we found that there was a significant rhythm of
18
Moreover, multiple histone modifications, including H2Bub1, coop- F-NaF uptake in 2-month-old mice, which reached a peak around
erate with BMAL1/CLOCK heterodimers in regulating gene tran- zeitgeber time (ZT) 18 and a nadir around ZT6 (Figure 1A). However,
scription. During the aging process, abnormal histone modification in 20-month-old mice, both the maximum and mean rates of 18F-NaF
impairs BMAL1/CLOCK heterodimer function in circadian- uptake were reduced compared with those in 2-month-old mice, and
controlled gene transcription and leads to morbidity.17 However, as the physiological variation was disrupted (Figure 1B), suggesting that
multiple histone modification enzymes or modulators are circa- circadian rhythm disruption occurred in the skeletal system in senile
dian-controlled genes, BMAL1 deficiency may also lead to dysfunc- osteoporosis.
tional histone modification during the aging process.18 As it remains
largely unknown whether reduced BMAL1 impairs the osteogenic To further establish circadian rhythm disruption, we tested the
differentiation of BM-MSCs through changes in histone modification expression of the circadian rhythm core components Bmal1 and
during aging, further investigation is needed. Clock in BM-MSCs from 2-month-old and 20-month-old mice
and from young and aged men. Interestingly, western blot results
Bone-targeting therapy remains a worldwide challenge. Drug agents showed that compared with that in the BM-MSCs of young mice
are frequently administered at high doses to reach the effective ther- or men, the expression of Bmal1 in BM-MSCs of aged mice or
apeutic level inside the diseased bone microenvironment, which men, respectively, was decreased, while the expression of Clock
may lead to severe side effects, including myelosuppression and was not (Figures 1C and 1D). Next, to examine whether the expres-
dose-limiting toxicity to healthy tissues. Thus, a drug delivery sys- sion of BMAL1 and CLOCK in osteoblasts was impaired during se-
tem with high bone affinity to reduce off-target side effects is nile osteoporosis, we collected bone specimens from 2-month-old
urgently needed. Adeno-associated virus (AAV) is a small and 20-month-old mice, patients with senile osteoporosis and young
(26 nm) nonenveloped parvovirus with a single-stranded genome patients with traffic injuries and analyzed bone sections (Figures 1E
of
4.7 kb.19 Because of its high transduction efficiency, persistent and 1F). According to the results of immunofluorescence staining, in
transgene expression, and lack of postinfection immunogenicity and the Ocn+ osteoblast lineage, Bmal1 expression was weaker in
pathogenicity, AAV is an attractive viral vector for use in gene ther- 20-month-old mice than in 2-month-old mice, whereas Clock
apy in the clinic.20 Previous studies have already confirmed that the expression showed no significant difference between the age groups
rAAV9 subtype could effectively drive DNA delivery into osteoblast (Figures 1G and 1H). The results demonstrated that the expression of
lineage cells in vivo.21 However, the prototype rAAV9 still has other BMAL1 was also lower in the OCN+ osteoblast lineage in the aged
organ tropisms, including that of cardiac muscle, skeletal muscle, patients than in the young patients, while CLOCK was not altered
lung alveoli, and liver. Thus, the modification of rAAV9 to confer (Figures 1I and 1J). Moreover, we further explored the expression
it a strongly osteophilic property and the ability to deliver bone-for- of BMAL1 and CLOCK in bone samples of ovariectomized (OVX)
mation genes into BM-MSCs may be a promising strategy for treat- mice, sham-operated mice, patients with postmenopausal osteopo-
ing senile osteoporosis. rosis, and age-matched controls, and the results demonstrated that
the expression of BMAL1 and CLOCK did not show a significant dif-
In this study, we demonstrated that the circadian rhythm was inter- ference (Figures S1A–S1D) in the OCN+ osteoblast lineage between
rupted in the skeletons of aging mice. BMAL1 but not CLOCK was the OVX mice and the sham-operated mice or between the patients
decreased in the osteoblast lineage in both human and mouse senile with postmenopausal osteoporosis and the age-matched controls.
osteoporotic specimens. BMAL1 targeted TTK as a circadian control Taken together, these results indicated that the circadian rhythm
gene to regulate the H2Bub1 level, while H2Bub1 in turn regulated the and BMAL1 expression were abnormal in the osteoblast lineage in
expression of BMAL1 at the transcriptional level. BMAL1-TTK- senile osteoporosis.
H2Bub1 formed a positive loop to maintain the osteogenic capacity
of BM-MSCs. Finally, we demonstrated that using bone-targeting
rAAV9 to enhance Bmal1 or Ttk could have a therapeutic effect on BMAL1 positively regulated the osteogenic differentiation of
senile osteoporosis and delayed bone repair in aging mice. Taken MSCs
together, our study indicated that targeting the BMAL1-TTK- To determine the role of BMAL1 in MSC osteogenic differentiation,
H2Bub1 axis via bone-targeting rAAV9 is a promising strategy for we first cultured human BM-MSCs in osteogenic medium (OM) for
treating senile osteoporosis. up to 14 days, and alizarin red S (ARS) staining showed a gradual

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Figure 1. Disruption of circadian rhythm and BMAL1 expression in the skeletal system in senile osteoporosis
(A) PET scans of 2-month-old and 20-month-old C57BL/6J mice after 18F-NaF tail vein injection at the indicated times. ZT0 is defined as the time the light was turned on (8
a.m.). (B) PET analysis of maximum and mean SUV. (C and D) Western blot analysis of the levels of the core rhythm components BMAL1 and CLOCK in BM-MSCs from
2-month-old and 20-month-old mice, patients with traffic injuries and patients with senile osteoporosis. (E and F) HE staining (scale bar, 100 mm) of bone specimens from
2-month-old and 20-month-old mice, patients with traffic injuries and patients with senile osteoporosis. (G and H) Immunofluorescence staining (scale bar, 100 mm) showed
Bmal1 and Clock expression in the Ocn+ osteoblast lineage in 2-month-old and 20-month-old mice (white arrows). (I-J) Immunofluorescence staining (scale bar, 100 mm)
showed BMAL1 and CLOCK expression in the OCN+ osteoblast lineage in young patients and patients with senile osteoporosis (white arrows). All data are presented as
mean ± SD; n = 3; *p < 0.05.

increase from days 0–14, while alkaline phosphatase (ALP) staining (Figures 2B and 2C). Furthermore, we analyzed the correlation of
peaked at 10 days (Figure 2A). The results of western blotting the BMAL1 expression levels with ARS and ALP staining and found
and quantitative real-time PCR (qRT-PCR) demonstrated that a strong positive relationship in the osteogenic differentiation of
BMAL1 expression was upregulated gradually during osteogenesis MSCs (Figure 2D).

