For example, apoptotic death of hepa toma cells expressing the oncogenic HBV X

Bioinfor matic analysis indicated supplier Amuvatinib that there are three N boxes, which is specific for Hes 1 binding, within 1 kb from the transcription start sites of human GADD45 promoters. To study whether Hes 1 actu ally binds to the GADD45 promoter, ChIP assay was performed. Results showed that Hes 1 apparently binds to the GADD45 promoter. The PCR product from ChIP assay was also observed by ChIP PCR and DNA electrophoresis. Among the three SUMOylation residues identified on Hes 1, Lys39 is located in the basic domain of Hes 1, which confers Hes 1 DNA binding activity during tran scriptional repression. This suggests that SUMOyla tion of Hes 1 may be associated with the DNA binding activity of Hes 1. To test this hypothesis, the DNA binding activity of Hes 1WT and Hes 1 3KR was ex amined by ChIP PCR.<br><br> Results revealed that Hes 1WT consistently binds to the GADD45 promoter, AT-406 価格 but Hes 1 3KR apparently decreased the binding activity to the GADD45 promoter. We next ex amined the effect of mutation of Lys39 alone on Hes 1 DNA binding. Result revealed that mutation of Lys39 alone decreased Hes 1 binding to GADD45 promoter for approximately 55%, but it is not sufficient to block Hes 1 DNA binding. This latter result suggests that Lys8 and Lys27, the target sumo sites on Hes 1, also play an important role in Hes 1 DNA bind ing. To further address this issue, we have examined the DNA binding activity of Hes 1WT and SUMO 1 fusioned Hes 1. Result from ChIP PCR revealed that SUMO 1 fusioned Hes 1 showed approximately three fold increase in DNA binding to GADD45 promoter than Hes 1WT did.<br><br> Next, we examined whether blockade of Hes 1 SUMOylation affects the promoter ac tivity of GADD45. Human GADD45 supplier AG-490 promoter was cloned from the genomic library of HEK293T cells and co transfected with different doses of the Flag Hes 1WT or Flag Hes 1 3KR plasmid to HEK293T cells for luciferase reporter assay. Results indicated that Flag Hes 1WT dose dependently sup pressed GADD45 promoter activity, but this effect was diminished by Flag Hes 1 3KR also in a dose dependent manner. Furthermore, results from quanti tative PCR indicated that transfection of Flag Hes 1WT plasmid decreased GADD45 mRNA level dose de pendently but this effect was partially reversed by Flag Hes 1 3KR transfection. The same results were found with GADD45 protein expression. Plasmid transfection and expression was con firmed by western blot against Flag.<br><br> Because GADD45 is a stress sensor, next we examined the effect of Hes 1 SUMOylation on GADD45 protein expression under the challenge of H2O2. Results revealed that H2O2 dramatically increased the expression of GADD45. This effect was decreased by Flag Hes 1WT transfection, but Flag Hes 1 3KR was less able to produce the same effect. Plasmid transfection and expression was confirmed by western blot against Flag. Because Hes 1 suppressed GADD45 promoter activity and expression, we expect that knockdown of Hes 1 expres sion should increase GADD45 promoter activity and ex pression. This issue was examined here. Two different sets of Hes 1 siRNA were transfected to HEK293T cells, respect ively. Results revealed that both Hes 1 siRNA transfec tions increased GADD45 promoter activity and endogenous GADD45 mRNA level.