Gestational choline supplementation improves cross-generational mood by epigenetic upregulation of Nr3c1 [ChIP-Seq]

Environmental cues during gestation could exert transgenerational effects on behavior mainly through changes in epigenetic marks such as DNA methylation. However, the role of histone modifications in the inheritance of acquired phenotypes remains largely unknown in mammals. Here, we report that gestational choline supplementation (GCS) in maternal mice exerts anxiolytic effects on male offspring across 2 generations. Correspondingly, GCS-induced H3K9 hyperacetylation in the Nr3c1 promoter in male hippocampus leads to upregulation of Nr3c1 and its encoded protein, glucocorticoid receptor (GR), across 2 generations through the male germline. Furthermore, inhibition of CREB-binding protein (CBP) histone acetyltransferase (HAT) function restored GCS-induced epigenetic and behavioral alterations, suggesting that CBP function as a HAT to increase Nr3c1 expression through H3K9 hyperacetylation. Thus, GCS-induced GR upregulation through CBP-mediated H3K9 hyperacetylation in the Nr3c1 promoter is associated with anxiolytic behavior in male offspring, highlighting the role of histone modification in acquired phenotypes across mammalian generations. Overall design: Libraries of ChIP DNA were constructed using the NEXT flex™ Illumina ChIP-Seq Library Prep Kit (Bioo Scientific, USA) following the manufacturer's instructions with modifications. Sequenced reads were mapped to the UCSC mouse mm10 reference genome using Bowtie (version 1.0.0) with specific settings(Langmead et al., 2009). We used the function “callpeak” in Model-based Alignment of ChIP-Seq version 2 (MACS2) (Feng et al., 2012) to call peaks of H3K9ac occupancy with default parameters. MACS2 compares the number of H3K9ac-enriched sequence tags in a given window (200 bp) with the number of tags in surrounding windows and with the number of tags in the same region of the input DNA library. Different peaks of H3K9ac occupancy between CON- and SUP-treated cells were called by the function “bdgdiff” in MACS2 and visualized by the Integrative Genomics Viewer(Thorvaldsdottir et al., 2013).