Nitric oxide inhibits ten-eleven translocation DNA demethylases to regulate 5mC and 5hmC across the genome

Abstract

DNA methylation at cytosine bases (5-methylcytosine, 5mC) is a heritable epigenetic mark regulating gene expression. While enzymes that metabolize 5mC are well-characterized, endogenous signaling molecules that regulate DNA methylation machinery have not been described. We report that physiological nitric oxide (NO) concentrations reversibly inhibit the DNA demethylases TET and ALKBH2 by binding to the mononuclear non-heme iron atom forming a dinitrosyliron complex (DNIC) and preventing cosubstrates from binding. In cancer cells treated with exogenous NO, or endogenously synthesizing NO, 5mC and 5-hydroxymethylcytosine (5hmC) increase, with no changes in DNA methyltransferase activity. 5mC is also significantly increased in NO-producing patient-derived xenograft tumors from mice. Genome-wide methylome analysis of cells chronically treated with NO (10 days) shows enrichment of 5mC and 5hmC at gene-regulatory loci, correlating with altered expression of NO-regulated tumor-associated genes. Regulation of DNA methylation is distinctly different from canonical NO signaling and represents a unique epigenetic role for NO.

Fig. 1. NO reversibly inhibits Fe(II)/2-OG-dependent DNA demethylases.

TET2 demethylase activity (a–e): a Conversion of 5mC (black line) to 5hmC (red line) when human TET2 was incubated with NO (Sper/NO; 0–500 μM; 3 h). Demethylase activity assay was initiated with addition of a 5mC-double-stranded DNA substrate, n = 3. b TET2 activity (5hmC formation) measured over a range of Sper/NO concentrations (0–1.5 mM), IC₅₀ = 165 μM Sper/NO, n = 2. c % inhibition of TET2: TET2 was incubated with 165 μM Sper/NO for 3 h, the reaction was started by the addition of one of three different DNA substrates for TET2 (DNA oligos containing 5mC, 5hmC, or 5fC), n = 2. d TET2 demethylase activity (5hmC product formation, ELISA, n = 3) was measured at 3 time points (1, 2, & 3 h) and under three different conditions: Panel 1 TET2 incubated without NO: Full Activity, Panel 2 TET2 incubated continuously with NO (300 μM Sper/NO): Full Inhibition, and Panel 3: TET2 incubated with NO for a short duration (25 μM DEA/NO): Inhibition and Recovery. The top bar graphs are the % product formation at each time point. The bottom graphs are the simulated [NO]_ss (red line) under each reaction condition, data (a–d) are mean ± SEM P-values determined by unpaired (a–c) or paired (d) two-tailed Student’s t-tests. e TET2 activity in the presence of freshly isolated genomic DNA (1 μg) and NO (Sper/NO 0–300 μM, 3 h), 5hmC measured by dot-blot hybridization using anti-5hmC antibody and total DNA measured by methylene blue, representative blot and densitometry shown, n = 3. f ALKBH2 activity: recombinant ALKBH2 was incubated with NO (Sper/NO 0–500 μM) and the fluorescent probe. ALKBH2 activity (demethylation of the fluorescent probe) was monitored for 1 h by fluorescence spectroscopy at 480 nm. g IC₅₀ determination from data in panel f, mean ± S.D., h Cellular extracts containing ALKBH2 were incubated with the fluorescent probe and the slow NO-releasing donor DETA/NO (t_1/2 = 22 h, 0–150 μM), demethylation was measured for 12 h (f, h: representative graphs, n = 3 separate experiments). NS not significant, AU arbitrary units. Source data are provided as a Source Data file.

