Figure 4. Knockdown of ARNTL, TNIP1 and RBCK1 prevents p21CIP1induction in BJtsLT cells. BJtsLT cells were infected at 32uC and shifted to 39uC for colony formation. After 14 days of culturing at 39uC cells were harvested, protein lysates were prepared and subjected to western blot for p53, CDK4 (control) and p21CIP1 (Figure 4a–c). Additionally, total RNA was isolated and used for QRT-PCR for p21CIP1 (Figure 4d). doi:10.1371/journal.pone.0004798.g004 p53 independent p21CIP1activation does not require ARNTL The results described above suggest a role for ARNTL in the regulation of p21CIP1 expression by p53. However they do not rule out that ARNTL controls p21CIP1activation in a general fashion, independent of p53. To test this possibility we made use of small molecule HDAC inhibitors (HDACi). These HDACi cause induction of p21CIP1 in both a p53 dependent and p53 independent manner [35–38]. In order to study the effect of ARNTL on p53 independent activation of p21CIP1, we made use of a HCT116 p53 knockout cell-line (HCT116 p532/2) and the HDACi PXD101 (BelinostatH) [32]. When these HCT116 p532/2 cells are treated with PXD-101, a strong induction of p21CIP1 is observed (Fig 5d and e). HCT116 p532/2 cells infected with a shRNA vector against p21CIP1 show reduced p21CIP1 protein levels after PXD-101 treatment. However, cells infected with a shRNA targeting ARNTL do not show any alteration in the induction of p21CIP1 protein levels following HDACi treatment. Thus the p53-independent induction of p21CIP1 by HDACi is not dependent on ARNTL. From this we conclude that ARNTL is not generally required for p21cip1 induction, but does affect the capacity of p53 to activate p21CIP1 expression. Discussion The screening of large-scale RNAi libraries has been used increasingly over the last years to identify the specific functions of shRNA Screen Modulators of p53 genes in cellular pathways, networks and mechanisms. Here we describe the screening of a complex RNAi library to identify genes that were previously unknown to regulate a p53-dependent cell cycle arrest. We have used the RNAi barcode technique to screen a human shRNA library containing ,24,000 vectors targeting ,8,000 genes. Using this approach, we were able to rapidly identify shRNAs that allow bypass of a p53 dependent cell cycle arrest. In total we confirmed that 32 out of the 37 genes that were identified by the barcode screen could indeed prevent cells from entering into a p53 dependent cell cycle arrest. However, only 5 of these 32 genes were targeted by two independent shRNAs. Two out of these five genes (TP53BP1 and p21cip1) are well-known to be involved in p53 signaling. However the other three genes (TNIP1, RBCK1 and ARNTL) were previously not known to be involved in the p53 pathway. The three newly identified genes all affect the induction of the p53 target gene p21CIP1 but no change in p53 protein stability is observed after ARNTL, TNIP1 or RBCK1 knockdown. Impor- tantly, p21CIP1 knockdown alone is sufficient to rescue cells from the p53 induced cell cycle arrest. This observation indicates that the rescue of the p53 induced cell cycle arrest by ARNTL, RBCK1 or TNIP1 knockdown is the result of a lack of p21CIP1 induction by p53. The activity of p53 has been mainly attributed to its role as transcription factor with tumor suppressive capacities. Therefore, we assessed if any of the genes identified in our screen had been linked to transcription before. TNIP1 was originally identified as an inhibitor of NF-kB signaling [22,39]. Although it was shown that TNIP1 over- expression inhibits the transcriptional activity of the NF-kB heterodimer it is believed that this is an indirect effect through an currently unknown mechanism. The ubiquitin E3 ligase RBCK1 has been reported to regulate and ubiquitinate several proteins [21,40– 42]. Although experiments have been performed that suggest a role for RBCK1 in transcription [43] a clear role for RBCK1 in regulating transcription has not been reported up till now. This picture is different for ARNTL which is known to be the central transcription factor in regulating circadian rhythm. The critical role of ARTNL in circadian rhythm was demonstrated by the construction of the knockout mouse. Mice that are deficient for ARNTL are unable to maintain a circadian rhythm in constant darkness [20]. In addition, the ARNTL knockout mouse also suffers from premature aging [44]. In recent years, many other processes have been shown to be regulated in a circadian fashion. Most importantly it was shown that the mammalian cell cycle is controlled by circadian rhythm [45]. The possible involvement of circadian rhythm in cancer results from studies of the Period 2 knockout mouse.This mouse is prone to develop tumors after radiation. Later it was shown that also the Period 1 protein can regulate cell cycle checkpoints [24–26]. Interestingly both Period 1 and 2 are bona-fide transcriptional targets of ARNTL. For the induction of target genes ARNTL must form a heterodimer with the CLOCK protein [28]. Target genes of the CLOCK/ARNTL heterodimer include the Period 1, 2 and 3 and Cryptochromes (Cry 1 & 2) [46]. The increased abundance of Period and Cryptochrome proteins [47] induces a negative feedback loop that ultimately shuts down transcription by the CLOCK/ARTNL heterodimer. When the concentration of Period and Crypto- chrome decreases due to proteasomal degradation the CLOCK/ ARNTL complex can initiate another round of transcription thereby completing a cycle of circadian rhythm. Another transcriptional target of the CLOCK/ARNTL is the CDK inhibitor p21CIP1 which is also regulated in a circadian manner [48]. We show here that ARNTL knockdown in human PLoS ONE | www.plosone.org 7 March 2009 | Volume 4 | Issue 3 | e4798