Transformation of 5-Carboxylcytosine to Cytosine Through C–C Bond Cleavage in Human Cells Constitutes a Novel Pathway for DNA Demethylation

Open AccessCCS ChemistryRESEARCH ARTICLE1 Apr 2021Transformation of 5-Carboxylcytosine to Cytosine Through C–C Bond Cleavage in Human Cells Constitutes a Novel Pathway for DNA Demethylation Yang Feng, Neng-Bin Xie, Wan-Bing Tao, Jiang-Hui Ding, Xue-Jiao You, Cheng-Jie Ma, Xiaoxue Zhang, Chengqi Yi, Xiang Zhou, Bi-Feng Yuan and Yu-Qi Feng Key Laboratory Analytical Chemistry Biology Medicine (Ministry Education), Sauvage Center Molecular Sciences, Department Chemistry, Wuhan University, 430072 Google Scholar More articles by this author , Xie Tao Ding You Ma Zhang State Protein Plant Gene Research, School Life Peking Beijing 100871 Peking-Tsinghua Academy Advanced Interdisciplinary Studies, Chemical Synthetic Functional Biomolecules Center, College Engineering, 100871. Yi Zhou *Corresponding author: E-mail Address: [email protected] https://doi.org/10.31635/ccschem.020.202000286 SectionsSupplemental MaterialAboutAbstractPDF ToolsAdd favoritesTrack Citations ShareFacebookTwitterLinked InEmail Active demethylation 5-methylcytosine (5mC) can be realized through ten-eleven translocation (TET) dioxygenase-mediated oxidation 5mC 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC), 5-carboxylcytosine (5caC), followed thymine glycosylase (TDG)-initiated base excision repair (BER). The TDG-BER pathway may lead the generation strand breaks, potentially compromising genome integrity. Alternatively, direct decarboxylation TET-produced 5caC is highly attractive because mechanism allows conversion cytosine without formation breaks. However, cleavage bond human cells remains an open question. We examined reaction cell extract live using 5caC-carrying hairpin substrate. After incubation with whole-cell protein or transfection into cells, we monitored transformation BER liquid chromatography–tandem mass spectrometry (LC-MS/MS) analyses at both mononucleotide oligodeoxynucleotide levels. Our results clearly showed providing evidence support novel active demethylation. Download figure PowerPoint Introduction 5-Methylcytosine CpG sites plays important role epigenetic regulation gene expression vertebrates.1,2 produced maintained (cytosine-5)-methyltransferases (DNMTs) via transfer methyl group from S-adenosylmethionine (SAM) C5 position genomic DNA.3 levels distributions are dynamic, current model mammals involves stepwise dioxygenases generate (5caC).4–11 Thymine (TDG) able recognize cleave N-glycosidic bonds 5fC produce abasic (AP-sites), which further processed (BER) machinery restore unmethylated cytosine.12–16 Although TET-TDG-BER best characterized vertebrates,17,18 it not account all scenarios. In particular, breaks as intermediates, compromise integrity.19 Additionally, was reported that mouse zygotes perturbed genetic deletion TDG, indicating presence initiated TET-mediated oxidation, but independent TDG-mediated BER.20 An alternative could possible 5caC. This realizes intermediates. Along line, previous study revealed undergo thiol-mediated acid-catalyzed C−C yield vitro.21 elimination formyl carboxyl groups requires transient addition nucleophile, example, thiol, C6 modified nucleobases 1,6 liberation formic acid CO2, respectively.21 corresponding nucleophile subsequently detached similar methylation employed DNMT proteins.22,23 Some bacterial mammalian also display potential activity vitro.24 Furthermore, biochemical data has demonstrated recombinant isoorotate decarboxylase, initially identified capable catalyzing 5-carboxyluracil uracil some fungi species, exhibited relatively weak cytosine.25 These