N6-methyladenine is incorporated into mammalian genome by DNA polymerase

Dear Editor, DNA N 6 -methyladenine (6mA), one of the most prevalent epigenetic base modi ﬁ cations in prokaryotes, 1 is recently found in multicellular eukaryotes. 2 – 8 This nucleobase may have epigenetic roles in regulation of retrotransposons, chromatin organization, and so on. 2 – 8 However, both our group 9 and Greer ’ s group 10 noticed that eukaryotic DNA is easily contaminated with a minute of bacterial DNA, which carries overwhelmingly abundant 6mA (~2% 6mA/dA). 1 This brings great challenges for accurate detection of DNA 6mA in eukaryotes in terms of both sample pretreatments and analytical technologies. 9,10 For example, inconsistent with the report of Wu et al., 8 Schiffers et al. failed to detect 6mA above background levels in mouse embryonic stem (mES) cells using sensitive ultra-high-performance liquid chromatography-quadruple mass spectrometry (UHPLC-MS/MS) analysis. 11 To date, it is of intensive interest to seek conclusive evidence to support the prevalence of this post-replicative adenine modi ﬁ cation in mammals. These issues prompted us to re-investigate DNA 6mA in mammalian cells. To provide robust and reliable data, a contamination-free UHPLC-MS/MS technology, which is being developed in our lab, was used for ultrasensitive and accurate detection of 6mA in mammals. We measured genomic 6mA in four cultured mammalian cell lines. We observed 6mA in three human cell lines, including HEK293T cells (~0.7 6mA per 10 7 dA), human mesenchymal stem cells (~2.0 6mA per 10 7 dA), and human embryonic stem cells (hES cells) (~4.0 6mA per 10 7 dA). We also detected 6mA in mES cells (~7.0 6mA per 10 7 dA)

The treatment of cultured cells with (deoxy)ribonucleoside [ 15 N 5 ]-dA, m 6 A, 6mA, [ 15 N 5 ]-6mA or [ 13 CD 3 ]-L-Methionine 1.0  10 5 mES cells were seeded in 6-well cell culture cluster (Corning), and cultured in 2.0 ml medium. The final concentration of treated (deoxy)ribonucleosides was used as stated, and the final concentration of treated [ 13 CD3]-L-methionine was 30 μg/mL. The cells were harvested for DNA extraction and UHPLC-MS/MS analysis after 4 days' treatment, or as stated.

Synchronization of cultured cells
The heavy stable isotope-labeled cells were synchronized by specific cell cycle inhibitors. Briefly, mES cells were arrested in G1, S, and G2/M phases by 16 h treatment of 400 μM mimosine or 100 nM palbociclib, 2.0 mM thymidine, and 0.4 3 μg/mL nocodazole, respectively. Transient transfection of 6mA-DNA oligo or m 6 A-RNA oligo and DNA extraction 1.0  10 5 mES cells were seeded into a six-well plate and cultured for 24 h before transfection using Lipofectamine RNAiMAX (Life Technologies) according to the manufacturer's instructions with minor modifications. 20 μL RNAiMAX reagent and 4 nmol of oligos were used and incubated with cells for 48 h. The oligos were purchased from Takara Bio (Dalian, China) and the sequences were listed in Supplementary information, Table S1.
To remove possible co-extracted oligo, ammonium acetate precipitation was performed. The genomic DNA that was purified by Promega Wizard genomic DNA purification kit was dissolved in 20 mM tris-HCl buffer (pH 8.0), then mixed with an equal volume of 5.0 M ammonium acetate (pH 5.2), and then added with three volume of ethanol and vortexed. Next, centrifuged by 12,000 rpm for 2 min at room temperature. The DNA pellet was washed with ethanol/water (7:3, v/v) three times, and dried in air. The dried DNA was then re-dissolved in ddH2O. The concentration of the purified DNA was determined with NanoDrop 2000 (Thermo Fisher Scientific, MA, USA) and the quality was evaluated with the ratio of absorbance at 260 nm and 280 nm.

SiRNA transient transfection followed by Synchronization
1.0  10 5 mES cells were seeded into a six-well plate and grew for 24 h. Then, the cells were transfected with 25 pmol siRNA (GenePharma, Suzhou, China) using Lipofectamine RNAiMAX (Life Technologies) following the manufacturer's guidelines and incubated for 30 h. Afterwards, mES cells were arrested at the G1 phase by the treatment of 400 μM mimosine for 16 h. The mRNA level of pol λ was measured by quantitative real time-PCR. The cells were harvested for DNA extraction and RNA extraction, respectively. The sequence of siRNAs used in this study were listed in Supplementary information, Table S2.

mRNA expression analysis
Total RNA was extracted from cells by TRIzol reagent (Life technologies Corporation). 1.0 µg RNA was reversely transcribed to cDNA with Promega reverse transcription system. Real-time PCR was performed using Promega GoTaq qPCR Master Mix following the manufacturer's protocol on Stratagene Mx3005P real-time PCR System (Agilent Technologies, CA, USA). The pol λ mRNA levels were normalized by the Gapdh mRNA levels. The RT-qPCR primer sequences were listed in Supplementary information, Table S3.

Generation of knockout cell lines by CRISPR/Cas9
Guide RNA oligos were annealed and cloned into a PX458 plasmid (Addgene plasmid #48138). mES cells were first seeded into a six-well plate at low density and grew for 12 h. Then the cells were transfected with 5.0 μg of the PX458-sgRNA plasmid. The transfection was performed using Lipofectamine 2000 (Life Technologies) according to the manufacturer's instructions. The transfected cells were further incubated for 24 h. Afterwards, the mES cells were digested into individual cells and sorted into 96-well plates according to GFP signal by BD Ariall flow cytometer (BD Biosciences, New Jersey, USA). Then, after three-days culturing, ~40 mES cell colonies were picked up for PCR screening. The PCR products were verified by T cloning vector sequencing using pClone007 Blunt Vector Kit following instructions (TsingKe biological technology, Beijing, China).
The sgRNA sequence and the sequence of PCR primers flanking guide RNA were listed in Supplementary information, Table S4.

