Leucine Zipper-Like Domain Is Required for Tumor Suppressor ING2-Mediated Nucleotide Excision Repair and Apoptosis

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FEBS Letters 580 (2006) 3787–3793

Leucine zipper-like domain is required for tumor suppressor ING2-mediated nucleotide excision repair and apoptosis

Yemin Wang1, Jing Wang1, Gang Li* Department of Dermatology and Skin Science, Jack Bell Research Centre, Vancouver Coastal Health Research Institute, University of British Columbia, 2660 Oak Street, Vancouver, BC, Canada V6H 3Z6

Received 8 February 2006; revised 24 May 2006; accepted 24 May 2006

Available online 9 June 2006

Edited by Laszlo Nagy

[5]. Several other motifs have also been predicted in ING pro- Abstract The plant homodomain (PHD) of ING2 was shown to regulate p53-dependent apoptosis through phosphoinositides sig- teins including the potential chromatin regulatory (PCR) do- naling. However, the role of a predicted leucine zipper-like main which may play a critical role in binding to HAT/ (LZL) motif in N-terminus of ING2 is unclear. Here, we show HDAC complexes [6]. However, previous studies on either that LZL motif is critical for the proper functions of ING2 in the founding member ING1b or its homology ING2 has pre- DNA repair, apoptosis and chromatin remodeling after UV irra- dominantly focused on the PHD zinc finger since it has been diation. Deletion of LZL domain also abrogated the association shown to be involved in chromatin remodeling and modulate between ING2 and p53, but not between ING2 and p300, sug- p53 function as a transcription factor and is also conserved gesting that ING2 modulates p53-dependent chromatin remodel- in three yeast and mouse ING homologues [7,8]. So far, the ing, apoptosis and DNA repair by functioning as a scaffold function of the N-terminal region of the ING proteins remains protein to mediate the interaction between p53 and p300. largely unclear. Only the ING1b has been known to bind to the 2006 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. proliferating cell nuclear antigen (PCNA) through a specific N- terminal sequence named the PCNA-interacting protein (PIP) Keywords: ING2; p53; Leucine zipper-like domain; DNA domain [9], which is found in proteins involved in growth inhi- repair; Apoptosis; Histone acetylation bition, cell cycle arrest, DNA replication and repair [10,11]. The ING2 gene encodes a 33-kDa protein, which shares 58.9% homology with ING1b [12]. It has been shown to negatively regulate cell growth in a p53-dependent manner through 1. Introduction induction of G1-phase cell cycle arrest and apoptosis [13] and interacts with phosphoinositides, Ptdlns(3)P and Ptdlns(5)P The ING (inhibitor of growth) family consists of five genes through PHD zinc finger that function in DNA damage-initi- that encode at least seven proteins which have been known ated stress signaling [14]. In addition, we have recently demon- as the regulators of transcription, apoptosis, cell cycle control, strated that ING2, like its homologue ING1b [15–17], DNA repair, senescence and angiogenesis [1,2]. It is also nota- significantly promotes UV-induced apoptosis [18] and en- ble that ING proteins both physically and functionally form hances nucleotide excision repair (NER) in melanoma cells complexes with several factors possessing intrinsic histone ace- in a p53-dependent manner by rapidly inducing histone H4 tyltransferase (HAT) and histone deacetylase complex acetylation, chromatin relaxation, and the recruitment of the (HDAC) activities, thus linking the function of ING tumor damage recognition factor XPA to the DNA photolesions suppressors to chromatin remodeling [1–3]. These proteins [19]. A leucine zipper-like (LZL) motif consisting of leucine share a conserved plant homeodomain (PHD) in the C-termi- residues spanning every seven amino acids has been predicted nus, which is a sequence encoding a specialized form of zinc in the N-terminal of ING2 [20], while no work has been done finger. This PHD zinc finger has been widely discovered in nu- to demonstrate its role in ING2 function yet. To further char- clear DNA-binding proteins with known or suspected roles in acterize the roles of the specific domains of ING2, especially regulating chromatin organization or gene expression [4]. The LZL domain, we constructed a panel of truncated ING2 ING proteins also possess the strong nuclear localization se- mutants to examine the putative role of sub-regions in ING2- quences (NLS). Three intrinsic nucleolar translocation se- mediated histone acetylation, chromatin assembly, DNA re- quences (NTS) are located within the NLS of ING1 and pair and apoptosis. Here we report that the LZL domain, ING2, which has been shown to target ING1 to the nucleolus but not the PHD motif, is critical for the proper functions of ING2 in DNA repair and apoptosis after UV irradiation. *Corresponding author. Fax: +1 604 875 4497. E-mail address: [email protected] (G. Li).

