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GO terms associated with upregulated genes bearing new ∙NO-induced H3K9ac peaks around promoter regions

Supplementary Materials and Methods. Figure S1. Nitric oxide induces significant changes in histone posttranslational modifications. Figure S2. NO-mediated gene expression changes are consistent with the induction of an oncogenic phenotype. Figure S3. Nitric oxide alters the distribution patterns of H3K9ac and H3K9me2 across the genome. Figure S4. NO-upregulated genes with a new promoter-associated H3K9ac peak are involved in critical tumorigenic pathways. Table S1. NO-mediated changes in global levels of histone acetylation. Table S2. List of NO-regulated miRNAs (1.5 Fold Change cut-off). Table S3. Control ChIP-PCR primer sequences. Table S4. List of transcription factors with binding motifs found exclusively around promoters of upregulated genes with a new NO-induced H3K9ac peak.

Nitrite represents an endocrine reserve of bioavailable nitric oxide (NO) that mediates a number of physiological responses including conferral of cytoprotection after ischemia/reperfusion (I/R). It has long been known that nitrite can react with non-heme iron to form dinitrosyliron complexes (DNIC). However, it remains unclear how quickly nitrite-dependent DNIC form in vivo, whether formation kinetics differ from that of NO-dependent DNIC, and whether DNIC play a role in the cytoprotective effects of nitrite. Here we demonstrate that chronic but not acute nitrite supplementation increases DNIC concentration in the liver and kidney of mice. Although DNIC have been purported to have antioxidant properties, we show that the accumulation of DNIC in vivo is not associated with nitrite-dependent cytoprotection after hepatic I/R. Further, our data in an isolated mitochondrial model of anoxia/reoxygenation show that while NO and nitrite demonstrate similar S-nitrosothiol formation kinetics, DNIC formation is significantly greater with NO and associated with mitochondrial dysfunction as well as inhibition of aconitase activity. These data are the first to directly compare mitochondrial DNIC formation by NO and nitrite. This study suggests that nitrite-dependent DNIC formation is a physiological consequence of dietary nitrite. The data presented herein implicate mitochondrial DNIC formation as a potential mechanism underlying the differential cytoprotective effects of nitrite and NO after I/R, and suggest that DNIC formation is potentially responsible for the cytotoxic effects observed at high NO concentrations., Graphical abstract fx1, Highlights • Dietary nitrite results in DNIC formation in many tissues, most notably the liver. • Nitrite-dependent DNIC accumulate within the mitochondrion. • NO generates greater DNIC formation in the mitochondrion than nitrite. • At high concentrations of NO DNIC formation is associated with mitochondrial injury.

Nitric oxide (NO), the endogenously produced free radical signaling molecule, is generally thought to function via its interactions with heme-containing proteins, such as soluble guanylyl cyclase (sGC), or by the formation of protein adducts containing nitrogen oxide functional groups (such as S -nitrosothiols, 3-nitrotyrosine, and dinitrosyliron complexes). These two types of interactions result in a multitude of down-stream effects that regulate numerous functions in physiology and disease. Of the numerous purported NO signaling mechanisms, epigenetic regulation has gained considerable interest in recent years. There is now abundant experimental evidence to establish NO as an endogenous epigenetic regulator of gene expression and cell phenotype. Nitric oxide has been shown to influence key aspects of epigenetic regulation that include histone posttranslational modifications, DNA methylation, and microRNA levels. Studies across disease states have observed NO-mediated regulation of epigenetic protein expression and enzymatic activity resulting in remodeling of the epigenetic landscape to ultimately influence gene expression. In addition to the well-established pathways of NO signaling, epigenetic mechanisms may provide much-needed explanations for poorly understood context-specific effects of NO. These findings provide more insight into the molecular mechanisms of NO signaling and increase our ability to dissect its functional role(s) in specific micro-environments in health and disease. This review will summarize the current state of NO signaling via epigenetic mechanisms (the “third pillar” of NO signaling).

