TRIM37–PARP1–TET1 axis maintains stemness and prevents osteoporosis by inhibiting DNMT1 alternative splicing via 5hmC regulation

Domain: biology
Published: 2025-12-16
Editorial outcome: minor_revision

Abstract
The significance of DNA hydroxymethylation in replicative senescence of mesenchymal stem cells (MSCs) and aging-related osteoporosis remains unknown. Here, we reveal 5hmC levels positively regulate MSC self-renewal and osteoblast differentiation. Mechanistically, PARP1 recruits TET1 to hydrolyze methylated nucleotides on DNMT1 exons, aiding CTCF in preventing DNMT1 alternative splicing in early MSCs. Additionally, ATM phosphorylates TRIM37 at Th203, promoting its nuclear entry and the monoubiquitination of PARP1, stabilizing the protein. CTCF or TRIM37 knockdown induces replicative senescence of MSCs with loss of full-length DNMT1. Co-treatment with resveratrol (ATM activator) and vitamin C (TET1 activator) rejuvenates late MSCs via the TRIM37/PARP1/DNMT1 pathway and alleviates osteoporosis in aged mice. Gene knockout experiments further reveal the participation of TRIM37 and PARP1 in MSC aging, contributing significantly to bone maintenance and repair in vivo. This study emphasizes the role of DNA hydroxymethylation in stemness, suggesting therapeutic strategies, especially for osteoporosis.

Introduction
Age-related osteoporosis presents a significant health challenge, impacting bone density and strength. Understanding the dynamic functions of mesenchymal stem cells or multipotential stromal cells (MSCs) within the bone cavity is crucial for maintaining bone mineral content1. The intricate interplay between aging-related osteoporosis and MSCs in bone homeostasis underscore the need for a comprehensive understanding.

MSCs, capable of self-renewal and differentiation into mesenchymal and non-mesenchymal cells2, exhibit genetic stability through in vitro culture expansion, making cell-based therapies increasingly promising. However, for clinical applications, generating a substantial number of MSCs with the desired phenotype and function relies on the expansion of large cultures. Nevertheless, culture-related modifications, such as in vitro aging, may lead to adverse reactions and unsatisfactory results3. Like other normal human somatic cells, MSCs divide approximately forty times until they are close to the “senescence”, after which they can no longer replicate, and are characterized by enlargement, changes in morphology, and senescence-associated β-galactosidase expression4. The concept of senescence5 was found to correlate with the length of the telomeric region at the end of chromosome6,7. In addition to telomere erosion, the senescence or senescence is caused by DNA damage response (DDR)-dependent and independent activation of cyclin-dependent kinase (Cdk) inhibitors, such as p21Waf1/Cip1 and p16Ink4a8, and other genetic and epigenetic modifications9–11. Like many other cells, MSC aging is caused by various factors10. However, the underlying mechanism for the sudden loss of replication after the cell reaches the senescence remains elusive.

Long-term culture causes MSCs to impair ataxia telangiectasia mutated (ATM) function and increase the accumulation of DNA double-strand breaks (DSBs)12, which are the most deleterious forms of DNA damage that cause genomic instability13. Moreover, accumulation of DNA damage during expansion may contribute to the loss of differentiation potential of expanded MSCs14. Epithelial-mesenchymal transition (EMT) regulator, zinc-finger E-box-binding homeobox 1 (ZEB1), which can induce stemness in cancer, promotes DDR and tumor radioresistance15. MSC aging is also associated with reduced radioresistance via the downregulation of DDR16. Deficiencies in DDR limit the function of aging hematopoietic stem cells (HSCs). However, it is unclear whether DDR itself or a specific DDR regulator is responsible for the properties associated with early stem cells, such as self-renewal and multi-differentiation.

Recent findings suggest that epigenetic regulators responsible for modifying histones, such as sirtuin (SIRT)317, SIRT618, and SIRT719, as well as those involved in DNA methylation, including methylcytosine oxidases ten-eleven translocation (TET)120 and TET221, play a vital role in preserving tissue-specific stem cells like HSCs22. TET2 is also required for pre-B cell differentiation to macrophage23. Intriguingly, aging-associated functional decline24 or cell lineage-specific functional change and oncogenic potential25 in MSCs are also regulated by epigenetic regulators, including DNA methyltransferase (DNMT)124 and DNMT3B25, which can be reversed by manipulating the epigenetic factors1,26 or culture conditions, such as reduced oxygen tension, to enhance MSC stemness and prevent senescence4,24. Recently, the decline in DNMT1 expression has been linked to the senescence-associated 3D genome reorganization27. The expression of DNMT1 plays a crucial role in MSC aging, being regulated by pluripotency factors and upregulated under hypoxic conditions24.

DNA 5-hydroxymethylcytosine (5hmC) modification, an oxidation product of the TET enzymes28, can potentially lead to DNA demethylation29,30, which is known to be associated with gene transcription. The dynamic changes in 5hmC levels have been well studied during embryonic31 or postnatal tissue-specific development process32 and aging33,34. Moreover, its roles in pluripotency35 and somatic reprogramming36 have been studied. Moreover, 5hmC is essential for the homeostasis of adult stem cells, such as the neural stem cells37 and HSCs38, and its dysregulation, caused by aging37 or mutation in TET enzymes, also predisposes a person to various diseases38. Through the development of a 5hmC tissue map by characterizing the genomic distribution of 5hmC in human tissues, 5hmC was found to be preferentially enriched on tissue-specific gene bodies and enhancers32. In mouse and human stem cells, 5hmC promotes co-transcriptional R-loop formation and regulates specific gene expression required for stem cell proliferation39. However, its role in maintaining the self-renewal and multi-differentiation potential of MSCs as well as the underlying molecular mechanisms remain to be studied.

In the current study, we first found that increased 5hmC levels are required to maintain the early passage characteristics of human bone marrow (BM)-derived MSCs. We further revealed that CTCF-mediated prevention of DNMT1 alternative splicing plays a key role in sustaining the proliferative capacity and differentiation potential of early passage BM-MSCs, which depends on the coordinated actions of TET1 and DNA damage response (DDR) components such as ATM, PARP1, and TRIM37. Mechanistically, PARP1 recruits TET1 to demethylate DNMT1 exons, facilitating CTCF-mediated splicing regulation, while ATM-mediated phosphorylation of TRIM37 promotes PARP1 stabilization. Knockdown of CTCF or TRIM37 induces replicative senescence. Importantly, combined treatment with an ATM activator (resveratrol) and a TET activator (vitamin C) rejuvenates late-passage MSCs, restores osteogenic capacity, and enhances bone regeneration in aged mice. These findings identify a TRIM37/PARP1/DNMT1 regulatory axis critical for MSC stemness and bone homeostasis, suggesting a therapeutic strategy for osteoporosis.

Results
Early human MSCs show higher levels of 5hmC, thereby exhibiting increased proliferation and differentiation potential

TET1/2 double knockout (DKO) mice had significantly reduced bone mineral density and bone volume/tissue volume ratio compared to the littermate controls40. Moreover, BM-MSCs isolated from TET1/2 DKO mice exhibited impaired bone formation compared to TET1/2 single KO or control mice, but did not exhibit any impaired proliferation and adipogenic differentiation capacities40. Although the 5hmC levels in BM-MSCs of TET1/2 DKO mice were lower than those of the TET1/2 KO or control group40, whether the 5hmC level plays an important role in replicative senescence and biological functions of expanded human BM-MSCs (henceforth referred to as MSCs, if not indicated) was not determined. We hypothesized that 5hmC levels may affect the senescence, proliferation, and differentiation capacities of MSCs. For this purpose, we first compared the 5hmC and 5mC levels between MSCs at early (P2-4) and late (P10-12) passages. Compared with early (passage) MSCs, late (passage) MSCs exhibit multiple hallmarks of cellular senescence, including enlarged and flattened morphology, increased expression of senescence markers (p16Ink4a and p21Waf1/Cip1), and reduced DNMT1 levels. Functionally, they show decreased proliferation and impaired differentiation into osteoblasts and adipocytes. Transcriptomic analysis reveals enrichment of the senescence-associated secretory phenotype (SASP) pathway, accompanied by elevated secretion of IL-1α and CXCL-8. Increased SA-β-gal+ staining further confirms the senescent phenotype (Supplementary Fig. 1)4. We observed an increase in the level of 5hmC in early MSCs using dot-blot (Supplementary Fig. 2a) and immunofluorescence (Fig. 1a) assays. In contrast, the level of 5mC was elevated in late MSCs (Supplementary Fig. 2a and Fig. 1a), consistent with findings from a previous study41,42.

We next demonstrated that treatment of early MSCs with 2-hydroxyglutarate (2-HG), a TET inhibitor (5 mM), reduced the level of 5hmC without affecting 5mC (Fig. 1b and Supplementary Fig. 2b). Additionally, this treatment suppressed their proliferative capacity (Fig. 1c) and differentiation potential into osteoblasts and adipocytes (Fig. 1d). These data suggest that the 5hmC level plays an important role in the maintenance of replication and biological functions of early MSCs.

TET1 knockdown reduces 5hmC levels, and suppresses the proliferation and differentiation potential of early human MSCs

We further used lentivirus-mediated knockdown (KD) to directly target and investigate the roles of TET1 and TET2 in maintaining early MSC properties (Fig. 1g), which are known to maintain the self-renewal and osteogenic differentiation potential of mouse BM-MSCs40. Our data further provides strong evidence that TET1 is the primary regulator of 5hmC levels in MSCs. KD of TET1, but not TET2, leads to a significant reduction in 5hmC levels, as demonstrated by both immunofluorescence (Fig. 1f) and dot blot analysis (Supplementary Fig. 2c). While dot blot results show that KD of either TET1 or TET2 can reduce 5hmC, the magnitude of reduction is substantially greater following TET1 KD (Supplementary Fig. 2c). Notably, immunofluorescence analysis reveals a more prominent and consistent decrease in nuclear 5hmC levels specifically upon TET1 depletion. These findings indicate that, although TET2 may contribute to 5hmC regulation, TET1 plays a dominant and non-redundant role in maintaining 5hmC levels in MSCs. Moreover, early MSCs with TET1 KD exhibited reduced cell growth (Fig. 1g) and decreased differentiation potential into osteoblasts and adipocytes (Fig. 1h) compared to cells transduced with scrambled (CTR) shRNAs. These findings suggest that TET1 is the primary enzyme responsible for increasing 5hmC levels in early MSCs, and that 5hmC is crucial for MSC growth and differentiation potential.

PARP1/TET1 complex regulates the proliferation and differentiation potential of early human MSCs

PARP1 interacts with target proteins involved in histone modification and DNA methylation43. For instance, it has been reported that PARP1 facilitates the establishment of H3K4me3 marks at the TET1 promoter and directly interacts with TET1, contributing to the progression of T-cell acute lymphoblastic leukemia44. To investigate the role of PARP1 in TET1-mediated elevation of 5hmC levels and the maintenance of early MSC properties, we first demonstrated an endogenous interaction between PARP1 and TET1 in early MSCs using co-immunoprecipitation (Fig. 2a). To confirm that this interaction is direct and independent of PARP1 enzymatic activity or poly(ADP-ribose) (PAR) levels, we treated cells with the PARP inhibitor PJ3445. Notably, the interaction between PARP1 and TET1 remained detectable by both immunoprecipitation and Duolink proximity ligation assay (PLA), even under PARP inhibition (Supplementary Fig. 3). Then, we showed that early MSCs from different individuals showed higher levels of PARP1 compared to late MSCs (Fig. 2b), We further analyzed the RNA-seq of gene expression profiles in early MSCs, late MSCs, and early MSCs with PARP1 KD or TET1 KD. Interestingly, hierarchical clustering analysis revealed that, with the exception of MSCs from donor 3, early MSCs from different individuals clustered together and were distinct from late MSCs. Notably, late MSCs clustered with early MSCs that had PARP1 or TET1 knocked down (Fig. 2c). Pathway analysis of 1026 differentially expressed genes in early MSCs compared to late MSCs revealed several altered pathways that are responsible for the early MSC phenotypes, including increased activity of “differentiation of osteoblast” and “adipogenesis of cells” (Supplementary Fig. 4, Supplementary Data 1). PARP1 KD downregulated 5hmC in early MSCs, while PARP1 overexpression upregulated 5hmC in late MSCs (Fig. 2d). In addition, PARP1 KD increased p16Ink4a and p21Waf1/Cip1 levels (Fig. 2e) and suppressed the proliferation and differentiation potentials in early MSCs (Fig. 2f, g), while PARP1 overexpression decreased p16Ink4a and p21Waf1/Cip1 levels and promoted the proliferation and differentiation potentials in late MSCs (Fig. 2e, h, i).

To further confirm the functional relationship between PARP1 and TET1 in maintaining MSC identity, we showed that TET1 KD effectively reduced TET1 protein levels despite robust PARP1 overexpression and reversed the associated decrease in senescence marker expression (Supplementary Fig. 5a). Consistently, PARP1-induced elevation of global 5hmC levels was abolished upon TET1 KD (Supplementary Fig. 5b). Functionally, TET1 KD impaired the proliferative capacity of PARP1-overexpressing (OE) MSCs (Supplementary Fig. 5c) and significantly reduced their differentiation potential into both osteoblasts and adipocytes (Supplementary Fig. 5d). Together, these data indicate that PARP1 interacts with TET1 to increase 5hmC levels and maintain early MSC properties.

MeDIP and ChIP-seq reveal multiple downstream targets of PARP1/TET1, including the alternative splicing of DNMT1

To identify the downstream targets with increased 5hmC levels of the PARP1/TET1 complex in early MSCs, we performed methyl DNA immunoprecipitation (MeDIP)-seq and hydroxymethyl DNA immunoprecipitation (hMeDIP)-seq analyses in early and late MSCs, and PARP1, and TET1 chromatin immunoprecipitation sequencing (ChIP-seq) analyses in early MSCs. Unfortunately, the hMeDIP-seq data were inconclusive, as the majority of 5hmC-enriched reads across the genome did not show significant enrichment over input controls. One possibility with fewer reads in hMeDIP-seq is that 5hmC in the DNA has been replaced by replication-dependent and independent DNA demethylation cycle46. Enriched DNA segments overlapped in MeDIP-seq (late > early), PARP1 ChIP-seq (ChIP > input), and TET1 ChIP-seq (ChIP > input) were analyzed and the overlapping gene numbers are shown in a Venn diagram (Fig. 3a). The triple overlapping enrichment included 20 genes targeted by the PARP1/TET1 complex (Supplementary Table 1), which showed reduced DNA methylation in early MSCs. The DNMT1 gene, a triple-overlapping gene, is essential for maintaining the self-renewal and undifferentiated state of early human MSCs24. Overexpression of DNMT1 in MSCs cultured under normoxic conditions led to reduced expression of p16Ink4 and p21Waf1/Cip1, increased proliferation rates, higher BrdU incorporation, and enhanced differentiation potential24. PARP1 and TET1 ChIP-seq analysis (Supplementary Fig. 6) and additional ChIP-PCR revealed that the region covering flanking to transcription start site (TSS, Chr19:10305629) of the DNMT1 gene was bound by PARP1 and TET1 in early MSCs (Fig. 3b). These data are similar to a previous study that showed the binding of PARP1 at or near the TSSs of genes positively regulated by PARP147. KD of PARP1 or TET1 reduced DNMT1 full-length protein levels (Fig. 3c). Interestingly, compared with early MSCs, the enriched MeDIP peaks of DNMT1 in the late MSCs were located exons 21, 30, 35, 36, 37, and 39, rather than the TSS site, which is the catalytic domain of DNMT1 (Fig. 3d). Because there is a direct mechanistic association between intragenic DNA methylation and alternative pre-mRNA splicing48, we checked the existence of the DNMT1 isoform with alternative splicing in this region in late MSCs (Fig. 3e) or in early MSCs with PARP1 or TET1 KD (Supplementary Fig. 7a) using 5’-RACE-PCR to amplify generated cDNA first strands. An additional band, significantly smaller than the full-length product, was observed when using primers flanking exons 25 and 38, but not with primers flanking exons 13 and 25 (Fig. 3e, Supplementary Fig. 7a). DNA sequencing of the shorter PCR product showed that it was an alternative splicing of DNMT1, resulting in exon exclusion from exon 30 to exon 37 (Supplementary Fig. 8). Unfortunately, the available antibodies against DNMT1 cannot recognize this isoform. Western blotting of cell lysates from early and late MSCs with or without pre-treatment with MG132 also failed to detect this isoform (Supplementary Fig. 9), suggesting that the mRNA or protein isoform was not stable. We identified differential alternative splicing events between different subgroups of MSCs by RNA-Seq and replicated multivariate analysis of transcript splicing (rMATS)49,50. Notably, the number of genes increased in alternative splicing events in late MSCs was slightly higher than that in early MSCs (Supplementary Table 2, Supplementary Data 2), and the number of genes increased in alternative splicing events in early MSCs with PARP1 or TET1 KD was slightly lower than that in early MSCs (Supplementary Table 2, Supplementary Data 3, 4). Among the 20 genes (Supplementary Table 1) targeted by the PARP1/TET1 complex, GUSBP1 and PDE4DIP increased in alternative splicing events in late MSCs when compared to early MSCs; GUSBP1 and LOC100506990 increased in alternative splicing events in early MSCs with TET1 KD when compared to early MSCs; and LOC100506990 increased in alternative splicing events in early MSCs with PARP1 KD when compared to early MSCs. These data indicate that the PARP1/TET1 complex-mediated loss of intragenic DNA methylation in DNMT1 prevents alternative splicing of DNMT1 in early MSCs.

