To assess the effect of PARP-1 on FOXO1-mediated transcription, we performed luciferase assays using a p27Kip1 promoter-driven luciferase construct. Overexpression of PARP-1 inhibited FOXO1-induced reporter activity in a dose-dependent manner (Fig. 2A, columns 5 and 6). Furthermore, we investigated whether enzymatic activity of PARP-1 is involved in this pirinixic acid repressive effect. PARP-1 (E988K) mutant repressed transcriptional activity of FOXO1 to the same extent as wild-type (Fig. 2A, columns 7 and cool . It should be noted that treatment with 3AB has no effect on PARP-1-mediated repression of the transcriptional activity of FOXO1 (data not shown). Next, we examined whether overexpression of PARP-1 could reduce endogenous p27Kip1 gene expression in HEK293T cells. In agreement with the result of luciferase assay, PARP-1 downregulated FOXO1-mediated p27Kip1 expression in a manner independent of its enzymatic activity (Fig. 2B).
Fig. 2.
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