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

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. RRBS libraries for the hippocampi of F1m-GCS and F1m-CON were constructed as previously described(Cai et al., 2015; Gu et al., 2011). Briefly, 500 ng of genomic DNA was digested with 20 units of MspI (NEB, USA) in a 20 μl reaction for 20 h at 37 °C. After purification, the digested products were blunt-ended, and then dA was added, followed by methylated-adapter (Illumina, USA) ligation. To obtain DNA fractions in 40-120 bp and 120-220 bp range of MspI-digested products, two ranges (160-240 bp and 240-340 bp) of adapter-ligated fractions were excised from 2% agarose gel. The size-selected DNA was bisulfite-treated for two rounds using EpiTect Bisulfite Kit (Qiagen, German) following the manufacturer’s standard protocol. The final libraries were generated by amplification with HiFi HotStart Uracil + ReadyMix PCR kit (KAPA, USA). The quality and quantity were analyzed by an Agilent 2100 Bioanalyzer (Agilent Technologies, USA) and qPCR, respectively. Illumina HiSeq 2000 was used to sequence the libraries according to the manufacturer’s protocol. After removal of the adaptor sequences and trimming of low quality sequences by using cutadapt, the reads from each of the biological replicates were aligned to mouse mm10 reference genome as well as the size-selected MspI fragments generated by our in silico simulation. Because of the strand specificity of DNA methylation, two rounds of alignments were carried out, i.e. the bisulfite converted reads were aligned to the genome sequences termed the “T genome” with each cytosine converted to thymine and in the meanwhile the reads were also aligned to the genome sequences termed the “A genome” with each guanine converted to adenosine. The alignments were carried out with BGI SOAPaligner verison2.21(Li et al., 2009) with default parameters, allowing up to five mismatches for successful mapping. Both replicates showed near complete (> 99%) bisulfite conversion of non-CpG cytosine (data not shown). The DNA methylation levels in Nr3c1 and Crebbp promoters in the hippocampi of F1m-GCS and F1m-CON mice were examined by DNA bisulfate conversion and PCR amplification and Sequencing using the EZ DNA Methylation-GoldTM Kit (Zymo Research, USA) following the manufacturer’s standard protocol.