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

DNA methylation at cytosine bases of eukaryotic DNA (5-methylcytosine, 5mC) is a heritable epigenetic mark that can regulate gene expression in health and disease. Enzymes that metabolize 5mC have been well-characterized, yet the discovery of endogenously produced signaling molecules that regulate DNA methyl-modifying machinery have not been described. Herein, we report that the free radical signaling molecule nitric oxide (NO) can directly inhibit the Fe(II)/2-OG-dependent DNA demethylases ten-eleven translocation (TET) and human AlkB homolog 2 (ALKBH2). Physiologic NO concentrations reversibly inhibited TET and ALKBH2 demethylase activity by binding to the mononuclear non-heme iron atom which formed a dinitrosyliron complex (DNIC) preventing cosubstrates (2-OG and O2) from binding. In cancer cells treated with exogenous NO, or cells endogenously synthesizing NO, there was a global increase in 5mC and 5-hydroxymethylcytosine (5hmC) in DNA, the substrates for TET, that could not be attributed to increased DNA methyltransferase activity. 5mC was also elevated in NO-producing cell-line-derived mouse xenograft and patient-derived xenograft tumors. Genome-wide DNA methylome analysis of cells chronically treated with NO (10 days) demonstrated enrichment of 5mC and 5hmC at gene-regulatory loci which correlated to changes in the expression of NO-regulated tumor-associated genes. Regulation of DNA methylation is distinctly different from canonical NO signaling and represents a novel epigenetic role for NO.

Introduction DNA methylation at the 5-carbon position of cytosine bases (5mC) is propagated through cell division and is considered a key epigenetic mechanism for cellular memory of transcriptional states.5mC at promoter CpG islands has been traditionally associated with repression of gene expression, Xchromosome inactivation, and genomic imprinting.Recent research has demonstrated a more complex relationship between DNA methylation and gene expression, that differs depending on the genomic context in which it occurs and is not restricted to transcriptional regulation/silencing at promoter regions.For example, 5mC methylation at enhancer regions can repress enhancer activity whereas gene body methylation can facilitate transcriptional elongation and is associated with actively transcribed genes.
Regardless of the functional consequences of 5mC on gene expression, speci c DNA methylation states are established and maintained in cells by the concerted activities of speci c methyl-modifying enzymes.
Despite the important gene-regulatory functions of DNA methylation, pathological alkylation damage from exposure to environmental or therapeutic alkylating agents or endogenous metabolic processes also causes DNA methylation.This type of alkylation DNA damage methylation occurs on any of the 4 DNA bases, but it is most prevalent on adenine (as 1-methyladenine (1mA)) and on cytosine (as 3methylcytosine (3mC)).To safeguard genomic integrity, repair pathways have evolved to recognize these cytotoxic alkylation-induced DNA lesions.ALKBH2 is a DNA repair enzyme in the AlkB family of enzymes that recognizes and demethylates 1mA and 3mC back to adenine and cytosine, respectively.CpG 5mC-DNA methylation and alkylation-mediated DNA methylation (1mA and 3mC) are both associated with numerous disease states including cancers.In cancer, hyper-or hypo-5mC DNA methylation can deactivate tumor suppressor genes or activate oncogenes, while 1mA and 3mC are mutagenic.
Despite the importance of methylated DNA bases and their oxidized derivatives in controlling normal gene expression or facilitating oncogenic transcriptional states, there is a lack of mechanistic knowledge regarding endogenous molecular regulators of DNA methyl-modifying machinery.Nitric oxide (NO) is an endogenously produced free radical signaling molecule that regulates protein function by either directly binding metal centers (the mechanism that activates soluble Guanylate Cyclase (sGC)) or by forming Snitrosothiols (RSNO) on cysteine residues.These signaling mechanisms underlie many NO-mediated physiological functions (i.e., immune defense, neurotransmission, and vasodilation).Nevertheless, the current understanding of NO signaling does not su ciently account for its diverse effects, especially in pathological conditions where NO synthesis and signaling are perturbed.This is particularly relevant in cancer, in which upregulated NO production, due to the induction of the inducible form of nitric oxide synthase (NOS2), is associated with altered tumor gene expression, poor patient outcomes, increased mortality, and resistance to chemotherapy across cancer types 1 , including triple-negative breast (TNBC) 2- 12 , lung [13][14][15] , prostate 16,17 , brain 18 , colon 19,20 , melanoma [21][22][23] , and liver 24, 25 .
Although dysregulated NO production and perturbations in DNA methylation patterns are both associated with many of the same pathologies, a causal mechanism linking NO synthesis to altered DNA methylation patterns has not been described.Our prior work demonstrated a novel role for NO as an endogenous epigenetic regulator of gene expression by controlling histone post-translational modi cations 26,27 and mRNA methylation (m 6 A) patterns 28 .Mechanistically, we demonstrated that physiologic NO concentrations could inhibit the catalytic activities of both histone lysine demethylases (KDM) and the RNA demethylase (fat mass and obesity associated protein (FTO) [29][30][31] ).As KDM, FTO, TET, and ALKBH2 all belong to the same family of Fe(II)/2-oxoglutarate(OG)-dependent oxygenases, we hypothesized that NO would similarly inhibit TET and ALKBH2 DNA demethylases.The catalytic activities of all Fe(II)/2-OG-dependent demethylases require the sequential binding of 2-OG and then molecular oxygen (O 2 ) to the mononuclear iron atom in the active site.Herein, we demonstrate that TET and ALKBH2 DNA demethylase activity is inhibited by the binding of two molecules of NO to the catalytic iron during the resting state of enzymes forming a dinitrosyliron complex (DNIC).
