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Nitric oxide inhibits ten-eleven translocation DNA demethylase activity to regulate 5mC and 5hmC across the genome [RNA-seq]

ABSTRACT: 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 in place of O2 to form a dinitrosyliron complex (DNIC). In cancer cells treated with exogeneous NO, or cells endogenously synthesizing NO, there was a global increase in DNA 5mC, a substrate for TET, that could not be attributed to increased DNA methyltransferase activity. 5mC was also elevated in NO-producing mouse xenograft and patient derived xenograft tumors. Genome-wide DNA methylome analysis of cells chronically treated with NO (10 days) demonstrated enrichment of 5mC at gene-regulatory loci which correlated to the expression of NO-regulated tumor-associated genes. Regulation of DNA methylation is distinctly different from canonical NO-signaling and represents a novel epigenetic regulatory mechanism of NO.

ORGANISM(S): Homo sapiens

PROVIDER: GSE248150 | GEO | 2024/05/01

REPOSITORIES: GEO

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Project description:Ten-eleven translocation (TET) proteins oxidize 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC), 5-formylcytsosine (5fC), and 5-carboxylcytosine (5caC). 5fC/5caC can be excised and repaired by the base excision repair (BER) pathway, implicating 5mC oxidation in active DNA demethylation. Genome-wide DNA methylation is erased in the transition from metastable states to ground state of embryonic stem cells (ESCs) and in migrating primordial germ cells (PGCs), while some resistant regions become demethylated only in gonadal PGCs. Understanding the mechanisms underlying global hypomethylation in naïve ESCs and developing PGCs will be useful for realizing cellular pluripotency and totipotency. In this study, we found that PRDM14, the PR-domain-containing transcriptional regulator, accelerates the TET-BER cycle, resulting in the promotion of active DNA demethylation in ESCs. Induction of PRDM14 expression rapidly removed the 5mC associated with transient elevation of 5hmC at pluripotency-associated genes, germline-specific genes, and imprinted loci but not across the entire genome, which resemble second wave of DNA demethylation in gonadal PGCs. PRDM14 physically interacts with TET1/TET2 and enhances the recruitment of TET1/TET2 at target loci. Knockdown of Tet1/Tet2 impaired transcriptional regulation and DNA demethylation by PRDM14. The repression of the BER pathway by administration of pharmacological inhibitors against APE1 and PARP1 and the knockdown of thymine DNA glycosylase (TDG) also impaired DNA demethylation by PRDM14. Furthermore, DNA demethylation induced by PRDM14 normally takes place in the presence of aphidicolin, which is an inhibitor of G1/S progression. Together, our analysis provides mechanistic insight into DNA demethylation in naive pluripotent stem cells and developing PGCs. To investigate the function of TET1/TET2 in transcriptional regulation by PRDM14 in ESCs, we exploited microarray analysis using total mRNA derived from Scramble, Scramble + PRDM14, Tet1/Tet2 KD, Tet1/Tet2 KD + PRDM14 mouse ESC.

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Nitric oxide inhibits ten-eleven translocation DNA demethylase activity to regulate 5mC and 5hmC across the genome [RNA-seq]

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