Involvement of CTCF in PARP1/TET1 complex-mediated loss of intragenic DNA methylation and prevention of DNMT1 alternative splicing

Since PARP1 interacts with CTCF51, a key insulator in intragenic chromatin looping and exon inclusion via RNA polymerase II pausing48,52, we hypothesized that CTCF facilitates the inclusion of unmethylated exons during transcription elongation in early MSCs. Similar to previous studies51, the immunoprecipitation and PLA assay revealed the interaction of the PARP1/TET1 complex with CTCF (Supplementary Fig. 3). Prediction of CTCF binding sites in 20 triple overlapping enrichment genes by CTCFBSDB 2.0 software53 revealed that most of the CTCF binding sites were restricted to intragenic regions (Supplementary Table 3). Moreover, CTCF-binding sites were found in exons 21, 27, 30, 37, 39, and 41 (Supplementary Table 4) of the DNMT gene, which were largely overlapped with the exons hypermethylated in late MSCs. Subsequent quantitative real-time PCR of CTCF-ChIP showed that, compared with that of early MSCs, the binding of CTCF in exons 30 and 37 of DNMT1 in late MSCs and TET1 or PARP1 KD in early MSCs was reduced, while no difference was observed between early and late MSCs in the H19 gene, the positive control region (Fig. 3f, Supplementary Fig. 7b). Furthermore, KD of CTCF in early MSCs reduced the protein level of full-length DNMT1 (Fig. 3g) and the mRNA level of DNMT1 in exon 34, but not in exon 13 (Fig. 3h), suggesting that CTCF forms intragenic chromatin loops including exon 34 and its KD induces alternative splicing. These data indicate the involvement of CTCF in PARP1/TET1 complex-mediated loss of intragenic DNA methylation and prevention of DNMT1 alternative splicing in early MSCs.

DNMT1 alternative spliced form promotes human MSC senescence and reduces stemness

To examine the biological function of the DNMT1 alternative spliced form with a truncated catalytic domain, we constructed a plasmid encoding full-length (Full-DNMT1) or alternative splicing form of DNMT1 (AS-DNMT1), which was then overexpressed in 293T cells. We then isolated and purified exogenous DNMT1 from 293T cells. The in vitro DNMT1 activity of Full-DNMT1 was 3.5 times that of AS-DNMT1 (Fig. 3i). In addition, AS-DNMT1 reduced the activity of Full-DNMT1 in a dose-dependent manner, both in vitro (Fig. 3i) and in vivo, as evidenced by the loss of 5mC levels (Fig. 3j). This suggests that AS-DNMT1 not only reduces the activity of DNMT1, but also competes to inhibit the activity of full-length DNMT1. Early MSCs were lentivirally transduced with a control vector (CV) or a vector containing AS-DNMT1, followed by western blotting analysis for the expression of senescence markers and functional analysis of proliferation and multi-differentiation potential. Compared with CV, overexpression of AS-DNMT1 increased the expression of a short-form DNMT1 (~135 kDa) and the levels of senescence markers, p16Ink4a and p21Waf1/Cip1 (Fig. 3k), but inhibited cell growth (Fig. 3l) and differentiation potential into osteoblasts and adipocytes (Fig. 3m). These results indicate that DNMT1 with alternative splicing in the catalytic domain not only loses DNMT1 activity, but also inhibits the activity of full-length DNMT1, thereby promoting MSC senescence and reducing stemness.

T203 phosphorylation is required for TRIM37 to stabilize PARP1 via K337 mono-ubiquitination

To identify the upstream proteins of PARP1, we compared phosphorylated proteins that interacted with PARP1 in early and late MSCs using immunoprecipitation (IP)-tandem mass spectrometry (MS/MS) analysis (Supplementary Table 5). We first demonstrated that phosphor-TRIM37 T203 was one of the enriched phosphopeptides associated with endogenous PARP1 in early MSCs (Fig. 4a) and confirmed the protein-protein interaction between exogenous PARP1 and TRIM37 in 293T cells co-transfected with plasmids encoding Flag-TRIM37 and HA-PARP1 (Fig. 4b). Because TRIM37 is an E3 ubiquitin-protein ligase that induces mono-ubiquitination of nuclear proteins, such as H2A54 and NEMO46, and results in H2A stabilization and nuclear export of NEMO in cancer cells, we then examined whether TRIM37 ubiquitinates PARP1. IP-MS/MS analysis revealed that Lys337 (K337) and Lys438 (K438) were the possible TRIM37-mono-ubiquitinated residues in PARP1 (Supplementary Table 6). Overexpression of Flag-TRIM37, His-ubiqutin, HA-PARP1/wild-type (WT), or mutants in 293T cells followed by immunoprecipitation with antibodies against HA revealed that TRIM37 interacted with and ubiquitinated PARP1 at K337, rather than with K438 (Fig. 4c). Moreover, K337 mono-ubiquitination caused by TRIM37 stabilized PARP1 (Fig. 4c). Furthermore, the function of T203 phosphorylation of TRIM37 was examined using 293T cells co-transfected with Flag-TRIM37/WT or T203A (non-phosphorylatable) inactive mutant or T203E (phosphomimetic) constitutively active mutant, His-ubiqutin, and HA-PARP1. When compared with cells transfected with WT Flag-TRIM37, TRIM37 binding, ubiquitination, and stabilization of PARP1 were significantly decreased in cells transfected with the T203A mutant (Fig. 4d) and a significant increase in cells transfected with the T203E mutant (Fig. 4e) in immunoprecipitation assay. Furthermore, lentiviral-mediated KD of TRIM37 was performed to confirm that PARP1 acts downstream of TRIM37. TRIM37 KD using two independent shRNAs targeting TRIM37 resulted in decreased protein levels of PARP1 and full-length DNMT1, along with increased expression of senescence markers (Fig. 4f). This was accompanied by reduced cell proliferation (Fig. 4g) and impaired differentiation into both osteoblasts and adipocytes (Fig. 4h) compared to cells transduced with control shRNAs. To determine the functional importance of TRIM37 and its phosphorylation at T203, we performed rescue experiments in TRIM37-KD MSCs using shRNA-resistant constructs encoding wild-type (WT), T203A, or T203E TRIM37. Re-expression of WT or T203E TRIM37 restored PARP1 protein levels, reduced senescence marker expression (Supplementary Fig. 10a), and rescued both proliferation (Supplementary Fig. 10b) and differentiation capacities into osteoblasts and adipocytes (Supplementary Fig. 10c). In contrast, re-expression of the T203A mutant failed to restore these functions (Supplementary Fig. 10). These results indicate that phosphorylation at T203 is critical for TRIM37-mediated regulation of PARP1 expression and MSC function.

ATM upregulates TRIM37 protein levels and nuclear interaction with PARP1 via T203 phosphorylation

According to a previous study showing that ATM promotes nuclear translocation of TRIM37 by phosphorylation55, we examined the role of ATM in the nuclear localization of the TRIM37-PARP1 complex. Early MSCs had higher levels of Ser1981 phosphor-ATM (p-ATM), total ATM, and TRIM37 than late MSCs (Fig. 5a). Treatment of early MSCs with ATM inhibitor KU-55933 reduced TRIM37, PARP1 and full length DNMT1 levels (Fig. 5b), while treatment of middle-passage (p7-9) MSCs with ATM activator resveratrol increased TRIM37, PARP1 and full length DNMT1 levels (Fig. 5c). In esophageal cancer cells, activated ATM kinase directly interacts with and phosphorylates TRIM37 in the cytoplasm, which induces translocation of TRIM37 into the nucleus55. Therefore, we checked the location of p-ATM, TRIM37, and PARP1 using immunofluorescence (Fig. 5d, e) and analyzed their association using the PLA assay (Fig. 5f, g). Compared with late MSCs, early MSCs increased the nuclear localization of p-ATM, TRIM37, and PARP1 (Fig. 5d, e). PLA revealed an increased association of TRIM37 with p-ATM and PARP1 (mainly in the nucleus) in early MSCs compared with late MSCs (Fig. 5f, Upper panel). Quantification data further revealed that these proteins have increased nuclear binding in early MSCs compared to late MSCs (Fig. 5f, Lower panel). Moreover, treatment with the ATM activator resveratrol enhanced the nuclear binding of TRIM37 with p-ATM and PARP1 in middle-passage MSCs (Fig. 5g). Interestingly, overexpression of a dominant-negative T203A TRIM37 mutant in 293T cells inhibited the nuclear translocation of exogenous TRIM37, while overexpression of a constitutively active T203E TRIM37 mutant promoted nuclear translocation of exogenous TRIM37 (Fig. 5h). These results indicate that ATM activation promotes the formation of the TRIM37/PARP1 complex and its nuclear translocation via phosphorylation of TRIM37 at T203.

ATM activator combined with TET activator maintains DNMT1 in full length and promotes MSC stemness

According to the above findings, the PARP1/TET1 complex maintains the 5hmC level of DNMT1, while the ATM/TRIM37 complex regulates the stability and nuclear localization of PARP1, thereby further promoting the proliferation and differentiation of early MSCs. We hypothesized that the combination of an ATM activator, resveratrol, and a TET activator, vitamin C, may improve the stemness of in vitro expanded MSCs. Compared with vehicle control and any single treatment, the combined treatment of MSCs increased growth rates in the logarithmic phase and the cumulative population-doubling level not only in medium supplemented with 10% fetal bovine serum (FBS) but also in serum-free medium containing 2% human platelet lysate (hPL) (Fig. 6a). Furthermore, compared with vehicle control and any single treatment, the combination treatment with resveratrol and vitamin C in medium containing 10% FBS and 2% hPL enhanced the differentiation potential of MSCs into osteoblasts (Fig. 6b) and adipocytes (Fig. 6c). As expected, combinational treatment with resveratrol and vitamin C also increased the DNMT1 full-length protein level and reduced senescence marker levels when MSCs were expanded to late passages (Fig. 6d). Together, these data suggest that the ATM/TRIM37 complex and PARP1/TET1 complex upregulate DNMT1 full-length protein levels, which is essential for preventing replicative senescence and maintaining early MSC properties.

The involvement of TRIM37 and PARP1 in MSC aging and function in vivo

To demonstrate the involvement of TRIM37 and PARP1 in MSC aging in vivo, BM-MSCs were isolated from B6 mice and minimally expanded and cell lysates were subjected to western blotting. BM-MSCs from mice less than 8 weeks increased in the levels of TRIM37, PARP1, PAR, total DNMT1, and decreased in the levels of senescence markers, p16Ink4a and p21Waf1/Cip1, than those from mice around 6 to 10 months (Supplementary Fig. 11a, b). To further demonstrate the involvement of TRIM37 and PARP1 in MSC function in vivo, BM-MSCs isolated from 8 weeks of both WT and TRIM37- and PARP1-deficient mice were subjected to comparisons of senescence marker expression, growth capacity and differentiation potentials. Compared with BM-MSCs from WT mice, BM-MSCs from TRIM37- or PARP1-deficient mice decreased in the levels of TRIM37, PARP1, and total DNMT1, increased in the expression of senescence markers, such as p16Ink4a, p21Waf1/Cip1 (Supplementary Fig. 11c, d) and senescence β-galactosidase staining (Supplementary Fig. 11e, f), and decreased in proliferation capacity (Supplementary Fig. 11g, h) and differentiation potentials into osteoblasts and adipocytes (Supplementary Fig. 11i, j). These data suggest the involvement of TRIM37 and PARP1 in MSC aging and function in vivo.

TRIM37 and PARP1 affect MSC ability to maintain bone homeostasis and contribute to in vivo bone repair

To link MSC function with bone formation and bone mineral content maintenance, we assessed distal femur bone parameters in mice at 12 weeks and over 53 weeks of age. Old mice demonstrated a decrease in BV/TV, BMD, Trabecular No., and an increase in Trabecular separation (Tb. Sp) compared to their counterparts at a younger age (Fig. 7b). Notably, young Trim37-deficient mice exhibited results similar to the changes associated with aging when compared to young WT mice (Fig. 7c). To further elucidate the roles of TRIM37 and PARP1 in new bone ingrowth in adults following injury, a single cortical drill hole was created in the medial side of the right femoral diaphysis in WT, TRIM37-deficient, and PARP1-deficient mice at 8 weeks of age. This procedure was followed by an intraperitoneal injection of calcein three weeks later and single-photon (Fig. 7a, d, e) or two-photon fluorescence imaging four weeks post-injury (Supplementary Fig. 12). In the control sham group, no fluorescence was observed in the right femurs of WT and genetically modified mice (Supplementary Fig. 12). However, continuous fluorescence throughout the entire drill hole was evident in WT young mice that underwent drill-hole surgery (Fig. 7d, e, Supplementary Fig. 12). In contrast, in aged mice and those lacking TRIM37 or PARP1, diminished and non-continuous fluorescence was observed near the drill-hole area (Fig. 7d, e, Supplementary Fig. 12). These findings collectively indicate the potential role of TRIM37 and PARP1 in the aging process of MSCs, influencing their ability to maintain bone homeostasis and contribute to in vivo bone repair.

The combination of an ATM activator and a TET activator promotes in vivo bone repair and exhibits anti-osteoporosis effects

Since ATM activator combined with TET activator maintains DNMT1 in full length and promotes MSC stemness, we therefore hypothesized that ATM activator combined with TET activator might rescue MSC osteogenic potential in vivo. We assessed left distal femur bone and fat parameters in 83-week-old female mice that had received daily treatment with resveratrol, vitamin C, or a combination of both starting at 75 weeks of age (Fig. 8a). Compared to the control and each of the single-treatment groups, the combination treatment group exhibited increased bone mineral density (BMD), bone volume fraction (BV/TV), trabecular number, and trabecular thickness, along with reduced bone surface-to-volume ratio (BS/BV) and trabecular separation (Tb. Sp) (Fig. 8b). However, fat content remained unchanged across all treatment groups (Supplementary Fig. 13). Following the creation of a single cortical drill hole on the medial side of the right femoral diaphysis at 79 weeks of age, calcein was administered intraperitoneally three weeks later, and single-photon fluorescence imaging was performed four weeks post-injury (Fig. 8c). Notably, the combination treatment group exhibited increased calcein fluorescence at the defect site in the right femoral shaft compared to the control and each single-treatment group, indicating enhanced bone repair capacity (Fig. 8c). Together, these findings suggest that the ATM/TRIM37 and PARP1/TET1 complexes cooperatively upregulate full-length DNMT1, which is critical for preventing replicative senescence, preserving early MSC characteristics in vitro, and promoting bone homeostasis, in vivo repair, and anti-osteoporotic effects.

Methods
Cell culture and reagent treatment
BM aspirates were collected from patients who received an orthopedic surgery in Taipei Veterans General Hospital with the approval of Institute of Review Board (#2012-05-007A). Informed consent was obtained from all subjects. Details regarding the donor information are shown in Supplementary Table 7. The protocols of MSC isolation and expansion were modified from previously described methods25. In brief, mononuclear cells were isolated from heparinized BM by density gradient centrifugation using Ficoll-Hypaque (Sigma-Aldrich, St. Louis, MO) of a density of 1.077 g/L at 400 x g, followed by seeding with complete medium [CM: Dulbecco’s Modified Eagle Medium (DMEM; Gibco-BRL, Gaithersburg, MD), supplemented with 10% fetal bovine serum (FBS, Gibco-BRL), 100 units/ml penicillin, 100 μg/ml streptomycin, and 2 mM L-glutamine (Invitrogen, Carlsbad, CA)] at 37°C under 5% CO atmosphere. Upon reaching semi-confluence, MSCs were retrieved and reseeded at an initial density of 4 × 103 cells/cm2 and the culture medium was changed twice per week. Mycoplasma contamination was checked every 1 month. Cells with mycoplasma contamination were discarded. For treatment with 2-hydroxyglutarate (2-HG, HY-100542, MedChem Express, Monmouth Junction, NJ), cells were cultured with 5 mM 2-HG in CM. For treatment with KU55933 (SML1109, Sigma-Aldrich, St. Louis, MO), cells were cultured with 10 μM KU55933 in CM. For treatment with resveratrol (R5010, Sigma-Aldrich), cells were treated with 5 μM resveratrol in CM. For treatment with vitamin C (A4544, Sigma-Aldrich), cells were cultured with 50 μg/ml vitamin C in CM. Cells were harvested and stained with Trypan Blue (Sigma-Aldrich) for counting cell numbers at each passage. Cumulative population doubling level (PDL) was calculated using (logN–logN0)/log 2, where N = cell number harvested during passaging and N0 = cell number at initial seeding.