In this study we focused on models of TNBC, as patients with this molecular subtype that harbor NOS2expressing tumors have a signi cantly higher mortality rate than patients with tumors that do not express NOS2.We demonstrate that cells exposed to endogenous or exogenous physiologic NO concentrations exhibit inhibition of TET demethylation that causes gene-speci c enrichment of 5mC/5hmC at known tumor-permissive gene-regulatory loci.Although the ability to correctly assign the transcriptional regulation of a particular gene to the local (or distant) presence of DNA methylation has remained surprisingly limited 32 , our data suggest that deleterious phenotypic effects of NO are partially determined by transcriptional reprogramming mediated by TET inhibition and the resultant changes in the DNA methyl landscape.

Results
Determination of NO as an inhibitor of TET and ALKBH2 demethylase activity in vitro.
In humans, active DNA demethylation is catalyzed by 2 members of the Fe(II)/2-OG-dependent family of oxygenases; TET (there are 3 isoforms: TET1, TET2 & TET3) and ALKBH2.To test whether NO could inhibit TET catalytic activity we incubated the puri ed catalytic domain of human TET2 enzyme with all cofactors (2-OG, Fe(II), and ascorbate), substrate (synthetic 5mC-DNA oligo), and with NO (the NO-donor Sper/NO, 50 -500 mM).After 1 hour incubation we measured the relative amounts of the substrate for TET2 (5mC) and its initial oxidation product (5hmC) at each NO (Sper/NO) concentration using a MALDI-TOF-MS-based assay 33 (Fig. 1A).Using a broader range of physiologic/pathologic NO concentrations, we determined that NO inhibited TET2 demethylase activity with half-maximal inhibitory concentration (IC 50 ) of 164.5 mM for Sper/NO (~1.5 mM NO) (Fig. 1B).Next, using the IC 50 concentration for Sper/NO, we tested whether NO could also inhibit the stepwise TET2-catalyzed oxidation of 5hmC to 5fC and, in a separate reaction, the conversion of 5fC to 5caC.When synthetic DNA oligos containing 5hmC were used as TET2 substrates, the enzymatic conversion of 5hmC to 5fC was inhibited by 23%.Using 5fC as the substrate, conversion to 5caC was inhibited by 20% (Fig. 1C).
To determine how the duration of NO exposure affected TET2 activity and if TET2 inhibition by NO was reversible, we compared the demethylase activity of TET2 alone with the activity of TET2 incubated with NO for either a short or a long exposure time.This was accomplished using DEA/NO, a short-acting NO donor (t ½ ≈ 2 min at 37 o C), or Sper/NO, a longer-acting NO donor (t ½ ≈ 37 min at 37 o C) 34 .By using the same concentrations of these two NO donors, we were able to expose the enzyme to the same amounts of NO (moles) but for different durations of time.TET2 activity was measured at 1 and 3 hours.When TET2 was not treated with NO, demethylation of 5mC to 5hmC was 100% complete within one hour ("None", Fig. 1D).When TET2 was incubated for a short period of time with NO, its demethylase activity was initially inhibited (~50% product formation at 1 h), but when NO was no longer present, TET2 was able to completely convert 5mC to 5hmC at 3 hours ("DEA/NO", Fig. 1D).Conversely, when TET2 was exposed to a continuous steady-state NO concentration for the duration of the experiment, the catalytic activity of TET2 was inhibited (~55%) within 1 hour and no further catalysis occurred during the remaining 2 hours of the experiment ("Sper/NO", Fig. 1D).These results suggest that TET2 inhibition by NO is reversible.Next, instead of using a synthetic 5mC-DNA oligo substrate, we tested NO-mediated TET2 inhibition using its biological substrate: genomic DNA (Fig. 1E).Again, NO inhibited TET2-catalyzed conversion of 5mC to 5hmC in a concentration-dependent manner.
Having established that NO could inhibit TET2, we tested whether NO could similarly inhibit ALKBH2, another Fe(II)/2-OG-dependent DNA demethylase.ALKBH2 activity was measured in real-time using a uorescence assay 35 .This assay used a uorogenic 1-methyladenine probe that has a >10-fold increase in uorescent signal intensity when the alkyl group is demethylated by ALKBH2.We incubated recombinant ALKBH2 and all cofactors (2-OG, Fe(II), ascorbate) with the methylated probe substrate and NO (Sper/NO, 100-500 mM) for 60 minutes (Fig. 1F).Under these conditions, NO inhibited ALKBH2 demethylase activity in a concentration-dependent manner with an IC 50 for Sper/NO of 165 mM (Fig. 1G).
Next, we isolated nuclear and cytosolic extracts containing endogenous ALKBH2 from cultured MDA-MB-231 breast cancer cells to test whether NO could similarly inhibit ALKBH2 derived from biological sources.The combined extracts were exposed to low steady-state concentrations of NO using the NO donor DETA/NO (t ½ ≈ 22 h, 50-150 mM) and demethylase activity was monitored for 12 h using the uorescent probe method (Fig. 1H).Over this range of DETA/NO concentrations we expect that the steady-state concentrations of NO correspond to low nM physiological levels (as we have previously measured 36,37 ).Under these conditions ALKBH2 demethylase activity was inhibited in a dose-dependent manner.Collectively, these results indicate that NO is a potent inhibitor of Fe(II)/2-OG-dependent DNA demethylases TET2 and ALKBH2.For subsequent studies we solely focused on TET enzymes because of their gene regulatory functions (rather than ALKBH2 which is part of the DNA damage response).
