A novel PHD-finger motif protein, p47ING3, modulates p53-mediated ...

Oncogene (2003) 22, 343–350

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A novel PHD-finger motif protein, p47ING3, modulates p53-mediated transcription, cell cycle control, and apoptosis Makoto Nagashima1,3, Masayuki Shiseki1,3, Remy M Pedeux1, Shu Okamura1, Mariko Kitahama-Shiseki1, Koh Miura1, Jun Yokota2 and Curtis C Harris*,1 1 Laboratory of Human Carcinogenesis, National Cancer Institute, NIH, Bethesda, MD 20892-4255, USA; 2Biology Division, National Cancer Center Research Institute, 1-1, Tsukiji 5-chome, Chuo-Ku, Tokyo 104, Japan

A candidate tumor suppressor gene, p33ING1, was previously identified by using the genetic suppressor element methodology. p33ING1 cooperates with p53 and plays a significant role in p53-mediated cellular processes. Recently, we have identified p33ING2, which shows a sequence homology similar to p33ING1 and modulates p53 function. In the present study, we identified and characterized another ‘ING family’ gene. The estimated molecular weight of the encoded protein is 46.8 kDa, thus, we named it p47ING3. The p47ING3 gene is located at chromosome 7q31.3 and consists of 12 exons that encode 418 amino acids. A computational domain search revealed a C-terminal PHD-finger motif. Such motifs are common in proteins involved in chromatin remodeling. p47ING3 is highly expressed in some normal human tissues or organs, including the spleen, testis, skeletal muscle, and heart. p47ING3 expression levels varied among cancer cell lines. p47ING3 overexpression resulted in a decreased population of cells in S phase, a diminished colony-forming efficiency, and induced apoptosis in RKO cells, but not in RKO-E6 cells with inactivated p53. p47ING3 activates p53-transactivated promoters, including promoters of p21/waf1 and bax. Thus, we have isolated a novel ING family gene, p47ING3, which modulates p53-mediated transcription, cell cycle control, and apoptosis. Oncogene (2003) 22, 343–350. doi:10.1038/sj.onc.1206115 Keywords: PHD-finger motif; ING1; ING3; p53 Introduction A candidate tumor suppressor gene, p33ING1, was originally cloned by the genetic suppressor element methodology (Garkavtsev et al., 1996). The p33ING1 gene is located at chromosome 13q33-34 and encodes a nuclear protein (Garkavtsev and Riabowol, 1997). Two additional alternative splicing variants of p33ING1 were

*Correspondence: CC Harris, Laboratory of Human Carcinogenesis, National Cancer Institute, NIH, 37 Convent Dr., Bldg 37 Rm 2C05, Bethesda, MD 20892-4255, USA; E-mail: [email protected] 3 These two authors contributed equally to this work. Received 29 April 2002; revised 1 October 2002; accepted 4 October 2002