Senescence-associated β-Galactosidase (SA-β-gal) staining
SA-β-gal staining was performed using a β-galactosidase staining kit (BioVision, Milpitas, CA) according to the manufacturer’s instructions. Briefly, cells were washed with PBS and fixed with 3.7% formaldehyde for 5 min at room temperature. After washing, the cells were incubated with β-gal staining solution (1 mg/mL, 5-bromo-4-chloro-3-indolyl-b-galactoside) overnight at 37°C. SA-β-gal-positive cells were observed and acquired at room temperature using an inverted microscope (Nikon Eclipse TE2000, Tokyo, Japan).

Proliferation assay
For proliferation assays, cells were plated in triplicate in 96-well tissue culture plates with growth media. At each time point, cells were seeded at a density of 1000 cells/well in 96-well plate, followed by recover and assay with a water-soluble tetrazolium salt, WST-1, 4-[3-(4 iodophenyl)−2-(4-nitrophenyl)−2H-5-tetrazolio] −1,3-benzene disulfonate), kit (Cayman Chemical Company, Ann Arbor, MI) at indicated time periods. Absorbance was measured at a wavelength of 450 nm.

Cell differentiation protocols
Cell differentiation was performed as previously described25. Before the initiation of differentiation, cells were seeded in CM at a density of 104 cells/cm2. For differentiation into osteoblasts and adipocytes, cells were induced in osteogenic induction medium [OIM: CM supplemented with 10−8 M dexamethasone, 50 µg/ml ascorbic acid-2 phosphate, 10 mM β-glycerophosphate (Sigma-Aldrich)] and adipogenic induction medium [AIM: CM supplemented with 50 mg/ml ascorbate-2 phosphate (Sigma), 10−7 M dexamethasone (Sigma-Aldrich), 50 mg/ml indomethacin (Sigma-Aldrich), and 10 mg/ml insulin (Sigma-Aldrich)], respectively. After induction in defined induction medium for 14 days, cells in OIM and AIM were stained with Alizarin red staining (ARS) and Oil Red O, respectively, followed by extraction and measurement of O.D. values of ARS at 550 nm and Oil Red O at 510 nm.

Quantitative analysis of 5hmC levels using dot blot
The procedure of dot-blot was modified from previously described method70. Briefly, genomic DNA samples were prepared in 2 N NaOH and 10 mM Tris-HCl, pH 8.5. The samples were spotted onto Hybond-N+ nylon membrane (GE Healthcare, Chicago, Illinois) using a 96-well dot-blot apparatus (Bio-Rad, Hercules, CA), baked in 80°C for 30 min, blocked with 5% skim milk for 1 h at room temperature, and incubated with anti-5-hydroxymethylcytosine antibody (39791, Active Motif, Carlsbad, CA) or anti-5-methylcytosine antibody (BI-MECY-0500, Eurogentec, Seraing, Belgium) at 4°C overnight, followed by incubation with species-specific HRP-conjugated secondary antibody, and dot signal was visualized with the ECL Plus chemiluminescence assay kit (GE Healthcare, Chicago, Illinois).

Genomic DNA preparation
Early-passage (P2-P3) and late-passage (P10-P11) MSCs were treated with RNase cocktail (Roche Diagnosis, Basel, Switzerland) for 5 min and subsequently incubated in 0.5% SDS and Proteinase K (100 ng/ul) for 2 h at 55°C. Genomic DNA was extracted with phenol:chloroform:isoamyl alcohol (25:24:1) and then chloroform using phase lock gel, followed by ethanol/NaCl precipitation overnight at −20˚C and two washes with 70% ethanol. The DNA pellets were resuspended in 30 μl nuclease-free MilliQ water, and the supernatant was transferred to low-binding tubes. Microplate Spectrophotometer (Multiskan™ GO, Thermo Fisher Scientific, Waltham, MA), and μDrop™ Plate (Thermo Fisher Scientific) were used to quantify DNA concentration.

RNA-seq and transcriptomic data analysis
RNA-seq experiments and data analysis were performed as previously described25. In brief, total RNA was isolated from three donors with the RNeasy Micro Kit (QIAGEN, Hilden, Germany). Illumina TruSeq RNA sample Prep kit (Illumina, San Diego, CA) was used to prepare RNA-Seq library. RNA-Seq experiments of early MSCs, late MSCs, TET-KD early-MSCs and PARP1-KD early-MSCs were performed for all three donors. More than 30 million (mean±standard deviation= 36,146,115±2,891,116) 100 bp paired-end reads for each RNA-Seq sample were generated using an Illumina HiSeq 2000 sequencer in National Center for Genome Medicine, Academia Sinica, Taiwan. Sequencing bases with low quality (Phred+33 quality score < 20) were trimmed using the TrimGalore program (Babraham Bioinformatics, Cambridge, UK) from the 5’- and 3’- ends of reads. After trimming, reads shorter than 50 bp were discarded. Then, low complexity reads (repeat sequences) were removed using the prinSeq program. The human genome assembly GRCh37/hg19 and NCBI RefSeq Genes were downloaded from the University of California Santa Cruz (UCSC) Genome Browser for genome mapping and gene annotation. Genome mapping was performed using Tophat (v2.0.10) with Bowtie (v.2.0.6.0) and Samtools (v.0.1.18.0). Cufflinks with GFF was used for transcript identification and counting. Quantification was done at gene-level. Normalization of gene expression level in individual sample was done by transforming count number to Fragments Per Kilobase of transcript per Million mapped read (FPKM). A total of 13326 genes with FPKM > 0 in at least half of the samples and with an average FPKM≥1. were selected for unsupervised hierarchical clustering of the genes and samples. Z-score was calculated for each gene across samples and presented in the heat map. Paired sample t-test was performed to compare the early MSCs to the late MSCs. Due to the small sample size, very few genes reached the genome-wide significance. Therefore, genes with differential expression in the early MSCs compared to the late MSCs were selected according to fold change. Differentially expressed genes should fulfilled the following two criteria: (1) mean fold change larger than or equal to 2 or smaller than or equal to −2, and (2) the three pairs of MSCs showed the same direction of fold change. A total of 1026 genes were identified. Activation or suppression of biological pathways by the altered expression of the 1026 genes was predicted using Ingenuity Pathway Analysis (IPA) (QIAGEN Inc) and quantified using the Activation Z-score metric. The enrichment of altered genes in biological pathways was calculated using Fisher Exact test with IPA. Only the pathways with P-values smaller than 0.05 for all the three donors are considered significant. RNA-seq raw data are accessible at NCBI GEO with accession number GSE178804. All relevant data are available from the authors with restrictions.

Alternative splicing analysis using rMATS
Differential alternative splicing (AS) events were identified between the two sample groups using rMATS version 4.1.1 (https://csibioinfo.nus.edu.sg/csingsportal/login/home.php)50, which detects five major types of AS events from RNA-Seq data (GSE178804) with replicates71. In each analysis, the experimental groups (late, PARP1 KD or TET1 KD MSCs) were compared to the reference group (early MSCs) to identify differentially spliced events with an associated change in the percent spliced in (ΔPSI) of these events. We computed P values of splicing events with a cutoff < 0.05 and then collected the splicing events with a |ΔPSI| of > 0.2. When the ΔPSI is a positive value, the number of AS events in the experimental group is more than the reference group.

5mC/5hmC DIP and high-throughput sequencing analysis
Aliquots of purified genomic DNA from early and late MSCs were first sonicated to ∼250 bp using Bioruptor (Diagenode, Seraing, Belgium), then end-repaired and ligated to Illumina PE adaptors using NEBNext DNA Library Prep Master Mix Set (New England Biolabs, Beverly, MA). In DIP assays, 10 μg of the adaptor-ligated genomic DNA was used as input, and 5 μl of anti-5-methylcytosine antibody (BI-MECY-0500, Eurogentec, Seraing, Belgium) or 5 μl of 5hmC antibody (39791, Active Motif, Carlsbad, CA) was added to immunoprecipitate modified DNA. Each of the immunoprecipitated DNA was amplified with forward primer and reverse primers in a 50 μl PCR reaction with 0.2× Sybr-Green I and 1× Phusion High-fidelity PCR Master Mix (New England Biolabs, Beverly, MA). PCR products that contained barcodes were sequenced after the standard first read using N2 Ind Seq primer. Raw data are accessible at NCBI GEO with accession number GSE178805. All relevant data are available from the authors with restrictions.
TrimGalore program (Babraham Bioinformatics, Cambridge, UK) was applied to clean the raw sequences to increase the quality of sequences for downstream analysis. After trimming, the sequence length of each read ranged from 36 bases to 101 bases. Interestingly, per sequence GC content of input control samples were generally all right, but the samples with methylated DNA immunoprecipitation (MeDIP) were tagged either warning or failed. This result suggested that the IP worked since the GC content was biased due to enrichment of methylated DNA fragments. The read sequences were aligned to human genome reference GRCh37/hg19 using Bowtie (v.2.0.10). Any read with more than two mapped ends were filtered out. The bam.files of MeDIP samples and input control samples were imported into PARTEK software for identification of methylated DNA peaks enriched in late passages of MSC. Data from late MSCs input was used as baseline to identify peaks enriched in samples with methylated DNA immunoprecipitation treatment. Firstly, sequencing peaks of each sample were detected by setting maximum fragment size at 200 bases with a widow size of 100 bases. Overlapping DNA fragments were merged. The peak cut-off FDR was set at 0.01. Then enriched peaks in late MSCs were identified by comparing sequencing peaks detected in late MSCs to paired early MSCs. DNA peaks whose q value is smaller or equals to 0.001 and scaled fold change is larger or equals to 4 were selected. The overrepresented peaks in late MSCs were annotated according to gene sections. Promoter region was defined as 5000 bp upstream of transcription start site (TSS, the first nucleotide of an mRNA according to RefSeq database) or 3000 bp downstream of TSS. By performing finding the nearest genomic features, 7633 genes were found to be hypermethylated at late-passage compared to early-passage of T7 MSC line.

Immunofluorescence
Cells were fixed with 4% paraformaldehyde. For 5hmC and 5mC detection, cells were treated with 2 N HCl for 30 min at 37°C, incubated with an anti-5hmC or anti-5mC monoclonal antibody, followed by incubation with a species-specific DyLightTM 488-conjugated secondary antibody (GTX213111-04, GeneTex, Inc., Irvine, CA), and counterstained with DAPI. For TRIM37 immunofluorescence, we used a mouse antibody against TRIM37 (sc-515044, Santa Cruz Biotechnology, Santa Cruz, CA) and reacted with a corresponding species-specific DyLightTM 488-conjugated secondary antibody. For pSer1981-ATM, PARP1 and FLAG-tag immunofluorescence, we used rabbit antibodies against pSer1981-ATM (GTX132146, GeneTex), PARP1 (#9532, Cell Signaling Technology, Danvers, MA) and FLAG-tag (GTX115043, GeneTex) and reacted with corresponding species-specific DyLightTM 594-conjugated secondary antibodies (GTX213110-05, GeneTex). Cells were counterstained with DAPI. Immunofluorescence was observed with a Leica TCSSP8 (Wetzlar, Germany) confocal system.

Proximity ligation assay
Cells were fixed with 4% formaldehyde in PBS for 10 min. Cells were then permeabilized and blocked against nonspecific binding with 0.1% Triton X-100 and 10% BSA in PBS for 1 h at room temperature. Primary antibodies, included anti-TRIM37, anti-pSer1981-ATM and anti-PARP1 antibodies, were then incubated with cells in 1% BSA in PBS at the indicated concentrations for 2 h at room temperature. Following the manufacturer-recommended procedure of Duolink® In Situ Red Starter Kit Mouse/Rabbit Kit (DUO92101-1KT, Sigma-Aldrich), cells were counterstained with DAPI and in situ protein-protein association was analyzed with a Leica TCSSP8 confocal system.

Lentiviral vector production and cell infection
The expression plasmids and the bacterial clones for PARP1 shRNA (TRCN0000007932), TET1 shRNA (TRCN0000075026), TET2 shRNA (TRCN0000144344), CTCF shRNA (TRCN0000218498 and TRCN0000230191), and TRIM37 shRNA [TRCN0000221060, TRCN0000370523 and TRCN0000370461 (3’UTR)] were provided by the RNAi core of the National Science Council in Taiwan. Lentiviral transfer vector pLAS2w.Ppuro carrying PARP1 (NM_001618.4) was used to generate stable PARP1 expression cells and lentiviral transfer vector (LV038, Applied Biological Materials Inc.) carrying wild-type TRIM37 or mutants (T203A or T203E) was used to generate stable TRIM37 expression cells. Lentiviral production was performed by transfection of 293T cells using Lipofectamine 3000 (L3000001, Invitrogen, Carlsbad, CA). Supernatants were collected 48 h after transfection and then were filtered. Subconfluent cells were infected with lentivirus in the presence of 100 μg/ml protamine sulfate (P4020, Sigma-Aldrich). At 24 h post-infection, we use puromycin (2 μg/ml) to select for infected cells for 48 h.

Western blotting
Cell extracts were prepared with M-PER (Pierce, Rockford, IL) or Subcellular Protein Fractionation Kit (Thermo Scientific, Waltham, MA) plus protease inhibitor cocktail (Halt™; Pierce, Rockford, IL) and protein concentrations were determined using the BCA assay (Pierce, Rockford, IL). Aliquots of protein lysates were separated on SDS–8 or 15% polyacrylamide gels and transferred to PVDF membrane filters, which were blocked with 5% blotting grade milk (Bio-Rad, Hercules, CA) in TBST (20 mM Tris-HCl [pH 7.6], 137 mM NaCl, 0.1% Tween 20) for 1 h. The filters were then incubated 1 h at room temperature with a 1:1000 dilution in TBST of antibodies against TET1 (GTX627420, GeneTex), TET2 (ab94580, Abcam, Cambridge, UK), PARP1 (#9532, Cell Signaling Technology), DNMT1 (#5032, Cell Signaling Technology, Danvers, MA), pSer1981-ATM (GTX132146, Gene Tex), TRIM37 (GTX114565, GeneTex), p16Ink4a (#92803, Cell Signaling Technology, Danvers, MA), p21Waf1/Cip1 (sc-6246, Santa Cruz Biotechnology), CTCF (GTX65926), HA tag (GTX115044), His tag (GTX628914), and Flag tag (GTX115043, GeneTex), reacted with corresponding secondary antibodies, and detected using a chemiluminescence assay (Millipore, Billerica, MA). The uncropped scans of the most important blots are shown in the Source Data file.

Co-immunoprecipitation assay
Cell extracts were prepared with Pierce IP lysis buffer (Thermo Scientific, Waltham, MA) plus protease inhibitor cocktail (Halt™; Pierce, Rockford, IL) and protein concentrations were determined using the BCA assay (Pierce, Rockford, IL). For TET1 and PARP1, the protein complexes were isolated by Dynabeads Protein G Immunoprecipitation Kit (Thermo Scientific, Waltham, MA). Anti-TET1 (GTX627420, GeneTex) or anti-PARP1 (#9532, Cell Signaling Technology, Danvers, MA) antibodies were crosslinked with protein G-magnetic beads by DSP (Proteochem, Inc., Denver, Colo.). The cell extracts were incubated with antibody-cross-linked protein G-magnetic beads for 30 min at room temperature and were collected by magnetic stand. For HA tag, cell extracts were incubated with Pierce Anti-HA Magnetic Beads (Thermo Scientific, Waltham, MA) for 30 min at room temperature and were collected by magnetic stand. The sample components were electrophoretically separated on SDS-PAGE for western blot analysis.

Phosphopeptides analysis by mass spectrometry
The TRIM37 or PARP1 phosphopeptides in anti-PARP1 immunoprecipitation from Early MSCs were performed in-gel digestion and detected on a nano-flow high-performance liquid chromatography system connected to an Orbitrap Q Exactive mass spectrometer. Each peptide fraction was autosampled and separated on a 15 cm analytical column (75 μm inner diameter) packed with 3 μm C18 beads with a 2 h gradient ranging from 5%–40% acetonitrile in 0.5% acetic acid at a flow rate of 250 nl/min. The Q Exactive mass spectrometer was operated in data-dependent acquisition mode, and all samples were analyzed with a “sensitive” acquisition method. All raw data analysis was performed with MaxQuant version 1.3.0.5 supported by the Andromeda peptide search engine.