The chelatable iron pool (CIP) is a small but chemically significant fraction of total cellular iron. While this dynamic population of iron is limited, it is redox active and capable of generating reactive oxygen species (ROS) that can lead to oxidative stress which is associated with various pathologies. Nitric oxide (•NO), is a free radical signalling molecule that regulates numerous physiological and pathological conditions. We have previously shown that macrophages exposed to endogenously generated or exogenously administered nitric oxide (•NO) results in its interaction with CIP to form dinitrosyliron complexes with thiol containing ligands (DNICs). In this study we assessed the consequences of DNIC formation in cancer cells as •NO is known to be associated with numerous malignancies. Incubation of cancer cells with •NO led to a time and dose dependent increase in formation of DNICs. The formation of DNICs results in the sequestration of the CIP which is a major source of iron for redox reactions and reactive oxygen species (ROS) generation. Therefore, we set out to test the antioxidant effect of •NO by measuring the ability of DNICs to protect cells against oxidative stress. We observed that cancer cells treated with •NO were partially protected against H(2)O(2) mediated cytotoxicity. This correlated to a concomitant decrease in the formation of oxidants when •NO was present during H(2)O(2) treatment. Similar protective effects were achieved by treating cells with iron chelators in the presence of H(2)O(2). Interestingly, •NO decreased the rate of cellular metabolism of H(2)O(2) suggesting that a proportion of H(2)O(2) is consumed via reactions with cellular iron. When the CIP was artificially increased by supplementation of cells with iron, a significant decrease in the cytoprotective effect of •NO was observed. Notably, •NO concentrations, at which cytoprotective and antioxidant effects were observed, correlated with concentration-dependent increases in DNIC formation. Collectively, these results demonstrate that •NO has antioxidant properties by its ability to sequester cellular iron. This could play a significant role in variety of diseases involving ROS mediated toxicity like cancer and neurodegenerative disorders where •NO has been shown to be an important etiologic factor.

Within the human body, nitric oxide (NO) can arise from several sources including enzymatic synthesis, dietary nitrate, and pharmacological agents. Regardless of its source, the phenotypic consequences of NO production are influenced by complex microenvironmental factors that determine the target molecules NO will react with. Classical signaling mechanisms of NO are mediated via interactions with soluble guanylyl cyclase (sGC) and other heme-containing proteins or through the formation of protein adducts (S-nitrosothiols, 3-nitrotyrosine, and dinitrosyliron complexes). These interactions usually result in a loss or gain of protein function, thereby altering cellular phenotype. In addition, multiple lines of evidence have emerged recently demonstrating that a significant proportion of phenotypic changes attributed to NO production may be downstream from epigenetic regulatory events. It is becoming clear that NO affects dozens of histone modifications as well as DNA adducts, thus altering the epigenetic programs controlling the gene expression patterns that dictate cell phenotype, fate, and differentiation. This chapter will give an overview of the major NO-driven epigenetic mechanisms using specific examples.

N-Myc downstream-regulated gene 1 (NDRG1) is a ubiquitous cellular protein that is up-regulated under a multitude of stress and growth-regulatory conditions. Although the exact cellular functions of this protein have not been elucidated, mutations in this gene or aberrant expression of this protein have been linked to both tumor suppressive and oncogenic phenotypes. Previous reports have demonstrated that NDRG1 is strongly up-regulated by chemical iron chelators and hypoxia, yet its regulation by the free radical nitric oxide (•NO) has never been demonstrated. Herein, we examine the chemical biology that confers NDRG1 responsiveness at the mRNA and protein levels to •NO. We demonstrate that the interaction of •NO with the chelatable iron pool (CIP) and the appearance of dinitrosyliron complexes (DNIC) are key determinants. Using HCC 1806 triple negative breast cancer cells, we find that NDRG1 is up-regulated by physiological •NO concentrations in a dose- and time-dependant manner. Tumor cell migration was suppressed by NDRG1 expression and we excluded the involvement of HIF-1α, sGC, N-Myc, and c-Myc as upstream regulatory targets of •NO. Augmenting the chelatable iron pool abolished •NO-mediated NDRG1 expression and the associated phenotypic effects. These data, in summary, reveal a link between •NO, chelatable iron, and regulation of NDRG1 expression and signaling in tumor cells.

