刘洪洋1,2, 魏蕊1,2, 李晓晓1,2, 邹康1,2,*
长期精原干细胞(spermatogonial stem cells, SSCs)有自发转化成多能性干细胞的潜力，推测与睾丸生殖细胞肿瘤的出现有关，尤其是p53缺失显著加剧SSCs的自发转化效率。能量代谢被证实与细胞多能性有强关联性。近期我们通过染色质可及性测序技术(Assay for Targeting Accessible-Chromatin with high-throughput sequencing, ATAC-seq)和转录组测序技术(RNA-seq)比较了野生型和p53缺失(p53−/−)小鼠SSCs染色质可及性及基因表达谱差异，揭示了SMAD3是SSCs向多能性转化中的一个关键转录因子，并观察到p53缺失后多个与能量代谢相关基因发生显著表达变化。为进一步揭示p53在多能性和能量代谢调控中的串扰作用，本文探究p53缺失后SSCs向多能性状态转化的能量调节机制。通过ATAC-seq和RNA-seq比较野生型和p53−/−小鼠SSCs的染色质可及性和转录组差异，结果显示，p53缺失后，与糖酵解正调控和电子传递及ATP合成相关的基因染色质可及性增强；编码糖酵解关键酶和调控电子传递相关酶的基因转录水平升高；转录因子SMAD3、SMAD4的靶基因，编码AMPK亚基的Prkag2染色质可及性增强且转录水平升高。以上结果提示，p53缺失激活了SSCs糖酵解关键酶基因，正调控糖酵解基因的染色质可及性增强，以提高糖酵解活性，促进向多能性转化；SMAD3/SMAD4介导的Prkag2基因转录促进AMPK进行能量稳态的调控，维持细胞能量稳态。这一结果对干细胞多能性转化及生殖腺肿瘤的临床防治具有一定的参考意义。
关键词： 精原干细胞; 多能性; p53; 能量代谢; ATAC-seq; RNA-seq
Effects and mechanism of p53 gene deletion on energy metabolism during the pluripotent transformation of spermatogonial stem cells
LIU Hong-Yang1,2, WEI Rui1,2, LI Xiao-Xiao1,2, ZOU Kang1,2,*
1 Germline Stem Cells and Microenvironment Lab, College of Animal Science and Technology, Nanjing Agricultural University, Nanjing 210095, China；2Stem Cell Research and Translation Center, Nanjing Agricultural University, Nanjing 210095, China
Previous studies have shown that long-term spermatogonial stem cells (SSCs) have the potential to spontaneously transform into pluripotent stem cells, which is speculated to be related to the tumorigenesis of testicular germ cells, especially when p53 is deficient in SSCs which shows a significant increase in the spontaneous transformation efficiency. Energy metabolism has been proved to be strongly associated with the maintenance and acquisition of pluripotency. Recently, we compared the difference in chromatin accessibility and gene expression profiles between wild-type (p53+/+) and p53 deficient (p53−/−) mouse SSCs using the Assay for Targeting Accessible-Chromatin with high-throughput sequencing (ATAC-seq) and transcriptome sequencing (RNA-seq) techniques, and revealed that SMAD3 is a key transcription factor in the transformation of SSCs into pluripotent cells. In addition, we also observed significant changes in the expression levels of many genes related to energy metabolism after p53 deletion. To further reveal the role of p53 in the regulation of pluripotency and energy metabolism, this paper explored the effects and mechanism of p53 deletion on energy metabolism during the pluripotent transformation of SSCs. The results of ATAC-seq and RNA-seq from p53+/+ and p53−/− SSCs revealed that gene chromatin accessibility related to positive regulation of glycolysis and electron transfer and ATP synthesis was increased, and the transcription levels of genes encoding key glycolytic enzymes and regulating electron transport-related enzymes were markedly increased. Furthermore, transcription factors SMAD3 and SMAD4 promoted glycolysis and energy homeostasis by binding to the chromatin of the Prkag2 gene which encodes the AMPK subunit. These results suggest that p53 deficiency activates the key enzyme genes of glycolysis in SSCs and enhances the chromatin accessibility of genes associated with glycolysis activation to improve glycolysis activity and promote transformation to pluripotency. Moreover, SMAD3/SMAD4-mediated transcription of the Prkag2 gene ensures the energy demand of cells in the process of pluripotency transformation and maintains cell energy homeostasis by promoting AMPK activity. These results shed light on the importance of the crosstalk between energy metabolism and stem cell pluripotency transformation, which might be helpful for clinical research of gonadal tumors.
Key words: spermatogonial stem cells; pluripotency; p53; energy metabolism; ATAC-seq; RNA-seq
刘洪洋, 魏蕊, 李晓晓, 邹康. p53缺失引发精原干细胞多能状态转化过程中能量代谢的改变及调控机制[J]. 生理学报 2023; 75 (1): 17-26.
LIU Hong-Yang, WEI Rui, LI Xiao-Xiao, ZOU Kang. Effects and mechanism of p53 gene deletion on energy metabolism during the pluripotent transformation of spermatogonial stem cells. Acta Physiol Sin 2023; 75 (1): 17-26 (in Chinese with English abstract).