Plasmid construction and cell infection
The pcDNA3-HA-PARP1, pcDNA3.1-Flag-TRIM37 and pcDNA3-His-Ubiquitin were gifts from Dr. Sheau-Yann Shieh72, Dr. Ping-Hui Tseng, and Heng-Hsiung Wu73, respectively. Human DNMT1-His (NM_001379.4) and alternative splicing DNMT1-His (3203-4521 deletion) constructs were generated by cloning specific coding sequences into the pcDNA3.1/V5-His vector (K4800-01, Invitrogen, Life Technologies, CA). Mutation of K337 or K438 on pcDNA3-HA-PARP1 and mutation of T203 on pcDNA3.1-Flag-TRIM37 were generated by QuikChange II XL Site-Directed Mutagenesis Kit (Agilent Technologies, Santa Clara, CA) using the following primers (Supplementary Table 8). Transient infection was performed by transfection of 293T human cell line using Lipofectamine 3000 (L3000001, Invitrogen, Carlsbad, CA). For primary MSCs, transient infection was performed by Neon transfection system (Invitrogen, Life Technologies, Carlsbad, CA).

DNMT1 activity assay
Full-length and alternative splicing form DNMT1 proteins generated in 293T cells were purified by Ni Sepharose 6 Fast Flow (GE Healthcare, Chicago, Illinois). The DNMT1 activity was determined with the EpiQuikTM DNA Methyltransferase Activity/Inhibition Assay Kit (Epigentek Inc., New York, NY) with a microplate reader at 450 nm. DNMT1 activity was calculated as OD/h/μg of protein.

Chromatin immunoprecipitation (ChIP) assay
To demonstrate the binding of PARP1, TET1 and CTCF protein to genomic DNA, the ChIP assay was performed using a commercial kit (Pierce, Rockford, IL) according to the manufacturer’s protocol with minor adjustments. The MSCs were grown to confluence, crosslinked with 1% formaldehyde for 8 min at room temperature, washed in ice-cold PBS containing protease inhibitors, then lysed on ice for 10 min in M-PER Extraction Reagent (Thermo Scientific, Waltham, MA) with phosphatase and protease inhibitors. DNA-protein complexes were sonicated to 200 and 500 base pairs. One aliquot of the soluble chromatin was stored at 20°C for use as input DNA, and the remainder was diluted 10 times in Pierce IP buffer (Thermo Scientific, Waltham, MA) containing phosphatase and protease inhibitors, and incubated overnight (4°C) with anti-PARP1 (#9532, Cell Signaling Technology, Danvers, MA), anti-TET1 (GTX627420, GeneTex) or anti-CTCF (GTX65930, GeneTex) antibodies. DNA-protein complexes were eluted with IP elution buffer. Cross-linking was reversed by incubation at 65°C for overnight. Proteins were removed with proteinase K, and DNA was extracted with phenol/chloroform, redissolved, and PCR-amplified with specific primers for DNMT1 exon 30, 34 and 37 (Supplementary Table 9).

ChIP-seq and data analysis
The immunoprecipitated DNA and input DNA were also subjected to next-generation sequencing using an Illumina HiSeq 2000 sequencer. The ChIP assay was performed using the Millipore chromatin immunoprecipitation (ChIP) assay kit (17-295, Millipore). The library preparation and sequencing procedure was performed following the manufacture’s instruction. After base-calling, trimming, and quality control, the high-quality reads (Phred+33 quality score > 20) were aligned to the human genome reference (GRCh37/hg19) using Bowtie (v.2.0.6.0) and Samtools (v.0.1.18.0). Bam files of T7E_INPUT, T7L_INPUT, T7_PARP1_ChIP, and T7_TET1_ChIP were imported into Partek software. The alignments per read of each sample past quality assurance and quality control. Sequencing peaks of each sample were detected by setting the maximum fragment size at 350 bp with a window size of 100 bp. Overlapping fragments were merged. The peak cut-off FDR was set at 0.001. Enriched PARP1 or TET1 binding sites were identified by comparing sequencing peaks detected in T7E_PARP1_ChIP or T7E_TET1_ChIP to T7E_INPUT. DNA segments with q value smaller or equals to 0.01 and scaled fold change larger or equals to 2 were selected. Additional filter criterion was non-overlapping between the selected segments with peaks detected in T7L_INPUT. A total of 249 peaks were specified to PARP1-ChIP and 960 peaks to TET1-ChIP. RefSeq transcripts database was used for annotation of the identified peak fragments. Promoter region was defined as 2000 bp upstream of transcription start site (TSS) or 1000 bp downstream of TSS. By performing finding the nearest genomic features, the 249 PARP1 binding peaks were co-localized with 117 genes and the 960 TET1 binding peaks were co-localized with 466 genes. The binding of PARP1 and TET1 to the promoter region, including TSS of the DNMT gene was enriched in early MSCs.

5’-Race PCR
Total RNA was extracted from early and late MSC using a RNeasy Micro Kit (QIAGEN) and was used for the first-strand cDNA synthesis by a SMARTer RACE 5’/3’ Kit (Takara Bio., Shiga, Japan). The first-strand reaction products in Tricine-EDTA buffer were stored at −80°C as the PCR templates. According to the cDNA sequences of DNMT1 (NM_001379.4), we designed gene-specific primers in Supplementary Table 9.

Animals and mouse BM MSC isolation
All animal experiments were conducted in accordance with the guidelines of the Animal Care Committee of China Medical University and the protocol was approved by Animal Care Committee of China Medical University (CMUIACUC-2019-374-1 and CMUIACUC-2021-374-1). All mice used in this study were on the C57BL/6 background. Male mice were used exclusively to avoid the influence of sex hormones on bone parameter assessment, except for experiments in Fig. 8 and Supplementary Fig. 13, in which female mice were used because osteoporosis models commonly employ females. The age of the animals used in each experiment is indicated in the corresponding figure legends. The strains used were as follows: C57BL/6-PARP1-deficient mice (129S-Parp1tm1Zqw/J)74 and their wild-type littermates (C57BL/6 background) obtained from The Jackson Laboratory (Bar Harbor, ME); and C57BL/6-Trim37em1(flox)Smoc and C57BL/6-Tg(Pgk1-RFP, -cre/ERT2)3Narl (RMRC13179) mice, which were kindly provided by Prof. Ping-Hui Tseng (National Yang Ming Chiao Tung University) and purchased from the National Laboratory Animal Center (Taipei, Taiwan). TRIM37-deficient mice were generated by cross-breeding C57BL/6-Trim37em1(flox)Smoc with C57BL/6-Tg(Pgk1-RFP, -cre/ERT2)3Narl mice. Mice were maintained under specific pathogen-free (SPF) conditions at 22±2°C with a 12 h light/dark cycle and ad libitum access to food and water. Health and welfare were monitored daily by trained animal care staff, and euthanasia was performed by CO₂ inhalation followed by cervical dislocation, in accordance with institutional and international guidelines.
For mouse BM MSC isolation, intact femurs and humeri were dissected carefully from the mice. The bones were cut at both ends, then a 26-gauge needle was inserted at the exposed end of the bones and the marrow plug was flushed out by 1% BSA in PBS. Cell suspension was collected through a 70-mm filter mesh to remove any cell clumps. MSCs were isolated and expanded with protocols described previously75. In brief, BM cells were cultured in 100-mm culture dishes with complete alpha MEM medium (with 16.6% FBS) and incubated at 37°C with 5% CO in a humidified chamber. After 12 h, the non adherent cells were removed on the surface of the dish by changing the medium. Thereafter, this step was repeated every 24 h for up to 72 h of initial culture. Within 4–8 days, the culture becomes more confluent and distinct fibroblastic cells reaches 65–70% confluence within 2 weeks. After 2 weeks of initiating culture, the culture was trypsinized with 0.05% trypsin/1 mM ethylenediaminetetraacetic acid for 2 min at 37°C. The trypsin was neutralized by adding 5 ml of complete medium, and culture in a 100-mm dish. The culture medium was changed every 3 days. Typically, cell confluence is achieved in 7 days.

Single-photon or two-photon evaluation of new bone growth in right femoral shaft drilling hole
TRIM37 deficiency was induced in mice at 6 weeks of age through intraperitoneal injection of 2 mg of tamoxifen (diluted in 100 μl of 20 mg/ml corn oil from Sigma-Aldrich) administered daily for a consecutive 5-day period. At 8 weeks of age, both young WT mice and mice deficient in TRIM37 or PARP1, as well as older mice (53 weeks), were anesthetized using 2.5–5% isoflurane in the presence of oxygen. A longitudinal incision, measuring 1 cm in size, was performed in the mid-portion of the right medial thigh of the mouse. Soft tissue retraction was accomplished using small retractors. A single medial cortical drill hole, created with a 1-mm pin, was carefully executed to avoid damage to the opposite cortex. Subsequently, the drill hole was rinsed with PBS, and the wound was closed using sutures. The animal was left move freely in the cage. Intraperitoneal injection of calcein (20 mg/kg) was performed 3 weeks later. Fluorescence microscopic evaluation was performed 4 weeks later. The femur bone was cleaned thoroughly, immersed in PBS and sophisticatedly attached on 6 cm dish for single-photon or two-photon microscopic observation.
For single-photon microscopic observation, the microscope system equipped with a Nikon AXR inverted microscope using a 2X objective (PLAN APO λ D 2x OFN25) was used. For two-photon microscopic observation, the microscope system was operated using a near-infrared femtosecond laser (Mira 900, Coherent) at the central wavelength of 810 nm, 76 MHz pulse repetition rate, and 200 fs pulse width for imaging. The laser power was controlled to 20 mW that is sufficient to produce second harmonic generation (SHG) and two-photon excitation fluorescence (TPEF), and also prevented photodamage during continuous illumination. Thus, the wavelength of SHG from collagen fibers is 405 nm, while the TPEF from collagen, elastin, FAD, and NADH is approximately ranging from 450 to 650 nm. All images were obtained by a laser scanning unit (Fluoview 300, Olympus), a pair of two objective lenses for both lasers focusing and collection of photons (UPlanSApo 20×/0.75, Olympus), and two photomultiplier tubes, respectively, for SHG and TPEF detection (R3896, Hamamatsu). SHG and TPEF were filtered from the intense excitation laser background by a combination of band-pass filter (FF01-405/10, Semrock) and color glass (BG39, Schott, Germany). And, then splitted by a dichroic mirror (FF435-Di01, Semrock) and forward detected. Note that we used a cube polarizing beam-splitter (GT10-B, Thorlabs) combined with a half (AHWP05M-980, Thorlabs) and a quarter (AQWP05M-980, Thorlabs) waveplates to demonstrate LP and CP imaging, respectively. Only the extinction ratio of linear polarization larger than 50:1 and the ellipticity of circular polarization (Imax/Imin) less than 1.1 after the focusing objective lens can then be used for the following two-photon imaging. The acquired images were mainly processed with ImageJ software. The collagen structure reconstructed through the second harmonic generation images showed porous collagen fiber inter-connected structures (white color) with fluorescent calcein (green area).

Measurement of bone and BM fat parameters by micro-CT
Mouse femurs were dissected and harvested, and subsequently, they were fixed in 10% neutral buffered formalin for a period of 72 h. Femurs were scanned at an isotropic voxel size of 4 μm (80 kV, 450 μA and 2000 ms integration time) by Extra-Ultra-High Resolution micro-CT system (U-CTX UHR, MILabs, Utrecht, Netherlands). Images were reconstructed and analyzed using MILabs Reconstruction 13.0 and Imalytics Preclinical software 3.1.0.7 (Gremse-IT GmbH, Aachen, Germany), respectively. Trabecular bone regions of distal femur were selected with reference to the growth plate (0.3–1.3 mm from growth plate). Bone mineral density, trabecular bone volume to tissue volume ratio, trabecular number, and increased trabecular spacing was determined based on calibration with phantoms of Imalytics Preclinical software, which were scanned under the same conditions as the bone samples. The calculated thresholds were bone (white) >800, BM fat (yellow) −100 to +80, and other tissues 80–800, and fat volume measured by Imalytics Preclinical software.

