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Leucine zipper-like domain is required for tumor suppressor ING2-mediated nucleotide excision repair and apoptosis
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
Abstract The plant homodomain (PHD) of ING2 was shown to regulate p53-dependent apoptosis through phosphoinositides signaling. However, the role of a predicted leucine zipper-like (LZL) motif in N-terminus of ING2 is unclear. Here, we show that LZL motif is critical for the proper functions of ING2 in DNA repair, apoptosis and chromatin remodeling after UV irradiation. Deletion of LZL domain also abrogated the association between ING2 and p53, but not between ING2 and p300, suggesting that ING2 modulates p53-dependent chromatin remodeling, apoptosis and DNA repair by functioning as a scaffold protein to mediate the interaction between p53 and p300. 2006 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. Keywords: ING2; p53; Leucine zipper-like domain; DNA repair; Apoptosis; Histone acetylation
1. Introduction The ING (inhibitor of growth) family consists of five genes that encode at least seven proteins which have been known as the regulators of transcription, apoptosis, cell cycle control, DNA repair, senescence and angiogenesis [1,2]. It is also notable that ING proteins both physically and functionally form complexes with several factors possessing intrinsic histone acetyltransferase (HAT) and histone deacetylase complex (HDAC) activities, thus linking the function of ING tumor suppressors to chromatin remodeling [1–3]. These proteins share a conserved plant homeodomain (PHD) in the C-terminus, which is a sequence encoding a specialized form of zinc finger. This PHD zinc finger has been widely discovered in nuclear DNA-binding proteins with known or suspected roles in regulating chromatin organization or gene expression [4]. The ING proteins also possess the strong nuclear localization sequences (NLS). Three intrinsic nucleolar translocation sequences (NTS) are located within the NLS of ING1 and ING2, which has been shown to target ING1 to the nucleolus
[5]. Several other motifs have also been predicted in ING proteins including the potential chromatin regulatory (PCR) domain which may play a critical role in binding to HAT/ HDAC complexes [6]. However, previous studies on either the founding member ING1b or its homology ING2 has predominantly focused on the PHD zinc finger since it has been shown to be involved in chromatin remodeling and modulate p53 function as a transcription factor and is also conserved in three yeast and mouse ING homologues [7,8]. So far, the function of the N-terminal region of the ING proteins remains largely unclear. Only the ING1b has been known to bind to the proliferating cell nuclear antigen (PCNA) through a specific Nterminal sequence named the PCNA-interacting protein (PIP) domain [9], which is found in proteins involved in growth inhibition, 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 induction of G1-phase cell cycle arrest and apoptosis [13] and interacts with phosphoinositides, Ptdlns(3)P and Ptdlns(5)P through PHD zinc finger that function in DNA damage-initiated stress signaling [14]. In addition, we have recently demonstrated that ING2, like its homologue ING1b [15–17], significantly promotes UV-induced apoptosis [18] and enhances nucleotide excision repair (NER) in melanoma cells in a p53-dependent manner by rapidly inducing histone H4 acetylation, chromatin relaxation, and the recruitment of the damage recognition factor XPA to the DNA photolesions [19]. A leucine zipper-like (LZL) motif consisting of leucine residues spanning every seven amino acids has been predicted in the N-terminal of ING2 [20], while no work has been done to demonstrate its role in ING2 function yet. To further characterize the roles of the specific domains of ING2, especially LZL domain, we constructed a panel of truncated ING2 mutants to examine the putative role of sub-regions in ING2mediated histone acetylation, chromatin assembly, DNA repair and apoptosis. Here we report that the LZL domain, 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.