found; thus, the original p33ING1 was renamed as p33ING1b, and the other two variants were named p47ING1a and p24ING1c (Garkavtsev et al., 1999; Saito et al., 2000). p33ING1 biological properties indicated that it is a negative growth regulator, and plays a role in senescence and apoptosis (Helbing et al., 1997; Garkavtsev and Riabowol, 1997). Two years after the initial publication, it was reported that p33ING1 cooperates with p53 and negatively controls cell growth (Garkavtsev et al., 1998). p33ING1 physically interacts with p53 and modulates p53-dependent transcriptional activation. The p53 gene is frequently inactivated in many types of human cancers (Hollstein et al., 1991, 1999; Greenblatt et al., 1994) and p53-mediated cellular processes play key roles in human carcinogenesis (Vogelstein et al., 2000). p33ING1 involvement in modulating p53 function suggests that it plays a significant role in human carcinogenesis. The precise mechanism by which p33ING1 modulates the p53-dependent pathway is not understood fully. Because p33ING1 has a PHD-finger motif in its C-terminal region, p33ING1 may be involved in chromatin remodeling and may modulate p53 function as a transcription factor. Three yeast proteins, Yngl, Yng2, and pho23, which have PHD-finger motifs at their C-terminal regions and have homology with human p33ING1, are associated with a histone acetyl transferase (HAT) complex (Loewith et al., 2000). Furthermore, p33ING1b associates with the mSin3/HDAC1 transcriptional represser complex, but p24ING1c does not (Skowyra et al., 2001). The N-terminus of p33ING1b is directly associated with SAP30, a component of HDAC/mSin3A complexes (Kuzmichev et al., 2002). These results suggest that ING proteins are involved in histone acetylation in chromatin remodeling. Recently, we identified the DNA damage-induced p33ING2 gene that shows high homology to p33ING1 (Nagashima et al., 2001). p33ING2 also controls cell growth negatively in a p53-dependent manner. In addition, we found that p33ING2 may modulate p53 function through p53 acetylation, and may play a role in DNA doublestrand break repair. In the present study, we identified and characterized a novel ‘ING family gene’ p47ING3, which shows high homology with p33ING1b and p33ING2.

A novel PHD-finger protein, p47ING3 M Nagashima et al

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Results Sequences of cDNA and a predicted amino acid, genomic structure, and chromosome localization of the p47ING3 gene Human EST clones were found to show high homology with the p33ING1b and p33ING2 cDNAs by a BLAST computational searching. Then, the entire cDNA sequence was determined by an additional computational search and rapid amplification cDNA end (RACE) method. The cDNA consists of 1807 nucleotides and encodes 418 amino acids whose estimated molecular weight is 46.8 kDa, thus, we named this novel gene as the p47ING3 gene (accession number AF074968). Figure 1 shows amino-acid sequences of p33ING1b and p33ING2, the p47ING3 protein. Similar to p33ING1b and p33ING2, the p47ING3 protein has a PHD-finger motif in its C-terminal region. The p47ING3 sequence was found in human PAC genomic clone DJ0872F07 (accession number AC004537). This human

PAC clone was indicated to be located at chromosome 7q31.3 by ‘human chromosome 7 mapping and sequence, National Human Genome Research Institute’. The cDNA sequence of p47ING3 was compared with the genomic sequence of the human PAC clone, DJ0872F07, and the exon/intron boundaries were assigned. The p47ING3 gene consists of 12 exons and the protein coding region spans exons 1–12 (nucleotides 112–1368) (Figure 2). p47ING3 expression in normal human organs or tissues p47ING3 expression in various normal human organs or tissues was examined by Northern blot analysis. Fulllength p47ING3 cDNA was used as a probe for the detection of p47ING3 transcripts. In addition to a major band (2.4 kb) that is most likely p47ING3 mRNA because p47ING3 cDNA consists of 1807 bp, two bands (4.4, 7.2 kb) were seen. These bands may be nonspecific bands or alternative-splicing p47ING3 transcripts

Figure 1 Comparison of amino-acid sequences of p33ING1b, p33ING2, and p47ING3 proteins. Asterisks indicate a putative PHD-finger motif (Cys4-His-Cys3) Oncogene


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Figure 2 Genomic structure of the p47ING3 gene. The p47ING3 gene consists of 12 exons. The amino-acid coding region spans exons 1–12 (nucleotides 112–1368)

Figure 3 p47ING3 expression in normal human tissues or organs. Northern blot analysis for p47ING3 expression in normal human tissues or organs by using MTN1 and two membranes (Clontech). The membranes were hybridized with p47ING3 cDNA, and then with b-actin cDNA

(Figure 3). In spleen, testis, skeletal muscle, and heart, the 2.4-kb transcript is highly expressed, and less expressed in lung and brain. p47ING3 negatively controls cell growth in a p53-dependent manner We investigated the effect of exogenous p47ING3 overexpression on cell growth and its p53 dependency using RKO cells and its isogenic subclone, RKO-E6 cells, in which p53 is inactivated with human papilloma viral oncoprotein E6 (Kessis et al., 1993). The effect of p47ING3 overexpression on the colony formation efficiency of RKO or RKO-E6 cells was examined.

