Supplementary Material for: Change of Epigenetic Control of Cystathionine Beta-Synthase Gene Expression through Dietary Vitamin B₁₂ Is Not Recovered by Methionine Supplementation

<i>Background/Aims:</i> Vitamin B₁₂ (B-12) is an essential cofactor for methionine synthase, and methionine is critical for the methylation of various biological molecules including DNA. Whether changes in B-12 levels can alter specific gene expression through DNA methylation and whether dietary methionine has any effect on general DNA methylation status still remains controversial. <i>Methods:</i> We raised severely B-12-deficient rats as well severely-B-12 deficient rats but supplemented with 5% methionine. mRNA levels of methionine cycle-related enzymes were analyzed. <i>Results:</i> Gene expression patterns changed under B-12-deficient conditions but were recovered by dietary methionine supplementation to B-12-deficient rats. However, cystathionine β-synthase mRNA levels, which had decreased under B-12-deficient conditions, did not recover with supplementary dietary methionine. The CpG island of the cystathionine β-synthase promoter was hypomethylated in B-12-deficient rats, and showed no recovery after methionine addition. <i>Conclusions:</i> Dietary B-12 can affect epigenetic machinery by regulating DNA methylation status and dietary methionine may have small effects on DNA methylation.

研究背景与目的：维生素B₁₂（Vitamin B₁₂，简称B-12）是甲硫氨酸合酶（methionine synthase）的必需辅因子，而甲硫氨酸对于包括DNA在内的多种生物分子的甲基化修饰至关重要。目前关于B-12水平变化是否可通过DNA甲基化调控特定基因表达，以及膳食甲硫氨酸是否对整体DNA甲基化状态存在影响，仍存在争议。研究方法：本研究构建了重度维生素B₁₂缺乏大鼠模型，同时设置另一组重度维生素B₁₂缺乏大鼠并予以5%甲硫氨酸膳食补充，对甲硫氨酸循环相关酶的信使RNA（mRNA）水平进行了检测分析。研究结果：维生素B₁₂缺乏条件下，基因表达模式发生改变，而该改变可通过对B-12缺乏大鼠补充膳食甲硫氨酸得以恢复。但原本在B-12缺乏条件下表达下调的胱硫醚β合酶（cystathionine β-synthase）mRNA水平，无法通过膳食甲硫氨酸补充得到恢复。在B-12缺乏大鼠中，胱硫醚β合酶启动子的CpG岛呈低甲基化状态，且在补充甲硫氨酸后未出现恢复趋势。研究结论：膳食维生素B₁₂可通过调控DNA甲基化状态影响表观遗传调控机制，而膳食甲硫氨酸对DNA甲基化的影响可能较为微弱。