General protocol for genomic DNA extraction and enzymatic digestion
The adherent cells were gently washed twice with 1  phosphate-buffered saline (PBS buffer, 8.1 mM Na2HPO4; 1.76 mM KH2PO4; 136.89 mM NaCl; 2.67 mM KCl), and detached by 1.0 min trypsin digestion. The genomic DNA was extracted according to the instructions of the Promega wizard® genomic DNA purification kit (Promega, WI, USA). The concentration of the extracted DNA fractions was determined with NanoDrop 2000 (Thermo Fisher Scientific, MA, USA) and the DNA quality was evaluated with the ratio of absorbance at 260 nm and 280 nm.
To obtain deoxyribonucleosides, per 5.0 µg DNA was digested with 0.5 U CIP (New England Biolabs), 0.25 U DNase I (New England Biolabs), and 0.25 U SVP (Worthington) at 37 °C overnight. To remove the digestive enzymes, DNA solution was ultrafiltered with ultrafiltration tubes (molecule weight cutoff: 3 KDa; Pall Corporation, Port Washington, NY, USA). The ultrafiltered solution was then subjected to UHPLC -MS/MS analysis.

UHPLC-MS/MS analysis
The UHPLC-MS/MS analysis was performed on an Agilent 1290 II UHPLC system coupled with an ESI-triple quadrupole mass spectrometer (6470, Agilent Technologies, CA, USA). A ZORBAX SB-Aq column (2.1 × 100 mm, 1.8 μm particle size, Agilent) was employed for the separation of deoxyribonucleosides.
The mass spectrometer was operated under positive ionization using multiple reactions monitoring (MRM) mode. ]-dG. The fragmentation voltage for all the MRM transitions were set at 90 V to allow efficient transit of precursor ions. Nitrogen gas was used for nebulization and desolvation. The nebulization gas pressure was set at 40 psi, and the temperature and the flow rate of desolvation gas were set at 300 °C and 9.0 L/min, respectively. High purity nitrogen (99.999%) was used as collision gas.
In vitro incorporation of 6mA in replicated DNA by high fidelity Taq DNA polymerase.
To explore whether DNA polymerase can incorporate 6mA during DNA synthesis or 5 replication in vitro, we performed a 6mA-involved polymerase chain reaction (PCR). 100 ng genomic DNA of mES cells was used as the template, and mixed with 0.5 μM of each primer and 5 μL HiFiTaq PCR buffer, 5 U HiFiTaq DNA polymerase (GeneStar, Shanghai, China), dCTP, dTTP, dGTP, and dATP or N6mdATP (50 μM each) in a total volume of 50 μL. The PCR products were analyzed by 1% agarose gel electrophoresis. The PCR products separated by the agarose gel electrophoresis were extracted from the gel using a PCR clean up kit (Promega, WI, USA), and the extracted DNA was analyzed for detection of incorporated 6mA by UHPLC-MS/MS.

Flow cytometry sorting of mES cells at the G1 and subG1 phase.
The mES cells were arrested at the G1 phase by a 16 h treatment of 400 μM mimosine, and then trypsinized to be dispersed into single-cell suspensions. The dispersed cells were washed with 1  PBS and collected by centrifugation. Re-suspended the collected cells in pre-chilled PBS, discarded the supernatant, and slowly dropwisely added 3.0 mL of pre-chilled absolute ethanol (final concentration: 75% v/v), then rested the cells overnight at 4 °C. Next, washed the fixed cells with pre-chilled PBS twice, removed PBS by centrifuge at 1000 rpm for 5 minutes, and added 200 μL of RNase A (50-100 μg/mL) and incubated at 37 °C for 30 min, then discarded the supernatant, and added 500 μL ethidium bromide (PI, 50 μg/mL) and incubated at 4 °C for 30 min in the dark. Finally, filtered the mES cells through a 200-mesh strainer and sorted the cells within 1 h following PI staining by a BD Ariall flow cytometer (BD Biosciences, NJ, USA). The flow cytometer was controlled using BD FACSDiva software. The sorted cells were collected by centrifugation, and genomic DNA was extracted for UHPLC-MS/MS analysis.

Western blotting analysis
The expression of Alkbh1 gene in Alkbh1 knock out cells was analyzed by western blot. Briefly, the total protein of wild type or Alkbh1 -/-cells was extracted by cold RIPA lysis buffer (Beyotime) with 1% PMSF, and then separated by NuPAGE 4-12% bis-tris gel in MOPS SDS running buffer (Invitrogen, California, USA), and then electrophoretically transferred onto 0.45 μm PVDF membrane (Millipore, Darmstadt, Germany). After blocking with 5% skim milk powder in TBST (20 mM Tris, pH 7.4, 150 mM NaCl, 0.1% Tween-20) for 1 h, the membrane was then incubated with a primary antibody (ab195376, Abcam, Cambridge, UK) diluted at 1:1000 in 5% milk at 4 °C overnight. Subsequently, the membranes were incubated with secondary antibody (goat anti-rabbit IgG, BS10007, Bioworld, MN, USA) for 1 h at room temperature. Then, the blot was imaged by Odyssey CLX infrared imaging system (LI-COR Biotechnology, Lincoln, NE, USA)