Possible involvement of DNA methylation in NKCC1 gene expression during postnatal development and in response to ischemia

In CNS, GABA_A receptor-mediated responses switch from depolarization to hyperpolarization during postnatal development. This switch is mediated by developmental down-regulation of inwardly directed Na ⁺-K⁺-2Cl^- co-transporter type 1 (NKCC1) and up-regulation of outwardly directed K⁺-Cl^- co-transporter type 2. While several factors have been shown to regulate K⁺-Cl ^- co-transporter type 2 expression, little is known about the mechanisms by which the expression of NKCC1 is regulated during postnatal development. Here, we report a novel epigenetic mechanism underlying the developmental regulation of NKCC1 gene expression in the rat cerebral cortex. In vitro DNA methylation of the NKCC1 promoter region, which contains a high density of cytosine-phosphodiester-guanine islands, significantly decreased the expression of NKCC1 mRNA, and the degree of methylation of the NKCC1 promoter region significantly increased during postnatal development. In addition, treatment with 5-aza-2'-deoxycytidine, a specific DNA methyltransferase inhibitor, elicited an increase in the expression of NKCC1 mRNA and protein in cortical slice cultures. Focal ischemic injury induced by the occlusion of the middle cerebral artery led to the re-expression of NKCC1 mRNA and protein even in the mature rat cortex. The re-expression of NKCC1 mRNA and protein in the injured cerebral cortex was related to a decrease in the methylation status of the NKCC1 promoter region. Our results indicate that epigenetic mechanisms, such as DNA methylation, might be involved in the regulation of NKCC1 gene expression during postnatal development as well as under pathological conditions.