Abstract
<jats:p>Backgrounds: Early-life environmental insults cause persistent neurodevelopmental abnormalities accompanied by transcriptional and epigenetic dysregulation despite removal of the original insult or postnatal intervention. However, transcriptomic and epigenomic responses to developmental insults and subsequent treatment during active neurodevelopment remain insufficiently characterized. Developmental iron deficiency (ID) provides a unique model for investigating this question because iron is an essential cofactor for TET DNA dioxygenases and developmental ID causes persistent behavioral and molecular alterations despite iron repletion. Results: We integrated the hippocampal transcriptome, DNA methylome (5mC), and hydroxymethylome (5hmC) in male rats at postnatal day 15 following developmental ID and postnatal iron treatment, using Oxford Nanopore sequencing for native DNA modification profiling. Developmental ID induced substantial transcriptional and epigenetic alterations associated with synaptic function, neurodevelopment, and neuroinflammation. Postnatal iron treatment induced a hierarchical response across molecular layers: while transcriptomic alterations largely normalized, 5mC showed only partial recovery, and 5hmC showed extensive de novo modifications. Recovered, persistent, and newly emerged epigenetic marks were associated with increasingly specialized biological functions, from broad neurodevelopmental processes to specific pathways. Furthermore, while 5mC enrichment was associated with transcriptionally suppressed pathways, 5hmC enrichment showed weaker coupling with concurrent transcriptomic activity, suggesting epigenetic poising rather than immediate transcriptional output. MergeOmics integration identified key driver genes showing post-treatment epigenetic regulation despite transcriptional recovery. Conclusions: Molecular recovery following developmental ID extends beyond transcriptomic normalization, involving persistent and extensive epigenetic remodeling. This study provides a framework for understanding molecular responses following early-life environmental insults and highlights the importance of delineating persistent regulatory reprogramming.</jats:p>