Analysis for the role of MAD1 in gene expression

• Main Authors: Chang-Ching Yeh, 葉昶慶
• Other Authors: Yue-Li Juang
• Format: Others
• Language: zh-TW
• Online Access: http://ndltd.ncl.edu.tw/handle/84827192236392226661

碩士 === 慈濟大學 === 分子生物暨人類遺傳學研究所 === 97 === Human MAD1 was originally identified by its binding to a viral oncoprotein, HTLV-1 Tax, which affects transcription of viral and human proteins(Jin DY. et al.,1998). MAD1 is a component of spindle assembly checkpoint which is a regulatory mechanism that prevents aneuploidy by delaying sister chromatid separation before all chromosomes have aligned at the metaphase plate (Musacchio A. et al.,2007). In previous study, RED was identified to be a MAD1-interacting protein (Li CH, 2002). In this study, when we tried to identify the RED-interacting region in MAD1 by yeast-two hybrid analysis, a peptide sequence of MAD1 from 301-340 had an ability to activate expression of the reporter gene HIS3. This prompted us to investigate whether MAD1 had a role in transcription. Using luciferase gene as a reporter, in MCF7, depletion of MAD1 resulted in a decrease in expression of luciferase gene from E-cadherin promoter. Consistently, depletion of MAD1 also resulted in a decrease in the protein levels of E-cadherin in MCF7 cells. Furthermore, in HeLa cells, ectopic overexpression of MAD1 caused a decrease in protein level of endogenous MAD1, and consistently a decrease in expression of luciferase reporter gene from the MAD1 promoter. Besides, MAD1 was coprecipitated with POlR2A (RNA polymerase II large subunit) from HeLa cell lysates by performing co-immunoprecipitation using anti-POlR2A antibody. Overall, these results reveal that MAD1 may play a role in gene expression at the transcriptional level.