Cell culture and reagent treatment
Bone marrow aspirates were collected from patients who received an orthopedic surgery in Taipei Veterans General Hospital with the approval of Institute of Review Board. Informed consent was obtained from all subjects. Details regarding the donor information are shown in Table S7. The protocols of MSC isolation and expansion were modified from previously described methods67. In brief, mononuclear cells were isolated from heparinized bone marrow by density gradient centrifugation using Ficoll-Hypaque (Sigma-Aldrich, St. Louis, MO) of a density of 1.077 g/L, followed by seeding into 6-well plate 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% CO2 atmosphere. At 9 days after seeding, MSCs were recovered and reseeded in 10-cm plastic dishes at an initial density of 4 × 103 cells/cm2 and the culture medium was changed twice per week. The medium was changed twice a week and a sub-culture was performed at 1:3 to 1:5 split every 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 5mM 2-HG in CM. For treatment with KU55933 (SML1109, Sigma-Aldrich, St. Louis, MO), cells were cultured with 10 mM KU55933 in CM. For treatment with resveratrol (R5010, Sigma-Aldrich), cells were treated with 5 mM resveratrol in CM. For treatment with vitamin C (A4544, Sigma-Aldrich), cells were cultured with 50 mg/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)/log2, where N= cell number harvested during passaging and N0= cell number at initial seeding.
Senescence-Associated β-Galactosidase (SA-β-gal)
Senescence-associated β-galactosidase (SA-β-gal) staining was performed using a β-galactosidase staining kit (BioVision, Milpitas, CA, USA) according to the manufacturer’s instructions. Briefly, MSCs at passage number 5, 9, and 13 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. The SA-β-gal-positive cells were observed in phase contrast images. SA-β-gal-positive cells were observed and acquired at room temperature using an inverted microscope (Nikon Eclipse TE2000, Tokyo, Japan) with a 20´/0.40 objective equipped with a camera (Nikon Corporation, Tokyo, Japan).
Proliferation assay
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 WST-1 kit (Cayman Chemical Company, Ann Arbor, MI) at indicated time periods. Absorbance was measured at a wavelength of 450 nm.
Cell differentiation protocols
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
Genomic DNA samples were prepared in 2N 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℃ 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 in 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 analysis for heatmap
Total RNA was isolated 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. Early MSCs, late MSCs, TET-KD MSCs and PARP1-KD MSCs RNA-Seq experiments are with three biological replicates. More than 30 million (mean ± standard deviation= 37,392,922 ± 3,100,588) 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. The human genome assembly hg38 including un-placed and un-localized scaffolds and RefGene annotation were downloaded from the University of California Santa Cruz (UCSC) Genome Browser on 2017.1.248. Sequencing bases with low quality were trimmed based on the Phred + 33 quality score (>20) from both of the 5’- and 3’- ends of reads. After trimming, reads were discarded, if they were shorter than 75 bp, or had one or more ambiguous base. The alignment, quantification, normalization, and differential expression analysis were performed by STAR 2.5.3a through Partek Flow (Partek Inc., St. Louis, MO), htseq-count 0.13.5, and edgeR 3.18.1, respectively. Genes with count-per-million (CPM) values above 1 in at least three samples were retained, and genes with no or low expression levels were discarded. False discovery rate (FDR) < 0.05 was set as a threshold to identify differentially expressed genes. 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)48, which detects five major types of AS events from RNA-Seq data (GSE178804) with replicates68. In each analysis, the experimental groups (late, PARP1KD or TET1KD 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.
5’mC/5’hmC DIP and High-Throughput Sequencing Analysis
Aliquots of purified genomic DNA from each situation of MSCs were first sonicated to ∼250 bp using Bioruptor (Diagenode, Seraing, Belgium), then end-repaired and ligated to Illumina PE adaptors using NEB Next 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 5’hmC 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 N2IndSeq primer. After base-calling, each sequence was decoded using an in-house Perl script which required at least five out of six matching positions in the barcode sequence. All sequencing reads were trimmed from 5’ end to obtain final 37 bases in length for further analysis. Raw data are accessible at NCBI GEO with accession number GSE178805. All relevant data are available from the authors with restrictions.
Immunofluorescence
Cells were fixed with 4% paraformaldehyde. For 5-hmC and 5-mC detection, cells were treated with 2N HCl for 30 min at 37°C, incubated with an anti-5-hmC or anti-5-mC 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 TCS SP8 (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 (DUO92101-1 KT, Sigma-Aldrich), cells were counterstained with DAPI and in situ protein-protein association was analyzed with a Leica TCS SP8 confocal system.
Lentiviral vector production and cell infection
The expression plasmids and the bacteria clone for PARP1 shRNA (TRCN0000007932), TET1 shRNA (TRCN0000075026), TET2 shRNA (TRCN0000144344), CTCF shRNA (TRCN0000218498 and TRCN0000230191), and TRIM37 shRNA (TRCN0000221060 and TRCN0000370523) were provided by the RNAi core of National Science Council in Taiwan. Lentiviral transfer vector pLAS2w.Ppuro carrying PARP1 (NM_001618.4) was used to generate stable PARP1 expression cells. Lentiviral production was performed by transfection of 293 T 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 48h.
Western blot
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), p16 (#92803, Cell Signaling Technology, Danvers, MA), p21(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 Shieh69, Dr. Ping-Hui Tseng, and Heng-Hsiung Wu70, 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 (Table S8). 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.
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 (Table S9), and high-throughput sequencing analysis.
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 Table S9.
Animals and mouse bone marrow MSC isolation
The animal study was carried out in accordance with the recommendations of 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). The C57BL/6-PARP1 deficient mice (129S-Parp1tm1Zqw/J ) and corresponding WT mice were purchased from The Jackson Laboratories (Bar Harbor, ME). The C57BL/6-Trim37em1(flox)Smoc and C57BL/6-Tg(Pgk1-RFP, -cre/ERT2)3Narl (RMRC13179) mice were gifted from Prof. Ping-Hui Tseng (National Yang Ming Chiao Tung University), and purchased from the National Laboratory Animal Center (Taipei, Taiwan). TRIM37 deficient mice were produced through cross-breeding C57BL/6-Trim37em1(flox)Smoc and C57BL/6-Tg(Pgk1-RFP, -cre/ERT2)3Narl mice.
For mouse bone marrow 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 previously71. In brief, bone marrow cells were cultured in 100-mm culture dishes with complete alpha MEM medium (with 16.6% FBS) and incubated at 37 °C with 5% CO2 in a humidified chamber. After 12 h, the nonadherent 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
At 8 weeks, wild-type (WT), TRIM37 deficient or PARP1 deficient B6 mice were anesthetized with 2.5–5% isoflurane with oxygen. A longitudinal incision with 1-cm in size was made in the mid portion of right thigh in the mouse, followed by retraction of soft tissue with small retractors. A single cortical drill hole was created by drilling a 1-mm pin from the outside and care was taken to prevent damage to the opposite cortex. The drill hole was then washed with PBS and wound was closed with 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 2´ objective (PLAN APO λD 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 split 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 Image J software. The type II collagen structure reconstructed through the second harmonic generation images showed porous collagen fiber inter-connected structures (white color) with fluorescent calcein (green area).
Statistical analysis and reproducibility
Data are presented as mean ± SD, statistical comparisons were performed by Student’s t-test or one-way analysis of variance (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.