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Figure 2. BMAL1 positively regulated the osteogenic differentiation of human BM-MSCs

(A) ARS, ALP staining, and quantification at different time points during osteogenic differentiation of MSCs. (B and C) BMAL1 protein (B) and mRNA (C) expression in MSCs
undergoing osteogenic differentiation as measured at different time points. GAPDH served as the internal control. Bar graphs showing the expression of BMAL1 relative to the
GAPDH level. (D) Correlation of expression levels of BMAL1 with ARS quantification and ALP activity during the osteogenic differentiation of MSCs. (E) ARS and ALP staining
(legend continued on next page)

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To further confirm the influence of BMAL1 on MSC osteogenesis, we levels. As shown by western blot analysis, the level of H2Bub1 was
used lentiviruses to diminish and overexpress BMAL1 in MSCs decreased after BMAL1 or CLOCK expression was diminished and
(Figures S2A and S2B). The osteogenic capacity of MSCs in different increased following overexpression of BMAL1 or CLOCK, while
groups was evaluated after osteogenic induction of MSCs for 14 days. the level of H2Aub1 showed no significant difference between control
The results of the ARS staining and ALP assays demonstrated that expression and over- or underexpression of either component
knocking down BMAL1 expression impaired the osteogenic capacity, (Figures 3D and S3A). The results of RNA-seq showed that following
whereas the overexpression of BMAL1 promoted osteogenesis (Fig- knockdown of BMAL1 or CLOCK, the expression of RNF20 and
ure 2E). We also tested the expression of the osteogenesis-associated RNF40, which act as classical factors monoubiquitinating histone
markers RUNX2, OSX, OPN, and OCN. The results of western blot- H2B,15 was not significantly altered (Figure 3E). The top 10 intersect-
ting and qRT-PCR demonstrated that diminished BMAL1 decreased ing genes with the most similar expression between the two groups
the expression of RUNX2, OSX, OPN, and OCN in MSCs during are listed according to log2 fold change (FC) and log10(q value)
osteogenic induction, while overexpressing BMAL1 increased the values, and TTK, which can regulate H2Bub1 levels according to
expression (Figures 2F, 2G, S2A, and S2B). In summary, these results the literature,23,24 is highlighted (Figure 3F) and shown in a volcano
indicated that BMAL1 positively regulated the osteogenic capacity of plot (Figure S4A). Together, these data suggested that the core circa-
MSCs in vitro. dian component BMAL1 regulated histone H2B monoubiquitination
by managing H2Bub1 level-regulated gene TTK expression.
MSCs were loaded onto hydroxyapatite/tricalcium phosphate
(HA/TCP) scaffolds and implanted into the dorsal subcutaneous BMAL1 targeted the circadian-controlled gene TTK to
space of nude mice. As shown by hematoxylin and eosin (HE) phosphorylate MDM2 and regulate H2Bub1 levels to affect the
and Masson staining, diminishing BMAL1 led to the reduced osteogenic capacity of MSCs
bone formation of MSCs on the scaffolds, while overexpression As mentioned above, the RNA-seq results suggested that diminishing
of BMAL1 significantly increased the formation of bone-like tissue. BMAL1 impaired TTK expression, which affected H2Bub1 levels. We
As shown by immunohistochemistry, the expression of collagen I then evaluated whether and how BMAL1 regulated TTK expression,
(Col I), a marker of bone formation, was decreased after knocking and the results of qRT-PCR and western blotting demonstrated that
down BMAL1 and increased by overexpressing BMAL1 (Fig- diminished BMAL1 decreased the expression of TTK in MSCs, while
ure 2H). In summary, BMAL1 positively regulated the osteogenic overexpressing BMAL1 increased the expression (Figures 4A and 4B).
differentiation of MSCs both in vitro and in vivo. BMAL1 regulation of gene expression relies on heterodimers consist-
ing of the circadian rhythm core components BMAL1 and CLOCK,
The core circadian component BMAL1 regulated histone H2B which bind to the E-box elements (50 -CANNTG-30 ) located within
monoubiquitination levels the promoters of circadian-controlled genes to activate their tran-
To determine how BMAL1 regulates the osteogenic capacity of MSCs scription.25 Through bioinformatic analysis, we identified candidate
as a part of the core circadian components, we performed RNA binding sites: two putative E-box motifs within the TTK promoter re-
sequencing (RNA-seq) to study the impact of diminishing the core gion (Figure 4C). Cleavage under targets and tagmentation (CUT&
component BMAL1 or CLOCK in MSCs. A heatmap showed distinct Tag) assays using primers targeting the E-box 1 or E-box 2 sites
expression patterns before and after knocking down each component showed that BMAL1 bound to these regions in the TTK promoter
(Figure 3 A). In Gene Ontology (GO) and Kyoto Encyclopedia of (Figure 4D). The results above demonstrated that BMAL1 regulated
Genes and Genomes (KEGG) analyses, the differentially expressed the circadian-controlled gene TTK by BMAL1/CLOCK dimers.
genes in either the Sh-BMAL1 or Sh-CLOCK group were enriched
in terms regulated by histone modification, including DNA replica- To further verify the effect of BMAL1 on MSC osteogenesis, which
tion, DNA unwinding, DNA repair, and homologous recombination, mediated H2Bub1 levels by regulating TTK expression, we used len-
and terms related to ubiquitination (Figure 3B). Furthermore, gene tiviruses to knock down and overexpress TTK in MSCs. The results of
set enrichment analysis (GSEA) of the RNA-seq data showed obvious qRT-PCR, western blotting, CUT&Tag-qPCR, ARS staining, and
enrichment of genes related to histone ubiquitination-related and ALP assays demonstrated that TTK positively regulated osteogenic
osteogenesis-related terms, including nuclear ubiquitin ligase com- capacity by affecting H2Bub1 levels (Figures S5A–S5D). Furthermore,
plex, regulation of ubiquitin protein ligase, osteoblast proliferation, we diminished BMAL1 while overexpressing TTK in MSCs, and the
bone growth, and bone cell development. (Figure 3C). As reported results of CUT&Tag-qPCR showed that overexpressing TTK rescued
previously, histone H2B monoubiquitination positively regulates the decreased recruitment of H2Bub1 to RUNX2 and OSX promoters
the osteoblastic differentiation of MSCs.16,22 Thus, we investigated by knocking down BMAL1 (Figure 4E). As expected, qRT-PCR, west-
whether the core components BMAL1 and CLOCK affect H2Bub1 ern blotting, ARS staining, ALP assays, HE staining, Masson staining,

of the MSCs infected with Sh-NC, Sh-BMAL1, OE-NC, or OE-BMAL1 lentiviruses on the 14th day of osteogenic differentiation. (F and G) Relative mRNA (F) and protein
(G) expression of the osteogenesis-associated markers RUNX2 and OSX in the MSCs infected with Sh-NC, Sh-BMAL1, OE-NC, or OE-BMAL1 lentiviruses on the 10th day of
osteogenic differentiation. (H) HE and Masson staining and Col I immunohistochemistry of transplanted HA/TCP embedded with the MSCs infected with Sh-NC, Sh-BMAL1,
OE-NC, or OE-BMAL1 lentiviruses. All data are presented as mean ± SD; n = 3 (MSCs derived from 3 different donors); *p < 0.05.