It is well established that nitric oxide ((•)NO) reacts with cellular iron and thiols to form dinitrosyliron complexes (DNIC). Little is known, however, regarding their formation and biological fate. Our quantitative measurements reveal that cellular concentrations of DNIC are proportionally the largest of all (•)NO-derived adducts (900 pmol/mg protein, or 45-90 μM). Using murine macrophages (RAW 264.7), we measured the amounts, and kinetics, of DNIC assembly and disappearance from endogenous and exogenous sources of (•)NO in relation to iron and O(2) concentration. Amounts of DNIC were equal to or greater than measured amounts of chelatable iron and depended on the dose and duration of (•)NO exposure. DNIC formation paralleled the upregulation of iNOS and occurred at low physiologic (•)NO concentrations (50-500 nM). Decreasing the O(2) concentration reduced the rate of enzymatic (•)NO synthesis without affecting the amount of DNIC formed. Temporal measurements revealed that DNIC disappeared in an oxygen-independent manner (t(1/2)=80 min) and remained detectable long after the (•)NO source was removed (24 h). These results demonstrate that DNIC will be formed under all cellular settings of (•)NO production and that the contribution of DNIC to the multitude of observed effects of (•)NO must always be considered.

The molecular circadian clock mechanism is highly conserved between mammalian and avian species. Avian circadian timing is regulated at multiple oscillatory sites, including the retina, pineal, and hypothalamic suprachiasmatic nucleus (SCN). Based on the authors' previous studies on the rat ovary, it was hypothesized that ovarian clock timing is regulated by the luteinizing hormone (LH) surge. The authors used the chicken as a model to test this hypothesis, because the timing of the endogenous LH surge is accurately predicted from the time of oviposition. Therefore, tissues can be removed before and after the LH surge, allowing one to determine the effect of LH on specific clock genes. The authors first examined the 24-h expression patterns of the avian circadian clock genes of Bmal1, Cry1, and Per2 in primary oscillatory tissues (hypothalamus and pineal) as well as peripheral tissues (liver and ovary). Second, the authors determined changes in clock gene expression after the endogenous LH surge. Clock genes were rhythmically expressed in each tissue, but LH influenced expression of these clock genes only in the ovary. The data suggest that expression of ovarian circadian clock genes may be influenced by the LH surge in vivo and directly by LH in cultured granulosa cells. LH induced rhythmic expression of Per1 and Bmal1 in arrhythmic, cultured granulosa cells. Furthermore, LH altered the phase and amplitude of clock gene rhythms in serum-shocked granulosa cells. Thus, the LH surge may be a mechanistic link for communicating circadian timing information from the central pacemaker to the ovary.

The role of nitric oxide (NO()) as a mediator of cancer phenotype has led researchers to investigate strategies for manipulating in vivo production and exogenous delivery of this molecule for therapeutic gain. Unfortunately, NO() serves multiple functions in cancer physiology. In some instances, NO() or nitric oxide synthase (NOS) levels correlate with tumor suppression and in other cases they are related to tumor progression and metastasis. Understanding this dichotomy has been a great challenge for researchers working in the field of NO() and cancer therapy. Due to the unique chemical and biochemical properties of NO(), it's interactions with cellular targets and the subsequent downstream signaling events can be vastly different based upon tumor heterogeneity and microenvironment. Simple explanations for the vast range of NO-correlated behaviors will continue to produce conflicting information about the relevance of NO() and cancer. Paying considerable attention to the chemical properties of NO() and the methodologies being used will remove many of the discrepancies in the field and allow for in depth understanding of when NO-based chemotherapeutics will have beneficial outcomes.