The RKO cells transfected with pcDNA3.1-ING3 vector formed a smaller number of colonies than those transfected with pcDNA3.1 (control) after a 2-week selection by hygromycin (Figure 4a). The effect of p47ING3 overexpression on the cell cycle profiles was then examined. Overexpression of p47ING3 in RKO cells resulted in a decreased proportion of cells in S phase and increased those in G0/G1 phase, compared with RKO cells transfected with the control (Figure 4b). p47ING3 overexpression in RKO-E6 cells also showed a decreased proportion of cells in S phase, but the effect was not as marked compared with that observed in RKO cells. We also investigated whether p47ING3 overexpression induces apoptosis in a p53-dependent Oncogene


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Figure 4 Negative regulation of cell proliferation by p47ING3. (a) Colony formation efficiency assay. RKO or RKO-E6 cells (2 104 cells/cm2) were seeded on 60 mm dishes, and transfected with pcDNA3.1-ING3 or pcDNA3.1 (control) vectors, which have a hygromycin-resistant gene. After 2 weeks of hygromycin selection (200 mg/ml), cells were fixed and stained by crystal violet for colony counting. Data represent the average of the triplicate experiments with standard deviation. Statistical analysis was carried out by Student’s t-test. (b) Effect of the overexpression of p47ING3 on cell cycle profiles. RKO or RKO-E6 cells were transfected with pcDNA3.1-p47ING3 or pcDNA3.1 (control) by the Lipofectamine Plus reagent (Invitrogen). Cells were cotransfected with the pEGFP-F vector (Clontech) as a marker for transfection. At 48 h after the transfection, cells were fixed and stained with propidium iodide and the DNA content of the cells was measured by a flow cytometer. Cell cycle profiles were analysed using the MODIFIT LT Program (Verify Software House, Topsham, ME, USA). At least 10 000 of the GFP-positive cells were analysed, (c) Apoptosis induction by p47ING3. RKO or RKO-E6 cells were plated into eight-well chamber slides, and transfected with the pcDNA3.1–ING3 or pcDNA3.1 (control) vectors. Cells were cotransfected with pEGFP-F vector as a marker for transfection. Cells were fixed at 24 h after transfection and fragmented DNA was detected by TUNEL assay. The slides were observed by fluorescent microscopy, and 100 GFP positive cells were analysed for apoptosis. Data represent the average of triplicate experiments with standard deviation Oncogene


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Figure 5 Sequence-specific transcriptional activity of p53. Promoter activities of p21/waf1 and bax genes were detected after cotransfection of p53-responsive promoter-luciferase constructs with pcDNA3.1 (control) or pcDNA3.1-ING3 vectors in RKO or RKO-E6 cells. The luciferase activities were measured with a luminometer, and normalized by the protein concentration. Each datum is shown as a relative value to the control. Results represent the means 7s.d. of six independent experiments

manner. A significant number of RKO cells went through apoptosis after p47ING3 overexpression compared with the control cells transfected with the control vector (Figure 4c). The number of apoptotic cells did not increase after p47ING3 overexpression in RKO-E6 cells. These results indicate that p47ING3 suppresses the cell growth through cell cycle control and the induction of apoptosis in a p53-dependent manner. p47ING3 activates p53-responsive promoters To test if p47ING3 modulates the transactivational activity of p53, the promoter activities of two p53regulated genes, p21/waf1 and bax, were examined after overexpression of the p47ING3 gene. The activities of the p21/waf1 and bax promoters were enhanced by the overexpression of p47ING3 in RKO cells, but not in RKO-E6 cells (Figure 5). p47ING3 does not affect post-translational modifications of p53 To determine the mechanism of p47ING3 modulation of p53 function, we investigated whether p47ING3 is involved in p53 post-translational modification. Our previous results indicated that p33ING2 overexpression increases p53 acetylation at Lys-382 residues, suggesting that p33ING2 negatively controls cell growth through the modulation of acetylation status of p53 lysine residues (Nagashima et al., 2001). The effect of the exogenous overexpression of p47ING3 on the p53 posttranslational modification was examined. Acetylation