Nitric oxide forms a dinitrosyl iron complex at the mononuclear non-heme iron atom in TET2.A critical step in the TET-catalyzed DNA demethylation reaction is binding O 2 to the non-heme iron atom 38 .We hypothesized that because of NO's structural and bonding similarity to O 2 that NO would compete with O 2 and inhibit TET by forming the more stable mononitrosyl complex at the iron site.To test whether NO interacts with the iron center, we conducted electron paramagnetic resonance (EPR) studies of TET2 treated with NO in the presence of all cofactors and substrate.We ran two identical reactions in parallel and stopped them at different time points (<1 and 20 minutes).EPR spectra at both time points showed that the enzyme does not form the expected S = 3/2 mononitrosyl complex (Fe(II)-NO), as shown by the absence of its intense g ┴ ~ 4.1 signal (Fig. S1A), and instead revealed the characteristic S = ½ signal of a non-heme dinitrosyl iron complex (DNIC, [Fe(NO) 2 ] 9 ), with g ┴ ~2.03, g || ~ 2.01 (Fig. 2A) 37,39,40 .The signature EPR spectrum of DNIC was almost undetectable when the TET2 enzyme was omitted from the complete reaction mixture (Fig. S1A & 2A), indicating that the observed DNIC signal is associated with the enzyme, and not with a complex formed in solution.Kinetically, the reaction was ~80% complete by the time all reactants were added, as shown by the small increase in the EPR intensity between samples frozen at 1 min and 20 min.
Density functional computations support the formation of a DNIC at the catalytic iron atom in TET2.Because the DNIC was formed instead of the mononitrosyl a series of density functional theory (DFT) computations were performed to investigate the relative a nity for binding of NO versus O 2 and water in a TET2 resting state.An active site model of TET2 was generated akin to that reported by Lu et al. 41 The charge of the model DNIC complex was adjusted to yield a neutral {Fe(NO) 2 } 9 electron count, i.e., d 6 Fe(II) + 2 NO p* e -+ 1 additional e-.Additional simulations were performed to assess NO binding to models in which 2-OG was ligated to the inner coordination sphere, but these were largely inconclusive apart from indicating that NO is bound more weakly to Fe(II) after ligation of 2-OG.The iron in the enzyme is coordinated by two histidine (His) and one aspartate (Asp) residue.To mimic the pertinent amino acid side chains, Asp was modeled by an acetate (OAc -) and His was modeled by N-methylimidizaole (Im).It was assumed that Asp and His model ligands would maintain a fac con guration.
The lowest energy coordination isomer had one NO trans to OAc and the other trans to Im.The OAc that mimics the Asp side chain forms a strong hydrogen bond with the ligated water.Given the experimental spectroscopic observations, it was investigated whether one of the NO ligands of Fe(OAc) (Im) 2 (NO) 2 (OH 2 ) could be displaced by either water or dioxygen; thus, computed DFT ground states of bond length of the optimized geometry of Fe(OAc)(Im) 2 (NO)(O 2 )(OH 2 ), (Fig. 2D), the DFT results suggest that O 2 does not readily displace NO, perhaps except at high O 2 partial pressures, and rationalizes the presence of only the di-nitrosyl complex as seen in the EPR spectra (Fig. 2A).
Based on the DFT results, further modeling was done for the DNIC core and the crystal structure of TET2 in complex with N-oxalyglycine (OGA), a 2-oxoglutarate analog 42 (PDB ID 4NM6, Fig. 2D).In the model, the NO molecules replace OGA and occupy similar positions to the OGA coordinating oxygens.One of these is close to Arg 1261, which may hydrogen bond to the NO and stabilize overall negative charge buildup on the NO ligands in the DNIC (Fig. 2E,F).
Nitric oxide increases 5mC in the DNA of human cancer cells.Having established that NO was a direct and potent inhibitor of TET enzymes under isolated conditions, the next step was to investigate whether NO could inhibit endogenous cellular TET enzymes and to determine if this would regulate nuclear DNA methylation.We selected four human cancer cell lines derived from aggressive tumor types that are known to express NOS2 and synthesize NO in vivo (2 triple negative breast (TNBC), 1 prostate, 1 brain).These cells do not synthesize NO in culture, so we treated them with DETA/NO (100 mM; 24 h), which resulted in low nM concentrations of NO, and measured 5mC-DNA (Fig. 3A).In all cell lines NO signi cantly increased global 5mC in DNA.Among NO-associated cancers, TNBC patients who harbor NOS2-expressing tumors have signi cantly worse prognoses.For this reason, we conducted all subsequent experiments using models of TNBC.
Under biological conditions, DNA methylation patterns are faithfully maintained over multiple cell generations as a form of epigenetic inheritance.Therefore, we developed a cell model to study DNA methylation responses to NO after multiple cell generations; this more accurately mimics the microenvironment of NOS2-expressing tumors in vivo where cells are exposed to chronic NO synthesis.We treated two TNBC cell lines with low physiologic steady-state concentrations of NO for 10 days (~12 cell doublings (NO did not alter the doubling rate)) and examined long-term "heritable" DNA methylation patterns.In the NO-treated cells, there was a signi cant increase in 5mC in DNA (Fig. 3B), and an increase in 5hmC in DNA, the rst oxidation product of TET (Fig. 3C).Cells not treated with NO had no change in 5mC/5hmC, suggesting 5mC increases were not attributable to epigenetic drift.To mimic the endogenous NO production observed in tumors, we transfected MDA-MB-231 cells with a human NOS2 gene (or empty vector control (VC)) (Fig. 3D).Accumulative NO synthesis was measured after 24 and 48 hours in both cell lines and NO was only detected in the NOS2-transfected cells, not in the cells transfected with the empty vector plasmid or in the NOS2-transfected cells treated with a pan-NOS inhibitor (L-NMMA) (Fig. 3E).5mC in DNA was also measured in both cell lines at 24 and 48 hours and 5mC was elevated only in the NO-producing cells but not the control cells or cells treated with L-NMMA (Fig. 3F).
