RAD6 regulates the dosage of p53 by a combination of transcriptional and posttranscriptional mechanisms.

Maintaining an appropriate cellular concentration of p53 is critical for cell survival and normal development in various organisms. In this study, we provide evidence that the human E2 ubiquitin-conjugating enzyme RAD6 plays a critical role in regulating p53 protein levels under both normal and stress conditions. Knockdown and overexpression of RAD6 affected p53 turnover and transcription. We showed that RAD6 can form a ternary complex with MDM2 and p53 that contributes to the degradation of p53. Chromatin immunoprecipitation (ChIP) analysis showed that RAD6 also binds to the promoter and coding regions of the p53 gene and modulates the levels of H3K4 and K79 methylation on local chromatin. When the cells were exposed to stress stimuli, the RAD6-MDM2-p53 ternary complex was disrupted; RAD6 was then recruited to the chromatin of the p53 gene, resulting in an increase in histone methylation and p53 transcription. Further studies showed that stress-induced p53 transcriptional activation, cell apoptosis, and disrupted cell cycle progression are all RAD6 dependent. Overall, this work demonstrates that RAD6 regulates p53 levels in a "yin-yang" manner through a combination of two distinct mechanisms in mammalian cells.