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(legend on next page)

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and immunohistochemistry demonstrated that diminishing BMAL1 of H2Bub1 and Pol II on BMAL1, which was examined by CUT&Tag-
impaired the osteogenic differentiation of MSCs, while overexpres- seq and CUT&Tag-qPCR, was decreased in both the Sh-RNF40 and
sion of TTK significantly rescued the osteogenic capacity of MSCs Sh-WAC groups, especially in colored region and at sites A–F
(Figures 4F–4I). (Figures 5F and 5G). Taken together, these observations suggest
that the expression of BMAL1 was positively modulated by
According to the literature, TTK regulates H2Bub1 levels by phos- H2Bub1 at the transcript level.
phorylating MDM2,23,24 while MDM2 acts as a regulatory factor in
differentiating osteoblasts.26 To confirm the role of MDM2 in MSC Bone-targeted BMAL1 or TTK rescue-treated senile
osteogenesis and the BMAL1-TTK-H2Bub1 axis, we knocked down osteoporosis
MDM2 in MSCs. The results of western blotting, ARS staining, and To establish the role of TTK and H2Bub1 in the impaired age-related
ALP assays showed that diminished MDM2 expression impaired osteogenic capacity, we examined TTK expression and H2Bub1 levels
the osteogenic differentiation of MSCs (Figures S6A and S6B). We in MSCs during senile osteoporosis using western blotting and immu-
conducted co-immunoprecipitation (IP)/western blot assays to nofluorescence staining. As expected, in BM-MSCs, the expression of
confirm that BAML1 can regulate TTK expression to modulate Ttk and the level of H2Bub1 were lower in 20-month-old mice than in
MDM2 phosphorylation and ultimately regulate H2Bub1 levels (Fig- 2-month-old mice and lower in aged patients than in young patients
ure S6C). Collectively, these results indicated that BMAL1 acts as a (Figures 6A–6F). To exclude the possible effect of RNF20/40 on
critical regulator of MSC osteogenesis by regulating TTK expression H2Bub1 level, we also examined RNF20/40 expression by western
to phosphorylate MDM2 and affect H2Bub1 levels. blotting and immunofluorescence staining. The results showed that
RNF20/40 expression did not significantly differ between younger
H2Bub1 positively modulated BMAL1 expression at the stages and senile osteoporosis (Figures S8A–S8F).
transcript level
The role of H2Bub1 in regulating part of the clock component has To evaluate whether BMAL1 and TTK can be used as therapeutic
been proposed,17 but the influence of H2Bub1 levels on BMAL1 targets for osteoporosis, we constructed bone-targeting recombinant
expression has never been reported. To answer this question, we per- adeno-associated virus 9 (rAAV9) as previously described21 to over-
formed integrative analyses to compare the Pol II and H2Bub1 chro- express Bmal1 or Ttk in vivo. To verify the therapeutic effect of Bmal1
matin immunoprecipitation sequencing (ChIP-seq) data of human and Ttk, we intravenously injected rAAV9-control/Bmal1/Ttk into
fetal osteoblasts (hFOB) 1.19 cells on days 0 and 10 of osteogenic dif- senile osteoporotic (18-month-old) mice, which were subjected to
ferentiation. The results revealed that Pol II and H2Bub1 occupancy procedures creating defects on the calvaria or femur and were sacri-
on BMAL1 was increased after osteogenic differentiation (Figure 5A). ficed for immunofluorescence staining and microcomputed tomogra-
To confirm whether H2Bub1 level regulates the expression of phy (micro-CT) analysis (Figure 7A). The tissue distribution of
BMAL1, we used lentiviruses to diminish the levels of RNF40 and rAAV9 was assessed by mNeonGreen expression, which appeared
WAC, which are parts of the obligate complex and monoubiquiti- in calvarias and femurs, showing that rAAV9 specifically infected
nated histone H2B,27 in MSCs (Figures S7A and S7B). The results these skeletal systems (Figure 7B). Additionally, fluorescence imaging
of qRT-PCR and western blotting demonstrated that diminished of different organs verified the effective bone-targeting delivery of
levels of RNF40 or WAC decreased BMAL1 expression and rAAV9 (Figure 7C). As shown by fluorescence microscopy, mNeon-
H2Bub1 levels (Figures 5B and 5C). Furthermore, to explore the Green was expressed primarily in osteoblasts in cortical and trabec-
impact of H2Bub1 on BMAL1, Pol II, and H2Bub1, we used CUT& ular bones (Figure 7D). These results demonstrated that the rAAV9
Tag-seq data from MSCs with decreased RNF40 or WAC. Heatmaps vector targets osteoblast lineage cells residing in the endosteal bone.
showing the general CUT&Tag-seq data signals revealed that loss of As shown by the results of immunofluorescence staining and western
RNF40 or WAC resulted in a significant reduction in Pol II and blotting, the expression of Bmal1 and Ttk and the levels of H2Bub1 in
H2Bub1 occupancy in the gene body (Figure 5D). GO biological pro- the rAAV9-Bmal1 group as well as Ttk expression and H2Bub1 levels
cess analysis revealed that the differentially expressed H2Bub1- or Pol in the rAAV9-Ttk group were increased in bone sections (Figures 7E–
II-occupied genes in both the Sh-RNF40 and Sh-WAC groups were 7G, S9A and S9B). Compared with those in the rAAV9-control group,
enriched in terms related to RNA transcription, osteoblast differenti- the mice in either the rAAV9-Bmal1 or rAAV9-Ttk group showed
ation, and circadian rhythm (Figure 5E). As expected, the occupancy accelerated calvarial- and femoral-defect healing as well as increased

Figure 3. The core circadian component BMAL1 regulated histone H2B monoubiquitination levels
(A) RNA-seq heatmap comparing the MSCs infected with Sh-NC, Sh-BMAL1, or Sh-CLOCK lentiviruses on the 7th day of osteogenic differentiation. (B) GO analysis of the
RNA-seq data between the Sh-NC and Sh-BMAL1 groups and the Sh-NC and Sh-CLOCK groups. Bar graph showing the p values of the enriched terms. (C) GSEA of
the RNA-seq data between the Sh-NC and Sh-BMAL1, Sh-NC, and Sh-CLOCK groups. (D) H2Bub1 and H2Aub1 levels in the MSCs infected with Sh-NC, Sh-BMAL1, or
Sh-CLOCK lentiviruses on the 10th day of osteogenic differentiation. H2B and H2A served as the internal controls. Bar graphs showing the relative levels. Data are presented
as mean ± SD; n = 3; *p < 0.05. (E) log2 FC and log10(q value) of differential RNF20/40 expression between the Sh-NC and Sh-BMAL1 groups and the Sh-NC and
Sh-CLOCK groups as obtained from the RNA-seq data. (F) Circos plot showing the terms with enriched genes and log2 FC and log10(q value). TTK, the regulator of histone
H2B monoubiquitination, is highlighted (G).

