# Nitric oxide inhibits Iron(II)2-Oxogluterate dependent demethylases to regulate DNA and mRNA methylation > **NIH NIH R01** · UNIVERSITY OF ILLINOIS AT CHICAGO · 2024 · $457,972 ## Abstract ABSTRACT- Nitric oxide (NO) is an essential signaling molecule that mediates dichotomous effects along a pathophysiological concentration gradient. Low levels maintain physiological homeostasis, while at high concentrations NO contributes to disease pathogenesis. Although mechanisms of physiologic NO signaling are relatively well established, these canonical pathways do not sufficiently explain how NO drives pathological alterations in gene expression. Epigenetic mechanisms, such as methylation of DNA and RNA, are central regulators of gene expression. Our recent publication and preliminary data demonstrate that NO inhibits DNA and mRNA demethylases, which causes the enrichment of DNA and mRNA methyl-adducts on genes transcriptionally regulated by NO. Specifically, our data demonstrate that a concentration gradient of NO differentially inhibits the activities of both DNA demethylases (TET; ten-eleven translocation, ALKBH2) and mRNA demethylases (FTO; fat mass and obesity associated protein, ALKBH5) by forming a dinitrosyl iron complex (DNIC) at the catalytic mononuclear iron atom. We found that each of these Fe(II)/2-oxoglutarate (2- OG)-dependent demethylases (2-ODD) had a different sensitivity to NO-dependent inhibition. In cells, this inhibition resulted in gene-specific enrichment of 5mC on DNA and m6A on mRNA; both of which are critical gene-regulatory methyl-modifications. However, it is unknow whether NO regulates methyl-adducts beyond m6A and 5mC and whether changes in DNA and mRNA methylation mechanistically drive NO-mediated changes in gene expression. Thus, we hypothesize that NO differentially inhibits DNA and RNA demethylases in a concentration-dependent manner to modulate the distribution of methyl-adducts on DNA and RNA, which in turn control the expression of specific NO-regulated genes. Aim 1 will use isolated 2-ODD enzymes to determine the structure-function relationship of NO bound to each enzyme. Kinetic studies will delineate the differential sensitivities of each enzyme to NO. These data will be applied to cellular models to define the effect of a pathophysiological gradient of NO, through demethylase inhibition, induces differential profiles of multiple methyl- adducts on DNA/mRNA. Aim 2 will use cell models to identify NO-regulated genes that are also enriched in DNA and mRNA methyl adducts. With CRISPR techniques we will modify DNA and mRNA methyl-sites and measure corresponding changes in gene expression to demonstrate a causal-link between NO-dependent DNA/mRNA methylation and gene expression. These studies will elucidate a novel molecular mechanism of signaling whereby NO regulates gene expression by inhibiting 2-ODD demethylases in a concentration-dependent manner thus providing a foundational understanding of the role of NO dysregulation in pathogenic gene expression. ## Key facts - **NIH application ID:** 10999570 - **Project number:** 1R01GM155946-01 - **Recipient organization:** UNIVERSITY OF ILLINOIS AT CHICAGO - **Principal Investigator:** DOUGLAS D. THOMAS - **Activity code:** R01 (R01, R21, SBIR, etc.) - **Funding institute:** NIH - **Fiscal year:** 2024 - **Award amount:** $457,972 - **Award type:** 1 - **Project period:** 2024-08-01 → 2026-07-31 ## Primary source NIH RePORTER: https://reporter.nih.gov/project-details/10999570 ## Citation > US National Institutes of Health, RePORTER application 10999570, Nitric oxide inhibits Iron(II)2-Oxogluterate dependent demethylases to regulate DNA and mRNA methylation (1R01GM155946-01). Retrieved via AI Analytics 2026-05-25 from https://api.ai-analytics.org/grant/nih/10999570. Licensed CC0. --- *[NIH grants dataset](/datasets/nih-grants) · CC0 1.0*