shRNA Screen Modulators of p53 Figure 5. ARNTL regulates p21CIP1 expression. a) Knockdown of ARNTL inhibits radiation induced p21CIP1 induction. U2OS cells were infected with the shRNA vectors as indicated. Cells were seeded and irradiated with 20 Gy of c-radiation. After o/n incubation cells were lysed and lysates were subjected to western blot using antibodies for p53, CDK4 (control) and p21CIP1. b) Knockdown of ARNTL can also rescue a p19-induced cell cycle arrest. U2OS cells were infected with the indicated shRNA vectors followed by a super-infection with p19ARF-RFP virus. Cells were seeded and incubated for three weeks. After three weeks the infected cells were fixed and stained. c) Knockdown of ARNTL in U2OS cells (Fig 5b) was quantified by QRT-PCR. d) ARNTL knockdown is not involved in p53 independent p21CIP1 induction. HCT116 wt and p532/2 cells were infected with knockdown vectors targeting p53, p21CIP1 and ARNTL. Cells were treated with 0.5 mM PXD101 for 16 hrs. Cells were then lysed and lysates were subjected to western analysis for p53, CDK4 (control) and p21CIP1. e) Quantification of p21 protein levels in the western blot in figure 5c using IMAGE J software. doi:10.1371/journal.pone.0004798.g005 cells can abrogate induction of p21CIP1 after p53 activation and overrides a p53-dependent cell cycle arrest. The effect on the induction of p21CIP1 is in contrast with previous reports on the p21CIP1 regulation in ARNTL knockout mice [48]. In these animals ARNTL is required for the circadian expression of p21CIP1. This discrepancy might be explained by differential regulation of the p21CIP1 promoter in mice or man. In particular, this difference may arise from stress signals differences from in vitro versus in vivo conditions. Nevertheless, our data clearly indicate that there is a link between the regulation of circadian rhythm and the control of p53 activity in human cells. Conclusions By screening a large scale RNAi library in human cells we have identified three novel genes that can regulate p53 function. Loss of expression for each of three genes results in a decreased ability of p53 to activate p21CIP1 expression. Importantly, we showed that ARNTL is required for the p53-dependent induction of p21CIP1 in PLoS ONE | www.plosone.org 8 March 2009 | Volume 4 | Issue 3 | e4798