Statistical analysis and reproducibility
Data are presented as mean±SD. Statistical comparisons were performed by unpaired two-tailed Student’s t-test, Tukey’s multiple comparisons test of one-way analysis of variance (ordinary one-way ANOVA), or two-way ANOVA and p values < 0.05 were considered significant. All calculations were performed using GraphPad Prism 8.0. All in vivo data are representative of at least 3 independent experiments as indicated.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "This is an insightful research focusing on the pathogenetic mechanisms of senescence of MSCs.This study emphasizes the\nrole of DNA hydroxymethylation in stemness, suggesting therapeutic strategies, especially for osteoporosis. The authors\ndemonstrate the critical role of TRIM37/PARP1/DNMT1 pathway by introducing splicing.Additionally, the research reveals\nthat expansion of MSCs in growth medium containing ATM and TET activators should be considered in future clinical\nsettings. This manuscript is recommended a manior revision before acceptance.\n1. This study focusing on the modification of 5hmC. As we all know, TET1 and DNMT1 also involve in the modofication of\n5mC. How about the corresponding change of 5mC in MSCs when disposed with inhibitors or shRNA. Maybe the autors\nshould claim this at the first part data to illuminate the change and the relationship with 5hmC.\n2.When refering to proliferation and differentiation potential of early human MSCs, osteogenesis and adiposis were\nperformed . Maybe chondrogeneis could be added to further clarify the differentiation potential of early human MSCs.\n3.The researchers reveal knockdown of CTCF in early MSCs reduced the protein level of full-length DNMT1 and the mRNA\nlevel of DNMT1 in exon 34, but not in exon 13, suggesting that CTCF knockdown caused alternative splicing. These data\nindicate\nthe involvement of CTCF in PARP1/TET1 complex-mediated loss of intragenic DNA methylation and prevention of DNMT1\nalternative splicing in early MSCs.AS is regarded as one innovative point of this study.The authors should provide more\ndetails and data on splicing mechanism.\n4. The researchers try to demonstrate the involvement of TRIM37 and PARP1 in MSC aging in vivo by using young, aged,\nand TRIM37 KO mice. One point is whether using ATM activator or vitamin C to old mice or TRIM37 KO micedoulc rescue\nthe aging process of mice in vivo? Maybe more experiments in vivo should be performed.\n5. The details of the figure designing could be further improved, such as the color of statistic panels and the structure of the\nfigures.\nOverall, this is an excellent research which will have a great potential in addressing the aging problem in future.", "reviewer_2": "In this paper, the authors showed that the phosphorylation of TRIM37 by ATM promotes nuclear translocation of PARP1 and\nprotein stabilization. In addition, PARP1 recruits TET1, which hydrolyzes methylated nucleotides on DNMT1 exons and\ninhibits alternative splicing of DNMT1. Bone density was reduced and bone regeneration was impaired in mice lacking\nTRIM37 or PARP1. The author demonstrated that TRIM37 and PARP1 are involved in MSC senescence and bone\nremodeling, and proposed a meaningful strategy for the treatment of osteoporosis. Overall, the paper is well organized, but\nthe most important part has already been reported that PARP1 and TET are involved in the maintenance of stemness. In\naddition, there are some questions which need to be addressed.\nMajor point\n・ The author should briefly describe the results obtained in this experiment in the Introduction section.\n・ Regarding the data from the experiment in which 5hmC was detected by immunofluorescence, the authors should include\nquantitative data of fluorescence intensity.\n・ In Figure 2, the authors claim “PARP1 interacts with TET1 to increase 5hmC levels and maintain early MSC properties”,\nbut the data to support this claim are insufficient, as they only show the results of knockdown or overexpression of PARP1.\nThe authors should show a functional relationship between PARP1 and TET1, for example, by examining whether PARP1\nOE-induced changes in MSC phenotype are cancelled by TET1 KD.\n・ In Figure 3, to show that “PARP1/TET1 complex-mediated loss of intragenic DNA methylation in DNMT1 prevents\nalternative splicing of DNMT1 in early MSCs”, the authors should check whether PARP1 KD and TET1 KD increase the\nalternative splicing of DNMT1.\n・ In Figure 4b, c, and d, the authors should show whether overexpression of full-length DNMT1 suppresses the proliferation\nand differentiation potential of MSCs as in the case of AS-DNMT1.\n・ In Figure 5, to show that “T203 phosphorylation is necessary for TRIM37 to stabilize PARP1 via K337\nmonoubiquitination, thereby maintaining early MSC properties”, the authors should show that re-expression of shRNA-\nresistant TRIM37 WT restores PARP1 expression, proliferation, and differentiation ability in TRIM37-depleted MSCs, but not\nT203A mutant expression.\n・ Regarding the immunofluorescence staining of the nuclear localization of p-ATM, TRIM37 in Figures 6d and 6e, the\nauthor should show the quantitative data of fluorescence intensity\n・ Schematic diagram should be provided to make it easier for the reader to understand.\nMinor point\n・ p21 and p16 should be written together with Waf1/Cip1 and Ink4a in superscript, respectively.\n・ In lines 62, 64, 66, 70, 93, “Hayflick limit” should be replaced with “senescence”.\n・ In lines 155, 205, 416, 1018, 1077, “PAPR1” should be “PARP1”.\n・ In Figure 2c, why does the effect of PARP1 KD or TET1 KD on the transcriptional profile differ significantly between\nDonor1 and Donor2?\n・ The author needs to indicate in Figure2e which cells in the image are early MSCs or late MSCs.\n・ In Extended Data Fig.3, the authors should clearly specify the range of TSS.\n・ In line 252, the authors should describe why they focused on phosphorylated proteins that interacted with PARP1 to\nidentify the upstream proteins of PARP1.\n・ In line 372, “5hMC” should be “5hmC”.", "reviewer_3": "In this manuscript, Chun-Te Ho and colleagues reported that 5hmC levels are higher in young MSCs, promoting MSC\nrenewal and osteogenesis. They described a mechanism in which the PARP1/TET1 complex hydrolyzes the nucleotide of\nDnmt1 exons to prevent Dnmt1 alternative splicing, producing a DNMT1 variant that suppresses full-length DNMT1. Also,\nthe stability and nuclear localization of PARP1 are regulated by the ATM/TRIM37 complex. This mechanism is innovative,\nand the mouse phenotype is interesting. However, the below major points weaken the rigor of this study.\nMajor points:\n1. This work described an interesting mechanism for the role of TRIM37 in regulating MSC property. However, the major\ncomponents in this mechanism, including TET1, DNMT1, and PARP1, all have a wide spectrum of downstream targets in\ncontrolling stemness, proliferation, and senescence. It is unclear how much this linear mechanism, as proposed by the\nmanuscript -- TRIM37 -> TET1/PARP1 stability and cellular localization –> DNMT1 alternative splicing -- contributes to the\nphenotype. To clarify this, many well-controlled rescue experiments are needed, which are lacking here.\n2. 2HG was used for TET inhibition in Fig. 1g to show that it produces a phenotype similar to TET1 KO in cell cultures.\nHowever, the specificity of 2HG is in question. 2HG is known to be a key metabolite that promotes several pathways, such\nas KDM family histone demethylase function, and broadly affects cellular processes.\n3. Vitamin C is widely used to promote in vitro MSC osteogenesis. Besides regulating TET function, it help removing histone\nH3K9me3 and K27me3 and can improve collagen production. It is unlikely to act solely via enhancing TET protein function.\n4. PJ34 inhibited PARP1 activity by targeting its catalytic domain, preventing the synthesis of PAR polymers, which should\nlead to the accumulation of DNA damage and subsequent cellular senescence. Do you see the difference in PAR level\nbetween early and late MSCs?\n5. In Fig 1a, the authors detected the global 5hmC level with dot-blot and observed a significant decrease in late MSCs.\nHowever, the dot-blot result of 5mC is not provided and quantified.\n6. In line 124, according to the early study, the authors mentioned that the TET1 and TET2 double knockout have lower\n5hmC levels in mouse BM-MSCs than the TET1/2KO or control group. TET2-KD 5hmC does not change 5hmC leve1 (Fig\n1h), while the expression level of TET2 seems to be higher than that of TET1 in the control MSC (Fig 1g). It is known that\nTET1 and TET2 have overlapping functions and exhibit distinct expression patterns in different cellular contexts. It is unclear\nwhether they target different genes and have a distinct role in MSC. How much TET2 contributes to the MSC properties and\naging was not assessed and discussed further. It would be helpful to have parallel TET2-KD experiments.\n7. The bioinformatics data needed to be analyzed and described more sufficiently. How many differentially expressed genes\nare observed from the RNA-seq? What are the altered pathways? Are those pathways relevant to the phenotypes? And can\nthe altered genes in these pathways be clustered well within each group? Does the heatmap of Fig 2C display CPM values\nor Z-scores? Additionally, are these values representative of all genes? A batch effect is noted from the late/KD cluster\nbetween donors 1 and 2. The hierarchical clustering showed that the TET1-KD and PARP1-KD tend to be closer to the late\nMSC.\n8. In line 202, the authors may provide a detailed description of the subgroups of MSC and list the number of genes in each\nsubset. The supplementary tables are difficult to navigate, so an instruction spreadsheet explaining the contents of each\nspreadsheet will be helpful.\n9. Extended Data Fig. denotes the colocalized ChIP-seq peaks of PARP1 and TET1 in the genome browser. Is that locus a\nTSS; if yes, how is TSS defined here? Please provide the peak coordinate in the browser view and figure legends.\n10. The decrease of DNMT1 in PARP1 and TET1 KD suggests the alternative splicing of DNMT1; has this been confirmed\nfrom RNA-seq data -- a genome browser view of the DMNT1 sequence would make this conclusion more convincing.\n11. In line 238. The spliced DNMT1 lost the DNMT1 activity. It was also observed that the addition of AS-DNMT1 abrupted\nthe activity of Full-DNMT1. This observation is interesting but needs further investigation/explanation. Does the\noverexpressed AS-DNMT1 change overall DNA methylation, and are those genes related to cellular senescence?\n12. Is DNMT1 the only target of this mechanism in alternative splicing? Considering TET1 affects the epigenome broadly, it\nis puzzling why only DNMT1 is selected for analysis.\nMinor points:\n1. In line 65, the authors said, “However, this does not explain why the telomers of cancer cells are paradoxically shorter\nthan those found in normal tissue.” This seems irrelevant to any part of the results.\n2. In line 152, the author mentioned, “PARP1 and TET2 contribute to an epigenetic program ... during somatic cell\nreprogramming”, but the objective is to investigate the PAPR1 and TET1 interaction in MSC. Please provide the known\nPARP1 and TET1 interaction.\n3. In Line 61, “analyzed the related heat maps” may be changed to “we further analyzed the RNA-seq of gene expression\nprofiles …”. Similarly, in the Method, line 533. The original texts sound like the RNA-seq was only used to generate a\nheatmap.\n4. Figure legend of Fig 3g, please provide the definition of #1 and #2\n5. Line 1099. Please add T203E.\n6. Please check text consistency, such as 5hmC vs. 5hmc, 5mC vs.5 mC.", "reviewer_4": "SIGNIFICANCE\nThis study attempts to create mechanistic connections between ATM, TRIM37, PARP1, TET1 and CTCF in modulating\nmethylation of the DNMT1 gene to mediate alternative splicing of DNMT1 to modulate the proliferative properties of MSCs.\nCRITIQUE\n1. One overall concern is that while the multiple mechanistic connections are theoretically feasible and somewhat supported\nby the data, experimental evidence for each connection is of poor quality, not well controlled and generally not compelling.\n2. The title severely overstates the findings. This paper does not present any direct data that TRIM37 mitigates osteoporosis.\nIn fact, nothing in this paper has even a remote bearing on osteoporosis. This problem of overstating the strength of the\nevidence is symptomatic for other findings in the paper.\n3. TRIM37 has a wide range of cellular activities and molecular functions. Effects on DNMT1 function by modifying\nalternative splicing of DNMT1 represents a very specific mechanism that is unlikely to be the only or principal target of\nTRIM37.\n4. The authors state that “5hmC levels positively regulates MSC self-renewal and osteoblast differentiation, which is at odds\nwith recent studies in Nat Comm that Vitamin C dependent 5hmC levels promote osteogenesis rather than stemness.\n5. All blot based methods (dot blots and western blots) are of very poor quality and these blots cannot be properly quantified.\n6. The paper contains a number of avoidable spelling errors and generally does not meet high standards of academic writing\nexpected for Nature brand papers (e.g., “Westering blotting”?)\n7. Extended Data:\na) Experimental evidence for senescence presented (see extended data, Figure 1) is rather minimal and insuffient.\nb) The quality of immunoprecipitations (see extended data, Figure 2) is not compelling and lack essential controls. The\nresulting conclusion that PARP1, TET1 and CTCF directly interact is not evident from the data shown.\nc) The ChIP-seq tracks (see extended data, Figure 3) claim to show TET1 and PARP1 binding to the TSS but there are\nupstream exons that are not annotated giving the appearance that the location is intragenic. Input background is high and\nthe level of enrichment appears to be limited.\nd) The compilation of data on effects of TRIM37 and PARP1 on MSC activity (see extended data, Figure 7) is of poor quality\nand not convincing.\ne) Effects of PARP1 knockout on bone healing in a drill hole model are not quantified and qualitative effects are not\napparent.\nf) Supplemental Table 7 shows that only three MSC donors were considered and these donors were neither age nor gender\nmatched."}, "author_response": "Point-by-Point Response\nReviewer #1\nThis is an insightful research focusing on the pathogenetic mechanisms of senescence of\nMSCs. This study emphasizes the role of DNA hydroxymethylation in stemness, suggesting\ntherapeutic strategies, especially for osteoporosis. The authors demonstrate the critical\nrole of TRIM37/PARP1/DNMT1 pathway by introducing splicing. Additionally, the research\nreveals that expansion of MSCs in growth medium containing ATM and TET activators\nshould be considered in future clinical settings. This manuscript is recommended a major\nrevision before acceptance.\n1. This study focusing on the modification of 5hmC. As we all know, TET1 and DNMT1 also\ninvolve in the modification of 5mC. How about the corresponding change of 5mC in MSCs\nwhen disposed with inhibitors or shRNA. Maybe the authors should claim this at the first\npart data to illuminate the change and the relationship with 5hmC.\nResponse: Thank you very much for your insightful comments. There are no corresponding\nchanges in 5mC levels in MSCs when treated with TET1 inhibitors or shRNA. Extended Data\nFig. 2b, 2c.\n2. When referring to proliferation and differentiation potential of early human MSCs,\nosteogenesis and adiposis were performed . Maybe chondrogeneis could be added to\nfurther clarify the differentiation potential of early human MSCs.\nResponse: Thank you very much for your insightful comments. Chondrogenesis is more\ncomplex. For example, leptin receptor (LepR)+ MSCs isolated from murine bone marrow\ngive rise to osteoblasts and adipocytes but not chondrocytes1. To date, no single MSC\npopulation has been shown to differentiate into osteoblasts, adipocytes, and chondrocytes\nsimultaneously. Since our study focuses on MSC senescence both in vitro and in vivo, we\ndid not include a chondrogenesis assay.\n3. The researchers reveal knockdown of CTCF in early MSCs reduced the protein level of\nfull-length DNMT1 and the mRNA level of DNMT1 in exon 34, but not in exon 13,\nsuggesting that CTCF knockdown caused alternative splicing. These data indicate the\ninvolvement of CTCF in PARP1/TET1 complex-mediated loss of intragenic DNA methylation\nand prevention of DNMT1 alternative splicing in early MSCs. AS is regarded as one\ninnovative point of this study. The authors should provide more details and data on splicing\nmechanism.\nResponse: Thank you for your insightful comments. To further investigate the splicing\nmechanism, we performed PARP1 and TET1 knockdown in early MSCs. Our results show\nthat their knockdown reduces CTCF binding to Exon 30 and Exon 37, supporting the role of\nCTCF in the PARP1/TET1 complex-mediated regulation of intragenic DNA methylation and\nthe prevention of DNMT1 alternative splicing in early MSCs (Extended Data Fig. 7).\n4. The researchers try to demonstrate the involvement of TRIM37 and PARP1 in MSC aging\nin vivo by using young, aged, and TRIM37 KO mice. One point is whether using ATM\nactivator or vitamin C to old mice or TRIM37 KO mice could rescue the aging process of\nmice in vivo? Maybe more experiments in vivo should be performed.\nResponse: Thank you for your insightful comments. We have now treated aged mice (75\nweeks old) with an ATM activator (resveratrol) and/or a TET activator (vitamin C). Our\nfindings indicate that the combination treatment enhanced bone parameters, including\nBMD, BV/TV, Tb.No., and Tb.Th., while reducing BS/BV and Tb.Sp. Additionally, it improved\nbone repair capacity after injury compared to the control and certain single-treatment\ngroups (Fig. 8).\n5. The details of the figure designing could be further improved, such as the color of\nstatistic panels and the structure of the figures.\nResponse: Thank you very much for your insightful comments. We have now improved the\ncolor of statistic panels and the structure of the figures, such as Fig. 1a-1d, 1f-1h, Fig. 2d,\n2f-2i, Fig. 3i-3n, Fig. 4g-4h, Fig. 5d-5h, and Fig. 7b-7e.\nOverall, this is an excellent research which will have a great potential in addressing the\naging problem in future.\nResponse: Thank you very much for your encouragement.