Supplementary Material for: A Methyl-Deficient Diet Modifies Early B Cell Development

A functional methyl group donor is essential for the epigenetic regulation of all biological events due to the importance of DNA methylation and histone methylation as an epigenetic marker. However, the epigenetic alterations in the immune system due to methyl donor deficiency are not well known. In this study, we tried to address this question by studying the lymphocyte development and DNA methylation changes caused by a methyl-deficient diet (MDD). We fed one group of C57BL/6J mice with a methyl-sufficient diet (MSD) and the other group with an MDD for 5 months. Flow cytometry analyses of their immune systems showed a decrease in B220+ IgM+ (immature B) cells and an increase in B220+ IgM– (pro/pre-B) cells in the bone marrow of mice fed an MDD. By means of an in vitro OP9 coculture system, we recognized that this B220+ IgM– cell fraction from the MDD has an intrinsic developmental defect. When we quantitatively measured the mRNA expression levels of transcription factors and recombination machinery related to B cell development in the B220+ IgM– cell fraction of their bone marrow, we found that <i>ADA, EBF1, DNTT </i>and <i>Pax5 </i>mRNA expression levels were significantly downregulated in mice fed with an MDD. In addition, there was a drastic decrease in histone methylation profile H3K4me3 in the <i>Pax5 </i>and <i>EBF1 </i>promoters in these B220+ IgM– B cells. However, CpG-DNA methylation profiles had not changed and this revealed that these two promoters are demethylated even under an MSD condition. We also found changed expression levels of the <i>Polycomb </i>group genes <i>(mel18</i>, <i>bmi1</i>, <i>Pc1</i>, <i>Pc2</i>, <i>Ring1A</i>, <i>Ring1B</i>, <i>Ph1)</i> on semi-quantitative RT-PCR. These results indicate that under an MDD condition, early B cell development in bone marrow is easily affected by epigenetic alterations.

功能性甲基供体（functional methyl group donor）是所有生命活动表观遗传调控的必需物质，这是由于DNA甲基化与组蛋白甲基化作为表观遗传标记（epigenetic marker）发挥着关键作用。然而，甲基供体缺乏所引发的免疫系统表观遗传改变目前仍未得到充分阐释。本研究通过探究甲基缺乏膳食（methyl-deficient diet, MDD）诱导的淋巴细胞发育与DNA甲基化变化，旨在解答这一科学问题。我们将C57BL/6J小鼠分为两组：一组饲喂甲基充足膳食（methyl-sufficient diet, MSD），另一组饲喂MDD，持续喂养5个月。对两组小鼠的免疫系统开展流式细胞术（flow cytometry）分析后发现，饲喂MDD的小鼠骨髓中，B220+ IgM+（未成熟B细胞）比例降低，而B220+ IgM–（原B/前B细胞）比例升高。借助体外OP9共培养系统，我们证实源自MDD组小鼠的B220+ IgM–细胞群存在固有的发育缺陷。通过定量检测骨髓B220+ IgM–细胞群中与B细胞发育相关的转录因子及重组调控机制的mRNA表达水平，我们发现，饲喂MDD的小鼠体内，*ADA、EBF1、DNTT* 及 *Pax5* 的mRNA表达水平显著下调。此外，在这些B220+ IgM– B细胞的*Pax5*与*EBF1*启动子区域，组蛋白甲基化修饰H3K4me3的水平出现显著下降。但CpG-DNA甲基化谱并未发生改变，这表明即便在MSD条件下，这两个启动子区域也处于去甲基化状态。我们还通过半定量逆转录聚合酶链反应（reverse transcription polymerase chain reaction, RT-PCR）检测发现，多梳蛋白家族（Polycomb group, PcG）基因*mel18、bmi1、Pc1、Pc2、Ring1A、Ring1B、Ph1*的表达水平发生了改变。上述结果表明，在MDD条件下，骨髓早期B细胞发育极易受到表观遗传改变的影响。