Park Hye-Young, Jeon Yoon-Kyung, Shin Hyun-Jin, Kim Il-Jin, Kang Hio-Chung, Jeong Sook-Jung, Chung Doo-Hyun, Lee Chang-Woo
Department of Molecular Cell Biology, Center for Molecular Medicine, Samsung Biomedical Research Institute, Sungkyunkwan University School of Medicine, Suwon 440-746, Korea.
Exp Mol Med. 2007 Apr 30;39(2):195-204. doi: 10.1038/emm.2007.22.
The BubR1 mitotic-checkpoint protein monitors proper attachment of microtubules to kinetochores, and links regulation of chromosome-spindle attachment to mitotic-checkpoint signaling. Thus, disruption of BubR1 activity results in a loss of checkpoint control, chromosomal instability caused by a premature anaphase, and/or the early onset of tumorigenesis. The mechanisms by which deregulation and/or abnormalities of BubR1 expression operate, however, remain to be elucidated. In this study, we demonstrate that levels of BubR1 expression are significantly increased by demethylation. Bisulfite sequencing analysis revealed that the methylation status of two CpG sites in the essential BubR1 promoter appear to be associated with BubR1 expression levels. Associations of MBD2 and HDAC1 with the BubR1 promoter were significantly relieved by addition of 5-aza-2'-deoxycytidine, an irreversible DNA methyltransferase inhibitor. However, genomic DNA isolated from 31 patients with colorectal carcinomas exhibited a +84A/G polymorphic change in approximately 60% of patients, but this polymorphism had no effect on promoter activity. Our findings indicate that differential regulation of BubR1 expression is associated with changes in BubR1 promoter hypermethylation patterns, but not with promoter polymorphisms, thus providing a novel insight into the molecular regulation of BubR1 expression in human cancer cells.
BubR1有丝分裂检查点蛋白监测微管与动粒的正确附着，并将染色体-纺锤体附着的调节与有丝分裂检查点信号传导联系起来。因此，BubR1活性的破坏会导致检查点控制丧失、由过早后期引起的染色体不稳定性和/或肿瘤发生的早期发作。然而，BubR1表达失调和/或异常发挥作用的机制仍有待阐明。在本研究中，我们证明去甲基化可显著增加BubR1的表达水平。亚硫酸氢盐测序分析显示，重要的BubR1启动子中两个CpG位点 的甲基化状态似乎与BubR1表达水平相关。添加不可逆的DNA甲基转移酶抑制剂5-氮杂-2'-脱氧胞苷后，MBD2和HDAC1与BubR1启动子的结合显著减弱。然而，从31例结直肠癌患者中分离的基因组DNA在约60%的患者中表现出+84A/G多态性变化，但这种多态性对启动子活性没有影响。我们的研究结果表明，BubR1表达的差异调节与BubR1启动子高甲基化模式的变化有关，而与启动子多态性无关，从而为人类癌细胞中BubR1表达的分子调节提供了新的见解。
