N6-methyladenosine in DNA promotes genome stability

Abstract

DNA base lesions, such as incorporation of uracil into DNA or base mismatches, can be mutagenic and toxic to replicating cells. To discover factors in repair of genomic uracil, we performed a CRISPR knockout screen in the presence of floxuridine, a chemotherapeutic agent that incorporates uracil and fluorouracil into DNA. We identified known factors, such as uracil DNA N-glycosylase (UNG), and unknown factors, such as the N6-adenosine methyltransferase, METTL3, as required to overcome floxuridine-driven cytotoxicity. Visualized with immunofluorescence, the product of METTL3 activity, N6-methyladenosine, formed nuclear foci in cells treated with floxuridine. The observed N6-methyladenosine was embedded in DNA, called 6mA, and these results were confirmed using an orthogonal approach, liquid chromatography coupled to tandem mass spectrometry. METTL3 and 6mA were required for repair of lesions driven by additional base-damaging agents, including raltitrexed, gemcitabine, and hydroxyurea. Our results establish a role for METTL3 and 6mA in promoting genome stability in mammalian cells, especially in response to base damage.

Keywords: 6mA; METTL3; MMR; N6-methyladenosine; U-BER; UNG; cancer biology; cell biology; human.

Figure 1.. Whole-genome CRISPR screen identifies N6-methyltransferases in repair of floxuridine-induced DNA lesions.

(A) Schematic of whole-genome CRISPR screen in HT-29 cells reported in B. (B) Volcano plot displaying MAGeCK gene level log₂(fold change) for each gene in treated and untreated arms versus −log₁₀(p-value). Cut-off displays genes with log₂(fold change) >|0.5| and −log₁₀(p-value) >2. Genes whose loss sensitizes cells to floxuridine skew to the left. Non-targeting guides are shown in light gray and fall below cut-off values. Essential genes also performed as expected, dropping out at later time points (data not shown). (C) KEGG pathway analysis for genes that sensitize cells to floxuridine with log₂(fold change) >|0.5| and −log₁₀(p-value) >2. Flox, floxuridine; FDR, false discovery rate.

Figure 1—figure supplement 1.. Generation of mCherry-tagged UNG KO DLD-1 cells.

(A) Percentage of reads from sequencing of DLD-1 cells after gene editing corresponding to gene transcripts that are unmodified, modified in in-frame, contain a frameshift, or are noncoding. (B) Representative immunoassay using the Simple Western Jess system showing protein expression levels of UNG in two HPRT-targeted and two UNG-targeted clones from A. α-Tubulin represents loading control. (C) Representative immunoblot showing protein expression levels of UNG2-mCherry (UNG) or mCherry empty vector (EV) constructs expressed in UNG KO DLD-1 cells. α-Tubulin represents loading control.

Figure 2.. Discovery-based proteomics identifies N6-methyltransferases at sites of UNG2-seeded condensates.

(A) Representative images from DLD-1 UNG KO cells expressing indicated mCherry-tagged cDNAs upon treatment with increasing concentrations of floxuridine at 64 hr post-treatment. (B) Quantification of experiment represented in A for percentage of cells with >5 mCherry foci. Error bars, mean ± SEM; ordinary one-way ANOVA with Dunnett’s multiple comparisons test with a single pooled variance, ***p ≤ 0.001, n = 3 biological replicates. Statistical tests performed within individual groups, EV or UNG2, respectively. (C) PONDR VSL2 plot of disorder for UNG2. (D) Representative schematic of mutant UNG2 cDNA constructs expressed in UNG KO DLD-1 cells in E. IDR-only cDNA lacks amino acids 93-313, ∆IDR cDNA lacks amino acids 1–92, and IDR-C cDNA moved amino acids 1–92 to the C-terminus. (E) Representative images from DLD-1 UNG KO cells expressing indicated mCherry-Cry2-tagged cDNAs without or with stimulation of blue light for 60 s. While ∆IDR and EV images display cytoplasmic foci, these lack the distinct nuclear foci patterning observed for UNG2, IDR, and IDR-C constructs. (F) Schematic of proximity biotinylation of IDR interaction partners in cells. (G) Venn diagrams of factors identified by stable isotope labeling of amino acids in cell culture (SILAC)-based mass spectrometry (MS) with 1 > log₂(L/H), reflecting a fourfold enrichment in UNG-IDR/Control, from two biological replicates. n.s., non-statistically significant; Flox, floxuridine; EV, empty vector; IDR, intrinsically disordered region.

