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

Abstract

Chen M, Xiong F, Zhang L. Promoter methylation of Egr-1 site contributes to fetal hypoxia-mediated PKCε gene repression in the developing heart. Am J Physiol Regul Integr Comp Physiol 304: R683-R689, 2013. First published February 20, 2013 doi:10.1152/ajpregu.00461.2012.-Fetal hypoxia causes protein kinase Cε (PKCε) 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 PKCε gene promoter contributes to sex dimorphism of hypoxia-induced programming of PKCε 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 PKCε 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 PKCε promoter was significantly increased in the male hearts by hypoxia, resulting in decreased Egr-1 binding affinity and reduced Egr-1 binding to the PKCε promoter. Nuclear Egr-1 levels were not affected by hypoxia. There was significantly higher abundance of estrogen receptor ␣ (ER␣) and ␤ (ER␤) isoforms in female than in male fetal hearts, which were not significantly altered by hypoxia. Both ER␣ and ER␤ 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 PKCε gene repression persisted in 3-mo-old adult male hearts in a sexdependent manner. The results indicate a key role for heightened methylation of the Egr-1 binding site in hypoxia-mediated programming of PKCε 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 PKCε gene expression patterns in the early developmental stage. hypoxia; heart; PKCε; DNA methylation; Egr-1 LARGE EPIDEMIOLOGICAL STUDIES have shown that adverse environment in utero causes increased risk of developing endocrine and cardiovascular disorders such as diabetes, hypertension, and ischemic heart disease in adult life (4,5,9,11,21,32). Hypoxia during gestation is one of the most common insults to the fetal development and is thought to be associated with fetal intrauterine growth restriction and increased risk of cardiovascular dysfunction in offspring (3,12,22,25,35). Recent studies in rats have found that maternal hypoxia results in developmental programming of heightened heart vulnerability to ischemia and reperfusion injury in male offspring in a sex-dependent manner (3,22). Among other mechanisms, it has been demonstrated that repression of protein kinase Cε (PKCε) gene in the developing heart is a congruent mechanism for programming of increased heart susceptibility to ischemia and reperfusion injury in offspring (2,19,24,35).DNA methylation is a chief mechanism for epigenetic modification of gene expression patterns that are...

Litter and sex effects on maternal behavior and DNA methylation of the Nr3c1 exon 17 promoter gene in hippocampus and cerebellum

“…This is of interest since we found previously that the Egr1 gene promoter region was hypomethylated in the NAc of single males compared to the other LGC groups (Kosten et al, 2014). Hypomethylation of the ‐10 site may alter transcription of the Nr3c1 exon 1 7 gene promoter as it has been shown previously that DNA methylation alters the binding of several TFs (Hwang et al, 2010; Chen et al, 2013). Although we did find differences in DNA methylation at specific sites in the Nr3c1 gene exon 1 7 promoter in the cerebellum, we found no change in GR expression.…”

Structural impact of complete CpG methylation within target DNA on specific complex formation of the inducible transcription factor Egr‐1

“…However, some studies suggest otherwise. For example, CpG methylation of Egr‐1 sites causes repression of the protein kinase Cε (PKCε) gene in the heart [23,24,26]. A decreased level of CpG methylation in the Egr‐1 target site causes hyperactive expression of the heparanase gene in bladder cancer [25].…”

“…The Egr‐1 target sequences contain two CpGs. It is known that methylation of these CpGs in the Egr‐1 target sites causes repression of some genes in vivo [23–26]. To better understand this effect, the impact of CpG methylation on DNA recognition by Egr‐1 should be investigated at both molecular and atomic levels.…”

EGR-mediated control of STIM expression and function

“…Briefly, within the mammalian genome, 70 to 80% of cytosines found within CpG sites (stands for 5’- C - p hosphate- G -3’) (CpG) sites are methylated. Given the EGR1 consensus sequence, methylation is common and has been correlated with reduced EGR1 binding and downregulation of EGR1 target genes in rodent models of cardiomyopathy and bladder cancer [25, 26]. However, X-ray crystallography and affinity studies showed that the EGR1 and WT1 zinc finger domains were able to accommodate fully methylated DNA, and their binding kinetics were insensitive to methylation [27, 28].…”