1 These authors contributed equally to this work. 2. Materials and methods

Abbreviations: HAT, histone acetyltransferase; HDAC, histone deace- 2.1. Cell lines and cell culture tylase; LZL, leucine zipper-like; NER, nucleotide excision repair; MMRU melanoma cells were cultured in Dulbecco’s Modiﬁed Eagle PCNA, proliferating cell nuclear antigen; PCR, potential chromatin Medium supplemented with 10% fetal bovine serum (Invitrogen, Miss- regulatory; PHD, plant homeodomain; PIP, PCNA-interacting pro- issauga, Ont., Canada), 100 U/ml penicillin, and 100 lg/ml streptomy- tein; wt, wild-type cin at 37 C in a humidiﬁed atmosphere of 5% CO2.

0014-5793/$32.00 2006 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.febslet.2006.05.065 3788 Y. Wang et al. / FEBS Letters 580 (2006) 3787–3793

2.2. Plasmids construction hypotonic fluorochrome buffer (0.1% Triton X-100, 0.1% sodium cit- The pcDNA3-ING2 plasmid (a gift from Dr. C.C. Harris) [14] was rate, 25 lg/ml RNase A, 50 lg/ml PI). After incubation at 4 C for digested with EcoRI and XbaI (New England Biolabs, Ipswich, MA), 30 min, the samples were then analyzed by a Coulter EPICS XL- and subcloned into p3 · FLAG vector (Sigma, St. Louis, MO) between MCL flow cytometer (Beckman Coulter, Miami, FL) to determine EcoRI and XbaI sites to get the p3 · FLAG-ING2 construct. The trun- the percentage of subdiploid DNA. cated ING2 sequences, DPHD, DLZL and DL+P, were generated with polymerase chain reaction from p3 · FLAG-ING2 using site-specific primers and subcloned into the p3 · FLAG between EcoRI and 2.8. Immunoprecipitation XbaI sites to get p3 · FLAG-DPHD, p3 · FLAG-DLZL and p3 · Cells were transfected with wt or mutant ING2 and harvested 48 h FLAG-DL+P constructs, respectively. The primers were: 50-GGAAT- post transfection. Cell lysates were prepared as described [18,19]. Ly- TCCGGTACCGAGCTCGGATCCA-30 (sense) and 50-GCTCTA- sates (300 lg of proteins) were pre-cleared with 60 ll of protein A GAGCCTACACTTGGTTGCATAAGCA-30 (anti-sense) for DPHD, or G agarose (50% slurry), incubated for 3 h at 4 C with 3 lgof 50-GGAATTCCATTGATGATGTCTACGA-30 (sense) and 50-GCT- monoclonal anti-p53 or p300 and then precipitated with fresh protein CTAGAGCAGAATTCTACTACCTCGA-30 (anti-sense) for DLZL, A or G beads (60 ll of 50% slurry). Mouse IgG (Santa Cruz Biotech- and 50-GGAATTCCGATAAAGCAAAGATGGA-30 (sense) and 50- nology) was used as a negative control. The beads were then pelleted GCTCTAGAGCAGAATTCTACTACCTCGA-30 (anti-sense) for by centrifugation at 10,000 · g for 1 min, washed thrice with PBS DL+P. All the constructs were sequenced across the newly created and boiled in 2 · Tris–glycine SDS sample buffer for Western blot junctions to confirm no reading frame shift induced by polymerase analysis using anti-p300 and anti-FLAG antibodies. chain reaction.