Figure 2—figure supplement 1.. UNG2 responds to uracil-based DNA damage in a manner partly dependent on its intrinsically disordered region.

(A) Percentage of EdU+ cells in indicated DLD-1 cells. Cells were pulsed with 10 μM EdU prior to fixation and staining with Invitrogen Click-It Alexa Fluor 488 Kit. HPRT indicates WT cells. These cells were targeted with a cutting control targeting to the intronic region of HPRT gene. (B) Representative images from DLD-1 UNG KO cells expressing indicated mCherry-tagged cDNAs upon treatment with increasing concentrations of RTX at 64 hr post-treatment. (C) Quantification of experiment represented in A for percentage of cells with >5 mCherry foci. Error bars, mean ± SEM; ordinary one-way ANOVA with Dunnett’s multiple comparisons test with a single pooled variance, **p ≤ 0.01, n = 3 biological replicates. Statistical tests performed within individual groups, EV or UNG2, respectively. (D) Representative images of mCherry staining in DLD-1 UNG KO cells expressing UNG2 or EV constructs upon treatment with indicated compounds at 64 hr. (E) Quantification of experiment represented in D for percentage of cells with >5 mCherry foci. Error bars, mean ± SEM; ordinary one-way ANOVA with Dunnet’s multiple comparisons test with a single pooled variance, **p ≤ 0.01, n = 3 biological replicates. (F) Representative images of yH2AX staining in DLD-1 UNG KO cells expressing UNG2-mCherry constructs upon treatment with indicated compounds at 64 hr from the same experiment as D, E. (G) Quantification of experiment represented in F for average mean nuclear intensity. Error bars, mean ± SEM; ordinary one-way ANOVA with Tukey’s multiple comparisons test with a single pooled variance, *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001, n = 3 biological replicates for all except HU where n = 2 biological replicates. (H) MTS cell proliferation in DLD-1 UNG KO cells expressing indicated mCherry-Cry2-tagged cDNAs upon treatment with floxuridine at increasing concentrations. Error bars, mean ± SEM. n = 4 biological replicates. (I) KEGG pathway analysis for genes that sensitize cells to floxuridine with log₂(foldchange) >|0.5| and −log₁₀(p-value) >2. RTX, raltitrexed; Flox, floxuridine; EV, empty vector; IDR, intrinsically disordered region; FDR, false discovery rate.

Figure 3.. METTL3 deposits N6-methyladenosine in DNA in response to agents that increase genomic uracil.

(A) Representative immunoblot images from DLD-1 cells nucleofected with ribonucleoproteins containing Cas9 and indicated guide RNAs (gRNA) as performed for B. α-Tubulin represents loading control. (B) MTS cell viability assay in the presence of floxuridine. Error bars, mean ± SEM, n = 2 biological replicates. (C) Growth curves in DLD-1 cells upon treatment with 15 μM METTL3 inhibitor and indicated drug concentration post-treatment. Error bars, mean ± SEM, n = 3 biological replicates. (D) Representative images from DLD-1 UNG KO upon treatment with floxuridine for 66 hr. Prior to staining with N6-methyladenosine antibody, indicated samples were treated with RNase A or DNase. (E) Quantification of experiment represented in D for a percentage of cells with >5 N6-methyladenosine foci. Error bars, mean ± SEM; repeated measures one-way ANOVA with Dunnet’s multiple comparisons test with a single pooled variance, *p ≤ 0.05, n = 5 biological replicates. (F) Schematic of the experiment shown in G. DLD-1 cells were treated with DMSO, 500 nM floxuridine, or 500 nM raltitrexed for 72 hr. Cells were washed, collected, and DNA was purified and digested with DNA degradase plus enzyme prior to separation and quantification by ultra-performance liquid chromatography–mass spectrometry (UPLC–MS/MS). (G) The ratio of 6mA analyte to dA analyte as detected in DNA of DLD-1 cells upon treatment with 500 nM floxuridine or 500 nM raltitrexed using UPLC–MS/MS. ****p ≤ 0.0001, n = 3 biological replicates. (H) Representative images of 6mA staining in DLD-1 UNG KO cells upon treatment with 500 nM of floxuridine and 30 μM METTL3 inhibitor at 64 hr. (I) Quantification of experiment represented in G for a percentage of cells with >10 6mA foci. Error bars, mean ± SEM; ordinary one-way ANOVA with Dunnett’s multiple comparisons test with a single pooled variance, *p ≤ 0.05, n = 3 biological replicates. HPRT, cutting control targeting intronic region of HPRT gene; Flox, floxuridine; RTX, raltitrexed; METTL3i, METTL3 inhibitor.

