Neuroepigenetic programming by the maternal environment. Part B: independent contribution of methylation and hydroxymethylation in the offspring's olfactory bulb following preconceptional weight loss in mice

Recent studies indicate that inadequate maternal diet can influence offspring physiology, metabolism and cognitive health, but the impact of the preconception period remains largely unexplored. We previously showed that maternal weight loss prior to conception affects metabolic outcomes and olfactory-driven behaviors in mouse offspring. To explore whether these intergenerational effects are linked to epigenetic mechanisms in the olfactory bulb (OB), we investigated changes in DNA methylation, specifically 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC), the latter being relatively abundant in the brain but still poorly characterized. We profiled DNA methylation and hydroxymethylation using Reduced Representation Bisulfite Sequencing (RRBS) and oxidative RRBS (oxRRBS) on OBs from offspring born to control or weight loss mothers, to enable a distinct quantification of 5mC and 5hmC levels at the same CpG sites. While 5mC was globally more abundant in the OB, a subset of CpGs showed high 5hmC levels that varied with maternal condition. Differential analysis identified more differential hydroxymethylated CpG (DhMCs, n = 2631) than differential methylated positions (DMC, n = 1834), indicating a stronger impact of maternal WL on hydroxymethylation. Both DMCs and DhMCs were enriched in genes involved in synaptic function, neuronal signaling, and energy metabolism. However, uniquely modified CpGs, i.e. those detected as modified in only one assay (RRBS or oxRRBS), revealed distinct functional profiles: uniquely differentially hydroxymethylated CpGs (uniqDhMCs) were enriched in genes related to neuronal activity and plasticity, while uniquely differentially methylated CpGs (uniqDMCs) were associated with chromatin remodeling and transcriptional regulation. These findings suggest that, although 5mC and 5hmC can impact similar pathways, they contribute differently to gene regulation in response to early-life nutritional environments. Our study highlights the feasibility of integrating 5mC and 5hmC analyses and uncovers their distinct genomic distributions. The maternal preconceptional weight loss induces lasting and specific epigenetic changes in the OB, notably through 5hmC-specific targeting on genes involved in neuronal activity. By distinguishing between shared and unique DMCs and DhMCs, we reveal complementary and functionally distinct contributions of 5mC and 5hmC to brain plasticity. These findings underscore the importance of integrating both epigenetic marks to fully understand how early-life environmental cues shape neural function and behavior.