Fig. 2. NO forms a DNIC at the mononuclear non-heme iron atom of TET2.

a Representative X-Band (77 K) EPR spectra of truncated TET2 protein incubated with NO (Sper/NO, 100 μM) and all substrates and cofactors for 1 min (red line) or 20 min (black line). (Blue line) is the complete reaction without TET2 at 20 min, n = 3. Spectrum is indicative of a non-Heme NO-bound DNIC. DFT computations (b–d): b Core of the ωB97xD/def2-svp optimized geometry of triplet dinitrosyl complex [Fe^II(OAc)(Im)₂(NO)₂(OH₂)]⁺, OAc = acetate, Im = N-methyl-imidazole. Bond lengths and angles in Å and °, respectively. c Core of the ωB97xD/def2-svp optimized geometry of [Fe(OAc)(Im)₂(NO)(O₂)(OH₂)]⁺ and (d) [Fe(OAc)(Im)₂(NO)(OH₂)₂]⁺. e Crystal structure of TET2 in complex with N-oxalylglycine (OGA), a 2-OG analog (PDB file 4nm6). OGA forms two critical hydrogen bonds with a conserved arginine (Arg 1261 in TET2). f Two NO molecules replace the two oxygens from OGA that coordinate with Fe(II). Shown also are Arg 1261, which may stabilize NO binding, a water molecule in the sixth coordination site, and coordinating residues His 1382, Asp 1384, and His 1881.

Fig. 3. NO increases 5mC on DNA from cancer cells and from tumors.

a Relative abundance of 5mC-DNA in cancer cells treated without (light bars) or with NO (dark bars, DETA/NO 100 μM, 24 h). b Relative abundance of 5mC-DNA and (c) 5hmC-DNA from TNBC cells that were chronically treated with NO for 10 days (DETA/NO 50 and 100 μM added every 48 h), (a–c, ELISA, mean ± SEM, n = 3). d Immunoblot for NOS2 protein in MDA-MB-231 empty vector control (VC), MDA-MB-231 NOS2-expressing, and MDA-MB-231 NOS2 + L-NMMA (1 mM), at 24 h. e Total NO synthesis as measured by nitrite, n = 6, mean ± SD), and (f) 5mC-DNA (ELISA) from MDA-MB-231 (VC) and MDA-MB-231 NOS2 + /- L-NMMA cells cultured for 24 and 48 hours, (n = 4, mean ± SD). g How inhibitors cycloleucine (CL), 5-Azacytidine (AZA), and NO interact with DNMT, MAT (methionine adenosyltransferase), and TET to affect 5mC-, 5hmC-DNA levels. h 5mC-DNA from MDA-MB-231 cells treated for 24 h +/- NO (100 μM DETA/NO, green bar), or +/- 1 mM AZA (purple bars), or +/- 1 mM CL (blue bars), ELISA, (n = 3, mean ± SD). Representative immunoblot and densitometry for (i) DNMT1, 3A, and 3B and (j) TET1, 2, 3, and ALKBH2 from MDA-MB-231 and MDA-MB-468 cells cultured for 10 days with NO (DETA/NO, 100 μM), n = 3 separate experiments. k 5mC-DNA levels from mice with NO-producing MDA-MB-231 xenograft tumors (dark green bars), and from tumors that did not synthesize NO (treated aminoguanidine (AG), light bars), ELISA, n = 7/group. l 5mC-DNA from mice with TNBC PDX tumors that did synthesize NO (dark green bars) and from tumors that did not synthesize NO (L-NMMA), ELISA, n = 3/group. Data are presented as box plots (center line at the median, upper and lower bounds are 75^th and 25^th percentile with whisker at 1.5 IQR. Each dot represents one mouse. For bar graphs data are mean ± S.E.M or ± SD, P-values were determined by unpaired two-tailed Student’s t-tests, NS = not significant, blue vertical line on immunoblots indicates splicing of lanes that were run on the same gel but were noncontiguous. Source data are provided as a Source Data file.

