Epigenetic regulation of glucocorticoid receptor expression in aorta from mice with hyperhomocysteinemia

Hyperhomocysteinemia (HHcy) is a risk factor for vascular disease but its underlying molecular pathology is not understood. Homocysteine is metabolically linked to the epigenetic process of DNA methylation. Tissue-specific changes in DNA methylation have been observed in HHcy but little is known about vascular tissue. The objective of this study was to determine if changes in the epigenetic regulation of glucocorticoid receptor (GR) expression (encoded by Nr3c1) in aorta are associated with HHcy. C57BL/6 mice heterozygous for disruption of the cystathionine-β-synthase gene (Cbs+/-) and controls (Cbs+/+) were fed a control or high methionine/low folate (HH) diet to induce HHcy. Cbs+/- and Cbs+/+ fed the HH diet had higher plasma total homocysteine levels (19.9 ± 3.2 and 7.0 ± 0.9 μM, respectively) than Cbs+/+ mice fed the control diet (2.7 ± 0.2 μM), and this was accompanied by lower Nr3c1 mRNA and lower GR protein in aorta. The Nr3c1 gene contains multiple first exons producing heterogeneous RNA transcripts expressed in a tissue-specific manner. We identified expression of two transcripts in aorta. Bisulfite pyrosequencing found increased methylation of the promoter regions for these transcripts at sites corresponding to Sp1 and Nrf1 binding sites. Chromatin immunoprecipitation found lower binding of Nrf1 to the Nr3c1 promoter but higher expression of Nrf1 protein in aorta from mice with HHcy. These findings show methylation and silencing of vascular Nr3c1 expression and suggest a role for epigenetic regulation of gene expression in HHcy.