2.3. Host-cell-reactivation assay 3. Results DNA repair was determined with the host-cell-reactivation assay [21,22] The pRL-CMV plasmid (Promega, Madison, WI) was dam- 3.1. The leucine zipper-like domain is required for ING2- aged with UVC at 400 J/m2 and co-transfected with gene of interest using Lipofectamine 2000 (Invitrogen). Forty hours after transfec- mediated repair of UV-damaged DNA in melanoma cells tion, the luciferase activity was measured with the Renilla luciferase as- The novel tumor suppressor ING2 has been shown to regu- say kit (Promega) on a 96-well plate format luminometer (Berthold late p53-mediated cell proliferation, apoptosis and DNA re- Technologies, Bad Wildbad, Germany). To estimate repair efficiency, pair [13,18,19]. To investigate the putative roles of the sub- the results were normalized to values obtained from undamaged plasmids. regions of ING2 in DNA damage responses, several truncated ING2 proteins were generated. As shown in Fig. 1A and B, 2.4. Western blotting ING2 DPHD, DLZL and DL+P mutants encoded 1–220, 63– Western analysis was performed as previously described [18,19]. The 280 and 136–280 amino acids, respectively. antibodies used in this study included anti-ING2, anti-p53, anti-b-ac- Since we have previously demonstrated that ING2 can facil- tin (Santa Cruz Biotechnology, Santa Cruz, CA), anti-acetylated his- itate the removal of bulky photolesions or DNA adducts tone H4, anti-p300 (Upstate, Charlottesville, VA) and anti-FLAG caused by UV light in melanoma cells and knockdown of (Applied Biological Materials Inc., Vancouver, BC, Canada) antibodies. Protein expression was quantified by densitometry using the Quan- ING2 renders cells resistant to DNA repair [13], a host-cell- tity One software (Bio-Rad, Mississauga, Ont., Canada). reactivation assay with luciferase reporter system was performed to investigate the role of different domains in ING2- 2.5. Micrococcal nuclease (MNase) digestion mediated DNA repair by co-transfecting a UV-damaged Cells in 100-mm dishes were harvested by scraping in 12 ml lysis pRL-CMV-luciferase plasmid with vector, wt ING2, DPHD, solution (10 mM Tris/HCl [pH 8], 10 mM MgCl2, 1 mM DTT) DLZL or DL+P into MMRU melanoma cells. The luciferase 30 min after UV irradiation. Then 0.3 ml of 2% NP-40 detergent was activity was used as an indicator of the extent of DNA repair. added to the cells followed by pipetting up and down 15 times to lyse the cells. After centrifugation at 1200 · g for 10 min, the supernatant The ING2 DPHD mutant, similar to the wt ING2, had a 2.8- was discarded and the nuclei pellet was resuspended in 200 ll MNase fold increase in DNA repair compared with control (P < 0.01, buffer (10 mM Tris/HCl [pH 8], 50 mM NaCl, 300 mM sucrose, 3 mM t-test), while the enhancement in DNA repair was abolished in MgCl2). MNase (Sigma) was added to the nuclei samples to digest the DLZL or DL+P-transfected cells (Fig. 1C), indicating that DNA for 5 min at 37 C. The reaction was terminated with the stop LZL domain is an important component for ING2 to exert solution (1% SDS, 20 mM EDTA). After centrifugation at 10,000 · g for 10 min, the aqueous phase was collected for DNA extraction with its role in DNA repair in melanoma cells. phenol:chloroform followed by precipitating with ethanol and analyz- ing on 1% agarose gel. 3.2. Leucine zipper-like domain is essential for ING2-mediated apoptosis induced by UV radiation 2.6. Hoechst staining Gozani et al. reported that PHD–phosphoinositide interac- Cells grown on coverslips in 6-well plates were transfected with wild- type (wt) or mutant ING2 for 24 h and then irradiated with UVB at tions directly regulated nuclear responses to DNA damage 400 J/m2. After another 24 h, cells fixed in fixation buffer (2% formal- and silencing of ING2 or abrogation of PHD domain reduced dehyde, 0.5% Triton X-100 in PBS, pH 7.2) and incubated for 45 min the responses to cell death stimuli [14]. On the other hand, at room temperature. The fixed cells were washed with 0.1% Triton X- ING1b mediated UV-induced apoptosis by binding to PCNA 100/PBS and stained with the 2.5 lg/ml Hoechst 33258 in PBS at room through the N-terminal PIP domain [9]. Recently, we also temperature for 5 min. The cells were then washed with PBS, mounted onto the slides with mounting media (Fisher Scientific, Nepean, Ont., showed that ING2 cooperated with p53 to regulate apoptosis Canada) and visualized under a fluorescent microscope (Zeiss, Chester, via the activation of both the mitochondrial/intrinsic and VA) for apoptotic bodies. Images were taken with a cooled mono 12- death-receptor/extrinsic apoptotic pathways in melanoma cells bit Retiga-Ex camera. [18]. In this study, we confirmed that overexpression of wt ING2 significantly induced UV-mediated apoptosis in MMRU 2.7. Flow cytometry melanoma cells as suggested by increased sub-G1 population Cells in 6-well plates were transfected with wt or mutant ING2 at 50% confluency, and irradiated with UVB at 400 J/m2 24 h after trans- in flow cytometry analysis (Fig. 2A). We also found that fection. After another 24 h, cells were collected by trypsinization, pel- full-length ING2 and its DPHD mutant induced significantly leted by centrifugation at 200 · g for 3 min and resuspended in 1 ml of higher rate of apoptosis (54.6% and 59.8%, respectively) Y. Wang et al. / FEBS Letters 580 (2006) 3787–3793 3789