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Figure 4. BMAL1 targeted the circadian-controlled gene TTK to regulate H2Bub1 levels to affect the osteogenic capacity of MSCs
(A and B) Relative mRNA (A) and protein (B) expression of TTK in the MSCs infected with Sh-NC, Sh-BMAL1, OE-NC, or OE-BMAL1 lentiviruses on the 10th day of osteogenic
differentiation. (C) The putative E-boxes in the TTK promoter region. (D) CUT&Tag-qPCR showed the percentage of BMAL1 occupancy on the TTK promoter. Data are shown
as the proportion of input level and normalized to the IgG control. (E) CUT&Tag-qPCR analysis showing the H2Bub1 occupancy on RUNX2 and OSX in the MSCs infected
with Sh-NC and OE-NC, Sh-BMAL1 and OE-NC or Sh-BMAL1 and OE-TTK lentiviruses that were undergoing osteogenic differentiation. (F and G) Relative mRNA expression
(F) and protein expression (G) of RUNX2 and OSX in MSCs infected with Sh-NC and OE-NC, Sh-BMAL1 and OE-NC, or Sh-BMAL1 and OE-TTK lentiviruses that were
undergoing osteogenic differentiation. Bar graphs showing the relative expression. (H) ARS and ALP staining of the MSCs infected with Sh-NC and OE-NC, Sh-BMAL1 and
OE-NC, or Sh-BMAL1 and OE-TTK lentiviruses on the 14th day of osteogenic differentiation. (I) HE and Masson staining and Col I immunohistochemistry of transplanted HA/
TCP embedded with the MSCs infected with Sh-NC and OE-NC, Sh-BMAL1 and OE-NC, or Sh-BMAL1 and OE-TTK lentiviruses. All data are presented as mean ± SD; n = 3;
*p < 0.05.

bone density and improvements in other femur parameters, including man worldwide older than 50 years will encounter bone fracture
BV/TV, Tb.Th, Tb.N, Tb.Sp, and Ct.Th, after infection with rAAV9 due to senile osteoporosis in their remaining lives.28 Senile osteopo-
(Figures 7H and 7I). Thus, rAAV9-Bmal1 and rAAV9-Ttk may be rosis and related fractures not only enhance the morbidity and
potential drugs for the treatment of senile osteoporosis as potent mortality of elderly individuals but also dramatically increase the
bone anabolic agents. financial burden on public health. Thus, further study to explore
the mechanism of the aging process in the skeletal system and
DISCUSSION conduct corresponding clinical translation research will not only
Senile osteoporosis is the most common age-related bone metabolic decrease the morbidity and mortality from senile osteoporosis-
disorder worldwide. According to the recent report of the National related fracture but will also reduce the economic burden on public
Osteoporosis Foundation (NOF), every second woman and fourth health.

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Figure 5. H2Bub1 positively modulated the expression of BMAL1 at the transcript level
(A) Signal traces of ChIP-seq data showing H2Bub1 and Pol II occupancy on BMAL1 in hFOB1.19 cells on day 0 or 7 of osteogenic differentiation. (B and C) Relative
mRNA expression (B) and protein expression (C) of BMAL1 in the MSCs infected with Sh-NC, Sh-RNF40, or Sh-WAC lentiviruses. Bar graphs showing the relative
expression. Data are presented as mean ± SD; n = 3; *p < 0.05. (D) CUT&Tag-seq average binding profiles and heatmaps depicting occupancy of H2Bub1 and Pol II in
the MSCs infected with Sh-NC, Sh-RNF40, or Sh-WAC lentiviruses. (E) GO biological process analyses of the CUT&Tag-seq data comparing between the Sh-NC and
Sh-RNF40 groups and the Sh-NC and Sh-WAC groups. Bar graph showing the p values of the enriched terms. (F) Signal traces of CUT&Tag-seq data showing H2Bub1
and Pol II occupancy on BMAL1 in the MSCs infected with Sh-NC, Sh-RNF40, or Sh-WAC lentiviruses. The colorful shadows showing regions with difference (G)
CUT&Tag-qPCR analysis showing the H2Bub1 and Pol II occupancy on BMAL1 sites A–F in the MSCs infected with Sh-NC, Sh-RNF40 or Sh-WAC lentiviruses. Data are
presented as mean ± SD; n = 3; *p < 0.05.

In contrast to postmenopausal osteoporosis, bone formation impair- showed severe adverse cardiovascular events in clinical trials.29
ment instead of osteoclastic hyperfunction is the major cause of senile Thus, a therapeutic strategy that can effectively promote BM-MSC-
osteoporosis. However, there are far fewer approaches to effectively mediated bone formation without adverse effects is still an
promote BM-MSC-mediated bone formation than inhibit osteo- unmet need.
clastic activity in the clinic for treating osteoporosis.29,30 Only two
anabolic agents, parathyroid hormone and parathyroid hormone- BMAL1/CLOCK heterodimers drive daily changes in the transcrip-
related protein, can promote osteoblast function to treat patients tion of circadian-controlled genes to control physiological system
with osteoporosis. However, these agents are costly, require frequent development and homeostasis.31 However, multiple studies have
injections and can promote the development of osteosarcomas. shown that during the aging process, the expression of BMAL1/
Recently developed agents, including an anti-clerostin antibody and CLOCK heterodimers is reduced, which impairs the physiological
a small-molecule inhibitor of cathepsin K, can increase bone mass function of various tissues and organs32–35; for example, BMAL1 defi-
and reduce fracture risk in osteoporosis. However, these drugs ciency has been found to contribute to the decline in visual function

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Figure 6. TTK expression and H2Bub1 levels were decreased in BM-MSCs in senile osteoporosis
(A and B) Western blot analysis of the levels of TTK and H2Bub1 in BM-MSCs from 2-month-old and 20-month-old mice, patients with traffic injuries and patients with senile
osteoporosis. (C and D) Immunofluorescence staining (scale bar, 100 mm) showed Ttk expression and H2Bub1 levels in the Ocn+ osteoblast lineage in 2-month-old and
20-month-old mice (white arrows). (E and F) Immunofluorescence staining (scale bar, 100 mm) showed TTK expression and H2Bub1 levels in the OCN+ osteoblast lineage in
young patients with traffic injuries and patients with senile osteoporosis (white arrows). All data are presented as mean ± SD; n = 3; *p < 0.05.

