SPATA12 and Its Possible Role in DNA Damage Induced by Ultraviolet-C

Our previous studies indicated that SPATA12, a novel spermatogenesis-associated gene, might be an inhibitor involved in spermatogenesis and tumorigenesis. To obtain a better understanding of the functions of SPATA12, a yeast two-hybrid screening system was used to search for interacting proteins, and chromodomain helicase DNA binding protein 2 (CHD2) was successfully identified. Bimolecular fluorescence complementation (BiFC) and subcellular co-localization assays further suggested a possible interaction between SPATA12 and CHD2 in the nuclei. CHD2 is known to be involved in the later stage of the DNA damage response pathway by influencing the transcriptional activity of p53. Thus, our hypothesis is that SPATA12 might play a role in DNA damage signaling. Western blotting results showed that SPATA12 expression could be induced in ultraviolet-C (UV-C) irradiated cells. Through reporter gene assays and the activator protein-1 (AP-1) decoy oligodeoxynucleotide method, we demonstrated that SPATA12 promoter activity could be up-regulated in response to UV-C radiation exposure and an AP-1 binding site in the SPATA12 promoter may have a role in transcriptional regulation of SPATA12. Using colony formation and host cell reactivation assays, it was demonstrated that SPATA12 might lead to inhibition of cellular proliferation in UV-C-irradiated DNA damage. Furthermore, SPATA12 was transfected into H1299, MCF-7 and HeLa cells, and flow cytometry (FCM) results suggested that there are some biological association between SPATA12 and p53 in UV-C-irradiated DNA damage. In addition, we investigated whether SPATA12 could up-regulate the expression of p53. Taken together, these findings indicate that SPATA12 could be induced under UV-C stress. During DNA damage process, AP-1 involves in the transcriptional up-regulation of SPATA12 in response to UV-C radiation and p53 involves in growth inhibitory effects of SPATA12 on UV-C irradiated cells.我们既往的研究显示，SPATA12作为一种新型精子发生相关基因，可能是参与精子发生与肿瘤发生过程的抑制因子。为深入阐明SPATA12的生物学功能，本研究采用酵母双杂交筛选系统搜寻其互作蛋白，并成功鉴定出染色质域解旋酶DNA结合蛋白2（chromodomain helicase DNA binding protein 2, CHD2）。双分子荧光互补（Bimolecular fluorescence complementation, BiFC）实验与亚细胞共定位分析进一步证实，SPATA12与CHD2可在细胞核内发生相互作用。已知CHD2可通过影响p53的转录活性参与DNA损伤应答通路的后期阶段。据此我们提出假说：SPATA12可能在DNA损伤信号通路中发挥作用。蛋白质印迹法（Western blotting）实验结果显示，紫外线C（ultraviolet-C, UV-C）照射可诱导细胞内SPATA12的表达。通过报告基因实验与激活蛋白1（activator protein-1, AP-1）诱饵寡脱氧核苷酸技术，我们证实UV-C辐射可上调SPATA12的启动子活性，且SPATA12启动子区域内的AP-1结合位点可能参与其转录调控过程。采用集落形成实验与宿主细胞复活实验证实，SPATA12可抑制UV-C照射所致DNA损伤后的细胞增殖。此外，我们将SPATA12分别转染至H1299、MCF-7及HeLa细胞中，流式细胞术（flow cytometry, FCM）结果提示，在UV-C照射所致的DNA损伤过程中，SPATA12与p53存在一定的生物学关联。另外，我们探究了SPATA12是否可上调p53的表达。综合上述实验结果，本研究表明SPATA12可在UV-C应激条件下被诱导表达。在DNA损伤过程中，AP-1可通过响应UV-C辐射而上调SPATA12的转录，而p53则参与SPATA12对UV-C照射细胞的生长抑制效应。