Department of Pathology and Laboratory Medicine, Child and Family Research Institute, University of British Columbia, Vancouver, BC, Canada.
Epigenetics. 2012 May;7(5):514-21. doi: 10.4161/epi.19836. Epub 2012 May 1.
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.
高同型半胱氨酸血症（HHcy）是血管疾病的一个风险因素，但它的潜在分子病理学尚未被理解。同型半胱氨酸在代谢上与 DNA 甲基化的表观遗传过程有关。在 HHcy 中已经观察到组织特异性的 DNA 甲基化变化，但对血管组织知之甚少。本研究的目的是确定糖皮质激素受体（GR）表达（由 Nr3c1 编码）的表观遗传调控是否与 HHcy 有关。C57BL/6 小鼠杂合子胱硫醚-β-合酶基因（Cbs+/-）的破坏和对照（Cbs+/+）被喂食对照或高蛋氨酸/低叶酸（HH）饮食以诱导 HHcy。Cbs+/-和 Cbs+/+喂食 HH 饮食的血浆总同型半胱氨酸水平（分别为 19.9±3.2 和 7.0±0.9 μM）高于喂食对照饮食的 Cbs+/+小鼠（2.7±0.2 μM），并且这伴随着主动脉中 Nr3c1 mRNA 和 GR 蛋白降低。Nr3c1 基因包含多个产生组织特异性表达的异质 RNA 转录物的第一外显子。我们在主动脉中鉴定出两种转录物的表达。亚硫酸氢盐焦磷酸测序发现，这些转录物的启动子区域的甲基化增加，对应于 Sp1 和 Nrf1 结合位点。染色质免疫沉淀发现，HHcy 小鼠主动脉中 Nrf1 与 Nr3c1 启动子的结合减少，但 Nrf1 蛋白的表达增加。这些发现表明血管 Nr3c1 表达的甲基化和沉默，并提示表观遗传调控在 HHcy 中的基因表达作用。