vitro studies suggest deformylation occur vivo. Recently, Carell et al.26 DNA, first line cells. unclear whether occurs A investigation suggested treatment 5caC-containing synthetic nuclear embryonic stem led cytosine,27 raising possibility Herein, have investigated extracts provide strong supporting idea Experimental Methods Chemicals reagents 2′-Deoxyadenosine (dA), thymidine (T), 2′-deoxycytidine (dC), 2′-deoxyguanosine (dG), 2′-deoxyadenosine 5′-monophosphate (dAMP), (TMP), (dCMP), 5’-monophosphate (dGMP) were purchased Sigma-Aldrich (Beijing, China). 5-Methyl-2′-deoxycytidine (5mdC), 5-hydroxymethyl-2′-deoxycytidine (5hmdC), 5-formyl-2′-deoxycytidine (5fdC), 5-carboxyl-2′-deoxycytidine (5cadC) Berry & Associates (Dexter, MI, USA). Venom phosphodiesterase I S1 nuclease, DNase I, calf-intestinal alkaline phosphatase (CIAP), marker obtained Takara Biotechnology Co. Ltd. (Dalian, SspI restriction endonuclease New England Biolabs (Ipswich, MA, Dulbecco’s Modified Eagle medium (DMEM) fetal bovine serum Thermo-Fisher Scientific (Waltham, Chromatographic grade methanol acetonitrile Merck (Darmstadt, Germany). substrates 40-mer (5caC-DNA) synthesized substrate contains site, biotin tag 3′ terminus, seven oxygen atoms replaced sulfur phosphodiester ( Supporting Information Figure Table S1). 5caC-DNA form structures carry enzyme region S2). dissolved TE buffer (10 mM Tris-HCl, 1 EDTA) final concentration 50 μM. μM) annealed structure containing 20 Tris-HCl (pH 7.0), NaCl, 10 MgCl2, DTT heating 95 °C 5 min, gradual cooling °C. digested 4 μL (5 U/μL), 6 10× CutSmart (NEB), incubated 37 2 h. resulting product analyzed 20% polyacrylamide gel electrophoresis. sequences other listed S1. Cell culture stable isotope labeling kidney epithelial (HEK293T), cervical carcinoma (HeLa), breast adenocarcinoma (MCF-7) China Type Culture Collection (Wuhan, DMEM 10% serum, 100 U/mL penicillin, μg/mL streptomycin 5% CO2 atmosphere. For isotope-labeling tracing (LC-MS/MS), HEK293T cultured supplemented μM 15N3-dC (Cambridge Isotope Laboratories, Tewksbury, USA) metabolically label DNA. passaged every days 15N3-dC. weeks, applied experiments. assay collected centrifugation 1500g under min then washed twice ice-cold phosphate-buffered saline (PBS) remove serum. Approximately × 107 lysed 200 RIPA lysis (Beyotime Biotech Inc, Shanghai, China) phenylmethylsulfonyl fluoride (Sigma-Aldrich, Beijing, assay, added pmol different time. isolated streptavidin-coated magnetic beads (PMG015, PuriMag Biotech, Xiamen, according manufacturer’s instructions. Briefly, mg/mL) pipetted nuclease-free tube 30 s separator. supernatant removed, suspended binding (20 pH 7.0, M EDTA, 0.02% Triton X-100). solution 15 25 separated separator buffer, H2O subsequent nuclease digestion digestion. Nuclease analysis nucleotides LC-MS/MS carried out report slight modification.28 aforementioned (attached on beads), (180 (0.001 3 (500 Tris–HCl, ZnSO4, added. Enzymatic continued adding 170 H2O, extracted chloroform twice.29 aqueous layer, released digestion, collected, lyophilized dryness, reconstituted analysis. conducted system consisting Shimadzu 8045 spectrometer (Kyoto, Japan) electrospray ionization (ESI) source coupled LC-30AD UPLC (Tokyo, Japan). chromatographic separation performed Thermo Accucore C18 column (150 mm 2.1 i.d., 2.6 μm) 35 Nucleotides 20-min gradient use NH4HCO3 (A) (B) mobile phases.30,31 flow rate set 0.3 mL min−1. 0–5 B, 5–10 5–80% 10–13 80% 13–16 80–5% 16–20 B employed. detected multiple-reaction monitoring (MRM) positive-ion mode. spectrometric parameters MRM optimized injection, conditions S2. oligodeoxynucleotides high-resolution LC/MS decarboxylated substrate, beads) H2O. mixture Then CIAP (30 U/μL) 37°C 138 twice. layer subjected High-resolution run LTQ-Orbitrap Elite (Thermo-Fisher Scientific, Waltham, equipped ESI Dionex UltiMate 3000 TEAA phases. 