Abstract 3075: A novel transcriptional role of spindle assembly checkpoint protein Cdc20 regulating the expression of mitotic ubiquitin carrier protein UbcH10

Abstract

Abstract Progression through mitosis requires precisely timed ubiquitin-dependent degradation of specific substrates. UbcH10, an ubiquitin-conjugating enzyme, plays a crucial role with anaphase promoting complex/ cyclosome (APC/C) in progression of and exit from mitotis. Similarly, spindle assembly checkpoint (SAC) protein Cdc20 plays an important role in transition from metaphase to anaphase by activating APC/C. Cdc20 itself gets ubiquitinated by UbcH10 and thus activated to bind with APC/C. Any defect in chromosome alignment activates SAC by blocking Cdc20 and thus anaphase transition is arrested. Earlier our lab reported that Cdc20 overexpression led to defective SAC resulting in aneuploidy. This prompted us to examine the regulation of UbcH10 expression in Cdc20 upregulated condition. Initially, we observed a correlation in the expression of both Cdc20 and UbcH10 in different primary tumors and cancer cell lines. Next, a dose-dependent increase in endogenous UbcH10 levels was found upon ectopic expression of Cdc20. Alternatively, CDC20 knockdown by siRNA led to downregulation of endogenous UbcH10. Activation of UBCH10 promoter-luciferase activity by Cdc20 indicated the regulation at transcriptional level. ChIP assay showed the presence of Cdc20 on UBCH10 promoter leading to chromatin remodelling by interacting with CBP/p300. APC/C-CBP/p300 complex is reported to be involved in transcription regulation. Here, we established the presence of APC/C on the UBCH10 promoter using ChIP assay. Further from ChIP and Co-IP assay we established that Cdc20 regulates APC/C and CBP/p300 association and thus modulates APC/C-CBP/p300 mediated transcriptional regulation of UBCH10. Also, we found cell cycle specific regulation of expression of UbcH10 by Cdc20. Finally, Cyclin B1 degradation assay showed physiological relevance of Cdc20 mediated UbcH10 regulation on mitotic progression. In summary, our data showed a novel role of Cdc20 as a transcription regulator and it regulates UbcH10 expression in a cell cycle specific manner. The co-regulated expression of both these proteins maintains proper mitotic progression which otherwise may results in aneuploidy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3075. doi:10.1158/1538-7445.AM2011-3075