Figure 3—figure supplement 1.. N6-methyladenosine foci in response to genomic uracil-inducing agents are not RNA:DNA hybrids.

(A) MTS cell proliferation in SW620 cells upon treatment with 15 μM METTL3 inhibitor and indicated compounds. Error bars, mean ± SEM, n = 3, technical replicates, representative of three biological replicates. Representative of three biological replicates. (B) Representative immunoblot images from DLD-1 cells nucleofected with ribonucleoproteins containing Cas9 and indicated guide RNAs (gRNA) as performed for C. Antibodies used for blotting indicated on the left. α-Tubulin represents loading control. (C) Growth curves in DLD-1 cells upon treatment of floxuridine. Error bars, mean ± SD, n = 3 technical replicates. (D) Representative images of immunofluorescence staining with anti-N6-methyladenosine antibody in DLD-1 cells after fixation and permeabilization but without pre-extraction in the presence or absence of floxuridine. (E) Agarose gel showing 1 μg of RNA extracted from whole cells with or without treatment with RNase A for 1 hr prior to loading on gel. (F) Representative images from DLD-1 cells upon treatment with floxuridine or raltitrexed for 66 hr. Prior to staining with anti-N6-methyladenosine antibody, indicated samples were treated with RNase H. (G) Quantification of experiment represented in F for a percentage of cells with >10 N6-methyladenosine foci. Error bars, mean ± SEM; ordinary one-way ANOVA with Tukey’s multiple comparisons test with a single pooled variance, *p ≤ 0.05, ****p ≤ 0.0001, n = 3 biological replicates for floxuridine treatment and n = 4 biological replicates for DMSO and raltitrexed treatment. (H) Quantification of mean nuclear DAPI staining from experiment in Figure 3D, E. Error bars, mean ± SEM; Krustal–Wallace with Dunn’s multiple comparisons test, **p ≤ 0.01, n = 5 biological replicates. (I) Representative images from DLD-1 cells upon treatment with raltitrexed for 66 hr. Prior to staining with anti-N6-methyladenosine antibody, indicated samples were treated with RNase A or DNase. (J) Quantification of experiment represented in I for percentage of cells with >5 N6-methyladenosine foci. Error bars, mean ± SEM; RM one-way ANOVA with Dunnet’s multiple comparisons test with a single pooled variance, ***p ≤ 0.001, n = 4 biological replicates. (K) Representative images from DLD-1 cells upon treatment with raltitrexed for 66 hr in the presence or absence of METTL3 inhibitor. (L) Quantification of experiment represented in K for percentage of cells with >10 6 mA foci. Error bars, mean ± SEM; ordinary one-way ANOVA with Dunnet’s multiple comparisons test with a single pooled variance, *p ≤ 0.05, ***p ≤ 0.001, n = 4 biological replicates. Flox, floxuridine; RTX, raltitrexed.

Figure 4.. 6mA promotes uracil repair upstream of UNG2 in uracil base excision repair.