Fig. 1. Effect of ING2 domains on nucleotide excision repair. (A) Schematic graph for the full-length ING2 and the PHD domain deletion (DPHD), LZL domain deletion (DLZL) and LZL plus PCR domain deletion (DL+P). LZL, leucine-zipper-like; PCR, potential chromatin regulatory; NLS, nuclear localization sequences; PDIM, phosphorylation-dependent interacting motif; PHD, plant homodomain. (B) Protein expression level of different ING2 construct in MMRU cells. MMRU cells were transfected with full-length ING2, DPHD, DLZL and DL+P constructs for 24 h before being harvested for detection of wt and mutant ING2 proteins by Western blot analysis. (C) Role of different regions of ING2 on nucleotide excision repair. MMRU cells were co-transfected with 400 J/m2 UVC-damaged pRL-CMV-luciferase and p3 · FLAG (V), p3 · FLAG-ING2, p3 · FLAG- DPHD, p3 · FLAG-DLZL, or p3 · FLAG-DL+P plasmids. The luciferase activity was determined 40 h after transfection and normalized by the results from undamaged pRL-CMV-luciferase plasmid transfected cells. Data represent means ± S.D. from triplicates.

Fig. 2. LZL domain of ING2 is essential for UV-induced apoptosis. MMRU cells were transfected with vector (V), ING2, or deletion mutants, irradiated with 400 J/m2 UVB, and subjected to propidium iodide staining and ﬂow cytometry analysis (A) or Hoechst staining analysis (B) 24 h after UV irradiation. The sub-G1 population from ﬂow cytometry analysis and the percentage of cells containing apoptotic bodies were quantitated and shown on the right panel of (A) or (B), respectively. Data represent means ± S.D. from triplicates. 3790 Y. Wang et al. / FEBS Letters 580 (2006) 3787–3793

(P < 0.001, t-test) compared with cells transfected with vector 3.3. Leucine zipper-like domain of ING2 plays a critical role in (24.4%), DLZL (27.2%), or DL+P (27.9%) (P < 0.001, t-test) the process of histone H4 hyperacetylation and chromatin after 400 J/m2 UVB irradiation (Fig. 2A). Cells were stained relaxation upon UV irradiation with Hoechst 33258, which binds to DNA, to further confirm Only recently has the role of histone modification in cell sig- that ING2-mediated cell death after UVB irradiation was the naling or facilitating DNA repair begun to be investigated [24]. result of apoptosis, not necrosis. Cells undergoing apoptosis The covalent acetylation or deacetylation of lysine residues are characterized by chromatin condensation, DNA fragmen- occurring within the N-terminal histone tails is believed to alter tation, and formation of apoptotic bodies [23]. Consistently, the chromatin structure and its accessibility to protein com- there were significantly more apoptotic cells in ING2 or DPHD plexes involved in transcription regulation, DNA repair or transfected MMRU cells (50.5% and 49.6%, respectively) after other cellular events [25,26]. p53 has been demonstrated to play UV irradiation compared to vector (18.7%), DLZL (15.5%) or an important role in modulating histone modifications after DL+P transfected cells (17.3%) (P < 0.001, t-test) (Fig. 2B). UV irradiation [27,28]. ING2 has the ability to modulate acet-