Figure 6 Post-translational modifications of p53 by p47ING3 overexpression. RKO cells were transfected with pcDNA3.1-ING2, pcDNA3.1-ING3, or pcDNA3.1 (control). Acetylation of p53 at Lys-382 was detected in anti-p53 (Ab-6 and Pab240) immunoprecipitates from RKO cells. p53 phosphorylation status at Ser-15, Ser-46, or Ser-392 was investigated in RKO cells by Western blot analysis. RKO cells treated with etoposide (20 mg/ml) for 8 h were used as a positive control for acetylated and phophorylated p53

status of p53 at Lys-382 was increased by the overexpression of p33ING2, but not in RKO cells transfected with p47ING3. The p53 phosphorylation levels at the Ser-15, Ser-46, or Ser-392 residues were also not changed significantly by p47ING3 overexpression (Figure 6).

The p47ING3 expression level is not altered by various genotoxic stresses The amount of p33ING2 protein is increased by some genotoxic stresses including etoposide and neocarzinostatin (Nagashima et al., 2001). We investigated the effect of the genotoxic stresses, including ionizing irradiation, cis-platinum, etoposide, bleomycin, adriamycin, and neocarzinostatin, on the p47ING3 expression level in C3ABR normal human lymphocytes. No significant Oncogene


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change in the p47ING3 expression level was observed (data not shown). Discussion We identified a novel ING family gene, p47ING3. p47ING3 suppresses the colony formation efficiency and induces apoptosis in RKO cells by a p53-dependent pathway. p47ING3 also activates p53-responsive promoters of the p21/waf1 and bax genes, indicating that p47ING3 modulates p53 function as a transactivator, and controls the expression of p53-responsive genes. Although p33ING1b physically interacts with p53 (Garkavtsev et al., 1998; Nagashima et al., 2001) and may modulate p53 function by direct interaction, we did not observe such an interaction between p47ING3 and p53 in vivo by coimmunoprecipitation experiments using either anti-p47ING3 or anti-p53 antibodies, followed by Western blot analysis (data not shown). p47ING3 has a PHD-finger motif at its C-terminal region similar to p33ING1 and p33ING2. Although the precise function of the PHD-finger motif is not fully clarified, it is found in proteins involved in chromatin remodeling. Recently, three yeast PHD-finger motif proteins, yngl, yng2, and pho23, were found to have a homology with human p33ING1 and to be located in HAT complexes (Nourani et al., 2001). Another report showed that pho23 forms a complex with Rpd3, which is a yeast histone deacetylase (HDAC) showing homology to mammalian class I histone deacetylase (Loewith et al., 2001). These results suggest that ING proteins may function by a similar mechanism. Recently, it was reported that p33ING1b forms a complex with HDAC1, but p47ING1a does not (Skowyra et al., 2001). HATs, including CBP/p300 and P/CAF, acetylate and HDACs, including Sir2, deacetylate p53 lysine residues and modulate p53 function (Gu and Roeder, 1997; Sakaguchi et al., 1998; Liu et al., 1999; Luo et al., 2001). It is still unclear how p53 acetylation is involved in the modulation of p53 function. One possibility is that p53 acetylation increases p53 sequence-specific DNA binding, resulting in the activation of p53 responsive promoters. p53 acetylation increases p53’s binding ability to oligonucleotides containing the p53 binding consensus sequence. However, in vivo chromatin immunoprecipitation analyses failed to show that p53 acetylation changes p53 sequence-specific p53 DNA binding (Barlev et al., 2001; Kaeser and Iggo, 2002). It seems likely that p53 acetylation modulates p53 function through other mechanisms, including the recruitment of transcriptional coactivators, and the alteration of p53 subcellular localization (Prives and Manley, 2001). ING proteins may bind to either HATs or HDACs, which are involved in the acetylation of p53 lysine residues, and may modulate p53 function through acetylation. In the previous study, we demonstrated that the overexpression of p33ING2 results in increased acetylation at the Lys-382 residue in p53 proteins (Nagashima et al., 2001), suggesting that p33ING2 may enhance HAT activity or inhibit HDAC activity. Therefore, HAT or Oncogene