Although it is generally accepted that the circadian clock provides a timing signal for the luteinizing hormone (LH) surge, mechanistic explanations of this phenomenon remain underexplored. It is known, for example, that circadian locomotor output cycles kaput (clock) mutant mice have severely dampened LH surges, but whether this phenotype derives from a loss of circadian rhythmicity in the suprachiasmatic nucleus (SCN) or altered circadian function in gonadotropin-releasing hormone (GnRH) neurons has not been resolved. GnRH neurons can be stimulated to cycle with a circadian period in vitro and disruption of that cycle disturbs secretion of the GnRH decapeptide. We show that both period-2 (PER2) and brain muscle Arnt-like-1 (BMAL1) proteins cycle with a circadian period in the GnRH population in vivo. PER2 and BMAL1 expression both oscillate with a 24-hour period, with PER2 peaking during the night and BMAL1 peaking during the day. The population, however, is not as homogeneous as other oscillatory tissues with only about 50% of the population sharing peak expression levels of BMAL1 at zeitgeber time 4 (ZT4) and PER2 at ZT16. Further, a light pulse that induced a phase delay in the activity rhythm of the GnRH-eGFP mice caused a similar delay in peak expression levels of BMAL1 and PER2. These studies provide direct evidence for a functional circadian clock in native GnRH neurons with a phase that closely follows that of the SCN.

Gonadotropin-releasing hormone (GnRH) neurons in the hypothalamus play an important role in reproductive function. These cells originate in the nasal compartment and migrate into the basal forebrain in association with olfactory/vomeronasal nerves in embryonic life in rodents. Here, we studied the role of neuropilins and their ligands, semaphorins, in the development of the olfactory-GnRH system. We focused onNeuropilin-2knock-out (Npn-2−/−) mice, because they are known to display defasciculation of olfactory nerves and reduced fertility. We found a significant decrease in the number of GnRH neurons in the hypothalamus and a marked reduction in their gonadal size. We then observed an abnormal increase of GnRH neurons in the noses ofNpn-2−/−mice, indicating that these cells failed to migrate into the forebrain. However, because neuropilins and semaphorins are involved in events of neuronal migration in the brain, we asked whether the observed reduction in GnRH neurons was directly attributable to the action of these molecules. Using fluorescence-activated cell sorting and reverse transcription-PCR on mRNA derived from embryonic green fluorescent protein (GFP)–GnRH transgenic mice, we found expression of class 3 semaphorins and their receptors (neuropilin-1/2 and plexin-A1) in GnRH neurons. Furthermore, double-immunofluorescence experiments showed that migrating GnRH neurons, as well as associated olfactory fibers, express Npn-2 in the nasal region. We then used a line of immortalized GnRH neurons (GN11 cells) that display the same expression patterns for semaphorins and their receptors as GFP–GnRH cells and found that class 3 semaphorins and vascular endothelial growth factors modulate their migratory activity. These studies provide support for the direct involvement of neuropilins and their ligands in the establishment of the GnRH neuroendocrine system.

Altered nitric oxide (•NO) metabolism underlies cancer pathology, but mechanisms explaining many •NO-associated phenotypes remain unclear. We have found that cellular exposure to •NO changes histone posttranslational modifications (PTM) by directly inhibiting the catalytic activity of JmjC-domain containing histone demethylases. Herein, we describe how •NO exposure links modulation of histone PTMs to gene expression changes that promote oncogenesis. Through high-resolution mass spectrometry, we generated an extensive map of •NO-mediated histone PTM changes at 15 critical lysine residues on the core histones H3 and H4. Concomitant microarray analysis demonstrated that exposure to physiologic •NO resulted in the differential expression of over 6,500 genes in breast cancer cells. Measurements of the association of H3K9me2 and H3K9ac across genomic loci revealed that differential distribution of these particular PTMs correlated with changes in the level of expression of numerous oncogenes, consistent with epigenetic code. Our results establish that •NO functions as an epigenetic regulator of gene expression mediated by changes in histone PTMs. Cancer Res; 75(24); 5299–308. ©2015 AACR.