\nReviewer #2\nIn this paper, the authors showed that the phosphorylation of TRIM37 by ATM promotes\nnuclear translocation of PARP1 and protein stabilization. In addition, PARP1 recruits TET1,\nwhich hydrolyzes methylated nucleotides on DNMT1 exons and inhibits alternative splicing\nof DNMT1. Bone density was reduced and bone regeneration was impaired in mice lacking\nTRIM37 or PARP1. The author demonstrated that TRIM37 and PARP1 are involved in MSC\nsenescence and bone remodeling, and proposed a meaningful strategy for the treatment of\nosteoporosis. Overall, the paper is well organized, but the most important part has already\nbeen reported that PARP1 and TET are involved in the maintenance of stemness. In\naddition, there are some questions which need to be addressed.\nResponse: We thank the reviewer for their thoughtful comment. We acknowledge that the\ninvolvement of PARP1 and TET2 proteins in the maintenance of pluripotency and\nreprogramming has been well-documented, particularly in the context of induced\npluripotent stem cell (iPSC) generation. Specifically, the study by Doege et al. (Nature,\n2012)2 demonstrated that PARP1 and TET2 are recruited to pluripotency loci such as\nNanog and Esrrb during early reprogramming stages. PARP1 was shown to regulate 5mC\nmodification, while TET2 catalyzed the conversion of 5mC to 5hmC, facilitating chromatin\nremodeling and transcriptional activation of pluripotency genes.\nHowever, our study expands on these earlier findings by uncovering a novel regulatory\nmechanism specific to adult stem cells—particularly MSCs. We provide evidence that\nTRIM37, through its phosphorylation by ATM, promotes the nuclear translocation and\nstabilization of PARP1 in adult MSCs. This process is crucial for the recruitment of TET1 to\nspecific exons of DNMT1, which regulates alternative splicing and influences MSC identity\nand function. This mechanism, not previously identified in the context of MSCs, links DNA\ndamage response signaling with epigenetic regulation, and highlights a critical pathway in\nMSC aging and bone regeneration.\nThus, while previous studies primarily focused on pluripotency induction in the\nreprogramming of somatic cells, our work provides a new dimension by revealing how\nPARP1 and TET1 cooperate to maintain stemness and regulate MSC function in the context\nof aging and bone remodeling. We hope this clarification underscores the novelty of our\nfindings and their potential relevance to regenerative medicine.\nMajor point\n・ The author should briefly describe the results obtained in this experiment in the\nIntroduction section.\nResponse: Thank you very much for the comments. “In the current study, we first found\nthat increased 5hmC levels are required to maintain the early passage characteristics of\nhuman bone marrow (BM)-derived MSCs. We further revealed that CTCF-mediated\nprevention of DNMT1 alternative splicing plays a key role in sustaining the proliferative\ncapacity and differentiation potential of early passage BM-MSCs, which depends on the\ncoordinated actions of TET1 and DNA damage response (DDR) components such as ATM,\nPARP1, and TRIM37. Mechanistically, PARP1 recruits TET1 to demethylate DNMT1 exons,\nfacilitating CTCF-mediated splicing regulation, while ATM-mediated phosphorylation of\nTRIM37 promotes PARP1 stabilization. Knockdown of CTCF or TRIM37 induces replicative\nsenescence. Importantly, combined treatment with an ATM activator (resveratrol) and a\nTET activator (vitamin C) rejuvenates late-passage MSCs, restores osteogenic capacity,\nand enhances bone regeneration in aged mice. These findings identify a\nTRIM37/PARP1/DNMT1 regulatory axis critical for MSC stemness and bone homeostasis,\nsuggesting a therapeutic strategy for osteoporosis.” has now added into the introduction\nsection. Page 6 Line 4-6\n・ Regarding the data from the experiment in which 5hmC was detected by\nimmunofluorescence, the authors should include quantitative data of fluorescence\nintensity.\nResponse: Thank you very much for the comments. We have now quantified the data, such\nas Fig. 1a, 1b, 1f and 2d.\n・ In Figure 2, the authors claim “PARP1 interacts with TET1 to increase 5hmC levels and\nmaintain early MSC properties”, but the data to support this claim are insufficient, as they\nonly show the results of knockdown or overexpression of PARP1. The authors should show\na functional relationship between PARP1 and TET1, for example, by examining whether\nPARP1 OE-induced changes in MSC phenotype are cancelled by TET1 KD.\nResponse: Thank you for your insightful comments. To address this concern, we\noverexpressed PARP1 in late MSCs, followed by transduction with either scrambled control\n(CTR) or TET1 shRNAs (TET1-KD). We then performed western blotting,\nimmunofluorescence for 5hmC detection, growth curve analysis, and\nosteogenesis/adipogenesis differentiation assays. Our results demonstrate that TET1\nknockdown in PARP1-overexpressing MSCs suppresses the effects of PARP1 overexpression\non senescence marker expression (Extended Data Fig. 5a), 5hmC levels (Extended Data\nFig. 5b), proliferation capacity (Extended Data Fig. 5c), and differentiation potential\n(Extended Data Fig. 5d). These findings confirm the functional relationship between PARP1\nand TET1 in regulating early MSC properties.\n・ In Figure 3, to show that “PARP1/TET1 complex-mediated loss of intragenic DNA\nmethylation in DNMT1 prevents alternative splicing of DNMT1 in early MSCs”, the authors\nshould check whether PARP1 KD and TET1 KD increase the alternative splicing of DNMT1.\nResponse: Thank you very much for the comments. Besides verifying the existence of the\nDNMT1 isoform with alternative splicing in this region in late MSCs (Fig. 3e), we have now\nexamined early MSCs with PARP1 KD or TET1 KD (Extended Data Fig. 7a). An additional\nband, significantly smaller than the full-length product, was observed using primers\nflanking exons 25 and 38. In addition, their knockdown reduces CTCF binding to Exon 30\nand Exon 37, supporting the role of CTCF in the PARP1/TET1 complex-mediated regulation\nof intragenic DNA methylation and the prevention of DNMT1 alternative splicing in early\nMSCs (Extended Data Fig. 7b).\n・ In Figure 4b, c, and d, the authors should show whether overexpression of full-length\nDNMT1 suppresses the proliferation and differentiation potential of MSCs as in the case of\nAS-DNMT1.\nResponse: Thank you very much for the comments for Figure 3b, c, and d in the revised\nmanuscript. Actually, we have shown this in 2012 Molecular Cell paper3. Overexpression of\nDnmt1 in MSCs expanded under normoxic conditions; decreased the expression of p16 and\np21; increased the proliferation rate, BrdU incorporation ratio, and differentiation potential.\nPlease refer to Supplementary Figure 6 in the Molecular Cell. We have now cited this in the\nmanuscript. Page 11 Line last 2 ~ Page 12 Line 1.\n・ In Figure 5, to show that “T203 phosphorylation is necessary for TRIM37 to stabilize\nPARP1 via K337 monoubiquitination, thereby maintaining early MSC properties”, the\nauthors should show that re-expression of shRNA-resistant TRIM37 WT restores PARP1\nexpression, proliferation, and differentiation ability in TRIM37-depleted MSCs, but not\nT203A mutant expression.\nResponse: We appreciate the reviewer's insightful comment regarding the role of T203\nphosphorylation in TRIM37 nuclear relocalization and its functional consequences. Our\nresults indicate that T203 phosphorylation enhances TRIM37 nuclear localization (Fig. 5h)\nand short-term proliferation (T203E > WT > T203A) (Extended Data Fig. 10b). However,\nPARP1 stabilization (Extended Data Fig. 10a) and differentiation ability (Extended Data Fig.\n10c) appear to be largely independent of nuclear localization efficiency, as both WT and\nT203E rescue PARP1 expression and differentiation in TRIM37-KD MSCs. This suggests that\nwhile phosphorylation of T203 facilitates TRIM37 nuclear relocalization, once in the\nnucleus, TRIM37's function in stabilizing PARP1 and supporting differentiation may not be\nstrictly phosphorylation-dependent. These findings highlight the possibility that distinct\nmechanisms regulate TRIM37’s roles in proliferation and differentiation. Further studies\ncould explore whether additional post-translational modifications influence TRIM37’s\ninteraction with PARP1 and its functional specificity in MSC fate decisions. We have also\nexpanded on this discussion in the revised manuscript (Page 26, Line last 3 ~ Page 27 Line\n11).\n・ Regarding the immunofluorescence staining of the nuclear localization of p-ATM,\nTRIM37 in Figures 6d and 6e, the author should show the quantitative data of fluorescence\nintensity\nResponse: Thank you very much for the comments on Figure 5d and 5e in the revised\nmanuscript. We have now quantified the data, such as Fig. 5d-5h.\n・ Schematic diagram should be provided to make it easier for the reader to understand.\nResponse: Thank you very much for the comments. We have revised the schematic\ndiagram in Fig. 7a to make it easier for the reader to understand.\nMinor point\n・ p21 and p16 should be written together with Waf1/Cip1 and Ink4a in superscript,\nrespectively.\nResponse: Thank you very much for the helpful comment. We have revised the text\nthroughout the manuscript to present p21Waf1/Cip1 and p16Ink4a in superscript as suggested.\n・ In lines 62, 64, 66, 70, 93, “Hayflick limit” should be replaced with “senescence”.\nResponse: Thank you very much for the comments. We have revised accordingly.\n・ In lines 155, 205, 416, 1018, 1077, “PAPR1” should be “PARP1”.\nResponse: Thank you very much for the comments. We have now revised them.\n・ In Figure 2c, why does the effect of PARP1 KD or TET1 KD on the transcriptional profile\ndiffer significantly between Donor1 and Donor2?\nResponse: Thank you very much for the comments. The differing effects of PARP1 KD or\nTET1 KD on the transcriptional profile between Donor1 and Donor2 could be attributed to\nseveral factors, including: genetic variability between donors; epigenetic differences;\nbaseline expression of PARP1, TET1, or other key regulators. Probably, cellular\nheterogeneity after ex vivo culture could also be a factor.\n・ The author needs to indicate in Figure2e which cells in the image are early MSCs or late\nMSCs.\nResponse: Thank you very much for the comments. We have now indicated in figure 2d\nand 2e which cells in the image are early MSCs or late MSCs.\n・ In Extended Data Fig.3, the authors should clearly specify the range of TSS.\nResponse: Thank you for your valuable comment. The transcription start site (TSS for\nDNMT1 was defined based on the first nucleotide of the mRNA that corresponds to the\n5’UTR according to the RefSeq database (NM_001130823.3; chr19:10305629). In\nExtended Data Fig. 6, the DNA region of promoter, exon 1, and part of intron 1 of the\nDNMT1 gene is enlarged to show physical coordinate of TSS flanking region and ChIP-seq\npeaks. We have also revised the figure legends: Extended Data Fig. 6. PARP1 and TET1\nChIP-seq analysis revealed that the flanking region of transcription start site (TSS, Chr19:\n10305629) of DNMT1 was enriched with PARP1 and TET1 in early MSCs.\n・ In line 252, the authors should describe why they focused on phosphorylated proteins\nthat interacted with PARP1 to identify the upstream proteins of PARP1.\nResponse: Thank you very much for the comments. We have focused on total associated\nprotein. Unfortunately, the data are not obvious. We then focused on phosphorylated\nproteins that interacted with PARP1. Actually, PARP1 activation involves in phosphorylation\ncascade, such as ERK signaling pathway4.\n・ In line 372, “5hMC” should be “5hmC”.\nResponse: Thank you very much for the comments. We have carefully checked and revised\nthe entire manuscript.\nReviewer #3 (Remarks to the Author):\nIn this manuscript, Chun-Te Ho and colleagues reported that 5hmC levels are higher in\nyoung MSCs, promoting MSC renewal and osteogenesis. They described a mechanism in\nwhich the PARP1/TET1 complex hydrolyzes the nucleotide of Dnmt1 exons to prevent\nDnmt1 alternative splicing, producing a DNMT1 variant that suppresses full-length DNMT1.\nAlso, the stability and nuclear localization of PARP1 are regulated by the ATM/TRIM37\ncomplex. This mechanism is innovative, and the mouse phenotype is interesting. However,\nthe below major points weaken the rigor of this study.\nMajor points:\n1. This work described an interesting mechanism for the role of TRIM37 in regulating MSC\nproperty. However, the major components in this mechanism, including TET1, DNMT1, and\nPARP1, all have a wide spectrum of downstream targets in controlling stemness,\nproliferation, and senescence. It is unclear how much this linear mechanism, as proposed\nby the manuscript -- TRIM37 -> TET1/PARP1 stability and cellular localization –> DNMT1\nalternative splicing -- contributes to the phenotype. To clarify this, many well-controlled\nrescue experiments are needed, which are lacking here.\nResponse: Thank you for your insightful comments regarding the role of TRIM37 in\nregulating MSC properties and the need for well-controlled rescue experiments to support\nour proposed mechanism. To address these concerns, we have performed additional\nexperiments in the new version to clarify the contributions of TRIM37-mediated\nstabilization of TET1/PARP1 and DNMT1 alternative splicing to the observed phenotype.\n1. Role of TRIM37 Nuclear Relocalization and Phosphorylation (T203) in MSC Function\nWe investigated the role of T203 phosphorylation in TRIM37 nuclear relocalization and its\nfunctional consequences. Our data show that T203 phosphorylation enhances TRIM37\nnuclear localization (Fig. 5h) and short-term proliferation (T203E > WT > T203A)\n(Extended Data Fig. 10b). However, nuclear localization does not appear to be the primary\ndeterminant of TRIM37’s function in PARP1 stabilization (Extended Data Fig. 10a) or MSC\ndifferentiation (Extended Data Fig. 10c). Both WT and T203E TRIM37 rescued PARP1\nexpression and differentiation in TRIM37-KD MSCs, indicating that while phosphorylation\nfacilitates TRIM37 nuclear relocalization, its role in stabilizing PARP1 and supporting\ndifferentiation is largely independent of nuclear localization efficiency. These findings\nsuggest that distinct mechanisms regulate TRIM37’s effects on proliferation and\ndifferentiation.\n2. PARP1/TET1 and CTCF Regulation of DNMT1 Alternative Splicing\nTo further investigate the splicing mechanism, we performed PARP1 and TET1 knockdown\nin early MSCs and analyzed CTCF binding at DNMT1 Exon 30 and Exon 37. Our results\nshow that knockdown of PARP1 and TET1 reduces CTCF binding at these sites, supporting\nthe role of the PARP1/TET1 complex in regulating intragenic DNA methylation and\npreventing DNMT1 alternative splicing in early MSCs (Extended Data Fig. 7). These\nfindings provide mechanistic evidence that TRIM37-mediated stabilization of PARP1 and\nTET1 contributes to DNMT1 splicing regulation and, consequently, MSC function. In these\nexperiments, we used scrambled shRNA as a control for shRNA against PARP1 and TET1,\nand H19 served as a control for Exon 30 and Exon 37.\n3. Functional Relationship Between PARP1 and TET1 in MSCs\nTo clarify the interdependence of PARP1 and TET1 in regulating MSC properties, we\noverexpressed PARP1 in late MSCs and performed TET1 knockdown. Our results\ndemonstrate that TET1 knockdown suppresses the effects of PARP1 overexpression on\nsenescence marker expression (Extended Data Fig. 5a), 5hmC levels (Extended Data Fig.\n5b), proliferation capacity (Extended Data Fig. 5c), and differentiation potential\n(Extended Data Fig. 5d). These findings confirm the functional relationship between PARP1\nand TET1 in regulating early MSC properties, further supporting the mechanistic pathway we\npropose. In these experiments, we used scrambled shRNA as a control for shRNA against\nTET1.\nTogether, these results reinforce that TRIM37 regulates MSC properties via stabilization of\nPARP1/TET1 and modulation of DNMT1 alternative splicing. While additional regulatory\nmechanisms may contribute to MSC function, our data demonstrate that this pathway\nplays a significant role in the observed phenotype.\nWe appreciate the reviewer’s suggestion for further validation, and we believe these\nadditional data strengthen our conclusions.\n2. 2HG was used for TET inhibition in Fig. 1g to show that it produces a phenotype similar\nto TET1 KO in cell cultures. However, the specificity of 2HG is in question. 2HG is known to\nbe a key metabolite that promotes several pathways, such as KDM family histone\ndemethylase function, and broadly affects cellular processes.\nResponse: Thank you very much for the comments. While 2HG was used as a TET inhibitor\nin Fig. 1g to demonstrate a phenotype similar to TET1 KO, we acknowledge that its\nspecificity is in question, as 2HG also inhibits KDM family histone demethylases in addition\nto TET enzymes, broadly affecting cellular processes. Both TET and KDM enzymes are\nFe(II)/α-KG-dependent dioxygenases involved in epigenetic regulation—TET enzymes\nregulate DNA demethylation, while KDM demethylases modify histone methylation.\nNotably, TET1 has been shown to interact with KDM2A and KDM6A, suggesting coordinated\nregulation of DNA and histone methylation. Given that 2HG inhibits both TET and KDM\nenzymes, the observed phenotype could result from broader epigenetic effects rather than\nTET inhibition alone. To address this concern, we performed TET1 knockdown experiments,\nwhich confirmed that TET1, rather than TET2, is the primary enzyme responsible for\nincreasing 5hmC levels.\n3. Vitamin C is widely used to promote in vitro MSC osteogenesis. Besides regulating TET\nfunction, it helps removing histone H3K9me3 and K27me3 and can improve collagen\nproduction. It is unlikely to act solely via enhancing TET protein function.\nResponse: Thank you very much for your insightful comments. Our current study\nhighlights the critical role of TET1 in intragenic DNA demethylation, which is essential for\nmaintaining MSC stemness during early MSC culture. Vitamin C enhances TET-mediated\n5hmC DNA hydroxymethylation at the promoters, enhancers, and super-enhancers of\nbone-specific genes, and it also regulates histone demethylation of H3K9me3 and\nH3K27me35. Notably, the deletion of the 5hmC writers, TET1 and TET2, in vitamin C-\nsufficient murine bone leads to severe skeletal defects that closely resemble those seen in\nvitamin C-deficient Gulo knockout mice, despite the preservation of normal histone\ndemethylation5. These findings challenge the sufficiency of histone demethylation in\nmaintaining bone growth and skeletal integrity, emphasizing that vitamin C’s role in\nosteogenesis extends beyond its impact on TET protein function alone.