during aging, and circadian rhythm disruption has been shown to osteogenic impairment is necessary to elucidate the mechanism of se-
play a role in osteoarthritis-like changes in chondrocyte activity.36 nile osteoporosis.
Consistent with these studies, our research demonstrated that
BMAL1 but not CLOCK was specifically decreased in osteoblast line- BMAL1/CLOCK heterodimers conservatively recognize the E-box
age cells of senile osteoporotic specimens from both human and element (CANNTG) in the promoter to initiate circadian-controlled
mouse but not postmenopausal osteoporotic specimens. Thus, we gene transcription in mammals.25 The transcription of circadian-
conclude that similar to that in multiple other organs, BMAL1/ controlled genes by BMAL1/CLOCK heterodimers also requires mul-
CLOCK heterodimer dysfunction is indispensable for the aging- tiple types of histone modifications. During aging, abnormal histone
related osteogenic impairment in senile osteoporosis. Further study modification impairs BMAL1/CLOCK heterodimer function in circa-
to reveal the mechanism of BMAL1 deficiency-mediated BM-MSC dian-controlled gene transcription, thereby leading to morbidity.32

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(legend on next page)

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However, as multiple histone modification enzymes or modulators et al.21 previously demonstrated that grafting the bone-targeting pep-
are circadian-controlled genes,37,38 BMAL1 deficiency may also lead tide motif (Asp-Ser-Ser)6 onto the AAV9-VP2 capsid protein could
to dysfunctional histone modification during the aging process. improve the affinity of AAV9 to osteoblast lineage cells while substan-
H2Bub1 in mammals is a type of histone modification that is essential tially reducing tropism to cardiac muscle, skeletal muscle, lung
for the early steps in transcriptional initiation and elongation. A stable alveoli, and liver. We also used this rational strategy to modify
level of H2Bub1 not only facilitates the transcriptional function of AAV9 to systemically deliver Bmal1 or Ttk to treat senile osteoporosis
BMAL1/CLOCK heterodimers but also is indispensable for the in aging mice. We obtained satisfactory results in which only a single
osteogenic capacity of BM-MSCs. H2Bub1 deficiency leads to gene systemic injection of this modified AAV9 carrying Bmal1 or Ttk plas-
transcription dysfunction and contributes to multiple diseases.39,40 mids could sufficiently improve the H2Bub1 level in osteoblast line-
However, whether H2Bub1 dysfunction plays a role in aging status, age cells in aging mice and further significantly promote bone
including senile osteoporosis, has never been explored. In this study, mass. These results indicated that targeting the BMAL-TTK-
we first discovered that BMAL1 can positively regulate H2Bub1 level H2Bub1 axis via bone-targeting AAV9 might have potential in treat-
by targeting the H2Bub1 modulator TTK as a circadian-controlled ing senile osteoporosis in the clinic.
gene. Knocking down BMAL1 significantly decreased the expression
of TTK and further reduced the level of H2Bub1 in BM-MSCs. Over- To our knowledge, our study is the first to demonstrate that BMAL1
expression of TTK rescued the H2Bub1 level as well as the osteogenic deficiency contributes to circadian rhythm disruption and impaired
capacity of BM-MSCs under BMAL1 deficiency. Accordingly, TTK- BM-MSC-mediated bone formation in senile osteoporosis. BMAL1
H2Bub1 deficiency was also observed in osteoblast lineage cells in positively regulated the osteogenic capacity of BM-MSCs by main-
senile osteoporotic specimens from both human and mouse. Interest- taining H2Bub1 levels by targeting the H2Bub1 modulator TTK as
ingly, we also confirmed that H2Bub1 can positively regulate the tran- a circadian-controlled gene. Furthermore, we are the first to discover
scription of BMAL1. Inhibiting H2Bub1 significantly decreased the that H2Bub1 in turn regulates BMAL1 expression at the transcrip-
expression of BMAL1. The results described above lead to the tional level. Thus, BMAL1-TTK-H2Bub1 forms a positive cycle to
intriguing possibility that BMAL1-TTK-H2Bub1 may form a positive maintain the osteogenic capacity of BM-MSCs. However, BMAL1
cycle to maintain the osteogenic capacity of BM-MSCs. However, deficiency deteriorated this cycle, which significantly impaired the
during the aging process, BMAL1 deficiency decreases H2Bub1, osteogenic capacity of BM-MSCs during the aging process and finally
which in turn further aggravates BMAL1 deficiency. BMAL1-TTK- led to senile osteoporosis. Finally, we demonstrated that only a single
H2Bub1 cycle impairment significantly reduces the osteogenic capac- injection to supplement BMAL1 or TTK in osteoblast lineage cells via
ity of BM-MSCs during the aging process and ultimately leads to bone-targeting rAAV9 could significantly promote bone mass and
senile osteoporosis (Figure 8). bone repair speed in senile mice. However, our study has some limi-
tations. First, we did not clarify the mechanism of BMAL1 deficiency
AAV vectors have a long track record of both safety due to their lack in osteoblast lineage cells in senile osteoporosis. Moreover, current
of pathogenicity and efficacy in treating multiple diseases in the bone-targeting rAAV9 still has some affinity for other organs. To
clinic.41 Moreover, unlike nonviral vectors, such as nanoparticles translate our research into the clinic, we will further explore the
and liposomes, which can be rapidly degraded and cleared in circula- mechanism of BMAL1 deficiency in senile osteoporosis and further
tion, AAVs are capable of persisting in postmitotic cells of host tissues modify this bone-targeting rAAV9 delivery system to improve its
for several years.42 As senile osteoporosis is a chronic disease bone affinity as well as reduce its off-target effects in the future.
throughout aging life, AAV is a relatively suitable delivery vehicle
for treating senile osteoporosis. Previous studies have confirmed MATERIALS AND METHODS
that the AAV9 subtype could target osteoblast lineage cells in the Animal models
bone to drive RNA delivery in vivo. However, there are still other or- Wild-type C57BL/6 mice at 2, 18, and 20 months of age and BALB/c-
gans, including cardiac muscle, skeletal muscle, lung alveoli, and liver, nu/nu mice at 2 months of age were purchased from the Laboratory
with a high affinity for rAAV9. Thus, delivery of DNA via rAAV9 to Animal Center of Sun Yat-sen University and housed in cages under
treat senile osteoporosis will still have multiple off-target effects. Yang standard animal housing conditions with 12 h light and 12 h dark

