Promoter methylation represses AT2R gene and increases brain hypoxic–ischemic injury in neonatal rats

Abstract

Perinatal nicotine exposure downregulated angiotensin II type 2 receptor (AT2R) in the developing brain and increased brain vulnerability to hypoxic–ischemic injury in male neonatal rats. We tested the hypothesis that site-specific CpG methylation at AT2R gene promoter contributes to the increased vulnerability of brain injury in the neonate. Nicotine was administered to pregnant rats from day 4 of gestation to day 10 after birth. Brain hypoxic–ischemic injury was induced in day 10 male pups. CpG methylation at AT2R promoter was determined in the brain by quantitative methylation-specific PCR. Nicotine exposure significantly increased the methylation of a single CpG−52 locus near the TATA-box at AT2R promoter. Electrophoretic mobility shift assay indicated that the methylation of CpG−52 significantly decreased the binding affinity of TATA-binding protein (TBP). Chromatin immunoprecipitation assay further demonstrated an increase in the binding of a methyl-binding protein and a decrease in TBP binding to AT2R promoter in vivo in neonatal brains of nicotine-treated animals. This resulted in AT2R gene repression in the brain. Intracerebroventricular administration of a demethylating agent 5-aza-2′-deoxycytidine abrogated the enhanced methylation of CpG−52, rescued the TBP binding, and restored AT2R gene expression. Of importance, 5-aza-2′-deoxycytidine reversed the nicotine-increased vulnerability of brain hypoxic–ischemic injury in the neonate. The finding provides mechanistic evidence of increased promoter methylation and resultant AT2R gene repression in the developing brain linking perinatal stress and a pathophysiological consequence of heightened vulnerability of brain hypoxic–ischemic encephalopathy in the neonate.

Keywords

• Nicotine
• AT2R
• Methylation
• Hypoxic–ischemic encephalopathy