Endoplasmic reticulum (ER) stress is the condition that unfolded or misfolded proteins accumulated in the ER which leads to the solubility stress. ER can activate the unfolded protein response (UPR) to restore the ER homeostasis. JAB1 was originally identified as the coactivator of AP-1 transcription factor. JAB1 was then discovered to mediate the cyclin-dependent-kinase inhibitor p27kip1 nuclear exportation and degradation. Previous studies demonstrate that ER stress may affect the regulation of JAB1, but the mechanism is still unknown. In this study, we want to investigate how JAB1 is regulated in ER stress. We applied tunicamycin, a protein N-glycosylation inhibitor, as the ER stress inducer. Western blot and reverse transcription PCR revealed that treatment with tunicamycin for 48 hours in Hep3B induced ER stress and repressed JAB1 protein and mRNA expression. Serial deletion of the JAB1 promoter activity assay revealed that the region from -405 bp to -223 bp may be responsive in the tunicamycin-induced ER stress. Computational prediction suggested that there are several candidate factors may join the regulation of JAB1 in this region. Site-directed mutation of JAB1 promoter assay revealed that the tunicamycin-induced ER stress repressed JAB1 promoter activity through the sites at -342/-338 and -331/-327 in JAB1 promoter. Chromatin immunoprecipitation assay suggested that tunicamycin-induced ER stress repressed the JAB1 promoter activity through increasing the SP1 and DNMT3b binding to the SP1 binding sites at -342/-338 and -331/-327 in JAB1 promoter. Methylation specific PCR showed that the SP1 binding sites at -342/-338 and -331/-327 in JAB1 promoter were methylated in tunicamycin-induced ER stress. Taken together, we demonstrated that tunicamycin-induced ER stress repressed the JAB1 gene expression in Hep3B through increasing the binding of SP1 and DNMT3b to the SP1 binding sites and inducing promoter methylation to repress JAB1 expression.
| Identifer | oai:union.ndltd.org:NSYSU/oai:NSYSU:etd-0727109-113910 |
| Date | 27 July 2009 |
| Creators | Chen, Chien-wei |
| Contributors | Hui-Chiu Chang, Wen-Chun Hung, Long-sen Chang |
| Publisher | NSYSU |
| Source Sets | NSYSU Electronic Thesis and Dissertation Archive |
| Language | English |
| Detected Language | English |
| Type | text |
| Format | application/pdf |
| Source | http://etd.lib.nsysu.edu.tw/ETD-db/ETD-search/view_etd?URN=etd-0727109-113910 |
| Rights | campus_withheld, Copyright information available at source archive |
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