Supplementary Material for: Epigenetic Regulation in Early Childhood: A Miniaturized and Validated Method to Assess Histone Acetylation

<b><i>Introduction:</i></b> Chronic inflammatory diseases including allergies and asthma are the result of complex interactions between genes and environmental factors. Epigenetic mechanisms comprise a set of biochemical reactions that regulate gene expression. In order to understand the cause-effect relationship between environmental exposures and disease development, methods capable of assessing epigenetic regulation (also) in large cohorts are needed. <b><i>Methods:</i></b> For this purpose, we developed and evaluated a miniaturized chromatin immunoprecipitation (ChIP) assay allowing for a cost-effective assessment of histone acetylation of candidate genes in a quantitative fashion. This method was then applied to assess H3 and H4 histone acetylation changes in cord blood (CB) samples from an established cohort of Australian children exposed in the fetal period to either very low or very high levels of maternal folate. <b><i>Results:</i></b> Our ChIP assay was validated for a minimum requirement of 1 × 10<sup>5</sup> target cells (e.g. CD4+ T cells). Very high levels of maternal folate were significantly associated with increased H3/H4 acetylation at <i>GATA3</i> and/or <i>IL9</i> promoter regions in CD4+ T cells in CB. <b><i>Conclusion:</i></b> We developed a ChIP method allowing reliable assessment of H3/H4 acetylation using 1 × 10<sup>5</sup> cells only. Practical application of this assay demonstrated an association between high maternal folate exposure and increased histone acetylation, corresponding to a more transcriptionally permissive chromatin status in the promoter regions of some Th2-related genes.