(A) Representative images of mCherry staining in DLD-1 UNG KO cells expressing UNG2-mCherry cDNAs upon treatment with 500 nM floxuridine and 30 μM METTL3 inhibitor at 64 hr. (B) Quantification of experiment represented in A for percentage of cells with >5 mCherry foci. Error bars, mean ± SEM; ordinary one-way ANOVA with Tukey’s multiple comparisons test with a single pooled variance, *p ≤ 0.05, ***p ≤ 0.001, n = 3 biological replicates. (C) Real-time quantitative PCR from A, B for UNG2 transcript levels normalized to tubulin controls. Error bars, mean ± SEM; Mann–Whitney t-test for the following pairs: DMSO versus METTL3 inhibitor and floxuridine versus floxuridine + METTL3 inhibitor. *p ≤ 0.05, n = 3 biological replicates except for the floxuridine only condition which includes n = 2 biological replicates. (D) Representative images of 6mA staining in DLD-1 UNG KO cells upon treatment with 500 nM floxuridine at 64 hr. HPRT indicates wild-type cells. These cells were targeted with a cutting control targeting the intronic region of HPRT gene. (E) Quantification of experiment represented in D for percentage of cells with >5 6 mA foci. Error bars, mean ± SEM; RM one-way ANOVA with Dunnet’s multiple comparisons test with a single pooled variance, *p ≤ 0.05, n = 5 biological replicates. Flox, floxuridine; METTL3i, METTL3 inhibitor.

Figure 4—figure supplement 1.. The presence of 6mA does not alter UNG-binding kinetics.

(A) Representative immunoblot of whole-cell lysates from DLD-1 cells in the presence or absence of METTL3 inhibitor blotted with UNG or β-actin antibodies. β-Actin represents loading control. (B) Equilibrium dissociation constants (KD) measured by biolayer interferometry for binding of UNG to indicated dsDNA templates. Mean ± SEM for n = 3 biological replicates displayed in the table. (C) Equilibrium dissociation constants (K_d) measured by biolayer interferometry for binding of UNG to indicated ssDNA templates. Mean ± SEM for n = 2 biological replicates displayed in the table.

Figure 5.. 6mA promotes genome repair of base damage beyond uracil incorporation.

(A–C) MTS cell viability in SW620 cells upon treatment with METTL3 inhibitor and indicated concentrations of drugs. Error bars, mean ± SEM, n = 3, technical replicates, representative of three biological replicates. (D) Representative images of 6mA staining in DLD-1 UNG KO cells upon treatment with indicated DNA damaging agents at 64 hr. (E) Quantification of experiment represented in D for percentage of cells with >5 6 mA foci. Error bars, mean ± SEM; ordinary one-way ANOVA with Dunnet’s multiple comparisons test with a single pooled variance, **p ≤ 0.01, n = 3 biological replicates for all except for the HU condition which includes n = 2 biological replicates. (F) Schematic of colony formation assay. SW620 cells, maintained in HAT media, were treated with 30 μM of METTL3 inhibitor for 7 days. 0.5 × 10⁴ METTL3 inhibitor-treated cells seeded in the presence of 5 μM 6-thioguanine (TG) and colony formation assay was assessed after 14 days. 0.5 × 10² METTL3 inhibitor-treated cells were seeded for untreated controls. (G) Representative dishes after 14 days of growth in 5 μM TG as described in F. (H) Quantitation of mutation frequency from G. Mutation frequency was calculated by normalizing to the untreated controls. Error bars, mean ± SEM; paired t-test, **p ≤ 0.01, n = 3 biological replicates. HU, hydroxyurea; Gem, gemcitabine; MMC, mitomycin C; METTL3i, METTL3 inhibitor.

Figure 5—figure supplement 1.. 6mA foci, but not UNG2 foci, correlate with DNA damage levels.

(A) Representative images of yH2AX staining in DLD-1 cells upon treatment with indicated compounds at 64 hr from the same experiment as Figure 5D, E. (B) Quantification of experiment represented in A for average mean nuclear intensity. Error bars, mean ± SEM; ordinary one-way ANOVA with Tukey’s multiple comparisons test with a single pooled variance, **p ≤ 0.01, ***p ≤ 0.001, n = 3 biological replicates for all except HU where n = 2 biological replicates. (C). Co-staining with anti-mCherry and anti-N6-methyladenosine antibodies in cells treated with indicated drugs. Green arrows indicate cells where both mCherry and 6mA staining are present and white arrows indicate cells where only 6mA staining is present. Flox, floxuridine; RTX, raltitrexed; HU, hydroxyurea; Gem, gemcitabine; MMC, mitomycin C.