Abstract:

Both Otx2 and the Wnt pathway are involved in nervous system development， and changes in H3K27me3 enrichment status are known to influence this process. This study investigated whether Otx2 regulates the Wnt pathway by modulating histone H3K27me3 modification. A stable C17.2 mouse neural stem cell line with Otx2 knockout was generated using CRISPR/Cas9. ChIP-Seq analysis identified DNA fragments bound by H3K27me3， and qPCR was used to assess Wnt-related gene expression changes after Otx2 knockout. GO and KEGG enrichment analyses revealed that differential genes associated with ChIP-Seq peaks between Otx2-knockout and control cells were predominantly enriched in pathways linked to nervous system development and function. Further analysis showed H3K27me3 enrichment at loci of Wnt5a， Wnt2， and Wnt16 genes in the Wnt pathway. Notably， Wnt5a， Wnt2， and Wnt5b expression levels were significantly reduced in Otx2-knockout cells compared to controls. These findings suggested that Otx2 regulates the Wnt pathway by altering H3K27me3 enrichment sites， providing mechanistic insights into neurological developmental abnormalities.

Key words: Otx2, H3K27me3, Wnt pathway

摘要：

Otx2与Wnt通路都参与神经系统的发育过程，H3K27me3富集状态的改变也会影响神经系统的发育。为探究Otx2是否可以通过改变组蛋白H3K27me3状态调控Wnt通路，采用CRISPR/Cas9系统构建稳定敲除Otx2基因的C17.2小鼠神经干细胞株，利用ChIP-Seq技术分析H3K27me3修饰特异性结合的DNA片段，通过qPCR技术检测Otx2基因敲除后Wnt基因的表达改变。经GO富集分析与KEGG富集分析结果显示，Otx2敲除组与对照组的ChIP-Seq差异富集峰所对应的差异基因主要集中于神经系统发育与功能相关的通路。Wnt通路中的Wnt5a、Wnt2、Wnt16基因位点发生H3K27me3富集情况改变。qPCR结果显示，相比于对照组，Otx2基因敲除组的Wnt5a、Wnt2、Wnt5b基因的表达量下降。结果表明，Otx2基因通过调节组蛋白H3K27me3的富集位点影响Wnt通路，可为神经系统发育异常相关研究提供理论参考。

关键词: Otx2基因, H3K27me3, Wnt通路