HDAC complexes containing ING family proteins, including p47ING3, may modulate p53 function through chromatin accessibility of genes transcriptionally regulated by p53 and/or through the alteration of acetylation status of lysine residues in p53 protein. Further investigations of the interactive effects of ING family proteins on chromatin remodeling and p53 post-translational modifications may reveal the multiple mechanisms controlling p53 function.

Materials and methods cDNA cloning A computational search was performed to find human EST clones showing high homology with p33ING1b and p33ING2 cDNAs by using a BLAST program (National Center for Biotechnology Information). To determine the entire cDNA sequence, the RACE method was carried out with the human placenta Marathon-Ready cDNA (Clontech, Palo Alto, CA, USA). Determination of genomic structures and chromosome localization Human genomic clones containing p47ING3 cDNA were searched by a BLAST program to determine the genomic structures of the p47ING3 gene. Exon–intron boundary sequences were determined by alignment of the p47ING3 cDNA sequence on the genomic sequence. Cell lines, expression vectors, and antibody A human colorectal cancer cell line, RKO, and its isogenic subclone, RKO-E6, with inactivated p53, were used for the colony formation efficiency assay, cell cycle analysis, apoptotic assay, and analysis for p53 post-translational modification. The coding region of p47ING3 cDNA was generated from human placenta Marathon-Ready cDNA by PCR. After cutting by restriction enzyme, cDNA was cloned into pcDNA3.1 (+)-Neo or pcDNA3.1 (+)-Hgr expression vectors (Invitrogen, Carlsbat, CA, USA), producing the pcDNA3.1-ING3 vector. The pcDNA3.1-ING2 expression vector we constructed previously (Nagashima et al., 2001) was also used in some experiments. Rabbit polyclonal antibody against p47ING3 was generated by the injection of chemically synthesized Keyhole limpet hemocyanin-conjugated oligopeptides, whose sequence corresponded to amino acids 141–157 in the p47ING3 protein. The antisera from immunized rabbits were purified with the oligopeptides coupled with SulfoLink (Pierce, Rockford, IL, USA). Northern blot analysis Human MTN-1 and -2 membranes (Clontech) were used for Northern blot analysis to examine the expression of p47ING3 in normal human tissues. p47ING3 cDNA was generated by PCR from human placenta Marathon-Ready cDNA and labeled with a-32p-dCTP by using the PrimeIT-RmT labeling kit (Stratagene, La Jolla, CA, USA) and then subjected to hybridization as the probe. Hybridization was performed in Hybrizol I solution (Oncor, Gaithersburg, MD, USA) at 551C for 16 h. The membranes were washed under stringent conditions, and then exposed to X-ray films. After stripping the probe, the membranes were subjected to hybridization with b-actin probe to compare the amounts of RNA in each lane.


349 Colony formation efficiency assay

Activity of p53-responsive promoters

Cells were plated on 60 mm dishes at 2 104 cells/cm2 and cultured for 24 h at 371C. Cells were transfected with 2 mg of pcDNA3.1-ING3 or pcDNA3.1 (control) vectors with the Lipofectamine Plus reagent (Invitrogen). Cells were cultured in selection media containing hygromycin (Sigma, St Louis. MO, USA) at a concentration of 200 mg/ml. After 2-week selection, cells were fixed on the dish, stained with crystal violet, and then the colonies were counted.