0.2 0–10 10–25 25–27 80%–5% 27–40 used. Full MS scans negative mode range m/z 500–2000 resolution 60,000. Data Xcalibur version 3.0 Decarboxylation transfected 300 Lipofectamine (Invitrogen, harvested time points after (6, 12, 24, 48 h). PBS three times PBS. adhesive surface residual medium. times. previously described method modification.32 500 HEPES 7.9, 1.5 KCl, 0.5 DTT, 0.15% NP40) kept ice allow swelling. sonication. Nuclei 1000g min. cytoplasmic fraction collecting supernatant. pellet 250 0.42 0.1% ice. 14,000 rpm °C, combined. Proteinase K (0.1 55 inactivate proteinase K. collected. procedures same those assay. TDG inhibition Juglone (Yuanye Bio-Technology, Closantel (Aladdin, inhibit activity33 utilized study. grown concentrations Closantel. Genomic Tissue Kit (Omega Bio-Tek Inc., Norcross, GA, To evaluate inhibitory effects Closantel, μg enzymatically assess attenuated activity, 24 points. procedures. Isolated LC-MS/MS, earlier. knockdown Knockdown siRNA targeting mRNA. Nontargeting used control. h, Total RNA efficiency knockdown. treated gDNA eraser (Takara, Dalian, degrade contaminated PrimeScriptTM RT reagent kit reverse transcription. Quantitative real-time polymerase chain (PCR) fulfilled CFX Connect Real-Time System (Bio-Rad Hercules, PCR (25 μL) contained transcription solution, forward primer μM), 12.5 SYBR Premix Ex Taq II China), 8.5 included 40 cycles s, 60° C 72 s. Glyceraldehyde 6-phosphate dehydrogenase (GAPDH) reference normalization. Sequences primers validated Western blotting. Briefly, buffer. Antibodies specifically (Abcam, ab154192, Cambridge, MA) GAPDH ab181602, 1∶1000 1∶8000 dilutions, respectively. Horseradish peroxidase-conjugated secondary goat anti-rabbit antibody ab6721, 1∶10,000 dilution. knockout generated CRISPR-Cas9 system.34 assays way wild-type Exploration endogenous decarboxylase DNMT1 orotidine (ODCase) toward ODCase labeled Dharmacon (catalog D-004605-01). Sangon (Shanghai, relative quantification mRNA ODCase. normalized GAPDH. (HEK293T continuously 15N3-dC) additional Results Discussion Direct lysate study, aimed explore end, carrying 5caC, 3’ atoms, vivo processing. isolation 5caC-DNA, 5′-monophosphorothioate (S-cadCMP) (S-dCMP) well LC-MS/MS. vein, upon S-dCMP, whereas original liberated S-cadCMP (Figure 1a). addition, molecular weight 44 Da larger than Thus, difference weights between S-dCMP fragments harnessed existence proportion | lysate. (a) Schematic illustration assessing (b) Extracted-ion chromatograms (c) Levels (normalized level dGMP substrate) (d) [M-4H]4− ions its (at 1870.67 1859.67, respectively). Negative control (NC1), heat-treated used; (NC2), instead Positive (PC), sequence context being cytosine. ion 1865.17 represents Na+ adduct [M-5H+Na]4−. (e) Quantitation time-dependent based S6. Shown red squares blue circles product, Three measurements performed, error bars represent standard deviations mean. duplex S2) respectively, 1b S3). nonspecific adsorption generally minimal S4), showing trace neglected. decrease S-cadCMP, accompanied progressive increase 1c S3), No detectable observed (NC1; used) (NC2; lysate) 1c). Upon > 95.4% eliminated, while comparable positive (PC; except cytosine; S1) 1c), high within next level. lysate, biotinylated products isolated, enzyme, ([M-4H]4−, 1870.67) (5caC-DNA heat-inactivated (PC modification) 1d). emanating zoomed-in spectrum shown S5) increased increasing 1e; Figures S6 S7). Similar nucleotide level, h 94.6% explored enzymes, 2a). 6-FAM-labeled