Figure 7. Bone-targeted Bmal1 or Ttk rescue-treated senile osteoporosis

(A) Diagram showing the workflow of rAAV9 injection in 18-month-old mice with calvarial and femoral defects and bone section analysis. (B) Immunoblot analysis showing
mNeonGreen expression in different organs of the mice injected with rAAV9. (C) Fluorescence images of different organs of mice injected with rAAV9. (D) Immunofluo-
rescence staining (scale bar, 100 mm) showing mNeonGreen-expressing osteoblasts in the femurs of the mice injected with rAAV9. (E) Immunofluorescence staining (scale
bar, 100 mm) showing Bmal1 expression in the Ocn+ osteoblast lineage in the mice injected with rAAV9-control or rAAV9-Bmal1 (white arrows). (F) Immunofluorescence
staining (scale bar, 100 mm) showing Ttk expression in the Ocn+ osteoblast lineage in the mice injected with rAAV9-control, rAAV9-Bmal1, or rAAV9-Ttk (white arrows). (G)
Immunofluorescence staining (scale bar, 100 mm) showing H2Bub1 levels in the Ocn+ osteoblast lineage in the mice injected with rAAV9-control, rAAV9-Bmal1, or rAAV9-Ttk
(white arrows). Data are presented as mean ± SD; n = 3; *p < 0.05. (H) Micro-CT analysis comparing the healing rates of calvarial and femoral defects in the mice injected with
rAAV9-control, rAAV9-Bmal1, or rAAV9-Ttk. (I) Representative micro-CT images showing the trabecular bone of mice with senile osteoporosis injected with rAAV9-control,
rAAV9-Bmal1, or rAAV9-Ttk. Bone morphometric analysis, including the analysis of BV/TV, Tb.Th, Tb.N, Tb.Sp, and Ct.Th., was performed. Data are presented as mean ±
SD; n = 5; *p < 0.05.

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 Molecular Therapy: Nucleic Acids

Figure 8. Model showing that the disruption of the BMAL1-TTK-MDM2-H2Bub1 positive loop led to the impaired osteogenic capacity of BM-MSCs in senile
osteoporosis

cycles. Mice in poor health were excluded from the study. All mouse Bone-targeting Bmal1/Ttk overexpression in vivo
experiments were approved by the animal ethics committee of Sun Bone-targeting rAAV9 overexpressing Bmal1/Ttk was designed and
Yat-sen University (SYSU-IACUC-2021-000811). constructed as previously described.21 In summary, the DNA
sequence encoding the bone-specific peptide motif DSS (Asp-Ser-
In vivo osteogenic induction Ser)6 was codon-optimized and inserted into the AAV9 capsid
For in vivo osteogenic induction, MSCs in the fourth passage infected protein VP2 to construct bone-targeting rAAV9 vectors that overex-
with lentiviruses (Sh-NC, Sh-BMAL1, Sh-TTK, OE-NC, OE-BMAL1, pressed Bmal1/Ttk in vivo.
and OE-TTK) were cultured in OM before use in the in vivo study.
After 7 days of osteogenic induction, MSCs (5  105) were trypsinized PET imaging
and seeded onto HA/TCP (Zimmer) for 24 h. HA/TCP loaded with PET imaging of mice was designed as previously described. In sum-
MSCs was implanted into the subcutaneous dorsal space of 8-week- mary, PET imaging was performed using an Inveon MM system
old BALB/c nu/nu mice. Specimens were harvested 8 weeks after (Siemens). A total of 120 mCi sodium fluoride labeled with fluorine-
implantation, and the animals were euthanized via cervical disloca- 18 was intraperitoneally injected into the animals 40 min before
tion. The grafts were retrieved for subsequent HE and Masson imaging for all four different time points. Quantification of bone
trichrome staining and histochemical analysis. metabolism was obtained using the maximum standardized uptake
value (SUVmax) and the mean standardized uptake value (SUV-
Calvarial and femoral bone defects in mice mean), which were normalized to body weight. SUV = CT/DInj/Ws.
Calvarial bone defects were created with an electric bone drill applied CT is the maximum/mean 18F-NaF radioactivity per unit volume
to the parietal bones of 18-month-old mice. Briefly, after anesthetiza- in the femur head, DInj is the injection dose, and WS is the mouse
tion, disinfection, and skin incision, the subcutaneous tissue was care- weight.
fully peeled away to expose the parietal bones. A 2.5 mm calvarial
defect was created with an electric bone drill 2 weeks after ejection, Isolation and culture of MSCs
and the incision was sutured with a thin thread. A 1.0 mm femoral MSC isolation
bone defect was created following the same method used to create cal- Bone marrow was extracted from the posterior superior iliac spine of
varial defects 8 weeks after ejection. Ten weeks after ejection, the healthy volunteers under sterile conditions. Then, MSCs were isolated
whole skulls and femurs were collected for micro-CT and subsequent and purified by density gradient centrifugation as described in our
analysis. previously reported methods.43 Briefly, MSCs were isolated and puri-
fied from bone marrow using density gradient centrifugation at
OVX mice 600  g for 30 min. The isolated MSCs were resuspended in a medium
For the generation of OVX mice, 2-month-old female wild-type composed of 90% Dulbecco’s modified Eagle’s medium (DMEM;
C57BL/6 mice underwent bilateral ovariectomy or sham operation. Gibco) and 10% fetal bovine serum (FBS; Hangzhou Sijiqing Biolog-
Mice were allowed 2 months to recover from the ovariectomy surgery ical Engineering Material Company) seeded in flasks and cultured in
and then were sacrificed and used in various assays. incubators at 37 C in a 5% CO2 atmosphere, and the culture medium

