Beijersbergen* Abstract A Large Scale shRNA Barcode Screen Identifies the Circadian Clock Component ARNTL as Putative Regulator of the p53 Tumor Suppressor Pathway Jasper Mullenders., Armida W. M. Fabius., Mandy Madiredjo, Rene´ Bernards, Roderick L. Division of Molecular Carcinogenesis, Centre for Biomedical Genetics and Cancer Genomics Centre, Netherlands Cancer Institute, Amsterdam, The Netherlands Background: The p53 tumor suppressor gene is mutated in about half of human cancers, but the p53 pathway is thought to be functionally inactivated in the vast majority of cancer. Understanding how tumor cells can become insensitive to p53 activation is therefore of major importance. Using an RNAi-based genetic screen, we have identified three novel genes that regulate p53 function. Results: We have screened the NKI shRNA library targeting 8,000 human genes to identify modulators of p53 function. Using the shRNA barcode technique we were able to quickly identify active shRNA vectors from a complex mixture. Validation of the screening results indicates that the shRNA barcode technique can reliable identify active shRNA vectors from a complex pool. Using this approach we have identified three genes, ARNTL, RBCK1 and TNIP1, previously unknown to regulate p53 function. Importantly, ARNTL (BMAL1) is an established component of the circadian regulatory network. The latter finding adds to recent observations that link circadian rhythm to the cell cycle and cancer. We show that cells having suppressed ARNTL are unable to arrest upon p53 activation associated with an inability to activate the p53 target gene p21CIP1. Conclusions: We identified three new regulators of the p53 pathway through a functional genetic screen. The identification of the circadian core component ARNTL strengthens the link between circadian rhythm and cancer. Citation: Mullenders J, Fabius AWM, Madiredjo M, Bernards R, Beijersbergen RL (2009) A Large Scale shRNA Barcode Screen Identifies the Circadian Clock Component ARNTL as Putative Regulator of the p53 Tumor Suppressor Pathway. PLoS ONE 4(3): e4798. doi:10.1371/journal.pone.0004798 Editor: Irene Oi Lin Ng, The University of Hong Kong, Hong Kong Received December 6, 2008; Accepted January 28, 2009; Published March 11, 2009 Copyright: 2009 Mullenders et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This research was funded by the Dutch Cancer Society (KWF) and by the EU 6th framework program TRANSFOG. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: r.beijersbergen@nki.nl . These authors contributed equally to this work Introduction The TP53 gene product is important in the cellular response to different types of stress [1,2]. A major physiological stress is DNA damage. DNA damage leads to activation of the ATM/ATR, CHK1/2 cascade, which in turn activates p53. Activation of p53 is achieved by increased stability and post translational modifications of the p53 protein. These modifications include phosphorylation, methylation [3], ubiquitination [4,5] and acetylation [6] leading to enhanced transcriptional activity of p53. Furthermore, oncogene activation can also lead to p53 activation through activation of the p19ARF protein. p19ARF inhibits MDM2, the major E3 ubiquitin ligase for p53 [7] leading to stabilization and activation of p53. Activation of p53 leads to transcriptional activation of a large set of p53 target genes, which in turn causes cell cycle arrest or apoptosis [8]. The p53 pathway is inactivated in almost all human cancers [9]. In about half of human cancers this is due to mutation or deletion of the TP53 gene itself. However in a significant fraction of human tumors, the p53 pathway is inactivated through alteration in cellular components acting up- or down-stream of p53. For example, amplification of the negative regulator of p53, MDM2, leads to accelerated degradation and inactivation of p53 [2,10]. As a model to screen for genes that modulate p53 function, we previously developed a human fibroblast cell line named BJtsLT [11]. These cells express a temperature-sensitive mutant of the SV40 large T antigen, which allows the cells to proliferate at the permissive temperature (32uC). However, when the cells are shifted to 39uC, the large T antigen is degraded and the cells enter a stable p53-dependent cell cycle arrest. We and others have previously described the construction and initial screening of shRNA libraries using the barcode technique [11–17]. The barcode technique allows the rapid identification of individual shRNA vectors from a large pool of shRNA vectors that produce a specific phenotype. This approach takes advantage of the fact that each shRNA vector contains a unique 19-mer sequence as part of the shRNA cassette, which can serve as a molecular ‘‘barcode’’ identifier. Briefly, cells are infected with the pooled shRNA library of some 24,000 vectors. The population of cells is then split into two separate populations. One population is PLoS ONE | www.plosone.org 1 March 2009 | Volume 4 | Issue 3 | e4798