Shen Tao, Li Yan, Liang Shuang, Chen Zhiguang
Department of Orthopedics, Shengjing Hospital of China Medical University, No. 36, Sanhao Street, Heping District, Shenyang, 110004 People's Republic of China.
Department of Cell Biology, Key Laboratory of Cell Biology, Ministry of Public Health and Key Laboratory of Medical Cell Biology, Ministry of Education, China Medical University, Shenyang, 110122 People's Republic of China.
Cancer Cell Int. 2020 Sep 22;20:459. doi: 10.1186/s12935-020-01553-9. eCollection 2020.
Centromere protein F (CENPF) is a key component of the kinetochore complex involved in mitosis, cell differentiation and cellular response to stresses. However, the alteration of CENPF in response to endoplasmic reticulum (ER) stress has not been well described. In the present study, we investigate CENPF regulation in response to ER stress.
Quantitative real-time polymerase chain reaction and western blotting were used to determine CENPF expression under ER stress. Luciferase activity analysis was performed to investigate the promoter regions contributing to CENPF transcription in response to TG. Chromatin immunoprecipitation (ChIP) and ChIP Re-IP assays were used to determine if X-box binding protein 1 (XBP1) and/or activating transcription factor 6α (ATF6α) bind in the CENPF promoter region. Cell apoptosis and proliferation were analyzed using TUNEL, cell growth and clonogenic assays.
CENPF expression is dramatically reduced under ER stress induced by thapsigargin (TG), brefeldin A (BFA), or tunicamycin (TM) and this downregulation of CENPF expression was dependent on XBP1 and ATF6α. Luciferase activity analysis of the truncated CENPF promoter indicates that regions from bases - 679 to - 488 and from - 241 to - 78 in the CENPF promoter were sensitive to TG treatment. Additionally, ChIP and ChIP Re-IP assays reveal that XBP1 and ATF6α were assembled on the same regions of CENPF promoter. Notably, we identify two XBP1 binding sequences at positions - 567 and - 192, to which XBP1 binding was enhanced by TG. Finally, CENPF overexpression inhibits cell apoptosis and promotes cell proliferation in response to ER stress.
In summary, these results demonstrate that ER stress plays a crucial role in CENPF expression, and XBP1 may up-regulate DNA-binding affinities after TG treatment to the promoter of CENPF. These findings may contribute to the understanding of the molecular mechanism of CENPF regulation.
着丝粒蛋白F（CENPF）是参与有丝分裂、细胞分化及细胞应激反应的动粒复合体的关键组成部分。然而，内质网（ER）应激时CENPF的变化尚未得到充分描述。在本研究中，我们探究了CENPF在ER应激反应中的调控机制。
采用定量实时聚合酶链反应和蛋白质免疫印迹法测定ER应激下CENPF的表达。进行荧光素酶活性分析以研究响应衣霉素（TG）时对CENPF转录有贡献的启动子区域。采用染色质免疫沉淀（ChIP）和ChIP再免疫沉淀（Re-IP）试验来确定X盒结合蛋白1（XBP1）和/或激活转录因子6α（ATF6α）是否结合于CENPF启动子区域。使用末端脱氧核苷酸转移酶介导的缺口末端标记法（TUNEL）、细胞生长和克隆形成试验分析细胞凋亡和增殖情况。
在毒胡萝卜素（TG）、布雷菲德菌素A（BFA）或衣霉素（TM）诱导的ER应激下，CENPF表达显著降低，且CENPF表达的这种下调依赖于XBP1和ATF6α。对截短的CENPF启动子进行荧光素酶活性分析表明，CENPF启动子中从碱基-679至-488以及从-241至-78的区域对TG处理敏感。此外，ChIP和ChIP Re-IP试验显示，XBP1和ATF6α组装在CENPF启动子的相同区域。值得注意的是，我们在-567和-192位置鉴定出两个XBP1结合序列，TG增强了XBP1与它们的结合。最后，CENPF过表达抑制ER应激诱导的细胞凋亡并促进细胞增殖。
总之，这些结果表明ER应激在CENPF表达中起关键作用，且TG处理后XBP1可能上调对CENPF启动子的DNA结合亲和力。这些发现可能有助于理解CENPF调控的分子机制。
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