580 Molecular Therapy: Nucleic Acids Vol. 31 March 2023

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was replaced every third day. When the cells reached 80%–90% Protein extraction and western blot analysis
confluence, the MSCs were digested using 0.25% trypsin containing The western blot protocols were reported previously. Briefly, the cells
0.53 mM EDTA and reseeded in new flasks or plates, and cells in were harvested and lysed in RIPA buffer containing protease and
the second through fifth passages were used for all experiments. phosphatase inhibitors for 30 min on ice. Lysates were obtained via
centrifugation at 14,000 rpm at 4 C for 10 min. Equal amounts of
Osteogenic induction each sample diluted in 5 sodium dodecyl sulfate (SDS) loading
MSCs (0.5  105 cells/well) were seeded in 12-well plates and cultured buffer were subjected to SDS-PAGE and were subsequently trans-
in DMEM (with 1,000 mg/L glucose) with 10% FBS. After 24 h, the cul- ferred to polyvinylidene fluoride membranes (Millipore). The mem-
ture medium was replaced with OM, consisting of DMEM (1,000 mg/L branes were blocked in 5% nonfat dry milk dissolved in TBST
glucose) with 10% FBS, 100 IU/mL penicillin, 100 IU/mL streptomycin, (150 mM NaCl, 50 mM Tris-HCl [pH 7.5], and 0.05% Tween 20)
0.1 mM dexamethasone, 10 mM b-glycerol phosphate, and 50 mM at room temperature for 1 h and then incubated overnight with
ascorbic acid (Sigma-Aldrich). The OM was changed every 3 days. primary antibodies at 4 C. After incubation with horseradish perox-
idase (HRP)-conjugated anti-mouse antibody (catalog no. 7076; Cell
Lentivirus construction and infection Signaling Technology) or HRP-conjugated anti-rabbit antibody (cat-
Protocols were reported previously.44 Lentiviruses encoding short alog no. 7074; Cell Signaling Technology) for 1 h at room temp-
hairpin RNA for BMAL1, TTK, RNF40, WAC, and MDM2 and nega- erature, the protein levels were detected using chemiluminescent
tive control lentivirus were generated by IGE (Guangzhou, China). reagents (catalog no. WBKLS0500; Millipore) according to the in-
BMAL1 and TTK overexpression lentiviruses and their vector structions provided by the manufacturer. The following primary an-
controls were also purchased from IGE. tibodies were used: anti-BMAL1 antibody (catalog no. 14020; Cell
Signaling Technology), anti-TTK antibody (catalog no. ab11108; Ab-
Detailed information on the reagents is shown in Table S1. cam), anti-CLOCK antibody (catalog no. ab3517; Abcam), anti-OCN
antibody (catalog no. 29560; Sab), anti-GAPDH (catalog no. 5174S;
ARS and ALP staining Cell Signaling Technology), anti-RUNX2 (catalog no. 12556S; Cell
Before staining, MSCs were first fixed with 4% paraformaldehyde Signaling Technology), anti-OSX (catalog no. ab209484; Abcam),
(PFA) for 30 min. anti-b-tubulin (catalog no. 2128; Cell Signaling Technology), anti-
OPN antibody (catalog no. 42036; Sab), anti-RNF20 antibody (cata-
ARS staining log no. ab181104; Abcam), anti-RNF40 antibody (catalog no.
For visualization of calcium deposition, MSCs were incubated with ab191309; Abcam), anti-WAC antibody (catalog no. ab109486;
1% ARS (pH 4.3) (catalog no. G8550; Solarbio) for 15 min at room Abcam), anti-H2B antibody (catalog no. 12364; Cell Signaling Tech-
temperature. The MSCs were rinsed three times with PBS to remove nology), anti-H2Bub1 antibody (catalog no. 5546; Cell Signaling
the nonspecific stain, and images were subsequently captured directly Technology), anti-H2A antibody (catalog no. 12349; Cell Signa-
with a microscope. For ARS quantification, 10% cetylpyridinium ling Technology) anti-H2Aub1 antibody (catalog no. 8240; Cell
chloride monohydrate (Sigma-Aldrich) was used to destain the cells Signaling Technology), anti-MDM2 antibody (catalog no.
for 1 h at room temperature. Then, 200 mL of the extraction mixture ab226939; Abcam), and anti-pan phosphoserine/threonine antibody
was transferred to a 96-well plate, and the spectrophotometric absor- (catalog no. AP1067; Abclonal).
bance was measured at 562 nm.
RNA isolation and qRT-PCR analysis
ALP staining Total RNA was extracted from MSCs using RNAiso Plus (catalog no.
MSCs were stained using a BCIP/NBT alkaline phosphatase kit (cat- 9109; TaKaRa), and the isolated RNA was reverse-transcribed into
alog no. C3206; Beyotime Institute of Biotechnology) following the cDNA using a PrimeScript RT reagent kit (catalog no. RR036A;
manufacturer’s instructions. Stained cells were photographed. For TaKaRa) according to the manufacturer’s recommendations. qRT-
the ALP activity assay, ALP activity kits (catalog no. A059-2; Nanjing PCR was then performed on a LightCycler R480 PCR system (Roche)
Jiancheng Biotech, Nanjing, China) were used according to the man- using SYBR Premix Ex Taq (catalog no. RR420A; TaKaRa). The
ufacturer’s instructions. In summary, MSCs were lysed in ice-cold results were analyzed using the 2-DDCt method, and the reference
radioimmunoprecipitation assay (RIPA) buffer containing protease gene GAPDH was used to normalize the amplification data obtained
and phosphatase inhibitors (Beyotime Institute of Biotechnology). for the target genes. Each qRT-PCR analysis was performed in tripli-
After centrifugation at 14,000 rpm and 4 C for 30 min, the superna- cate. The primer sequences are listed in Table S1.
tants were incubated with reaction buffer at 37 C for 15 min. After
addition of the stop solution, the absorbance was measured at Histology
405 nm. A Pierce bicinchoninic acid (BCA) protein assay kit (Thermo The HA/TCP grafts loaded with MSCs and bone tissues were
Fisher Scientific) was used to detect the total protein concentration. collected, fixed in 4% PFA, and then decalcified for 10 days in
ALP activity was normalized to the total protein content at the end 10% EDTA (pH 7.4). After decalcification, the specimens were de-
of the experiment and reported as units per gram of protein per hydrated and subsequently embedded in paraffin. Sections (5 mm
15 min (U/gpro/15 min). thickness) were stained with HE stain (catalog no. AR1180; Boster)

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 Molecular Therapy: Nucleic Acids

and Masson stain (catalog no. G1340-100; Solarbio) according to library was amplified with phi29 to generate DNA nanoballs (DNBs)
the manufacturers’ instructions. The slides were subjected to with more than 300 copies of a molecule. The DNBs were loaded
immunohistochemistry with anti-collagen I (catalog no. ab34710; into a patterned nanoarray, and single-end 50 base reads were gener-
Abcam). ated with a BGISeq500 platform (BGI-Shenzhen).

