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.