ATAC and histone H3K9me3 landscapes reveal long-term epigenomic effects of fetal-neonatal iron deficiency in rat hippocampus [ChIP-seq]

Background: Iron deficiency (ID) during the fetal-neonatal period results in long-term neurodevelopmental impairments associated with pervasive hippocampal gene dysregulation. Prenatal choline supplementation partially normalizes these effects, suggesting an interaction between iron and choline in hippocampal transcriptome regulation. To understand the regulatory mechanisms, we investigated epigenetic marks of genes that are poised to be activated (ATAC-seq) or repressed (H3K9me3 ChIP-seq) in iron-repleted adult rats having experienced fetal-neonatal ID exposure with or without prenatal choline supplementation. Results: Fetal-neonatal ID was induced by limiting maternal iron intake from gestational day (G) 2 through postnatal day (P) 7. Half of the pregnant dams were given supplemental choline (5.0 g/kg) from G11-18. This resulted in 4 groups at P65 (Iron-sufficient [IS], Formerly Iron-deficient [FID], IS with choline [ISch], and FID with choline [FIDch]). Hippocampi were collected from P65 iron-repleted male offspring and analyzed for chromatin accessibility and H3K9me3 enrichment. 22% and 24% of differentially transcribed genes in FID- and FIDch-groups, respectively, exhibited significant differences in chromatin accessibility, whereas 1.7% and 13% exhibited significant differences in H3K9me3 enrichment. These changes mapped onto gene networks regulating synaptic plasticity, neuroinflammation, and reward circuits. Motif analysis of differentially modified genomic sites revealed significantly stronger choline effects than early-life ID and identified multiple epigenetically modified transcription factor binding sites. Conclusions: This study reveals genome-wide, stable epigenetic changes and epigenetically modifiable gene networks associated with specific chromatin marks in the hippocampus, and lays a foundation to further elucidate iron-dependent epigenetic mechanisms that underlie the long-term effects of fetal-neonatal ID, choline, and their interactions.背景：胎儿-新生儿期的铁缺乏（Iron Deficiency, ID）会引发长期神经发育损伤，且伴随广泛的海马基因表达失调。产前胆碱补充可部分逆转这些效应，提示铁与胆碱在海马转录组调控中存在相互作用。为阐明其调控机制，本研究针对曾暴露于胎儿-新生儿期铁缺乏环境、且伴或不伴产前胆碱补充的铁充足成年大鼠，对处于激活预备状态（转座酶可及性测序，ATAC-seq）或受抑制状态（组蛋白H3赖氨酸9三甲基化染色质免疫共沉淀测序，H3K9me3 ChIP-seq）的基因的表观遗传标记进行了分析。 结果：通过限制孕鼠自妊娠第2天（G2）至出生后第7天（P7）的铁摄入，构建胎儿-新生儿期铁缺乏模型。其中一半孕鼠自妊娠第11至18天（G11-18）给予5.0 g/kg的胆碱补充剂，最终在大鼠出生后第65天（P65）将子代分为4组：铁充足组（Iron-sufficient, IS）、既往铁缺乏组（Formerly Iron-deficient, FID）、胆碱干预铁充足组（IS with choline, ISch）以及胆碱干预既往铁缺乏组（FID with choline, FIDch）。收集P65月龄的铁充足雄性子代大鼠的海马组织，分析其染色质可及性与H3K9me3富集水平。既往铁缺乏组（FID）与胆碱干预既往铁缺乏组（FIDch）中，分别有22%与24%的差异表达基因的染色质可及性存在显著差异，而分别有1.7%与13%的差异表达基因的H3K9me3富集水平存在显著差异。上述差异位点富集于调控突触可塑性、神经炎症与奖赏通路的基因网络中。对差异修饰基因组位点的基序分析显示，胆碱的调控效应显著强于早期生命期铁缺乏，并鉴定出多个经表观遗传修饰的转录因子结合位点。 结论：本研究揭示了海马中与特定染色质标记相关的全基因组范围的稳定表观遗传变化，以及可经表观遗传调控的基因网络，为进一步阐明胎儿-新生儿期铁缺乏、胆碱及其相互作用的长期效应背后的铁依赖性表观遗传机制奠定了基础。