Gao Xiaomin, Shi Xiang, Zhou Shujian, Chen Chen, Hu Cai, Xia Qian, Li Xinlin, Gao Wen, Ding Ying, Zuo Qisheng, Zhang Yani, Li Bichun
Key Laboratory of Animal Breeding Reproduction and Molecular Design for Jiangsu Province, College of Animal Science and Technology, Yangzhou University, Yangzhou, China.
College of Animal Science and Technology, Institutes of Agricultural Science and Technology Development, Yangzhou University, Yangzhou, China.
J Cell Physiol. 2022 Sep;237(9):3640-3650. doi: 10.1002/jcp.30822. Epub 2022 Jul 5.
Detailed analysis of the regulatory mechanism of spermatogonia stem cell (SSCs) genesis can provide a novel strategy for the application of SSCs in the fields of transgenic animal production and regenerative medicine. Previous studies in this study showed that WNT signaling can positively regulate the formation of SSCs, but the exact regulatory mechanism is not clear. Here, we predicted the target gene of the Wnt/TCF7L2 pathway, namely TDRD1, by bioinformatics analysis. Functional studies revealed that overexpression of TDRD1 during RA-induced SSCs formation in vitro significantly upregulated the expression of reproductive marker genes (Integrinβ1 and Integrinα6), and further flow cytometric analysis also confirmed that the formation efficiency of SSCs was significantly increased after overexpression of TDRD1; while interference with TDRD1 showed the exact opposite result. The in vivo experiments were consistent with the results of the in vitro experiments. Interestingly, although Wnt/TCF7L2 can promote the formation of SSCs, its function must be dependent on the expression of TDRD1, which was also repeatedly demonstrated as a target gene of the Wnt/TCF7L2 signaling pathway. Mechanistically, we found a large number of CpG sites in the TDRD1 promoter, and BSP analysis also confirmed that DNA methylation modifications in the TDRD1 promoter were significantly higher in embryonic stem cells than in SSCs, and further dual-luciferase reporter system assays revealed that low DNA methylation modification levels could enhance TDRD1 promoter activity; although previous studies demonstrated that TCF7L2 could enrich in the TDRD1 promoter region, the binding of the two was dependent on low DNA methylation modification. Taken together, we confirmed that low DNA methylation mediates Wnt/TCF7L2 regulation of TDRD1 to promote the formation of SSCs, providing a basis for SSCs in improving animal productivity.
深入分析精原干细胞（SSCs）发生的调控机制可为SSCs在转基因动物生产和再生医学领域的应用提供新策略。本研究之前的研究表明，WNT信号可正向调控SSCs的形成，但确切的调控机制尚不清楚。在此，我们通过生物信息学分析预测了Wnt/TCF7L2通路的靶基因，即TDRD1。功能研究表明，在体外视黄酸诱导SSCs形成过程中过表达TDRD1可显著上调生殖标记基因（整合素β1和整合素α6）的表达，进一步的流式细胞术分析也证实，过表达TDRD1后SSCs的形成效率显著提高；而干扰TDRD1则呈现相反结果。体内实验结果与体外实验一致。有趣的是，尽管Wnt/TCF7L2可促进SSCs的形成，但其功能必须依赖于TDRD1的表达，这也反复证明TDRD1是Wnt/TCF7L2信号通路的靶基因。机制上，我们在TDRD1启动子中发现了大量CpG位点，亚硫酸氢盐测序分析也证实，胚胎干细胞中TDRD1启动子的DNA甲基化修饰显著高于SSCs，进一步的双荧光素酶报告系统检测表明，低DNA甲基化修饰水平可增强TDRD1启动子活性；尽管先前研究表明TCF7L2可富集于TDRD1启动子区域，但二者的结合依赖于低DNA甲基化修饰。综上所述，我们证实低DNA甲基化介导Wnt/TCF7L2对TDRD1的调控以促进SSCs的形成，为利用SSCs提高动物生产力提供了依据。
