DNA damage is associated with hyperhomocysteinemia (HHcy) and neural tube defects (NTDs). Additionally, HHcy is a risk factor for NTDs. Therefore, this study examined whether DNA damage is involved in HHcy-induced NTDs and investigated the underlying pathological mechanisms involved. Embryonic day 9 (E9) mouse neuroectoderm cells (NE4C) and homocysteine-thiolactone (HTL, active metabolite of Hcy)-induced NTD chicken embryos were studied by Western blotting, immunofluorescence. RNA interference or gene overexpression techniques were employed to investigate the impact of Menin expression changes on the DNA damage. Chromatin immunoprecipitation-quantitative polymerase chain reaction was used to investigate the epigenetic regulation of histone modifications. An increase in γH2AX (a DNA damage indicator) was detected in HTL-induced NTD chicken embryos and HTL-treated NE4C, accompanied by dysregulation of phospho-Atr-Chk1-nucleotide excision repair (NER) pathway. Further investigation, based on previous research, revealed that disruption of NER was subject to the epigenetic regulation of low-expressed Menin-H3K4me3. Overexpression of Menin or supplementation with folic acid in HTL-treated NE4C reversed the adverse effects caused by high HTL. Additionally, by overexpressing the Mars gene, we tentatively propose a mechanism whereby HTL regulates Menin expression through H3K79hcy, which subsequently influences H3K4me3 modifications, reflecting an interaction between histone modifications. Finally, in 10 human fetal NTDs with HHcy, we detected a decrease in the expression of Menin-H3K4me3 and disorder in the NER pathway, which to some extent validated our proposed mechanism. The present study demonstrated that the decreased expression of Menin in high HTL downregulated H3K4me3 modifications, further weakening the Atr-Chk1-NER pathway, resulting in the occurrence of NTDs.

DNA 损伤与高同型半胱氨酸血症 (HHcy) 和神经管缺陷 (NTD) 相关。此外，HHcy 是 NTD 的一个危险因素。因此，本研究探讨了 HHcy 诱导的 NTD 是否涉及 DNA 损伤，并探讨了相关的潜在病理机制。通过蛋白质印迹、免疫荧光研究胚胎第 9 天 (E9) 小鼠神经外胚层细胞 (NE4C) 和同型半胱氨酸硫内酯 (HTL，Hcy 的活性代谢物) 诱导的 NTD 鸡胚胎。采用RNA干扰或基因过表达技术来研究Menin表达变化对DNA损伤的影响。染色质免疫沉淀-定量聚合酶链反应用于研究组蛋白修饰的表观遗传调控。在 HTL 诱导的 NTD 鸡胚胎和 HTL 处理的 NE4C 中检测到 γH2AX（DNA 损伤指标）增加，并伴有磷酸-Atr-Chk1-核苷酸切除修复 (NER) 途径的失调。基于之前的研究，进一步的研究表明，NER 的破坏受到低表达 Menin-H3K4me3 的表观遗传调控。在 HTL 处理的 NE4C 中过度表达 Menin 或补充叶酸可以逆转高 HTL 引起的不利影响。此外，通过过表达Mars基因，我们初步提出了一种HTL通过H3K79hcy调节Menin表达的机制，随后影响H3K4me3修饰，反映了组蛋白修饰之间的相互作用。最后，在10例患有HHcy的人类胎儿NTD中，我们检测到Menin-H3K4me3表达下降和NER通路紊乱，这在一定程度上验证了我们提出的机制。 本研究表明，高HTL中Menin表达的减少下调了H3K4me3修饰，进一步削弱了Atr-Chk1-NER通路，导致NTD的发生。