Fig. 4. NO is associated with aggressive cancer phenotypes and causes transcriptional changes in breast cancer.

a Patient survival as a function of NOS2 gene expression and hormone receptor status using Kaplan-Meier Plotter (public database includes 7830 unique breast cancer samples). (b–d) Cell migration and invasion measured in real time by the xCELLigence® DP system. b MDA-MB-231 cells were plated and allowed to adhere for 20 h before the addition of NO (100 μM DETA/NO). c MDA-MB-231 empty vector control (VC) and MDA-MB-231 NOS2-expressing cells were plated, and migration was monitored for 40 h. d Invasion: MDA-MB-231 cells were plated on a Matrigel® matrix and incubated for 20 h before the addition of NO (100 μM DETA/NO), (b–d, data lines represent the mean +/- SD, n = 2). e NOS2 gene expression in tumors from TNBC patients who responded to chemotherapy and patients who did not respond to chemotherapy (n = 164). The two cohorts were compared using ROC Plotter platform, Mann–Whitney test. (f–h) mRNA-Seq was conducted on samples from MDA-MB-231 and MDA-MB-468 cells treated with or without NO (DETA/NO 100 μM; 10 days, n = 3 biological replicates for each cell type). f Volcano plots of NO-mediated mRNA changes. Significantly up- and down-regulated genes are indicated in red, (significance indicated as log₂ fold > 1 or <−1 and -log₁₀ P-values > 0.05) Differential expression analysis was completed using quasi-likelihood F-tests (glmQLFTest function) in edgeR v3.40.2. P-values from all tests were adjusted to generate FDR (False Discovery Rate) with built-in Benjamini-Hochberg method. g Venn diagram demonstrates the number of significant NO-regulated genes that overlap between the two cell types. h Multidimensional scaling (MDS) analysis of gene expression from mRNA-Seq data sets. Source data are provided as a Source Data file.

Fig. 5. NO increases 5mC and 5hmC in DNA at specific genomic locations.

Oxidative reduced representation bisulfite sequencing (oxRRBS) was conducted on samples from MDA-MB-231 and MDA-MB-468 cells treated with or without NO (DETA/NO 100 μM; 10 days, n = 2 biological replicates). a, b percent change of hyper- and hypo- differentially methylated positions (DMP) and hyper- and hypo-differentially hydroxymethylated positions (DhMP) across all 8 annotated sites in both cell types, each dot represents one annotated site, center line at the median, upper bound at 75th percentile, lower bound at 25th percentile. c Annotated CpG sites of NO-mediated hyper-DMP and hyper-DhMP (DMP and DhMP = P-value < 0.05, mean difference in abs (beta value) of ≥ 0.1 according to RnBeads). d NO-mediated hyper- and hypo-differentially methylated CpG positions and e hyper- and hypo-differentially hydroxymethylated CpG positions at functional elements (5’UTR = 5’untranslated region (5’UTR), 3’UTR = 3’untranslated region (5’UTR), SE = Super enhancers, and TE = typical enhancers). Comparing differences in the magnitude of (f) DMP and (g) DhMP at functional elements between both cell types. Source data are provided as a Source Data file.

Fig. 6. NO-dependent changes in 5mC and 5hmC are associated with transcriptional changes in NO-responsive genes.

a, b oxRRBS data from MDA-MB-231 and MDA-MB-468 cells treated with or without NO for 10 days. β-values (5mC or 5hmC) at Enhancers, Gene Bodies, and Promoters (as determined by RnBeads) are ranked from lowest to highest and are paired with the expression changes (RNA-seq) in their associated genes. a Changes in gene expression and in 5mC in DNA from NO-treated MDA-MB-231 and MDA-MB-468 cells. b Changes in gene expression and in 5hmC in DNA from NO-treated MDA-MB-231 and MDA-MB-468 cells. Specific tumor-permissive genes that are associated with 5mC (c) and with 5hmC (d) at their promoter regions. Left panels Cellular Responses to NO, are representative genes shown with their difference in β-value at their promoter region (oxRRBS data) and their expression level (mRNA-seq) in response to NO. Right side, Clinical Correlations, are scatter plots demonstrating correlations between NOS2 expression and the indicated gene in tumors from patients with aggressive breast cancer. Far right column, Patient Survival TNBC, are Kaplan-Meier plots of TNBC patient survival and the expression levels high (red line) or low (black line) of the indicated gene.