An J, Zhang X, Qin J, Wan Y, Hu Y, Liu T, Li J, Dong W, Du E, Pan C, Zeng W
College of Animal Science and Technology, Northwest A&F University, Shaanxi, China.
College of Veterinary Medicine, Northwest A&F University, Shaanxi, China.
Cell Death Dis. 2014 Apr 24;5(4):e1196. doi: 10.1038/cddis.2014.171.
Self-renewal and differentiation of spermatogonial stem cells (SSCs) are the foundation of spermatogenesis throughout a male's life. SSC transplantation will be a valuable solution for young male patients to preserve their fertility. As SSCs in the collected testis tissue from the patients are very limited, it is necessary to expansion the SSCs in vitro. Previous studies suggested that histone methyltransferase ERG-associated protein with SET domain (ESET) represses gene expression and is essential for the maintenance of the pool of embryonic stem cells and neurons. The objective of this study was to determine the role of ESET in SSCs using in vitro cell culture and germ cell transplantation. Cell transplantation assay showed that knockdown of ESET reduced the number of seminiferous tubules with spermatogenesis when compared with that of the control. Knockdown of ESET also upregulated the expression of apoptosis-associated genes (such as P53, Caspase9, Apaf1), whereas inhibited the expression of apoptosis-suppressing genes (such as Bcl2l1, X-linked inhibitor of apoptosis protein). In addition, suppression of ESET led to increase in expression of Caspase9 and activation of Caspase3 (P17) as well as cleavage of poly (ADP-ribose) polymerase. Among the five ESET-targeting genes (Cox4i2, spermatogenesis and oogenesis Specific Basic Helix-Loop-Helix 2, Nobox, Foxn1 and Dazl) examined by ChIP assay, Cox4i2 was found to regulate SSC apoptosis by the rescue experiment. BSP analyses further showed that DNA methylation in the promoter loci of Cox4i2 was influenced by ESET, indicating that ESET also regulated gene expression through DNA methylation in addition to histone methylation. In conclusion, we found that ESET regulated SSC apoptosis by suppressing of Cox4i2 expression through histone H3 lysine 9 tri-methylation and DNA methylation. The results obtained will provide unique insights that would broaden the research on SSC biology and contribute to the treatment of male infertility.
精原干细胞（SSCs）的自我更新和分化是男性一生精子发生的基础。SSC移植将是年轻男性患者保留生育能力的一种有价值的解决方案。由于从患者收集的睾丸组织中的SSCs非常有限，因此有必要在体外扩增SSCs。先前的研究表明，组蛋白甲基转移酶SET结构域相关蛋白（ESET）可抑制基因表达，并且对于维持胚胎干细胞和神经元库至关重要。本研究的目的是通过体外细胞培养和生殖细胞移植来确定ESET在SSCs中的作用。细胞移植试验表明，与对照组相比，ESET基因敲低减少了有精子发生的生精小管数量。ESET基因敲低还上调了凋亡相关基因（如P53、Caspase9、Apaf1）的表达，而抑制了凋亡抑制基因（如Bcl2l1、X连锁凋亡抑制蛋白）的表达。此外，ESET的抑制导致Caspase9表达增加、Caspase3（P1）激活以及聚（ADP-核糖）聚合酶的裂解。通过染色质免疫沉淀分析检测的五个ESET靶向基因（Cox4i2、生精和卵子发生特异性碱性螺旋-环-螺旋2、Nobox、Foxn1和Dazl）中，通过拯救实验发现Cox4i2调节SSC凋亡。BSP分析进一步表明，Cox4i2启动子位点的DNA甲基化受ESET影响，这表明ESET除了通过组蛋白甲基化外，还通过DNA甲基化调节基因表达。总之，我们发现ESET通过组蛋白H3赖氨酸9三甲基化和DNA甲基化抑制Cox4i2表达来调节SSC凋亡。获得的结果将提供独特的见解，拓宽对SSC生物学的研究，并有助于男性不育症的治疗。