\n4. PJ34 inhibited PARP1 activity by targeting its catalytic domain, preventing the synthesis\nof PAR polymers, which should lead to the accumulation of DNA damage and subsequent\ncellular senescence. Do you see the difference in PAR level between early and late MSCs?\nResponse: Thank you for your comment. We observed that PAR levels in murine MSCs\nfrom 24-week-old mice were significantly lower than those from 4- or 8-week-old mice\n(Extended Data Fig. 11a). Although PJ34 treatment effectively reduced TET1 PARylation by\ninhibiting PARP1’s catalytic activity and blocking PAR polymer synthesis, it did not disrupt\nthe interaction between TET1, PARP1, and CTCF (Extended Data Fig. 3). These findings\nsuggest that TET1 associates with PARP1 and CTCF independently of PARP1’s enzymatic\nfunction.\nWhile PJ34 treatment may potentially lead to increased DNA damage and subsequent\ncellular senescence due to impaired PARP1 function, the treatment duration in our\nexperiments was short-term. As a result, we did not observe overt signs of DNA damage or\nsenescence within this timeframe.\n5. In Fig 1a, the authors detected the global 5hmC level with dot-blot and observed a\nsignificant decrease in late MSCs. However, the dot-blot result of 5mC is not provided and\nquantified.\nResponse: Thank you very much for the comments. We have now provided and quantified\nthe dot-blot results for 5mC in early and late MSCs (Extended Data Fig. 2a). Our analyses\nrevealed an increase in 5hmC levels in early MSCs, as shown by both immunofluorescence\n(Fig. 1a) and dot-blot assays (Extended Data Fig. 2a). In contrast, 5mC levels were\nelevated in late MSCs (Extended Data Fig. 2a, Fig. 1a), aligning with findings from previous\nstudies6,7.\n6. In line 124, according to the early study, the authors mentioned that the TET1 and TET2\ndouble knockout have lower 5hmC levels in mouse BM-MSCs than the TET1/2KO or control\ngroup. TET2-KD 5hmC does not change 5hmC level (Fig 1h), while the expression level of\nTET2 seems to be higher than that of TET1 in the control MSC (Fig 1g). It is known that\nTET1 and TET2 have overlapping functions and exhibit distinct expression patterns in\ndifferent cellular contexts. It is unclear whether they target different genes and have a\ndistinct role in MSC. How much TET2 contributes to the MSC properties and aging was not\nassessed and discussed further. It would be helpful to have parallel TET2-KD experiments.\nResponse: Thank you very much for the comments. We appreciate the reviewer's\nsuggestion regarding the importance of performing TET2 knockdown experiments in\nparallel with TET1 knockdown to fully dissect their roles in early MSC properties. Our\ncurrent data already provide strong evidence that TET1, but not TET2, knockdown\nsignificantly reduces 5hmC levels, as demonstrated by both immunofluorescence and dot\nblot analysis. While dot blot suggests that both TET1 and TET2 knockdowns reduce 5hmC,\nthe immunofluorescence results indicate a more prominent effect for TET1. These results\nsuggest that TET1 plays a dominant role in regulating 5hmC levels in MSCs.\nThe discrepancy between immunofluorescence and dot blot results in TET2 knockdown\nlikely arises due to differences in detection sensitivity and the nature of the assays. Dot\nblot provides a global, quantitative measurement of total 5hmC levels and is more\nsensitive to small reductions, whereas immunofluorescence captures regional 5hmC\ndistribution and may not detect subtle, widespread changes. This suggests that TET2\ninfluences 5hmC at a genome-wide level but does not cause strong focal reductions in\nnuclear 5hmC staining. Given that TET1 knockdown significantly affects both dot blot and\nimmunofluorescence, our results support the dominant role of TET1 in regulating 5hmC in\nMSCs.\nPerforming additional TET2 knockdown experiments in early MSCs would be a substantial\nundertaking, requiring extensive time and resources. Given that our key findings\nemphasize TET1’s role, we believe our current data sufficiently support our conclusions.\nHowever, if the reviewer considers Tet2’s contribution critical to our study’s main\nconclusions, we could consider conducting a limited set of TET2 knockdown experiments in\nMSCs in future follow-up studies.\n7. The bioinformatics data needed to be analyzed and described more sufficiently. How\nmany differentially expressed genes are observed from the RNA-seq? What are the altered\npathways? Are those pathways relevant to the phenotypes? And can the altered genes in\nthese pathways be clustered well within each group? Does the heatmap of Fig 2C display\nCPM values or Z-scores? Additionally, are these values representative of all genes? A batch\neffect is noted from the late/KD cluster between donors 1 and 2. The hierarchical\nclustering showed that the TET1-KD and PARP1-KD tend to be closer to the late MSC.\nResponse: Thank you very much for the comments. The analysis of the RNA-seq data is\ndescribed in details in the Method section in the revised manuscript. There are 1026\ndifferentially expressed genes (DGEs) by comparing early MSCs to late MSCs from the\nthree donors. Pathway analysis of these DGEs revealed several altered pathways that are\nresponsible for the early MSC phenotypes. The top 20 activated biological functions,\nincluding differentiation of osteoblast and adipogenesis of cells, and top 20 suppressed\nbiological functions are presented in Extended Data Fig. 4 in the revision. Moreover, the\nenrichment of the DGEs in the altered pathways within each group is evident by P-value\n(Dataset S1). The heatmap of Fig. 2c displays Z-scores and the information is stated in the\nFigure legend. We recognize the individual difference among the three donors, especially\ndonor 3 has a quite distinct expression profile from donors 1 and 2. Interestingly, donor 3\nis older than donor 1 and donor 2 (Table S7). Although batch effect did exit when RNA-seq\nassays were performed in different flow cells, the TET1-KD and PARP1-KD tending to be\ncloser to the late MSCs is more relevant to biological effect than batch effect, as the early\nMSC, late MSC, TET1-KD, and PARP1-KD of donor 1 were subjected to NGS in the same\nflow cell. In Figure 2c, the hierarchical clustering showed that the TET1-KD and PARP1-KD\ntend to be closer to the late MSCs of donor 1 or 2, strongly suggesting that knocking down\nthese genes may drive MSCs toward a late-stage phenotype.\n8. In line 202, the authors may provide a detailed description of the subgroups of MSC and\nlist the number of genes in each subset. The supplementary tables are difficult to navigate,\nso an instruction spreadsheet explaining the contents of each spreadsheet will be helpful.\nResponse: Thank you very much for the helpful comments. We have provided a schematic\ndiagram for each subset in Supplementary Table 2 to aid interpretation, following the\nformat used in a previous study8. Supplementary Table 2 includes the counts of five types\nof alternative splicing events identified by rMATS: SE (skipped exon), A5SS (alternative 5′\nsplice site), A3SS (alternative 3′ splice site), MXE (mutually exclusive exons), and RI\n(retained intron). Please note that the analysis was performed using a cutoff of FDR < 0.05\nand |ΔPSI| > 0.1. To improve usability, we will also provide an instruction sheet for\nSupplementary Table 2, as well as Datasets S2, S3, and S4, summarizing the contents and\nstructure of each spreadsheet.\n9. Extended Data Fig. denotes the colocalized ChIP-seq peaks of PARP1 and TET1 in the\ngenome browser. Is that locus a TSS; if yes, how is TSS defined here? Please provide the\npeak coordinate in the browser view and figure legends.\nResponse: Thank you for your valuable comment. The transcription start site (TSS for\nDNMT1 was defined based on the first nucleotide of the mRNA that corresponds to the\n5’UTR according to the RefSeq database (NM_001130823.3; chr19:10305629). In\nExtended Data Fig. 6, the DNA region of promoter, exon 1, and part of intron 1 of the\nDNMT1 gene is enlarged to show physical coordinate of TSS flanking region and ChIP-seq\npeaks. We have also revised the figure legends: Extended Data Fig. 6. PARP1 and TET1\nChIP-seq analysis revealed that the flanking region of transcription start site (TSS, Chr19:\n10305629) of DNMT1 was enriched with PARP1 and TET1 in early MSCs.\n10. The decrease of DNMT1 in PARP1 and TET1 KD suggests the alternative splicing of\nDNMT1; has this been confirmed from RNA-seq data -- a genome browser view of the\nDMNT1 sequence would make this conclusion more convincing.\nResponse: Thank you very much for the comments. We performed genome browser\nanalysis of the DNMT1 locus using RNA-seq data in Integrative Genomics Viewer (IGV), but\ndid not observe any promising alternatively spliced transcripts. One possible explanation is\nthat the alternatively spliced DNMT1 transcripts were not stable and therefore not captured\nby RNA-seq. Consistently, Western blotting of cell lysates from early and late MSCs, with or\nwithout pretreatment with MG132, also failed to detect this isoform (Extended Data Fig.\n9), further suggesting that the mRNA or protein isoform is unstable.\n11. In line 238. The spliced DNMT1 lost the DNMT1 activity. It was also observed that the\naddition of AS-DNMT1 abrupted the activity of Full-DNMT1. This observation is interesting\nbut needs further investigation/explanation. Does the overexpressed AS-DNMT1 change\noverall DNA methylation, and are those genes related to cellular senescence?\nResponse: Thank you very much for the thoughtful comments. Compared to the control\nvector (CV), overexpression of AS-DNMT1 led to a reduction in global 5mC levels in 293T\ncells (Fig. 3j), suggesting a dominant-negative effect on DNA methylation. Consistently, in\nMSCs, AS-DNMT1 overexpression resulted in upregulation of the senescence-associated\ngenes p21 and p16, along with impaired proliferation and differentiation potential\ncompared to CV (Fig. 3k–3m). These genes, p21 and p16, are canonical markers of cellular\nsenescence. Their upregulation in AS-DNMT1–overexpressing MSCs supports the idea that\nAS-DNMT1 contributes to senescence-associated phenotypes through epigenetic\nderegulation. These findings indicate that AS-DNMT1 may functionally interfere with full-\nlength DNMT1, potentially linking alternative splicing to stem cell aging. Further\nmechanistic studies are warranted to elucidate the precise pathways involved.\n12. Is DNMT1 the only target of this mechanism in alternative splicing? Considering TET1\naffects the epigenome broadly, it is puzzling why only DNMT1 is selected for analysis.\nResponse: Thank you very much for the insightful comments. We agree that TET1 broadly\ninfluences the epigenome and may impact the splicing of multiple genes. To identify\npotential targets, we examined CTCF binding motifs within the regions showing triple\noverlapping enrichment from MeDIP-seq, PARP1 ChIP-seq, and TET1 ChIP-seq, which\nyielded 20 candidate genes (Table S3). Among these, DNMT1 and LPA (Lipoprotein A)\nexhibited the highest number of predicted CTCF motifs. We chose to focus on DNMT1\nbecause of its well-established role in maintaining mesenchymal stem cell (MSC)\nstemness, a key factor in preserving stem cell identity3. While other genes may also be\ninvolved, DNMT1 was prioritized due to its strong functional relevance to our study.\nMinor points:\n1. In line 65, the authors said, “However, this does not explain why the telomers of cancer\ncells are paradoxically shorter than those found in normal tissue.” This seems irrelevant to\nany part of the results.\nResponse: Thank you very much for the comments. We have now deleted this description\nin the revised version.\n2. In line 152, the author mentioned, “PARP1 and TET2 contribute to an epigenetic\nprogram ... during somatic cell reprogramming”, but the objective is to investigate the\nPAPR1 and TET1 interaction in MSC. Please provide the known PARP1 and TET1 interaction.\nResponse: Thank you very much for your valuable comment. We have now included\nprevious studies that describe the interaction between PARP1 and TET1. “For instance, it\nhas been reported that PARP1 facilitates the establishment of H3K4me3 marks at the TET1\npromoter and directly interacts with TET1, contributing to the progression of T-cell acute\nlymphoblastic leukemia9” Please refer to Page 9 Line 8 – Page 9 Line11.\n3. In Line 61, “analyzed the related heat maps” may be changed to “we further analyzed\nthe RNA-seq of gene expression profiles …”. Similarly, in the Method, line 533. The original\ntexts sound like the RNA-seq was only used to generate a heatmap.\nResponse: Thank you very much for the insightful comment. We have revised the\nmanuscript accordingly. Specifically, the sentence in Line 61 has been modified to “we\nfurther analyzed the RNA-seq of gene expression profiles…” to better reflect the scope of\nthe analysis. In the Methods section, the original title “RNA-seq analysis for heatmap” has\nbeen revised to “RNA-seq and transcriptomic data analysis” to clarify that the RNA-seq\ndata were used for comprehensive transcriptomic analysis, not solely for generating\nheatmaps. Please refer to Page 33 Line 6.\n4. Figure legend of Fig 3g, please provide the definition of #1 and #2\nResponse: Thank you very much for the helpful comment. We have revised the figure\nlegend in the manuscript to clarify that “#1” and “#2” refer to two different shRNA\nconstructs targeting CTCF.\n5. Line 1099. Please add T203E.\nResponse: Thank you very much for the comments. We have revised them in the Figure 5h\nlegend in the revised manuscript.\n6. Please check text consistency, such as 5hmC vs. 5hmc, 5mC vs.5 mC.\nResponse: Thank you very much for your comment. We have carefully revised the\nmanuscript to ensure consistency in the formatting of terms such as 5mC and 5hmC.\nReviewer #4 (Remarks to the Author):\nSIGNIFICANCE\nThis study attempts to create mechanistic connections between ATM, TRIM37, PARP1,\nTET1 and CTCF in modulating methylation of the DNMT1 gene to mediate alternative\nsplicing of DNMT1 to modulate the proliferative properties of MSCs.\nCRITIQUE\n1. One overall concern is that while the multiple mechanistic connections are theoretically\nfeasible and somewhat supported by the data, experimental evidence for each connection\nis of poor quality, not well controlled and generally not compelling.\nResponse: We appreciate the reviewer’s concern regarding the quality, control, and\nstrength of the experimental evidence supporting our proposed mechanistic connections.\nIn response to this overarching critique—as well as to specific comments from all four\nreviewers—we have extensively revised the manuscript to significantly enhance both the\nrigor and clarity of our findings.\nSpecifically, we have conducted additional well-controlled experiments to directly address\neach mechanistic link. For example:\nThe levels of 5hmC and 5mC, originally detected by immunofluorescence, are now further\nvalidated using dot-blot analysis with quantification (Extended Data Fig. 2).\nWe demonstrate that re-expression of shRNA-resistant TRIM37 wild-type (WT) and T203E\nmutant restore PARP1 expression, as well as MSC proliferation and differentiation capacity,\nwhile expression of the T203A mutant does not (Extended Data Fig. 10).\nWe treated aged mice (75 weeks old) with an ATM activator (resveratrol) and/or a TET\nactivator (vitamin C). This combinatorial treatment significantly improved bone\nparameters—such as BMD, BV/TV, Tb.N, and Tb.Th—and reduced BS/BV and Tb.Sp.\nMoreover, it enhanced bone repair following injury compared to both control and single-\ntreatment groups (Fig. 8).\nThese additional data now provide strong, experimentally substantiated support for the\nmechanistic model proposed. We have also strengthened the statistical analyses and\nquantification throughout.\nWe believe these comprehensive revisions have substantially improved the manuscript.\nThe experiments are now rigorously controlled, the results are robust, and the mechanistic\nconclusions are compelling.\n2. The title severely overstates the findings. This paper does not present any direct data\nthat TRIM37 mitigates osteoporosis. In fact, nothing in this paper has even a remote\nbearing on osteoporosis. This problem of overstating the strength of the evidence is\nsymptomatic for other findings in the paper.\nResponse: We respectfully disagree with the reviewer’s statement that our findings lack\nrelevance to osteoporosis. Our in vivo data clearly support the conclusion that TRIM37 is\ncritical not only for maintaining skeletal homeostasis and preventing age-related bone loss,\nbut also for preserving bone repair capacity—both of which are hallmark features of\nosteoporosis.\nFirst, TRIM37-deficient mice exhibit a pronounced osteoporotic phenotype, characterized\nby significant reductions in bone mineral density (BMD), bone volume fraction (BV/TV),\nand trabecular number (Tb.N), along with increased trabecular separation (Tb.Sp) (Fig.\n7b). Furthermore, these mice show impaired bone repair capacity following cortical bone\ninjury (Fig. 7e).\nSecond, and importantly, TRIM37 deficiency compromises the bone’s intrinsic ability to\nheal. In a cortical defect model, TRIM37-deficient mice demonstrated delayed bone\nhealing. However, treatment of aged mice (75 weeks old) with an ATM activator\n(resveratrol) and/or a TET activator (vitamin C) not only improved structural bone\nparameters (BMD, BV/TV, Tb.N, Tb.Th, BS/BV, Tb.Sp) (Fig. 8a), but also significantly\nenhanced bone regeneration at the defect site compared to vehicle or single-agent\ntreatments (Fig. 8b). Notably, the combination treatment led to the greatest improvement\nin bone repair capacity following cortical injury (Fig. 8c).\nTogether, these findings provide compelling biological and functional evidence that TRIM37\ndeficiency contributes to an osteoporosis-like condition, and that modulation of the\ndownstream ATM/TET pathway can partially rescue both bone loss and regenerative\ndeficits. We believe the current title accurately reflects the significance of our study,\nthough we remain open to revising it should the editors prefer a more conservative\nphrasing.\n3. TRIM37 has a wide range of cellular activities and molecular functions. Effects on\nDNMT1 function by modifying alternative splicing of DNMT1 represents a very specific\nmechanism that is unlikely to be the only or principal target of TRIM37.