Fig. 3. LZL domain ING2 plays a critical role in histone H4 hyperacetylation and chromatin relaxation upon UV irradiation. (A) MMRU cells were transfected with vector (V), ING2, or deletion mutants, irradiated with 200 J/m2 UVC before being harvested for Western blot analysis of acetylated histone H4 (Ac-H4) expression 5 min after UV irradiation. The fold induction of acetylated histone H4 was showed in the right panel. (B) DNA was extracted from MMRU cells transfected with vector (ctrl), ING2 and the deletion constructs with or without UVC irradiation (200 J/m2), and subjected to micrococcal nuclease digestion with diﬀerent concentration of the nuclease (0, 0.1, or 1 U). The percentage of DNA fragments below 2 kb is quantitated with Quantity One software. Data represent means ± S.D. from triplicates. The experiments were repeated three times with similar results. Y. Wang et al. / FEBS Letters 580 (2006) 3787–3793 3791

Fig. 4. LZL domain of is required for ING2-mediated association between p53 and p300. MMRU cells were transfected with vector (V), ING2 or DLZL, irradiated with 400 J/m2 UVC. After 15 min, cells were harvested and protein extracts were used for immunoprecipitation with p53 (A) or p300 (B) antibody and immunoblotted with anti-FLAG, anti-p300, and anti-p53 antibodies. (C), Western blot analysis of 10% input protein extracts for immunoprecipitation. ylation levels through regulating the activity of coactivator and/ decondensation, and NER. We have previously shown that or corepressor complexes [14]. We also found that ING2 is PHD region of ING1b plays an essential role in NER as the involved in histone acetylation and chromatin relaxation to PHD deletion mutant is deficient in repair of UV-damaged mediate the repair of UV-damaged DNA [19]. To explore if DNA [17]. Due to the structural similarity between ING1b deletion of ING2 LZL domain eliminates the role of ING2 in and ING2, we sought to investigate whether PHD of ING2 histone acetylation and chromatin relaxation, we examined is also involved in the DNA repair process. Our data indicated the level of histone H4 acetylation and performed the micrococ- that unlike ING1b, the PHD region of ING2 is dispensable, cal nuclease digestion assay in MMRU cells after transfection while the LZL domain is critical for NER (Fig. 1C). The rea- with mutant ING2 constructs and irradiation with 200 J/m2 son for different domains of ING family members required for UVC. Consistent with the DNA repair experiments, full-length the DNA repair process is unclear. It is possible that distinct ING2 as well as DPHD mutant, enhanced histone H4 acetyla- domain of each ING member is required for the interactions tion by 1.8-fold compared to control cells after UV irradiation, with specific HAT and/or HDAC complexes to induce histone while overexpression of ING2 DLZL or DL+P mutants did not acetylation and chromatin relaxation. affect histone H4 acetylation (Fig. 3A). Moreover, chromatin We also observed that LZL motif, but not PHD domain, is was more sensitive to the micrococcal nuclease (1 U treatment) essential for ING2 to enhance UV-medicated apoptosis in mel- in cells overexpressing wt ING2 or DPHD mutants (86% and anoma MMRU cells (Fig. 2). Gozani et al. previously showed 85% of the DNA fragments <2 kb, respectively) upon UVC that mutation in the PHD region of ING2 abolished ING2-in- irradiation compared to cells transfected with vector (15%), duced apoptosis in human fibrosarcoma HT1080 cells [14]. DLZL (22.5%) or DL+P (23.2%) (Fig. 3B). This discrepancy may be due to cell type-specific responses. We has recently demonstrated that ING2 not only activated 3.4. Leucine zipper-like domain is required for ING2-mediated p53-dependent pathway, but also activated the Fas pathway association between p53 and p300 to induce apoptosis in MMRU cells [18], while ING2 did Our previous data demonstrated that ING2-mediated DNA not mediate Fas ligand-induced apoptosis in HT1080 cells repair and apoptosis after UV irradiation require functional [12]. This cell type-specific response was further addressed by p53 [18,19]. Nagashima et al. and Pedeux et al. reported that knockdown of ING2 which did not render MMRU cells resis- ING2 regulates cell proliferation and replicative senescence tant to apoptosis, while RNAi-treated HT1080 cells were by acetylation-mediated stabilization of p53 [13,29]. In this highly resistant to apoptosis [14]. Moreover, another ING study, we investigated the role of LZL domain of ING2 in family member, ING3, promoted UV-induced