Methylation of lysine residues on histone tails is an important epigenetic modification that is dynamically regulated through the combined effects of methyltransferases and demethylases. The Jumonji C domain Fe(II) α-ketoglutarate family of proteins performs the majority of histone demethylation. We demonstrate that nitric oxide ((•)NO) directly inhibits the activity of the demethylase KDM3A by forming a nitrosyliron complex in the catalytic pocket. Exposing cells to either chemical or cellular sources of (•)NO resulted in a significant increase in dimethyl Lys-9 on histone 3 (H3K9me2), the preferred substrate for KDM3A. G9a, the primary methyltransferase acting on H3K9me2, was down-regulated in response to (•)NO, and changes in methylation state could not be accounted for by methylation in general. Furthermore, cellular iron sequestration via dinitrosyliron complex formation correlated with increased methylation. The mRNA of several histone demethylases and methyltransferases was also differentially regulated in response to (•)NO. Taken together, these data reveal three novel and distinct mechanisms whereby (•)NO can affect histone methylation as follows: direct inhibition of Jumonji C demethylase activity, reduction in iron cofactor availability, and regulation of expression of methyl-modifying enzymes. This model of (•)NO as an epigenetic modulator provides a novel explanation for nonclassical gene regulation by (•)NO.

S-Nitrosothiol (RSNO) formation is one manner by which nitric oxide (•NO) exerts its biological effects. There are several proposed mechanisms of formation of RSNO in vivo: auto-oxidation of •NO, transnitrosation, oxidative nitrosylation, and from dinitrosyliron complexes (DNIC). Both free •NO, generated by •NO donors, and S-nitrosocysteine (CysNO) are widely used to study •NO biology and signaling, including protein S-nitrosation. It is assumed that the cellular effects of both compounds are analogous and indicative of in vivo •NO biology. A quantitative comparison was made of formation of DNIC and RSNO, the major •NO-derived cellular products. In RAW 264.7 cells, both •NO and CysNO were metabolized, leading to rapid intracellular RSNO and DNIC formation. DNIC were the dominant products formed from physiologic •NO concentrations, however, and RSNO were the major product from CysNO treatment. Chelatable iron was necessary for DNIC assembly from either •NO or CysNO, but not for RSNO formation. These profound differences in RSNO and DNIC formation from •NO and CysNO question the use of CysNO as a surrogate for physiologic •NO. Researchers designing experiments intended to elucidate the biological signaling mechanisms of •NO should be aware of these differences and should consider the biological relevance of the use of exogenous CysNO. Antioxid. Redox Signal. 17, 962–968.

N-Myc downstream-regulated gene 1 (NDRG1) is a ubiquitous cellular protein that is up-regulated under a multitude of stress and growth-regulatory conditions. Although the exact cellular functions of this protein have not been elucidated, mutations in this gene or aberrant expression of this protein have been linked to both tumor suppressive and oncogenic phenotypes. Previous reports have demonstrated that NDRG1 is strongly up-regulated by chemical iron chelators and hypoxia, yet its regulation by the free radical nitric oxide ((•)NO) has never been demonstrated. Herein, we examine the chemical biology that confers NDRG1 responsiveness at the mRNA and protein levels to (•)NO. We demonstrate that the interaction of (•)NO with the chelatable iron pool (CIP) and the appearance of dinitrosyliron complexes (DNIC) are key determinants. Using HCC 1806 triple negative breast cancer cells, we find that NDRG1 is up-regulated by physiological (•)NO concentrations in a dose- and time-dependant manner. Tumor cell migration was suppressed by NDRG1 expression and we excluded the involvement of HIF-1α, sGC, N-Myc, and c-Myc as upstream regulatory targets of (•)NO. Augmenting the chelatable iron pool abolished (•)NO-mediated NDRG1 expression and the associated phenotypic effects. These data, in summary, reveal a link between (•)NO, chelatable iron, and regulation of NDRG1 expression and signaling in tumor cells.

Despite a central circadian role in Drosophila for the transcriptional regulator Timeless ( dTim ), the relevance of mammalian Timeless ( mTim ) remains equivocal. Conditional knockdown of mTim protein expression in the rat suprachiasmatic nucleus (SCN) disrupted SCN neuronal activity rhythms, and altered levels of known core clock elements. Full-length mTim protein (mTIM-fl) exhibited a 24-hour oscillation, where as a truncated isoform (mTIM-s) was constitutively expressed. mTIM-fl associated with the mammalian clock Period proteins (mPERs) in oscillating SCN cells. These data suggest that mTim is required for rhythmicity and is a functional homolog of dTim on the negative-feedback arm of the mammalian molecular clockwork.