RKO cells were plated into six-well plates at 2 104 cells/cm2 and cultured for 24 h at 371C. Cells were transfected with 500 ng of pcDNA3.1-ING3 or pcDNA3.1 (empty vector) with the Lipofectamine Plus reagent. At 24 h after the transfection, the activity of p53 as a transactivator was assessed by cotransfection with two vectors, WWP-Luc-p21 (El-Deiry et al., 1992) or PGL-Luc-BAX (Miyashita and Reed, 1995), which contain p53-responsive element-reporter gene constructs.

Cell cycle analysis RKO or RKO-E6 cells were plated on 100 mm dishes at 2 104 cells/cm2 at 24 h prior to the transfection. RKO or RKO-E6 cells were transfected with 4 mg of pcDNA3.1p47ING3 or pcDNA3.1 (control) by Lipofectamine Plus reagent (Invitrogen). Cells were cotransfected with 400 ng of pEGFP-F vector (Clontech) as a marker for transfection. At 48 h after transfection, cells were harvested, washed with PBS, and then fixed with 70% ethanol for more than 3 h. After ethanol was removed by centrifugation, pellets were resuspended with PI-RNase solution, containing 20 mg/ml of propidium iodide (Sigma) and 100 mg/ml of RNase A (Sigma), and incubated for 30 min at room temperature. The DNA content of the cells was measured by the FACSCalibur flow cytometer (Beckton Dickinson, Flanklin Lakes, NJ, USA). Cell cycle profiles were analysed using the MODIFIT LT Program (Verify Software House, Topsham, ME, USA). At least 10 000 of the GFP-positive cells, which are considered to be transfection-positive cells, were analysed for cell cycle. Detection of apoptotic cells RKO or RKO-E6 cells were plated into eight-well chamber slides at 1 104 cells/cm2 and cultured for 24 h. Cells were transfected with 50 ng of pcDNA3.1-ING3 or pcDNA3.l (control) vectors with the Lipofectamine Plus reagent. Cells were cotransfected with 5 ng of the pEGFP-F vector as a marker for transfection efficiency. Cells were fixed at 24 h after transfection and fragmented DNA was detected by the TUNEL assay kit (Oncogene Research Products, Cambridge, MA, USA) according to the manufacturer’s instructions. Positive cells were counted by fluorescent microscopy.

Detection of post-translational modifications of p53 RKO cells were seeded on a 100 mm dish at 2 104/cm2 and cultured for 24 h. The cells were transfected with pcDNA3.1-ING2, pcDNA3.1-ING3, or pcDNA3.1 (control) vectors. The pcDNA3.1-ING2 vector was used in our previous study (Nagashima et al., 2001). Cells were harvested at 24 h after transfection. In the experiments, to check p53 acetylation, trichostatin A treatment was carried out for 3 h before cell harvest. To detect acetylated p53, p53 was immunoprecipitated by agarose-conjugated anti-p53 antibodies, Ab-6 (Calbiochem, San Diego, CA, USA) and Pab 240 (Santa Cruz Biotechnology). Samples were subjected to Western blot analysis to detect the acetylated p53 at the Lys-382 residue using antiacetylated p53 antibody (Calbiochem). For the detection of phosphorylated p53 at Ser-15 or Ser-392 residues using antiphospho-p53specific antibodies (Cell Signaling Technology, Beverly, MA, USA) whole-cell lysates were used for the Western blot analysis. For the detection of phosphorylated p53 at Ser46, a specific antibody, which was provided by Dr E Appella, was used.

Acknowledgements We thank Drs B Vogelstein and W El-Deiry for providing the WWW-Luc-p21 and PGL3-Luc-Bax vectors, Dr E Appella for providing a specific antibody for phosphorylated p53 at Ser-46 and advice on preparing the anti-p47ING3 antibody, and Ms D Dudek for editorial and graphic assistance.

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