5mC is catalytically installed on DNA by DNA methyltransferase enzymes (DNMT) which, along with TET enzymes, maintain steady-state 5mC levels.NO-dependent increases in 5mC could therefore be due to increased DNA methyltransferase activity rather than inhibition of TET DNA demethylase activity.To test this, we treated MDA-MB-231 cells with NO and either a DNA methyltransferase 1 (DNMT1) inhibitor (5-Azacytidine (AZA)), or a competitive inhibitor of methionine adenosyltransferase (MAT) (cycloleucine (CL)).CL depletes the cell of S-adenosylmethionine (SAM), the substrate for DNA methyltransferases (Fig. 3G).In cells treated with either AZA or CL alone, a signi cant reduction in global 5mC levels was observed as expected 43 , but when NO was present during either CL or AZA treatment, 5mC levels remained elevated (Fig. 3H).Another potential explanation for increases in 5mC would be if NO was changing the expression levels of DNA methyl-modifying enzymes (i.e., increasing DNMT or decreasing TET expression).To examine this possibility, we treated two TNBC cell lines with NO for 10 days and measured the protein expression levels of DNA methyltransferases (DNMT1, DNMT3a, DNMT3b) and DNA demethylases (TET1,2,3, and ALKBH2) (Fig. 3I,J).For all enzymes there was almost no change in protein expression in response to NO after 10 days.Together these data further support the hypothesis that NO-mediated increases in 5mC result from inhibition of TET demethylases and are not a result of changes in the expression levels of DNA methyl-modifying enzymes or increased DNA methyltransferase activity.
Nitric oxide increases 5mC in DNA from tumors in vivo.To investigate whether NO could increase 5mC in vivo we used a mouse xenograft model of NOS2-expressing cell-line derived tumors.Mice bearing NOS2expressing MDA-MB-231 xenograft tumors were divided into two groups; half were treated with aminoguanidine (AG), a selective inhibitor of NOS2, and the other half were treated with saline (control).
After 37 days of treatment, the tumors were removed, the DNA extracted, and 5mC-DNA was quanti ed (Fig. 3K).5mC in DNA from the NO-producing tumors was signi cantly greater than in the tumors where NO synthesis was inhibited.As further con rmation that NO regulates 5mC in vivo we measured 5mC-DNA in NOS2-positive patient derived xenograft (PDX) tumors (Fig. 3L).In this experiment the control group received a vehicle saline injection, and the treatment group was administered the pan-NOS inhibitor NG-monomethyl-L-arginine (L-NMMA) daily.After 40 days the tumors were excised and 5mC was measured.Again, in the NO-producing PDX tumors, 5mC was signi cantly higher than in the tumors where NO synthesis was inhibited (Fig. 3L).
NOS2 expression and NO production drive aggressive cancer phenotypes.Clinically, NOS2 expression in tumors is associated with worse patient outcomes and poor responses to therapy.We used Kaplan-Meier Plotter 44 to analyze transcriptomic datasets of metastatic breast cancer patients found in GEO, EGA and TCGA.When we examined NOS2 expression in 3 breast cancer patient groups (all subtypes, basal-like, and ER -/PR -) we found that high NOS2 expression was associated with decreased overall survival (con rming previous reports 11 ) (Fig. 4A).To experimentally determine whether NO would result in a more aggressive cell phenotype in vitro, we measured cell migration and invasion (two hallmarks of cancer) in real time of TNBC cells exposed to NO.In cells exposed to exogenous NO or in cells endogenously synthesizing NO, the rates of cell migration and invasion were increased compared to untreated control cells (Fig. 4B-D), consistent with what we and others have shown previously 6, 45 .Another phenotype associated with tumor aggressiveness is resistance to chemotherapy.Using the ROC plotter platform 46 , we analyzed the expression levels of NOS2 as a predictive biomarker of e cacy of any chemotherapy in TNBC patients (n = 164; response based on relapse-free survival at 5 years).Patient "non-responders" to therapy had signi cantly higher NOS2 expression than patient "responders" to therapy (Fig. 4E).
Nitric oxide regulates gene expression in TNBC cells.With the observation that NO directly inhibits TET demethylase activity leading to global increases in 5mC, we sought to decipher whether this was functionally associated with transcriptional changes in NO-regulated genes that may drive aggressive phenotypes.First, we quanti ed changes in transcription by performing RNA-sequencing on samples from two TNBC cell lines treated chronically (10 days) with NO.In both cell lines, NO signi cantly up-and down-regulated several hundred genes compared to untreated control cells (Fig. 4F,G).There were 880 signi cantly differentially expressed genes in the MDA-MB-231 cells with FDR (False Discovery Rate) <0.05 (454 upregulated log2FC>1, 426 downregulated log2FC <-1) and 765 signi cantly differentially expressed genes in the MDA-MB-468 cells (451 upregulated log2FC>1, 314 downregulated log2FC <-1).Although there was only a 12 -14% overlap in common genes transcriptionally regulated by NO between the two cell types (90 in total), this may be due to signi cant differences in their basal transcriptional pro les (control cells not treated with NO).Multidimensional scaling (MDS) analysis illustrated how the expression pro les differed far more between the two cell types than between the NO treated and control cells (Fig. 4H).Despite only modest overlap in speci c genes transcriptionally regulated by NO in both cell types, Gene Set Enrichment Analysis (GSEA) of the 90 genes differentially expressed in the same direction identi ed several KEGG pathways relevant to cancer progression (Table S1).
Nitric oxide increases 5mC and 5hmC differentially at speci c genomic features.To determine if NOmediated changes in 5mC/5hmC were functionally associated with changes in gene expression, we identi ed the locations of 5mC/5hmC on a genome-wide scale at single-nucleotide resolution by performing oxidative reduced representation bisul te sequencing (oxRRBS) on samples from two TNBC cell lines that were chronically treated with NO for 10 days.Between the NO-treated cells and the untreated cells we identi ed differentially methylated positions (DMPs) and differentially hydroxymethylated positions (DhMPs, 5hmC), de ned as: p < 0.05 and difference in β-value > 0.1.On a global scale, we found that both 5mC and 5hmC were increasing in both cell types in the NO-treated cells compared to the untreated control cells, consistent with the ELISA data in Figure 3 that NO increases 5mC/5hmC in cells.Although there were net increases in 5mC and 5hmC, these changes were dynamic in that both increases and decreases in 5hmC and 5mC were observed at all annotated regions (Fig. 4I).When we examined annotated CpG sites (islands, shores, shelves, and open seas) 47 we found that the majority (>60%) of DMPs and (>45%) DhMPs were occurring at open sea positions (Fig. 4J).We then focused on speci c functional elements (Super enhancers (SE), 5' UTR, 3' UTR, Typical enhancers (TE), promoters, exons, intergenic regions, and introns) to determine if they also exhibited speci c patterns of 5mC/5hmC enrichment.In both cell types, and at all genic annotations, we identi ed hyper-and hypo-DMPs and DhMPs (Fig. 4K, L), with the majority located at introns.The locations of DMPs and DhMPs were similar in both cell types, but the numbers of differentially methylated positions tended to be greater in the MDA-MB-468 cells (Fig. 4M).