apoptosis only p53 stability and the interactions among ING2, p53 and through Fas pathway in MMRU cells [30], despite that p300. We found that deletion of LZL domain is required for ING3 modulates p53-dependent apoptosis in RKO cells [31]. the physical binding between ING2 and p53 and the associa- ING2 has been known to enhance the p300-dependent acet- tion between p53 and p300 (Fig. 4A). However, LZL domain ylation of p53, which stabilizes p53 and plays an important is not required for interaction between ING2 and p300 role in the cellular responses to DNA damage agents [13,29]. (Fig. 4B). Furthermore, we confirmed that overexpression of Our results indicated that LZL deletion mutant abrogated ING2 increased p53 level, while ING2 DLZL mutant failed ING2-mediated association between p53 and p300 (Fig. 4A). to induce p53 (Fig. 4C). In addition, we found that LZL domain is required for the association of ING2 and p53, but not the association of ING2 and p300 (Fig. 4A and B). Leucine zipper domain is 4. Discussion heptad repeats of leucines with an average of six repeats and a minimum of only four heptad repeats to make up a func- We have previously demonstrated that ING2 cannot only tional leucine zipper [32]. Although the N-terminal sequence enhance UV-induced apoptosis, but also increase the repair of ING2 does not stringently match this motif, this leucine zip- of UV damaged DNA by promoting global histone acetylation per-like (LZL) region has been suggested to help target partic- and chromatin relaxation [18,19]. In this study, we reported ular HAT or HADC complexes to chromatin considerably that the LZL motif, instead of the PHD region, plays a critical more dynamic [20]. Therefore, it will be important to clarify role for the interaction between ING2 and certain HAT or how LZL domain of ING2 binds to p53 and targets it for HDAC proteins to regulate histone H4 acetylation, chromatin HAT or HADC complexes (e.g p300, mSin3A), since ING2 3792 Y. Wang et al. / FEBS Letters 580 (2006) 3787–3793 has been found in both HAT and HADC complexes [3].On [9] Scott, M., Bonnefin, P., Vieyra, D., Boisvert, F.M., Young, D., the other hand, the PCR domain is a novel predicted motif lo- Bazett-Jones, D.P. and Riabowol, K. (2001) UV-induced binding cated after LZL domain of ING2. It is also found in other of ING1 to PCNA regulates the induction of apoptosis. J. Cell Sci. 114, 3455–3462. family members and may play a critical role in binding to [10] Warbrick, E. (1998) PCNA binding through a conserved motif. HAT/HDAC complexes since it linked ING1b to the Sin3/ Bioessays 20, 195–199. HDAC complex through direct interaction with SAP30 [22]. [11] Feng, X., Hara, Y. and Riabowol, K. (2002) Different HATS of The results from this study demonstrated no significant differ- the ING1 gene family. Trends Cell Biol. 12, 532–538. [12] Shimada, Y., Saito, A., Suzuki, M., Takahashi, E. and Horie, ence in the biological functions between the cells transfected M. (1998) Cloning of a novel gene (ING1L) homologous to with DLZL and DL+P constructs, suggesting deletion of ING1, a candidate tumor suppressor. Cytogenet. Cell Genet. 83, LZL motif alone is enough to eliminate ING2 function as a 232–235. regulator of apoptosis and DNA repair. However, there is [13] Nagashima, M., Shiseki, M., Miura, K., Hagiwara, K., Linke, no direct evidence showing that the putative PCR domain is S.P., Pedeux, R., Wang, X.W., Yokota, J., Riabowol, K. and Harris, C.C. (2001) DNA damage-inducible gene p33ING2 not required for ING2 function. One possibility is that PCR negatively regulates cell proliferation through acetylation of region may mediate the direct interaction between ING2 and p53. Proc. Natl. Acad. Sci. USA 98, 9671–9676. p300 or other HAT/HDAC proteins. Further experiments [14] Gozani, O., Karuman, P., Jones, D.R., Ivanov, D., Cha, J., are required to test this hypothesis. Lugovskoy, A.A., Baird, C.L., Zhu, H., Field, S.J., Lessnick, S.L., Villasenor, J., Mehrotra, B., Chen, J., Rao, V.R., Brugge, J.S., Taken together, ING2 may possibly function as a scaffold Ferguson, C.G., Payrastre, B., Myszka, D.G., Cantley, L.C., protein that mediates the interaction between p53 and p300, Wagner, G., Divecha, N., Prestwich, G.D. and Yuan, J. (2003) thereby inducing the acetylation level of p53 and p53-depen- The PHD finger of the chromatin-associated protein ING2 dent chromatin remodeling, apoptosis and DNA repair pro- functions as a nuclear phosphoinositide receptor. Cell 114, 99– cess. 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