Spine section biopsies RNA-seq data analysis

We recruited three male patients with compression fractures of the The sequencing data were filtered with SOAPnuke (version 1.5.2)45
vertebra who had been diagnosed with senile osteoporosis and three by removing reads containing sequencing adaptors, a low-quality
male patients with fractures of the spine who had experienced traffic base rate (base quality less than or equal to 5) greater than 20%, or
accidents as a control group. All of the patients needed treatment for an unknown base (“N” base) rate greater than 5%. Then, clean reads
percutaneous vertebroplasty or subtotal vertebrectomy. The charac- were obtained and stored in FASTQ format. The clean reads were
teristics of the study subjects are presented in Table S2. The other mapped to the hg38 reference genome for coverage calculation and
bone sections biopsied were obtained as described.44 The specimen quality control using HISAT2 (version 2.0.4).46 Bowtie2 (version
was obtained during the surgery. 2.5.1)47 was used to align the clean reads to the reference coding
gene set, and the gene expression levels were calculated with RSEM
Immunofluorescence software (version 1.2.12).48 A heatmap was drawn using pheatmap
For antigen retrieval, bone tissue sections were deparaffinized and (version 1.0.8) (https://CRAN.R-project.org/package=pheatmap) ac-
rehydrated, immersed in 10 mM citrate buffer (pH 6.0), and micro- cording to the gene expression in different samples. Differential
waved for 15 min. Bone sections were permeabilized with 0.5% expression analysis was performed using DESeq2 (version 1.4.5)49
Triton X-100 for 20 min and blocked with 10% fetal bovine serum with a cutoff Q value of %0.05.
in PBS for 1 h. An anti-BMAL1 antibody (catalog no. ab230822;
Abcam), an anti-TTK antibody (catalog no. ab11108; Abcam), an KEGG (https://www.kegg.jp/) and GO (http://www.geneontology.
anti-CLOCK antibody (catalog no. ab3517; Abcam), an anti-OCN org/) enrichment analyses of annotated differentially expressed genes
antibody (catalog no. 29560; Sab), an anti-OCN antibody (catalog were performed using the R package clusterProfiler (version 3.11).
no. MAB1419; Novus), or an anti-H2Bub1 antibody (catalog no. The significance levels of the terms and pathways were corrected ac-
5546; Cell Signaling Technology) was incubated with the samples cording to the Q value on the basis of a rigorous threshold (Q value %
overnight at 4 C. The secondary antibodies used were anti-rabbit 0.05) through the Bonferroni method. GSEA was carried out using
Alexa 488 (catalog no. 2975; Cell Signaling Technology), anti-mouse OmicStudio tools (https://www.omicstudio.cn/tool) according to
Alexa 488 (catalog no. 4408; Cell Signaling Technology), anti-rabbit the instructions.
Alexa 555 (catalog no. 4413; Cell Signaling Technology), and anti-
mouse Alexa 555 (catalog no. 4409; Cell Signaling Technology). CUT&Tag assay, qPCR, and sequencing
Antifade mounting medium with DAPI (catalog no. P0131; Beyo- CUT&Tag assay
time) was used for mounting. The samples were viewed under a The CUT&Tag assay was performed using the NovoNGS CUT&Tag
laser scanning confocal microscope at wavelengths of 488 nm 3.0 High-Sensitivity Kit (catalog no. N259-YH01; NovoProtein).
(green, BMAL1, TTK, CLOCK, H2Bub1), 555 nm (red, OCN), MSCs (1.0  105) were washed twice with 0.5 mL of wash buffer,
and 405 nm (blue, DAPI). Images were captured using a Nikon mixed with ConA beads and incubated with an anti-BMAL1 antibody
Eclipse Ni-E confocal microscope. (catalog no. 14020; Cell Signaling Technology), an anti-H2Bub1 anti-
body (catalog no. 5546; Cell Signaling Technology), or an anti-RNA
RNA-seq library preparation and sequencing polymerase II CTD antibody (catalog no. ab26721; Abcam) overnight
Total RNA was extracted using RNAiso Plus (catalog no. 9109; at 4 C. Goat anti-rabbit IgG antibody (NovoProtein) was diluted at
TaKaRa) according to the manufacturer’s instructions. Oligo(dT)- 1:500 and added to the sample; then, the sample was incubated for
attached magnetic beads were used to purify mRNA. Purified mRNA 1 h at room temperature (RT). The cells were washed and incubated
was fragmented into small pieces with fragment buffer at the appro- with pAG-Tn5 for 1 h at RT. Then, MgCl2 was added to activate tag-
priate temperature. Then, first-strand cDNA was generated using mentation for 1 h at 37 C. Next, DNA was isolated by NovoNGS
random hexamer-primed reverse transcription, and second-strand DNA Extract Beads and dissolved in TE buffer. DNA was amplified
cDNA was synthesized. Next, an A-Tailing Mix and RNA Index Adap- with N5 and N7 primers, which can be enriched by PCR to form
tors were added and incubated for end repair. The cDNA fragments ob- sequencing-ready libraries. After PCR, libraries were purified with
tained in the previous step were amplified by PCR, and the products NovoNGS DNA Clean Beads, and library quality was assessed using
were purified with AMPure XP Beads and then dissolved in EB solu- the Agilent Bioanalyzer 2100 system.
tion. The product was validated with an Agilent Technologies 2100
bioanalyzer for quality control. The double-stranded PCR products CUT&Tag-qPCR
obtained in the previous step were heated, denatured, and circularized Part of the purified DNA from the CUT&Tag assay was diluted 10
with a splint oligo sequence to generate the final library. Single-strand times and used for CUT&Tag-qPCR analysis. Primer sequences are
circle DNA (ssCirDNA) was formatted in the final library. The final listed in Table S1.

582 Molecular Therapy: Nucleic Acids Vol. 31 March 2023

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CUT&Tag sequencing DATA AVAILABILITY

The clustering of the index-coded samples was performed on a All data generated or analyzed during this study are included in this
cBot Cluster Generation System using TruSeq PE Cluster Kit v3- published article (and its supplemental information files). The data
cBot-HS (Illumina) according to the manufacturer’s instructions. from RNA-seq and CUT&Tag-seq have been deposited in NCBI’s
The library preparations were sequenced on the Illumina Gene Expression Omnibus database under GEO accession number
NovaSeq platform at Novogene Science and Technology (Beijing, GSE210426.
China), and 150 bp paired-end reads were generated.
SUPPLEMENTAL INFORMATION
CUT&Tag/ChIP-seq data analysis Supplemental information can be found online at https://doi.org/10.
CUT&Tag/Chip-seq data analysis was performed as previously 1016/j.omtn.2023.02.014.
described with slight modification.50 In summary, raw reads were
filtered and trimmed to remove adaptor sequences and low-quality ACKNOWLEDGMENTS
reads using TrimGalore (version 0.6.6) with the parameters -q 20 This study was supported by the Shenzhen Key Medical Discipline
–phred33 –stringency 3. Clean reads were mapped to the human Construction Fund (ZDSYS20190902092851024), the National Natu-
genome version hg38 using Bowtie2 (version 2.5.1)51 with the pa- ral Science Foundation of China (82172385, 82172349, and
rameters -p 6 -q. Only the uniquely mapped reads were retained. 82002267), and the Shenzhen Science and Technology Program
All peak calling was performed with MACS2 using ‘macs2 -q 0.05 (RCBS20200714114909007 and JCYJ20210324115007019).
–call-summits –nomodel –shift 100 –extsize 200 –keep-dup all’.
For simulations of peaks called per input read, aligned and dedupli- AUTHOR CONTRIBUTIONS
cated BAM files were used without any additional filtering. Normal- S.H. designed the study. L.J.T., X.P., Y.W., Y.Z., Y.G., Y.F., X.X., S.Z.,
ization (in reads per kilobase per million [RPKM]) of the CUT& L.J.J., C.Y., Z.Z., Z.Y., and L.Z. performed experiments and analyzed
Tag-seq data was performed using the bamCoverage command, the data. L.J.T., X.P., and Y.W. wrote the manuscript. F.P., C.Q., and
and heatmaps were plotted using the computeMatrix and plotHeat- L.D. provided technical assistance. W.Y. and X.Z. provided valuable
map commands in deepTools (version 2.3.6.0).52 The normalized comments.
CUT&Tag-seq data were visualized using the Integrative Genomics
Viewer (IGV). DECLARATION OF INTERESTS
The authors declare no conflict of interests.
The raw data obtained from hFOB1.19 H2Bub1 and RNAPII ChIP-
seq data from the GSE82295 dataset were downloaded. We generated
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