Promoter methylation of Egr-1 site contributes to fetal hypoxia-mediated PKCε gene repression in the developing heart

Fetal hypoxia causes protein kinase C epsilon (PKC epsilon) gene repression in the heart resulting in heightened ischemic injury in male offspring in a sex-dependent manner. The present study tested the hypothesis that heightened methylation of the early growth response factor-1 (Egr-1) binding site at PKC epsilon gene promoter contributes to sex dimorphism of hypoxia-induced programming of PKC epsilon gene repression in the developing heart. Pregnant rats were divided into normoxic and hypoxic (10.5% O-2 from day 15 to 21 of gestation) groups. Hypoxia selectively decreased PKC epsilon mRNA and protein abundance in the heart of male, but not female, near-term (21 days) fetuses. Methylation of the CpG site at the Egr-1 binding site of PKC epsilon promoter was significantly increased in the male hearts by hypoxia, resulting in decreased Egr-1 binding affinity and reduced Egr-1 binding to the PKC epsilon promoter. Nuclear Egr-1 levels were not affected by hypoxia. There was significantly higher abundance of estrogen receptor alpha (ER alpha) and beta (ER beta) isoforms in female than in male fetal hearts, which were not significantly altered by hypoxia. Both ER alpha and ER beta bind to the Egr-1 binding site with significant greater levels in the female fetal hearts. The increased methylation with reduced Egr-1 binding and PKC epsilon gene repression persisted in 3-mo-old adult male hearts in a sex-dependent manner. The results indicate a key role for heightened methylation of the Egr-1 binding site in hypoxia-mediated programming of PKC epsilon gene repression in the developing heart and suggest a novel protective mechanism of ER by binding to the Egr-1 binding site in epigenetic regulation of PKC epsilon gene expression patterns in the early developmental stage.