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Analysis transcriptional mechanism and physiological function of a BTB-oontaining zinc finger protein, CIBZ

含BTB锌指蛋白CIBZ的转录机制和生理功能分析

基本信息

批准号：
18590268
负责人：
MATSUDA Eishou
金额：
$ 2.57万
依托单位：
Nara Institute of Science and Technology
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2006
资助国家：
日本
起止时间：
2006 至 2007
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-18590268/
关键词：
Transcription BTB CtBP apoptosis caspase p53 PARP MEF CASTing法細胞周期ノックアウトマウス

项目摘要

We previously identified and characterized a murine BTB-containing protein, CIBZ (ZBTB38 in human), that interacts with CtBP and binds to methylated CpGs. However, its physiological function remained unknown. As CtBP is reportedly involved in p53-independent programmed cell death, we examine here whether CIBZ is associated with apoptosis. We found that CIBZ was highly expressed in proliferating C2C12 cells, but that its expression levels decreased upon induction of apoptosis by serum starvation. Knockdown of CIBZ by siRNA in C2C12 cells induced apoptosis, as determined by an increase of annexin V/PI labeling, activation of caspase-3, and cleavage of PARP. CIBZ inhibition also activated caspase-7 and caspase-9, suggesting that CIBZ-associated apoptosis occurs through the mitochondrial pathway. Notably, knockdown of CIBZ in p53 -/- MEF cells also activated caspase-3 and cleavage of PARP, indicating that CIBZ-associated apoptosis is mediated by a p53-independent pathway; however, since both common and distinct targets are regulated by CIBZ-and CtBP-associated apoptosis, we conclude that more than one pathway is involved. Finally, using mutagenesis and an in vitro caspase cleavage assay, we show that CIBZ is a novel substrate of caspase-3, and identify two caspase-3 recognition sites. These findings indicate, collectively, that CIBZ plays an important role by participating in the negative regulation of apoptosis in murine cells.

我们之前鉴定并表征了一种含有鼠BTB的蛋白质CIBZ（人类ZBTB 38），它与CtBP相互作用并结合甲基化CpGs。然而，其生理功能仍然未知。据报道，CtBP参与p53非依赖性程序性细胞死亡，我们在这里研究CIBZ是否与细胞凋亡有关。我们发现CIBZ在增殖的C2 C12细胞中高度表达，但其表达水平在血清饥饿诱导凋亡后降低。C2 C12细胞中通过siRNA敲低CIBZ诱导凋亡，如通过膜联蛋白V/PI标记的增加、半胱天冬酶-3的活化和PARP的裂解所确定的。CIBZ抑制还激活半胱天冬酶-7和半胱天冬酶-9，表明CIBZ相关的细胞凋亡通过线粒体途径发生。值得注意的是，敲低CIBZ在p53 -/- MEF细胞也激活caspase-3和PARP的裂解，表明CIBZ相关的细胞凋亡是由p53非依赖性途径介导的;然而，由于共同和不同的目标是由CIBZ和CtBP相关的细胞凋亡，我们得出结论，不止一个途径参与。最后，利用诱变和体外caspase裂解实验，我们表明CIBZ是caspase-3的一种新底物，并确定了两个caspase-3识别位点。这些发现共同表明，CIBZ通过参与小鼠细胞凋亡的负调控而发挥重要作用。