Determination of 5mC-and 5hmC-associated transcriptional changes.Having demonstrated that NO increased 5mC/5hmC in DNA at genomic loci relevant to regulation of gene expression, and that NO produced signi cant transcriptional changes, we attempted to link changes in DNA methylation to the changes in gene expression.We identi ed overlaps between signi cantly expressed genes (RNA-seq) and their b-values (the degree of CpG methylation) at that gene or at gene regulatory loci associated with that gene (i.e.promoters, enhancers, super enhancers).For both control and NO treatment groups there was a clear negative correlation between 5mC b-values at promoters and gene expression in both cell types (Extended data Fig. 2).We next identi ed speci c genes that had signi cant transcriptional changes in response to NO (upregulated or downregulated) and also had signi cant changes in methylation (increase or decrease in b-value for 5mC or 5hmC) at speci c gene-regulatory genomic loci (Fig. 5 A-C, Extended data Tables 2 & 3).Although the correlations between methylation status (hyper or hypo, 5mC or 5hmC) of speci c gene-regulatory regions and the direction of transcriptional changes were not 100%, certain trends did emerge.For example, increases of 5mC/5hmC at promoters was more associated with downregulated genes whereas gene body enrichment of 5mC/5hmC was more associated with upregulated genes.Increased 5mC/5hmC at typical enhancers correlated to downregulation of associated genes.Although links between many of the differentially expressed genes in Fig. 5 A-C and cancer are unknown or have yet to be established, some of them have an experimental or clinical association with breast cancer progression (Fig. 5D, E).On the left sides of Figures 5D & E ("Cellular Responses to NO") are select genes that are transcriptionally regulated by NO and show signi cant changes in their b-values at the promoter regions for these genes (Fig. 5D is 5mC, 5E is 5hmC).On the right side of these gures ("Clinical Correlation") are results from analysis of publicly available data sets of gene expression from tumors of patients with aggressive breast cancers 48 .Genes that were transcriptionally regulated by NO in our cell culture models also had directionally similar gene expression changes in NOS2-expressing patient tumors.Kaplan Meier plots demonstrate the correlation between the NO-regulated gene and patient survival 44 .Although a direct relationship between these NO-regulated genes and cancer progression have yet to be documented in the scienti c literature, some reports demonstrate that the genes upregulated by NO (GJC2, CPA4, SMG8, COL5A2, POLQ) [49][50][51][52][53] and the genes downregulated by NO (SYTL1, PRR15L) 54,55 are associated with deleterious outcomes when up-or downregulated in the same direction in cancer patients.These data demonstrate that genes that exhibit changes in their promoter 5mC/5hmC status and are transcriptionally regulated by NO in breast cancer cells in vitro show similar trends in vivo in patient tumors, suggesting a link with NO-regulated genes and the association between poor patient outcomes in breast cancer.

Discussion
Here we demonstrate that physiologic NO concentrations directly inhibit TET and ALKBH2 via a novel mechanism involving DNIC assembly at the catalytic non-heme iron atom.Moreover, we nd that in cancer cells exposed to exogenous NO, or in cells endogenously synthesizing NO, 5mC/5hmC in DNA is increased, and that 5mC and 5hmC are enriched on gene-regulatory loci (i.e., promoter, enhancer regions) of speci c NO-regulated genes.
There have been several excellent studies that provided insight and mechanisms through which NO in uences DNA methylation patterns 56 , predominantly by modulating the expression levels of DNA methyl-modifying enzymes (i.e., methyltransferases or demethylases).For example, Switzer et al. demonstrated that NO caused a decrease in DNA methylation via S-nitrosation of DNA methyltransferase 1 (DNMT1) which induced DNMT1 degradation 57 .Similarly, another study demonstrated that, in the vascular wall, NO led to an overall decrease in DNA methylation by increasing TET activity and decreasing DNMT activity 58 .Conversely, others have shown that NO enhances the enzymatic activity of DNA methyltransferases (DNMTs) and downregulates TET enzymes to cause an increase in DNA methylation 59 .While our study does not refute these previous ndings, we provide multiple lines of biochemical evidence to demonstrate that NO can directly inhibit the catalytic activities of TET enzymes leading to increased 5mC/5hmC on DNA.
There are no structures of NO bound to TET/ALKBH2 and very little data on NO binding to any member of this family of proteins with the exception of two examples of the crystalline form demonstrating formation of the mono-NO at the iron site 60,61 .The non-heme Fe(II) in TET enzymes bind 2-OG in a bidentate manner during their catalytic cycle which leaves one coordination site available for O 2 binding, or as we initially hypothesize for NO binding.Intriguingly, EPR and modeling studies demonstrated that NO preferentially formed a DNIC and not a mononitrosyl, suggesting NO binds to the enzyme in its resting state prior to 2-OG coordination.This means that NO may compete with 2-OG and not O 2 to prevent the interaction of TET with all its DNA substrates allowing their differential accumulation.Kinetic experiments, conducted at "room air" oxygen concentrations (~220 mM), revealed that low physiologic concentrations of NO (< nM in the case of ALKBH2) were su cient to inhibit demethylase activity in vitro and in vivo.This indicates that TET enzymes have a much higher a nity for NO than O 2 .We also found that NO signi cantly inhibited TET's initial oxidation step (5mC Þ 5hmC) more than its subsequent oxidation steps (5hmC Þ 5fC Þ 5caC).Although further mechanistic studies are needed to delineate factors contributing to the differential buildup of TET substrates, these differences may partially be a function of differences in the catalytic rates for each of its oxidation steps, with the rst step, conversion of 5mC Þ 5hmC, being the fastest 62 .
