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A Functional Genome-Wide Rnai Screen Identifies TAF1 As A

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A Functional Genome-Wide Rnai Screen Identifies TAF1 As A 5250–5259 Nucleic Acids Research, 2008, Vol. 36, No. 16 Published online 6 August 2008 doi:10.1093/nar/gkn506 A functional genome-wide RNAi screen identifies TAF1 as a regulator for apoptosis in response to genotoxic stress Junko Kimura, Su Tien Nguyen, Hanshao Liu, Naoe Taira, Yoshio Miki* and Kiyotsugu Yoshida* Department of Molecular Genetics, Medical Research Institute, Tokyo Medical and Dental University, Yushima 1-5-45, Bunkyo-ku, Tokyo 113-8510, Japan Received June 18, 2008; Accepted July 22, 2008 Downloaded from ABSTRACT dissection of specific signaling pathways. Indeed, in com- bination with high-throughput assays, genome-wide Evasion from apoptotic cell death is a character- RNAi studies have uncovered novel gene functions in http://nar.oxfordjournals.org/ istic of cancer; genes that modulate this process various biological processes (2). Several of these studies may be optimal for therapeutic attack. Identifying were aimed at the identification of genes essential for cell key regulators of apoptosis is thus a central goal division, cell cycle progression, endocytosis, tumor trans- in cancer therapy. Here, we describe a loss-of- formation and apoptosis (3–10). Through these studies, function screen that uses RNA interference libraries the power of this approach for the identification of func- to identify genes required for induction of apoptosis. tional modules has been demonstrated. We used a short-hairpin RNA expressing vector Regulation of apoptosis is critical in many fundamental cellular processes. Because defective regulation of apopto- with high gene-expression silencing activity that at University of California, Los Angeles on April 4, 2015 contained fetal brain cDNAs. Survived cells from sis provokes human disease, particularly cancers (11–13), genotoxic stress were isolated to determine knock- a global survey of genes essential for apoptosis in human down of molecules that are crucial for induction cells is thus not only advance the understanding of a fun- damental biological process but also delivers novel diag- of apoptosis. We identified TBP-associated factor 1 nostic and therapeutic targets for cancer. In particular, the (TAF1), a gene previously implicated as an essential signals that induce apoptosis in response to genotoxic component of transcription machinery. Depletion stress are largely unknown. of TAF1 was associated with substantial atten- In this study, we used an improved short-hairpin uation of apoptosis induced by oxidative as well RNA (shRNA) library with genome-wide coverage (14). as genotoxic stress. Microarray analysis further We performed transfection of the shRNA library and demonstrated that a number of genes were tran- high-throughput cell-survival analysis to detect cells that scriptionally declined in cells silenced for TAF1. escaped from death, which were further analyzed by Surprisingly, knocking down TAF1 exhibited a TUNEL assays. We identified TBP-associated factor 1 marked decrease in p27Kip1 expression, allowing (TAF1), an essential component of transcription machin- ery. Microarray and apoptosis analyses demonstrated that cells resistant from oxidative stress-induced apo- Kip1 ptosis. These results suggest that TAF1 regulates TAF1-mediated p27 expression is involved in the induction of apoptosis in response to genotoxic stress. apoptosis by controlling p27Kip1 expression. Our Using this approach, we uncovered a new gene implicated system provides a novel approach to identifying can- in the apoptosis process, including transcriptional regula- didate genes that modulate apoptosis. tory networks that govern cell death in mammalian cells. INTRODUCTION MATERIALS AND METHODS Gene silencing by RNA interference (RNAi) has devel- Cell culture oped a powerful tool for loss-of-function studies (1). Large-scale RNAi has facilitated the search for genes Human 293T embryonal kidney cells, HeLa cervical required for diverse biological processes enabling stepwise cancer cells and MCF-7 breast cancer cells were cultured *To whom correspondence should be addressed. Tel: +81 3 5803 5826; Fax: +81 3 5803 0242; Email: [email protected] Correspondence may also be addressed to Yoshio Miki. Tel: +81 3 5803 5825; Fax: +81 3 5803 0242; Email: [email protected] ß 2008 The Author(s) This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/ by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. Nucleic Acids Research, 2008, Vol. 36, No. 16 5251 in Dulbecco’s modified Eagle medium containing 10% (50 mM Tris–HCl, pH 7.6, 150 mM NaCl, 1 mM Na3VO4, heat-inactivated fetal bovine serum, 100 U/ml penicillin, 1mM PMSF, 1mM DTT, 10mg/ml aprotinin, 1 mg/ml 100 U/ml streptomycin and 2 mM L-glutamine. U2OS leupeptin, 10 mM NaF, 1 mg/ml Pepstatin A, 0.05% deoxy- osteosarcoma cells were cultured in RPMI 1640 medium cholic acid and 1% NP-40). Lysates were centrifuged at containing 10% heat-inactivated fetal bovine