A Large Scale shRNA Barcode Screen Identifies the Circadian Clock Component ARNTL as Putative Regulator of the p53 Tumor Suppressor Pathway

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 p21(CIP1). 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

Contrasting roles of histone 3 lysine 27 demethylases in acute lymphoblastic leukaemia

T-cell acute lymphoblastic leukaemia (T-ALL) is a haematological malignancy with a dismal overall prognosis, including a relapse rate of up to 25%, mainly because of the lack of non-cytotoxic targeted therapy options. Drugs that target the function of key epigenetic factors have been approved in the context of haematopoietic disorders, and mutations that affect chromatin modulators in a variety of leukaemias have recently been identified; however, ‘epigenetic’ drugs are not currently used for T-ALL treatment. Recently, we described that the polycomb repressive complex 2 (PRC2) has a tumour-suppressor role in T-ALL. Here we delineated the role of the histone 3 lysine 27 (H3K27) demethylases JMJD3 and UTX in T-ALL. We show that JMJD3 is essential for the initiation and maintenance of T-ALL, as it controls important oncogenic gene targets by modulating H3K27 methylation. By contrast, we found that UTX functions as a tumour suppressor and is frequently genetically inactivated in T-ALL. Moreover, we demonstrated that the small molecule inhibitor GSKJ4 (ref. 5) affects T-ALL growth, by targeting JMJD3 activity. These findings show that two proteins with a similar enzymatic function can have opposing roles in the context of the same disease, paving the way for treating haematopoietic malignancies with a new category of epigenetic inhibitors.National Institutes of Health (U.S.) (Grant R37-HD04502

TET1 is a tumor suppressor of hematopoietic malignancy

The methylcytosine dioxygenase TET1 (‘ten-eleven translocation 1’) is an important regulator of 5-hydroxymethylcytosine (5hmC) in embryonic stem cells. The diminished expression of TET proteins and loss of 5hmC in many tumors suggests a critical role for the maintenance of this epigenetic modification. Here we found that deletion of Tet1 promoted the development of B cell lymphoma in mice. TET1 was required for maintenance of the normal abundance and distribution of 5hmC, which prevented hypermethylation of DNA, and for regulation of the B cell lineage and of genes encoding molecules involved in chromosome maintenance and DNA repair. Whole-exome sequencing of TET1-deficient tumors revealed mutations frequently found in non-Hodgkin B cell lymphoma (B-NHL), in which TET1 was hypermethylated and transcriptionally silenced. Our findings provide in vivo evidence of a function for TET1 as a tumor suppressor of hematopoietic malignancy.National Institutes of Health (U.S.) (5RO1HD045022)National Institutes of Health (U.S.) (5R37CA084198

Miz1 and HectH9 regulate the stability of the checkpoint protein, TopBP1

The Myc-associated zinc-finger protein, Miz1, activates transcription of the p21cip1 gene in response to UV irradiation. Miz1 associates with topoisomerase II binding protein1 (TopBP1), an essential activator of the Atr kinase. We show here that Miz1 is required for the recruitment of a fraction of TopBP1 to chromatin, for the protection of TopBP1 from proteasomal degradation and for Atr-dependent signal transduction. TopBP1 that is not bound to chromatin is degraded by the HectH9 (Mule, ARF-BP1 and HUWE1) ubiquitin ligase. Myc antagonizes the binding of TopBP1 to Miz1; as a result, expression of Myc leads to dissociation of TopBP1 from chromatin, reduces the amount of total TopBP1 and attenuates Atr-dependent signal transduction. Our data show that Miz1 and Myc affect the activity of the Atr checkpoint through their effect on TopBP1 chromatin association and stability

ARNTL regulates p21^CIP1 expression.

<p>a) Knockdown of ARNTL inhibits radiation induced p21^CIP1 induction. U2OS cells were infected with the shRNA vectors as indicated. Cells were seeded and irradiated with 20 Gy of γ-radiation. After o/n incubation cells were lysed and lysates were subjected to western blot using antibodies for p53, CDK4 (control) and p21^CIP1. 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 (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0004798#pone-0004798-g005" target="_blank">Fig 5b</a>) was quantified by QRT-PCR. d) ARNTL knockdown is not involved in p53 independent p21^CIP1 induction. HCT116 wt and p53^−/− cells were infected with knockdown vectors targeting p53, p21^CIP1 and ARNTL. Cells were treated with 0.5 µM PXD101 for 16 hrs. Cells were then lysed and lysates were subjected to western analysis for p53, CDK4 (control) and p21^CIP1. e) Quantification of p21 protein levels in the western blot in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0004798#pone-0004798-g005" target="_blank">figure 5c</a> using IMAGE J software.</p

Knockdown of ARNTL, TNIP1 and RBCK1 prevents p21^CIP1induction in BJtsLT cells.

<p>BJtsLT cells were infected at 32°C and shifted to 39°C for colony formation. After 14 days of culturing at 39°C cells were harvested, protein lysates were prepared and subjected to western blot for p53, CDK4 (control) and p21^CIP1 (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0004798#pone-0004798-g004" target="_blank">Figure 4a–c</a>). Additionally, total RNA was isolated and used for QRT-PCR for p21^CIP1 (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0004798#pone-0004798-g004" target="_blank">Figure 4d</a>).</p

List of shRNA vectors that were selected by the criteria as in figure 1c. Depicted are shRNAs that were used in validation experiments.

<p>Under rescue; + means a validated shRNA, − means not validated. <i>M</i> indicates the ²log (ratio Cy5/Cy3), A the ²log(√intensity Cy3×Cy5).</p

shRNA barcode screen identifies mediators of the p53 dependent cell cycle arrest.

<p>a) Schematic outline of the BJtsLT genetic screen. BJtsLT cells were infected with the NKI shRNA library and were either left at 32°C or shifted to 39°C. After 7 days the cells at 32°C had reached confluency and were harvested. Cells at 39°C were harvested after 23 days after which they had formed visible colonies. b) Analysis of the relative abundance of shRNAs recovered from the BJtsLT barcode experiment. Data are normalized and plotted as <i>M</i>, the ²log (ratio Cy5/Cy3), versus <i>A</i> (²log(√intensity Cy3×Cy5)). The data are the average of two independent hybridization experiments performed in duplicate with reversed colour. A red box is drawn around the top 100 enriched shRNAs at 39°C. c) Schematic overview of selection criteria used to select hits from the shRNA barcode screen for further validation.</p