Regardless of the mechanism of TET inhibition, in vitro (cells culture) and in vivo (cell-derived and patient derived xenograft) models demonstrated that NO was associated with global increases in 5mC and 5hmC, which is consistent with other reports demonstrating that TET2,3 knockdown in cancer cells resulted in an increase in 5mC and 5hmC 63 .Moreover, since TET inhibition by NO is reversible this suggests that NO could act as a "molecular switch" to turn on and off demethylase activity as a mechanism of dynamic regulation of DNA methylation.Cellular expression of NOS isoforms, which synthesize NO at different rates, could be the key determinant of whether NO signals epigenetically, through changes in DNA methylation, or predominantly through canonical mechanisms.
To our knowledge, these are the rst studies to look at transcriptional changes in cells exposed to chronic (10 day) physiologic low steady-state NO concentrations which more accurately re ects the cellular microenvironment of NOS2-expressing tumors where NO synthesis is constitutively turned on.Although we measured thousands of genes that were transcriptionally changing in response to NO, the overlap between speci c genes in two transcriptionally diverse cell types (MDA-MB-231, -468) was relatively small.A major question is whether TET inhibition by NO to increase 5mC/5hmC is a speci c mechanism to mediate these transcriptional changes or whether this represents a more generalized phenomenon associated with dysregulated gene expression.In this regard our ability to link promoter or gene body methylation (5mC or 5hmC) to the regulation of speci c genes did not prove to be 100% consistent across genomic regions or cell lines.
We found that there were NO-regulated genes associated with increases or decreases in promoter 5mC and 5hmC in both cell types.For example, 5mC methylation increased at the promoter regions of GJC2 and AAK1 genes which were transcriptionally upregulated by NO.Interestingly, 5mC methylation was also increased at the promoter regions of the genes SYTL1 and PRR15L, which were transcriptionally downregulated by NO.At gene bodies, 5mC was largely associated with increased gene expression.The strongest association was the enrichment of 5mC at typical enhancers in MDA-MB-668 cells which correlated strongly to downregulation of associated genes.5hmC is often found in active gene bodies, and linked to gene transcription or translation 64 .5hmC is usually present at transcription start sites (TSS) of genes with high CpG promoters, marked by bivalent histone modi cations, suggesting a role in regulating gene expression by modulating chromatin accessibility or inhibiting repressor binding 65 .We noted increases in gene expression correlated with 5hmC losses in typical enhancers (MDA-MB-468).
Although linking 5mC/5hmC to changes in gene expression in our models proved to be complicated, we did nd that many of the genes transcriptionally regulated by NO in cell culture showed similar directional expression changes in hundreds of clinical samples of NOS2-expressing tumors.Moreover, the directional changes in these NO-regulated methylated genes correlated to decreased patient survival for patients harboring NOS2 expressing tumors.For decades it has been widely accepted that DNA methylation at CpG promoter regions is associated with transcriptional repression.Yet, numerous recent studies challenge this by demonstrating the persistent inability to attribute gene expression causality to methylation at speci c DNA loci 66 .Several studies have shown an inverse correlation between DNA methylation of the rst intron and gene expression across multiple tissues and species 67 .Numerous other studies have demonstrated that gain of promoter methylation is associated with increased gene expression 68- 70 .For example, a large study of prostate cancers (1,117 samples) found that hypermethylated genes were strongly associated with increased gene expression 71 .Further complicating the issue are the multiple studies demonstrating that both transcription factor binding and changes in gene expression can in some cases precede DNA demethylation 72,73 .DNA methylation appears to be highly context speci c, even in a single cancer type like TNBC, the focus of this study.The correlations between TET expression, DNA methylation, and tumor progression are highly dependent on both the TET isoform (1, 2, or 3) and the type of breast cancer.For example, TET1 expression and 5hmC levels are decreased in tumor samples from luminal A, luminal B, and HER2-positive subtypes compared to normal breast tissue samples and correlate with larger tumors, advanced stage, lymph node status, and poor patient survival [74][75][76][77] .Conversely, in TNBC, TET1 expression is increased in tumor tissue samples compared to normal breast tissue samples, and its high expression correlates with poor patient outcomes 78, 79 .Taken together, we favor a more stochastic view of NO-mediated gene expression changes where NO inhibition of TET may predominantly lead to indiscriminate disruption of DNA methylation that cancers exploit to regulate tumor-permissive gene expression programs.This may ultimately bene t cancer cell survival by creating transcriptional diversity among populations of cells independent of their genetic transcriptomic background.This theory is somewhat supported by our data demonstrating that although NO-dependent transcriptomic changes were vastly different within two TNBC cell lines, similarities were noted at the level of tumor pathway analysis.
In conclusion, this study demonstrates that NO is an endogenous regulator of TET activity and DNA methylation.This represents a novel and unprecedented functional role for NO in regulating steady-state DNA methylation (and hydroxymethylation) levels.How changes in DNA 5mC/5hmC at speci c loci regulate the expression of NO-responsive genes and how this mechanism synergizes with or antagonizes other canonical NO signaling mechanisms is still an open question.In cancer, further mechanistic studies are needed to fully understand the functional consequences of NO-mediated TET inhibition in relation to transcriptional malleability, transcriptional heterogeneity, and phenotypic plasticity; all associated with more aggressive tumors, worse patient prognosis, and resistance to chemotherapies.The ndings presented herein have been in the context of cancers (breast), but we suspect that the fundamental discovery that NO inhibits TET enzymes to change DNA methylation patterns is a contributing factor to numerous diseases where there is dysregulated NO synthesis and aberrant DNA methylation patterns 80 .
