DNA hydroxymethylation of NCX1 heart promoter by GATA6/TET3 epigenetic complex participates in neuroprotection induced by hypoxic preconditioning

Na⁺ -Ca²⁺ exchanger 1 (NCX1), an antiporter that regulates the homeostasis of calcium and sodium ions contributes to neuroprotection elicited by ischemic preconditioning. In the brain, NCX1 is transcriptionally regulated by two gene promoters: NCX1-Br (brain) and NCX1-Ht (heart). Notably, the epigenetic mechanism involved in the methylation of cytosine (C) to form 5-methylcytosine (5mC) in neurons represses NCX1-Ht, but not NCX1-Br promoter activity. Importantly, hydroxylation of 5mC via ten eleven translocases (TET) Enzymes leads to 5-hydroxymethylcytosine (5hmC), a marker of transcriptional activation. Herein, in ascorbic acid-treated SH-SY5Y cells TET3 isoform promoted 5hmC formation at NCX1-Ht promoter, but not at the level of NCX1-Br promoter, thus inducing NCX1 mRNA and protein up-regulation. Notably, TET3 physically interacted with the transcription factor GATA-binding factor 6 (GATA6), but not with RE1-silencing transcription factor (REST), and site-direct mutagenesis of GATA binding sites present on NCX1-Ht sequence blocked GATA6 and TET3 binding on NCX1 gene. Furthermore, GATA6 and TET3 protein levels were up-regulated in cortical neurons exposed to the neuroprotective hypoxic preconditioning (PC) stimulus followed by oxygen and glucose deprivation (OGD/Rx), that caused an increase of DNA hydroxymethylation on NCX1-Ht promoter, an effect not observed in neurons exposed to OGD/Rx alone. Collectively, this study showed that in neurons the exposure to PC followed by OGD/Rx 72 h elicited the formation of GATA6/TET3 complex that, by binding NCX1-Ht promoter, epigenetically activates NCX1 gene transcription. Given the NCX1 neuroprotective role in PC followed by OGD/Rx, activating this epigenetic pathway could be a potential therapeutic target for stroke treatment.