serum and 15 000g for 5 min at 48C, and the supernatants were sepa- antibiotics. rated by SDS–PAGE and transferred to nitrocellulose mem- branes. The membranes were incubated with anti-Flag Cell transfection (Sigma-Aldrich, St. Louis, MO, USA), anti-TAF1 (Santa Kip1 Plasmid DNA was transfected by using FuGENE 6 trans- Cruz Biotechnology, Santa Cruz, CA, USA), anti-p27 fection reagent (Roche, Basel, Switzerland). TAF1, (Santa Cruz Biotechnology), anti-Notch2 (Developmental p27Kip1 and caspase-3 gene-specific siRNAs were pur- Studies Hybridoma Bank, Iowa City, IA, USA), antitubulin chased by Invitrogen, Carlsbad, CA, USA (Stealth (Sigma-Aldrich) or anti-PCNA (Santa Cruz Biotechnology). RNAi). Transfection of siRNAs was performed using Immune complexes were incubated with secondary anti- bodies and visualized by chemiluminescence (PerkinElmer). Lipofectamine RNAi MAX (Invitrogen). Downloaded from Construction of shRNA expression library TUNEL assays A shRNA library was constructed as previously described Cells cultured in poly-D-lysine-coated 4-well chamber (14). The library was generated from human fetal brain slides were transfected with plasmids or siRNAs and cDNAs. DNA fragments in the library theoretically cover then treated with H2O2 for 24 h. Apoptotic cells were http://nar.oxfordjournals.org/ a genome-wide transcriptome. detected by TUNEL assays using a DeadEnd Fluorometric TUNEL System (Promega). To detect apoptotic cells TM Construction of plasmids expressing GFP-TAF1, the FluoroLink Cy5-dUTP TM (GE Healthcare, Buckinghamshire, England) was used TAF1 cDNA was amplified by PCR using the PfuUltra instead of Fluorescein-12-dUTP. High-Fidelity DNA polymerase (Stratagene, La Jolla, CA, USA) from pBlueBacHis250 and cloned into the pEGFP- Microarray analysis C1 vectors. p27Kip1 cDNA was amplified by PCR from 293T cDNA and cloned into pcDNA3-Flag (15,16). Total RNA was isolated from cells using an RNeasy kit (Qiagen). Total RNA (5 mg) was used to start the protocol at University of California, Los Angeles on April 4, 2015 Measurement of cell viability of One-Cycle cDNA Synthesis and to label cRNA, follow- ing the manufacturer’s protocol (Affymetrix, Santa Clara, Cells were cultured in 96-well plates and individually CA, USA). Before making a cocktail solution, 20 mgof transfected with shRNA expression vector. After treat- biotin-labeled cRNA was fragmented to 35–200 bases, and ment with H2O2 or etoposide for 24 h, MTS assays were 15 mg of cRNA fragment was used to prepare the cocktail performed by adding 20 ml CellTiter 96 AQueous One solution. The solution was applied into a GeneChip Human Solution Reagent (Promega, Madison, WI, USA) directly Genome U133 plus 2.0 array (Affymetrix) and hybridized into 80 ml culture media. After incubation for 3 h at 378C for 16 h at 458C. After hybridization, the arrays were in a humidified 5% CO2 atmosphere, the absorbance was washed and stained using Fluidic station 450 according to measured at 490 nm with the use of a multilabel counter protocol EukGE-WS2v5_450 and were then scanned using (PerkinElmer, Waltham, MA, USA). the Affymetrix GeneChip Scanner 3000. Analysis of the data was performed as previously described (20). RT–PCR analysis Total RNA was isolated from cells using RNeasy kit Quantitative real-time RT–PCR analysis (Qiagen, Hilden, Germany). First strand cDNA synthesis Total RNA (5 mg) was reverse transcribed into cDNA using and following PCR were performed with 500 ng of total a Superscript III First-Strand Synthesis System for RT– RNA using a SuperScript III One-Step RT–PCR System PCR (Invitrogen), following the manufacturer’s protocol. with Platinum Taq DNA Polymerase (Invitrogen) accord- For TAF1 gene expression, the nucleotide sequence of ing to the manufacturer’s protocol. For TAF1 gene 0 0 0 5 -CAACACAAACCAGTGACCAGAG-3 was used as expression, the nucleotide sequence of 5 -GGTATGATA 0 0 the sense primer, and 5 -CCAGAATGCCTTAGCTTC TGCTGGGTGTC-3 was used as the sense primer, and 0 0 0 CA-3 was used as the antisense primer. For Notch2 gene 5 -CAAGAGTGGCTGCAAAACCT-3 was used as the expression, the nucleotide sequence of 50-TCCTCTTCT antisense primer. For GAPDH gene expression, the 0 0 GCCTGCCTTTG-3 was used as the sense primer, and nucleotide sequence of 5 -AAGGCTGTGGGCAAGGT 50-TACCTTTCCCTTCCCCACCT-30 was used as the anti- CATCCCT-30 was used as the sense primer, and 50-TTA Kip1 0 sense primer. For p27 gene expression, the nucleotide CTCCTTGGAGGCCATGTGGGC-3 was used as the sequence of 50-TGGCATGTTTTGTGCATTTG-30 was antisense primer. The reaction products were separated used as the sense primer, and 50-TTGGCTCAGTATGC on 2% agarose gels. AACCTTTT-30 was used as the antisense primer. For Actin gene expression, the nucleotide sequence of 50-GTG Immunoblot analysis GCCGAGGACTTTGATTG-30 was used as the sense Immunoblot analysis was performed as described elsewhere primer, and 50-TGGACTTGGGAGAGGACTGG-30 was (17–19). Briefly, cells were suspended with the lysis buffer used as the antisense primer. The PCR reaction was 5252 Nucleic Acids Research, 2008, Vol.
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