\nResponse: We appreciate the reviewer’s comment regarding the diverse functions of\nTRIM37 and the specificity of its effects on DNMT1. However, we respectfully submit that\nour data clearly demonstrate a mechanistically specific and functionally significant role for\nTRIM37 in regulating PARP1 stability through mono-ubiquitination at K337, and that this\nprocess is critically dependent on phosphorylation of TRIM37 at T203.\nTo identify upstream regulators of PARP1, we performed immunoprecipitation-coupled\nmass spectrometry (IP-MS/MS), which revealed phospho-TRIM37 (T203) as one of the\nmost enriched phosphoproteins associated with PARP1 in early MSCs (Fig. 4a). This\ninteraction was confirmed biochemically in 293T cells co-transfected with Flag-TRIM37 and\nHA-PARP1 (Fig. 4b). As TRIM37 is known to function as an E3 ubiquitin ligase, we\nexamined whether it ubiquitinates PARP1. Our data demonstrate that TRIM37 mediates\nmono-ubiquitination of PARP1 specifically at Lys337 (Fig. 4c), and that this modification\nstabilizes PARP1.\nImportantly, this mono-ubiquitination and stabilization require phosphorylation of TRIM37\nat T203. In 293T cells, the non-phosphorylatable T203A mutant showed markedly reduced\nability to bind, ubiquitinate, and stabilize PARP1, while the phosphomimetic T203E mutant\nsignificantly enhanced these processes (Fig. 4d–e). Moreover, in functional rescue\nexperiments using TRIM37-knockdown MSCs, only re-expression of wild-type or T203E\nTRIM37—not T203A—restored PARP1 protein levels, enhanced proliferation, reduced\nsenescence, and rescued osteogenic and adipogenic differentiation (Extended Data Fig.\n10). These findings firmly establish that T203 phosphorylation is not a secondary or non-\nspecific event, but is essential for TRIM37’s role in regulating PARP1 function and\ndownstream MSC biology.\nFurthermore, we demonstrate that ATM kinase acts upstream by promoting TRIM37\nphosphorylation at T203, enhancing its nuclear localization and interaction with PARP1.\nEarly-passage MSCs display higher levels of activated ATM, TRIM37, and nuclear PARP1\ncompared to late-passage MSCs (Fig. 5a–e). Proximity ligation assays confirm enhanced\nnuclear association of TRIM37 with both phospho-ATM and PARP1 (Fig. 5f–\ng). Pharmacological activation of ATM by resveratrol further increased this nuclear complex\nformation. Conversely, overexpression of the T203A TRIM37 mutant impaired nuclear\ntranslocation of TRIM37, while the T203E mutant promoted it (Fig. 5h).\nTaken together, these data provide compelling mechanistic evidence that phosphorylation\nof TRIM37 at T203 is a critical regulatory event that enables its E3 ligase activity toward\nPARP1 at K337, thereby stabilizing PARP1 and supporting MSC proliferation and\ndifferentiation. This mechanism operates independently of TRIM37’s other functions and\nhighlights a highly specific, ATM-regulated pathway central to MSC function and\nsenescence.\n4. The authors state that “5hmC levels positively regulates MSC self-renewal and\nosteoblast differentiation, which is at odds with recent studies in Nat Comm that Vitamin C\ndependent 5hmC levels promote osteogenesis rather than stemness.\nResponse: Our results showed that old female mice daily administrated with Vit C and\nresveratrol only promoted osteogenesis but not adipogenesis in vivo, however MSCs\ntreated with Vit C and resveratrol promoted both osteogenesis and adipogenesis in vitro\n(direct effect).\n1. Vitamin C Enhances the Generation of Mouse and Human Induced Pluripotent Stem\nCells. Cell Stem Cell 1: 71-79, 2010.\n2. Ascorbic acid phosphate stimulates type IV collagen synthesis and accelerates adipose\nconversion of 3T3-L1 cells. Experimental Cell Research. Volume 187, Issue 2, April\n1990, Pages 309-314.\n3. Effect of ascorbic acid on bone marrow-derived mesenchymal stem cell proliferation\nand differentiation. Journal of Bioscience and Bioengineering.\n105: 586-594, 2008. Figure 7 & 8\n5. All blot based methods (dot blots and western blots) are of very poor quality and these\nblots cannot be properly quantified.\nResponse: We sincerely appreciate the reviewer’s feedback regarding the quality and\nquantification of our blot-based methods. We have carefully reviewed our data and have\nnow quantified all dot blot results using densitometric analysis with appropriate\nnormalization controls (Extended Data Fig. 2). These quantified data are now included in\nthe revised manuscript to improve clarity and rigor. We hope this addresses the reviewer’s\nconcern and appreciate the opportunity to further strengthen our study.\n6. The paper contains a number of avoidable spelling errors and generally does not meet\nhigh standards of academic writing expected for Nature brand papers (e.g., “Westering\nblotting”?)\nResponse: We appreciate the reviewer’s feedback regarding the language and presentation\nof our manuscript. We have carefully proofread the revised version to correct any spelling\nerrors, including the mentioned typo, and have improved the overall clarity and readability\nto meet the high standards expected for Nature-brand papers. We sincerely thank the\nreviewer for highlighting this, as it has helped us further refine our manuscript.\n7. Extended Data:\na) Experimental evidence for senescence presented (see extended data, Figure 1) is rather\nminimal and insufficient.\nResponse: We thank the reviewer for this valuable comment. To more comprehensively\ndemonstrate senescence in late-passage MSCs, we have expanded our experimental\nvalidation and clarified the evidence provided in Extended Data Fig. 1, which now includes\nmultiple, widely accepted hallmarks of cellular senescence:\nMorphological changes (Fig. 1a): Late-passage MSCs exhibit enlarged and flattened\nmorphology, a classical feature of senescent cells.\nSenescence markers (Fig. 1b): Western blot analysis shows a consistent decrease in\nDNMT1 and an increase in p16Ink4a and p21Waf1/Cip1, both well-established markers of\nsenescence.\nFunctional decline (Fig. 1c–d): Late-passage MSCs display reduced proliferation and\nimpaired differentiation potential into osteoblasts and adipocytes, both indicative of cellular\naging.\nTranscriptomic evidence (Fig. 1e): RNA-seq analysis followed by GSEA reveals significant\nenrichment of the senescence-associated secretory phenotype (SASP) pathway in late-\npassage MSCs.\nSecretory profile (Fig. 1f): Conditioned medium from late MSCs shows elevated secretion\nof key SASP components, including IL-1α and CXCL-8, as measured by ELISA.\nSA-β-galactosidase activity (Fig. 1g): Increased SA-β-gal⁺ cell percentage in late MSCs\nfurther confirms senescence at the cellular level.\nTogether, these multiple independent lines of evidence (morphological, molecular,\nfunctional, transcriptomic, and biochemical) provide a robust and multidimensional\ncharacterization of senescence in late-passage MSCs. We hope this additional clarification\nand the strengthened figure address the reviewer’s concern comprehensively.\nb) The quality of immunoprecipitations (see extended data, Figure 2) is not compelling and\nlack essential controls. The resulting conclusion that PARP1, TET1 and CTCF directly\ninteract is not evident from the data shown.\nResponse: We thank the reviewer for this thoughtful and constructive comment. In\nExtended Data Fig. 3, we show that anti-TET1 immunoprecipitates both PARP1 and CTCF,\nwith consistent results observed under both control and PJ34-treated conditions.\nAdditionally, Fig. 2a includes a co-immunoprecipitation experiment supporting the\ninteraction between PARP1 and TET1. IgG immunoprecipitations were included as negative\ncontrols to assess nonspecific binding.\nWe acknowledge, however, that co-immunoprecipitation does not distinguish between\ndirect and indirect interactions. To further address this, we are currently performing\nproximity ligation assays (PLA) to visualize the spatial proximity of endogenous PARP1,\nTET1, and CTCF in situ. In untreated cells, PLA signals for TET1–PARP1 and TET1–CTCF are\nprimarily nuclear, whereas PJ34 treatment leads to additional cytoplasmic PLA signals. This\ncytoplasmic redistribution upon PARP1 inhibition is consistent with observations reported in\nprevious studies 10. While this redistribution may reflect a disruption of nuclear retention or\nchromatin association—potentially due to PARP1’s role in stabilizing chromatin interactions\nand anchoring nuclear proteins, such as TET1 and CTCF—we would like to emphasize that\nthe underlying mechanism of this relocalization is beyond the scope of the present study\nand will not be further investigated. Nevertheless, we will include these additional PLA\nresults in the revised manuscript or supplementary materials (Extended Data Fig. 3b) to\nprovide stronger support for the spatial proximity and potential interaction among PARP1,\nTET1, and CTCF.\nc) The ChIP-seq tracks (see extended data, Figure 3) claim to show TET1 and PARP1\nbinding to the TSS but there are upstream exons that are not annotated giving the\nappearance that the location is intragenic. Input background is high and the level of\nenrichment appears to be limited.\nResponse: We thank the reviewer for their insightful comments regarding the ChIP-seq\ntracks shown in Extended Data Fig. 6 in the revised version. We would like to clarify and\naddress the points raised as follows:\nFirst, regarding transcript annotation: we acknowledge that the DNMT1 gene locus\ncontains alternative upstream exons and TSSs that are not consistently annotated in all\ngenome builds or databases. In the updated figure, we now include expanded RefSeq\ntranscript tracks, which show that the observed PARP1 and TET1 binding peaks\n(highlighted in orange) are located immediately upstream of exon 1 of a major DNMT1\nisoform, and thus are consistent with proximal promoter and TSS binding, not intragenic\nbinding.\nSecond, we appreciate the comment regarding the appearance of intragenic binding. The\nearlier visual ambiguity was due in part to limited annotation in the displayed tracks. We\nhave corrected this in the revised figure, which supports that the peaks align with the\ncanonical promoter region.\nThird, concerning input signal and background, we recognize that the input tracks showed\nmoderate baseline levels. To improve clarity, we have now reprocessed the ChIP-seq data\nwith normalized tracks and adjusted dynamic ranges. Importantly, ChIP enrichment over\ninput is clearly seen in both TET1 and PARP1 tracks compared to the corresponding inputs,\nespecially at the promoter region of DNMT1 (highlighted in orange).\nLastly, while the absolute enrichment may appear modest, we note that TET1 and PARP1\nare chromatin regulators with generally broader and lower-amplitude binding profiles,\nwhich is consistent with previous reports 11,12. Nevertheless, we agree with the reviewer\nthat careful presentation is necessary, and we have now updated the figure and legend to\nclearly indicate TSS positions, highlight the peak regions, and explain the biological\nrelevance of these binding events.\nWe hope these clarifications and figure revisions adequately address the reviewer’s\nconcern and improve the transparency and rigor of our ChIP-seq analysis.\nd) The compilation of data on effects of TRIM37 and PARP1 on MSC activity (see extended\ndata, Figure 7) is of poor quality and not convincing.\nResponse: We sincerely thank the reviewer for this valuable comment. We acknowledge\nthat the original data presented in Extended Data Figure 7 may not have been of sufficient\nquality to convincingly support the conclusions regarding the roles of TRIM37 and PARP1 in\nMSC activity. In response to this concern, we have significantly revised and expanded our\ndataset. The updated and more robust data are now presented in Figure 4 and Extended\nData Figures 10 and 11.\nIn these new figures, we show that lentiviral knockdown of TRIM37 using two independent\nshRNAs led to decreased PARP1 and full-length DNMT1 protein levels, increased expression\nof senescence markers (Fig. 4f), impaired cell proliferation (Fig. 4g), and reduced\nosteogenic and adipogenic differentiation capacity (Fig. 4h) compared to scrambled shRNA\ncontrols.\nTo further strengthen our mechanistic insight, we performed rescue experiments using\nshRNA-resistant constructs encoding wild-type (WT), T203A (non-phosphorylatable), or\nT203E (phosphomimetic) TRIM37 in TRIM37-knockdown MSCs. Re-expression of WT or\nT203E TRIM37 restored PARP1 expression, reduced senescence marker expression, and\nrescued both proliferation and differentiation potentials. In contrast, the T203A mutant\nfailed to rescue these phenotypes (Extended Data Fig. 10), highlighting the functional\nimportance of T203 phosphorylation in TRIM37-mediated regulation of MSC biology.\nTo address the in vivo relevance, we isolated minimally expanded BM-MSCs from young\n(<8 weeks) and older (6–10 months) B6 mice. Western blotting revealed age-associated\ndecreases in TRIM37, PARP1, PAR, and DNMT1, along with increased levels of p16Ink4a and\np21Waf1/Cip1 (Extended Data Fig. 11a, b). Furthermore, BM-MSCs isolated from TRIM37- or\nPARP1-deficient mice exhibited reduced TRIM37, PARP1, and DNMT1 protein levels,\nelevated senescence markers (Extended Data Fig. 11c–f), and significantly impaired\nproliferation (Extended Data Fig. 11g, h) and differentiation potential (Extended Data Fig.\n11i, j) compared to WT controls.\nCollectively, these improved and extended datasets provide compelling evidence for the\nrole of TRIM37 and PARP1 in regulating MSC senescence and functional decline both in\nvitro and in vivo.\ne) Effects of PARP1 knockout on bone healing in a drill hole model are not quantified and\nqualitative effects are not apparent.\nWe sincerely appreciate the reviewer’s comment regarding the assessment of bone healing\nin the drill hole model. To address this concern, we have now included quantitative analysis\nof new bone formation using fluorescent densitometry of calcein labeling, as presented in\nthe updated Supplemental Fig. 1. This quantification, based on ImageJ analysis of\nintegrated fluorescence density (IntDen), complements the qualitative imaging data\nobtained via two-photon microscopy and SHG. To further improve clarity, we have also\nincluded magnified images to better visualize the bone regeneration signals. These\nadditions have been incorporated into the revised manuscript to enhance both the clarity\nand scientific rigor. We thank the reviewer again for this constructive suggestion.\nf) Supplemental Table 7 shows that only three MSC donors were considered and these\ndonors were neither age nor gender matched.\nWe thank the reviewer for pointing out the limited number of MSC donors and the lack of\nstrict age or gender matching in Supplemental Table 7. We acknowledge this as a limitation\nof the study. Nonetheless, even with three donors, our findings yielded biologically\nmeaningful and reproducible insights, consistent with previous studies (e.g., Nat Commun.\nin the revised manuscript as a study limitation.\nTake RNA-seq as an example: although only three donors were used, the transcriptomic\nprofiles still revealed consistent and biologically relevant patterns. We recognize that\ndonor-specific differences exist—for instance, Donor 3, who is older than Donors 1 and 2\n(Table S7), showed a distinct gene expression profile. However, this variation may reflect\ntrue biological aging effects rather than being solely a technical artifact.\nAlthough batch effects are a valid concern, especially since RNA-seq libraries were\nsequenced on different flow cells, the observed clustering of TET1-KD and PARP1-KD\nsamples with late-passage MSCs suggests that biological signals outweighed technical\nvariation. Notably, for Donor 1, early MSCs, late MSCs, TET1-KD, and PARP1-KD samples\nwere processed in the same sequencing run, minimizing technical confounders. In Figure\n2c, hierarchical clustering showed that TET1-KD and PARP1-KD samples closely resemble\nlate MSCs from Donor 1 or 2, indicating that knockdown of these genes drives a late-stage\nor senescent-like molecular phenotype.\nWe have also revised the manuscript to clearly acknowledge the limited donor number and\naddress donor variability while highlighting the biological consistency observed, particularly\nin RNA-seq analyses. “This study analyzed MSCs from three human donors who were not\nstrictly age- or gender-matched, which may contribute to inter-donor variability. Despite\nthis, key findings—such as transcriptomic shifts in TET1- and PARP1-KD MSCs resembling\nlate-passage MSCs—were consistent across donors. Notably, Donor 1 samples, including\nearly, late, and knockdown MSCs, were sequenced in the same flow cell, minimizing batch\neffects and reinforcing the biological relevance of the clustering patterns. While Donor 3\ndisplayed a distinct gene expression profile, likely related to older age, this variation\nhighlights the natural heterogeneity in human MSCs. Nevertheless, future studies with\nlarger, age- and gender-matched donor cohorts will be essential to validate and extend\nthese results.” Page 28 Line 13 – Page 29 Line 2.\nReferences\nreceptor-expressing mesenchymal stromal cells represent the main source of\nbone formed by adult bone marrow. Cell Stem Cell 15, 154-168,\ndoi:10.1016/j.stem.2014.06.008 (2014).\nreprogramming by Parp1 and Tet2. Nature 488, 652-655,\ndoi:10.1038/nature11333 (2012).\ndirectly regulate Dnmt1 to maintain self-renewal and undifferentiated state in\nmesenchymal stem cells. Mol Cell 47, 169-182,\ndoi:10.1016/j.molcel.2012.06.020 (2012).\nPharmacol Sci 28, 556-560, doi:10.1016/j.tips.2007.08.005 (2007).\nmineralization. Nat Commun 13, 5883, doi:10.1038/s41467-022-32915-8\n(2022).\nbone marrow mesenchymal stem cells in long-term culture. Exp Mol Med 44,\n503-512, doi:10.3858/emm.2012.44.8.057 (2012).\n(1991).\nepidermal differentiation. Cell Rep 43, 113814,\ndoi:10.1016/j.celrep.2024.113814 (2024).\nleukemia and can be antagonized via PARP inhibition. Leukemia 35, 389-403,\ndoi:10.1038/s41375-020-0864-3 (2021).\nglioma-conditioned brain microvascular endothelial cells. Eur J Pharmacol\n761, 55-64, doi:10.1016/j.ejphar.2015.04.026 (2015).\nwhy? Mol Cell 49, 825-837, doi:10.1016/j.molcel.2013.01.038 (2013).\ngenome. Nature 489, 57-74, doi:10.1038/nature11247 (2012)."}, {"round": 2, "reviewer_comments": {"reviewer_1": "The author has adequately addressed my concerns. I suggest that it be published.", "reviewer_2": "The authors have carefully and appropriately addressed nearly all of my comments. The revised manuscript has improved in\nclarity and rigor. I believe it is suitable for publication in its current form.", "reviewer_3": "The concerns from previous review was sufficiently addressed."}, "author_response": ""}]

Decision letter