Abstract
p53 regulates a number of genes through transcriptional activation and repression. p53-dependent mitotic checkpoint has been described, but the underlying mechanism is still obscure. Here we examined the effect of p53 on the expression of a human mitotic checkpoint protein, Mitosis Arrest Deficiency 1 (MAD1), in cultured human cells. The expression of MAD1 was reduced when the cells were overexpressing exogenously introduced wild-type p53. The same reduction was also observed when the cells were treated with anticancer agents 5-fluorouracil and cisplatin or were irradiated with UV. Consistently, MAD1 promoter activity diminished in a dose-dependent manner when induced by p53, indicating that p53 repressed MAD1 at a transcriptional level. Intriguingly, several tumor hot spot mutations in p53 (V143A, R175H, R248W, and R273H) did not abolish the ability of p53 to repress MAD1 expression. By serial truncation of the MAD1 promoter, we confined the p53-responsive element to a 38-bp region that represents a novel sequence distinct from the known p53 consensus binding site. Trichostatin A, a histone deacetylase inhibitor, relieved the p53 transrepression activity on MAD1. Chromatin immunoprecipitation assay revealed that p53, histone deacetylase 1, and co-repressor mSin3a associated with the MAD1 promoter in vivo. Taken together, our findings suggest a regulatory mechanism for the mitotic checkpoint in which MAD1 is inhibited by p53.
Highlights
MMP-1 and -13, presenilin-1, MAP-4, and stathmin genes (9 – 12)
We mapped the p53-responsive element in the Mitosis Arrest Deficiency 1 (MAD1) promoter. p53 repression activity on MAD1 is decreased upon addition of histone deacetylase inhibitor, trichostatin A
We first queried whether the expression vector alone without p53 could lead to the decrease of MAD1 expression. p53 was cloned previously into an expression vector carrying a CMV promoter [50]
Summary
MMP-1 and -13, presenilin-1, MAP-4, and stathmin genes (9 – 12). the repression mechanisms are relatively obscure. Down-regulation of MAD1 Protein Expression by Wild-type p53—A genome-wide SAGE analysis revealed that human mitotic checkpoint gene MAD1 is induced by p53 in the colorectal cancer cell line DLD-1 [39].
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