Moreover, this discovery raises the possibility that NO could regulate DNA methylation to control gene expression under physiological settings which should be explored further.Our previous work demonstrated that NO is an endogenous regulator of histone posttranslational modi cations 26,27 and mRNA methylation 28 , and here we show how NO regulates DNA methylation.Therefore, in addition to its canonical roles in cell signaling and gene expression, NO should be recognized as a dominant regulator of the epigenetic landscape 81 .
group received the NOS inhibitor aminoguanidine at a concentration of 0.5 g/L in lter-sterilized drinking water.After 6 weeks, the mice were sacri ced and subjected to imaging.All animal protocols were approved and followed the principles outlined in the Guide for the Care and Use of Laboratory Animals by the Institute of Laboratory Animal Resources, National Research Council.

Patient-derived xenograft mouse studies
PDX in vivo studies were conducted by the Chang Lab at Houston Methodist Hospital in 3 PDXs derived from primary human TNBC tumors: BCM-5998, BCM-3107, and BCM-3807.These PDXs were transplanted into the cleared mammary fat pad of mice.Upon reaching an average tumor volume of 150-250 mm 3 , the mice were randomly assigned to either the treatment or control group.For treatment, mice received intraperitoneal injections of L-NMMA (Santa Cruz Biotechnology) and amlodipine in sterile PBS (100 µL total/animal).L-NMMA was administered at 400 mg/kg on the rst day and 200 mg/kg on subsequent days.Amlodipine (Major Pharmaceuticals NDC 0904-6371-61) was given at a dose of 10 mg/kg along with each L-NMMA treatment to counteract the elevated blood pressure associated with eNOS inhibition.The control group received sterile PBS via intraperitoneal injection (100 µL/animal).All animal procedures were approved by the Houston Methodist Hospital Research Institute Animal Care and Use Review O ce.

Western blot
Whole cell lysates were obtained using the RIPA Lysis Buffer System (Santa Cruz), which includes lysis buffer, phenylmethylsulfonyl uoride, protease inhibitor cocktail, and sodium orthovanadate.The protein concentration was determined using the Lowry assay (Biorad DC protein assay).Subsequently, 30-40 µg of lysate was loaded into each well of a 10-well 10% Mini-PROTEAN TGX precast gel with Laemmli sample buffer and β-mercaptoethanol.Electrophoresis was conducted at 115 V for 1 hour.Protein transfer to a PVDF membrane was accomplished using the iBlot™ Transfer System (Invitrogen).The membrane was then blocked and incubated overnight at 4°C with the primary antibody in 5% milk in PBS-Tween.Following secondary antibody incubation, each blot was imaged using the FluorChem E system (ProteinSimple).Chemiluminescent substrate coating, either SuperSignal™ West Femto Maximum Sensitivity Substrate (Thermo Scienti c) or SuperSignal™ West Pico PLUS Chemiluminescent Substrate (Thermo Scienti c), was applied before imaging.Densitometry was conducted using Fiji (ImageJ).The list of antibodies used is provided below.E. Coli expression/puri cation and i n vitro TET2 enzymatic assays TET2 enzyme was expressed and puri ed from BL21 E. Coli (as described 33 ) using a truncated 54.64 kD TET2 (1099-1936 with residues 1481-1843 replaced by a 15-residue GS linker).In vitro TET2 enzymatic assay was conducted by incubating 10 µM of an 8-nucleotide double-stranded DNA substrate (5'-CAC-X-GGTG-3', where X is 5mC, 5hmC, or 5fC) with 5 µM of puri ed TET2 and varying concentrations of Sper/NO or DEA/NO.The reaction was allowed to proceed for 1-3 hours at 37C in a 25 µL assay also containing 50 mM HEPES (pH 8.0), 100 mM NaCl, 100 µM Fe(NH4)2(SO4)2, 2 mM ascorbate, 1 mM DTT, 1 mM ATP, and 1 mM 2-KG.The DNA was desalted by adding 5 µL of AG® 50W-X8 Cation Exchange Resin (BioRad, Cat # 143-5441) directly into the biochemical mixture and agitated followed by incubation for 5 min at room temperature.The samples were then centrifuged at 10,000 rpm for 2 min. 1 µL of sample was mixed with 1 µL of 3-Hydroxypicolinic Acid + Ammonium Citrate Dibasic matrix on a MALDI plate and the oxidized products were analyzed by MALDI-TOF mass spectrometry (Brukerultra eXtreme™ MALDI-TOF/TOF spectrometer) using re ectron negative ionization mode and a mass range of 2400-2500.

Density functional theory simulations and modeling
Density functional theory simulations employed the Gaussian 16 code 92 to investigate the stability of nitrosyl complexes of Fe(II) active site models of TET2.As per the study of Lu et al., 41 the wB97xD functional was employed as this Hamiltonian incorporates dispersion effects 93 , in conjunction with the def2-svp basis set 94 for geometry optimizations and vibrational frequency calculation.For more accurate reaction energies, a single point calculation with a larger basis set -def2-tzvpp -was employed at the optimized stationary points.For the model complexes investigated all reasonable charge, spin and coordination states were examined.The results presented herein focus on the ground states identi ed for the proposed reaction intermediates.All complexes were fully optimized in the absence of any geometric or symmetry constraints.Quoted thermodynamic values assumed a temperature of 298.15K and a pressure of 1 atm; enthalpic and entropic corrections used unscaled vibrational frequencies obtained at the wB97xD/def2-svp level of theory.An SMD continuum solvation model (solvent = water) was employed for closer congruence with experimental conditions 94 .
