Description: Our previous study demonstrated that pregnancy increased large-conductance Ca 2+ -activated potassium channel β1 subunit (BKβ1) expression and large-conductance Ca 2+ -activated potassium channel activity in uterine arteries, which were abrogated by chronic hypoxia. The present study tested the hypothesis that promoter methylation/demethylation is a key mechanism in epigenetic reprogramming of BKβ1 expression patterns in uterine arteries. Ovine BKβ1 promoter of 2315 bp spanning from –2211 to +104 of the transcription start site was cloned, and an Sp1 –380 binding site that contains CpG dinucleotide in its core binding sequences was identified. Site-directed deletion of the Sp1 site significantly decreased the BKβ1 promoter activity. Estrogen receptor-α bound to the Sp1 site through tethering to Sp1 and upregulated the expression of BKβ1. The Sp1 binding site at BKβ1 promoter was highly methylated in uterine arteries of nonpregnant sheep, and methylation inhibited transcription factor binding and BKβ1 promoter activity. Pregnancy caused a significant decrease in CpG methylation at the Sp1 binding site and increased Sp1 binding to the BKβ1 promoter and BKβ1 mRNA abundance. Chronic hypoxia during gestation abrogated this pregnancy-induced demethylation and upregulation of BKβ1 expression. The results provide evidence of a novel mechanism of promoter demethylation in pregnancy-induced reprogramming of large-conductance Ca 2+ -activated potassium channel expression and function in uterine arteries and suggest new insights of epigenetic mechanisms linking gestational hypoxia to aberrant uteroplacental circulation and increased risk of preeclampsia.

Description: Previous in vivo study demonstrated that chronic hypoxia during gestation was associated with estrogen receptor-α (ER-α) gene repression in ovine uterine arteries. Yet, it remains undetermined whether hypoxia had a direct effect and if DNA methylation played a causal role in hypoxia-mediated ER-α gene repression. Thus, this study tested the hypothesis that prolonged hypoxia has a direct effect and increases promoter methylation resulting in ER-α gene repression and inhibition of estrogen-mediated adaptation of uterine vascular tone. Uterine arteries isolated from nonpregnant and pregnant sheep were treated ex vivo with 21.0% O 2 and 10.5% O 2 for 48 hours. Hypoxia significantly increased ER-α promoter methylation at both specificity protein-1 and upstream stimulatory factor binding sites, decreased specificity protein-1 and upstream stimulatory factor binding to the promoter, and suppressed ER-α expression in uterine arteries of pregnant animals. Of importance, the effects of hypoxia were blocked by a methylation inhibitor 5-aza-2'-deoxycytidine. In addition, hypoxia abrogated steroid hormone–mediated increase in ER-α expression and inhibited the hormone-induced increase in large-conductance Ca 2+ -activated K + channel activity and decrease in myogenic tone in uterine arteries of nonpregnant animals, which were reversed by 5-aza-2'-deoxycytidine. The results provide novel evidence of a direct effect of hypoxia on heightened promoter methylation that plays a causal role in ER-α gene repression and ablation of steroid hormone–mediated adaptation of uterine arterial large conductance Ca 2+ -activated K + channel activity and myogenic tone in pregnancy.