Smad3 regulates the transcriptional activity of amelotin gene promoter in ameloblasts
XU Zhen-zhen
ZHANG Li
WANG Yu-min
LI Dong-liang
GAO Yan
GAO Yu-guang
Abstract：AIM:To study the regulatory effects of Smad3 on the promoter activity of Amelotin gene in amelo-blasts. METHODS:Luciferase reporter gene vectors containing different lengths of Amelotin promoters were constructed by gene cloning and then the constructs were co-transfected into ameloblasts with Smad3 respectively. Dual luciferase a-nalysis was used to observe the effects of Smad3 on the transcriptional activity of Amelotin gene promoter in ameloblasts. On the basis of above results, the new functional promoter region was found. Electrophoretic Mobility Shift Assay( EM-SA) was carried out to identify the interaction between Smad3 and Smad3 binding site on the Amelotin promoter. The Smad3 binding site on Amelotin promoter were mutated by site-specific mutagenesis and the effect of Smad3 on the tran-scriptional activity of the wild or mutant type Amelotin promoter was observed by Dual luciferase analysis. RESULTS:Transfection of Smad3 increased the Amelotin gene promotor (-160~ +196) transcriptional activity(P<0. 05). Blas-ting analysis showed that promoter sequence(-160~ -1)of Amelotin gene between human and mouse was homologous. EMSA identified the interaction between Smad3 and Smad3 binding site on Amelotin promoter. Mutation of Smad3 bind-ing site did not change the transcriptional activity of Amelotin promoter(P>0. 05). CONCLUSION: Smad3 enhances the transcriptional activity of Amelotin promoter by interacting with Smad3 binding site on Amelotin promoter.
Keywords：Smad3amelotinEMSAdual luciferase reporter assay [Chinese Journal of Conservative Dentistry201525(6):329
Publication Date：2015-01-01
Online Publishing Date：2025-08-15(First online date of this platform, not the publication date of the document)
Pages：6( 329-334 )