The DNIC was modeled into PDB le 4NM6 42 using the modeling program COOT 95 .The model was energy minimized using REFMAC 96 as implemented in CCP4i 97 .The NO bond length was restrained to 1.12 Å and the Fe-NO bond lengths restrained to 2.0Å.

Declarations
Data Availability RNA-sequencing and RRBS-sequencing data are stored in the NCBI GEO repository under accession number GSE248151.Source data are provided with this paper.

Fe
(OAc)(Im) 2 (NO)(OH 2 ) 2 and Fe(OAc)(Im) 2 (NO)(O 2 )(OH 2 ) were sought.The lowest energy geometries are shown in Fig. 2C.In both cases, geometry optimizations initiated from six-coordinate, pseudo-octahedral starting guesses yielded minima with a weakly bound exogenous ligand.For Fe(OAc)(Im) 2 (NO)(OH 2 ) 2 , the NO ligand is ejected from the inner coordination sphere upon geometry optimization, Fe … NO = 4.13 Å.For Fe(OAc)(Im) 2 (NO)(O 2 )(OH 2 ), the complex barely maintains an octahedral geometry with the dioxygen very weakly bonded (Fe … O 2 ~ 2.95 Å).The tenuous nature of O 2 binding in Fe(OAc)(Im) 2 (NO)(O 2 )(OH 2 ) is further indicated by the near complete lack of any spin delocalization from the O 2 to the complex (r spin (O 2 ) = 1.99 e -) and the computed free energy for NO/H 2 O exchange, DG = +23.0kcal/mol, indicating that NO binds much more tightly than water.The NO/O 2 exchange free energy is essentially thermoneutral, DG = -0.3kcal/mol.In conjunction with the weak O 2 binding indicated by the long Fe … O 2

Figures
Figures

Figure 1 NO
Figure 1

Figure 5 NO
Figure 5 91om temperature for 25 min.The demethylase assay was initiated with 1 µg of genomic DNA, isolated from HEK293T cells, and incubated at 37°C for 3 hours.After incubation, ¼ volume of 2 M NaOH-50 mM EDTA was added before addition of 1:1 ice cold 2 M ammonium acetate.Immobilin-P upper and lower values of a given curve; logIC50: same log units as X; Hill Slope: Slope factor or Hill slope, unitless.5mCand5hmCELISADNAwasextractedfromcellsortissuesusingtheQIAGENDNEasyBlood&Tissue kit and quanti ed using the BioTek Take3 system.Global %5mC and %5hmC were quanti ed by ELISA assay (Epigentek P-1030-96, P-1032-96).First, 100 ng DNA was bound to the bottom of each assay well.The wells were washed, and detection complex solution was added containing a 5mC or 5hmC antibody.After a 50minute incubation, wells were washed, and color developer solution was added before measuring absorbance at 450 nm.%5mC and %5hmC were calculated as a proportion of the total DNA.Libraries were prepared with Kapa Hyper Stranded mRNA library kit (Roche).The libraries were pooled; quantitated by qPCR and sequenced on one SP lane for 101 cycles from one end of the fragments on a NovaSeq 6000.Fastq les were generated and demultiplexed with the bcl2fastq v2.20Conversion Software (Illumina).Library preparation and sequencing was conducted by the University of Illinois at Urbana Champaign Roy J. Carver Biotechnology Center in triplicate, with a total of 476 million reads 100 nt in length.Read quality was assessed using MultiQC.Reads were aligned to hg38 GENCODE human genome (release 22) using STAR 2.5.2a 82orted using samtools83.Gene counts were generated using HTSeq84.Differential expression analysis was completed using edgeR v3.40.285.Gene Set Enrichment Analysis was completed using OmicPath Genomic DNA was extracted and puri ed from cells using the DNEasy Blood & Tissue kit (QIAGEN) with RNase step.The libraries were prepared with the Ovation® RRBS Methyl-Seq with TrueMethyl® oxBS from Tecan.Th libraries were pooled; quantitated by qPCR and sequenced on one S1 lane for 101 cycles from one end of the fragments on a NovaSeq 6000.Fastq les were generated and demultiplexed with the bcl2fastq v2.20Conversion Software (Illumina).Oxidative reduced representation bisul te sequencing library preparation and sequencing was performed by the UIUC core in duplicate with a total of 924 million reads 20-100 nt in length.Red quality was assessed using MultiQC 86 .Sequences were trimmed using TrimGalore (https://github.com/FelixKrueger/TrimGalore).Reds were aligned to hg38 using bowtie2 within Bismark (mapping e ciency ~ 62%) to count 5mC and 5hmC marks in a CpG context.Methylation analysis was performed using Bismark 87 and Rnbeads88.Differential methylation analysis was performed using RnBeads, Dnmtools 89 , and MethylKit90.CpG annotation was performed with the annotatr package91from BioConductor.
ascorbate, 100 mM NaCl, 1 mM ATP, and 50 mM HEPES.Samples were ash frozen in liquid nitrogen either immediately after adding NO or 20 minutes after.X-band continuous wave EPR spectroscopy (a) at 77 K was conducted using a modi ed Varian E4 spectrometer equipped with a quartz nger dewar that operates at liquid nitrogen temperature, and (b) at 10 K using a Bruker ESP 300 spectrometer equipped with an Oxford Instruments ESR 910 continuous helium ow cryostat.Experimental parameters: for Fe(II)-NO signals 10 K, M.W. frequency 9.37 GHz, modulation amplitude 5 G; for DNIC spectra T = 77 K, M.W. frequency 9.135 GHz, modulation amplitude 10 G.RNA sequencing (RNA-seq) and analysis RNA was isolated using the RNAqueous™-4PCR kit (Ambion) and treated with DNase I (Ambion) to avoid genomic DNA contaminations.(https://github.com/CBIIT-CGBB/OmicPath).