Abbreviations: HAT, histone acetyltransferase; HDAC, histone deacetylase; LZL, leucine zipper-like; NER, nucleotide excision repair; PCNA, proliferating cell nuclear antigen; PCR, potential chromatin regulatory; PHD, plant homeodomain; PIP, PCNA-interacting protein; wt, wild-type
2. Materials and methods 2.1. Cell lines and cell culture MMRU melanoma cells were cultured in Dulbecco’s Modified Eagle Medium supplemented with 10% fetal bovine serum (Invitrogen, Mississauga, Ont., Canada), 100 U/ml penicillin, and 100 lg/ml streptomycin 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 2.2. Plasmids construction The pcDNA3-ING2 plasmid (a gift from Dr. C.C. Harris) [14] was digested with EcoRI and XbaI (New England Biolabs, Ipswich, MA), and subcloned into p3 · FLAG vector (Sigma, St. Louis, MO) between EcoRI and XbaI sites to get the p3 · FLAG-ING2 construct. The truncated 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 XbaI sites to get p3 · FLAG-DPHD, p3 · FLAG-DLZL and p3 · FLAG-DL+P constructs, respectively. The primers were: 5 0 -GGAATTCCGGTACCGAGCTCGGATCCA-3 0 (sense) and 5 0 -GCTCTAGAGCCTACACTTGGTTGCATAAGCA-3 0 (anti-sense) for DPHD, 5 0 -GGAATTCCATTGATGATGTCTACGA-3 0 (sense) and 5 0 -GCTCTAGAGCAGAATTCTACTACCTCGA-3 0 (anti-sense) for DLZL, and 5 0 -GGAATTCCGATAAAGCAAAGATGGA-3 0 (sense) and 5 0 GCTCTAGAGCAGAATTCTACTACCTCGA-3 0 (anti-sense) for DL+P. All the constructs were sequenced across the newly created junctions to confirm no reading frame shift induced by polymerase chain reaction. 2.3. Host-cell-reactivation assay DNA repair was determined with the host-cell-reactivation assay [21,22] The pRL-CMV plasmid (Promega, Madison, WI) was damaged with UVC at 400 J/m2 and co-transfected with gene of interest using Lipofectamine 2000 (Invitrogen). Forty hours after transfection, the luciferase activity was measured with the Renilla luciferase assay kit (Promega) on a 96-well plate format luminometer (Berthold Technologies, Bad Wildbad, Germany). To estimate repair efficiency, the results were normalized to values obtained from undamaged plasmids. 2.4. Western blotting Western analysis was performed as previously described [18,19]. The antibodies used in this study included anti-ING2, anti-p53, anti-b-actin (Santa Cruz Biotechnology, Santa Cruz, CA), anti-acetylated histone H4, anti-p300 (Upstate, Charlottesville, VA) and anti-FLAG (Applied Biological Materials Inc., Vancouver, BC, Canada) antibodies. Protein expression was quantified by densitometry using the Quantity One software (Bio-Rad, Mississauga, Ont., Canada). 2.5. Micrococcal nuclease (MNase) digestion Cells in 100-mm dishes were harvested by scraping in 12 ml lysis solution (10 mM Tris/HCl [pH 8], 10 mM MgCl2, 1 mM DTT) 30 min after UV irradiation. Then 0.3 ml of 2% NP-40 detergent was 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 was discarded and the nuclei pellet was resuspended in 200 ll MNase buffer (10 mM Tris/HCl [pH 8], 50 mM NaCl, 300 mM sucrose, 3 mM MgCl2). MNase (Sigma) was added to the nuclei samples to digest the DNA for 5 min at 37 C. The reaction was terminated with the stop solution (1% SDS, 20 mM EDTA). After centrifugation at 10,000 · g for 10 min, the aqueous phase was collected for DNA extraction with phenol:chloroform followed by precipitating with ethanol and analyzing on 1% agarose gel. 2.6. Hoechst staining Cells grown on coverslips in 6-well plates were transfected with wildtype (wt) or mutant ING2 for 24 h and then irradiated with UVB at 400 J/m2. After another 24 h, cells fixed in fixation buffer (2% formaldehyde, 0.5% Triton X-100 in PBS, pH 7.2) and incubated for 45 min at room temperature. The fixed cells were washed with 0.1% Triton X100/PBS and stained with the 2.5 lg/ml Hoechst 33258 in PBS at room temperature for 5 min. The cells were then washed with PBS, mounted onto the slides with mounting media (Fisher Scientific, Nepean, Ont., Canada) and visualized under a fluorescent microscope (Zeiss, Chester, VA) for apoptotic bodies. Images were taken with a cooled mono 12bit Retiga-Ex camera. 2.7. Flow cytometry 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 transfection. After another 24 h, cells were collected by trypsinization, pelleted by centrifugation at 200 · g for 3 min and resuspended in 1 ml of
Y. Wang et al. / FEBS Letters 580 (2006) 3787–3793 hypotonic fluorochrome buffer (0.1% Triton X-100, 0.1% sodium citrate, 25 lg/ml RNase A, 50 lg/ml PI). After incubation at 4 C for 30 min, the samples were then analyzed by a Coulter EPICS XLMCL flow cytometer (Beckman Coulter, Miami, FL) to determine the percentage of subdiploid DNA. 2.8. Immunoprecipitation Cells were transfected with wt or mutant ING2 and harvested 48 h post transfection. Cell lysates were prepared as described [18,19]. Lysates (300 lg of proteins) were pre-cleared with 60 ll of protein A or G agarose (50% slurry), incubated for 3 h at 4 C with 3 lg of monoclonal anti-p53 or p300 and then precipitated with fresh protein A or G beads (60 ll of 50% slurry). Mouse IgG (Santa Cruz Biotechnology) was used as a negative control. The beads were then pelleted by centrifugation at 10,000 · g for 1 min, washed thrice with PBS and boiled in 2 · Tris–glycine SDS sample buffer for Western blot analysis using anti-p300 and anti-FLAG antibodies.
3. Results 3.1. The leucine zipper-like domain is required for ING2mediated repair of UV-damaged DNA in melanoma cells The novel tumor suppressor ING2 has been shown to regulate p53-mediated cell proliferation, apoptosis and DNA repair [13,18,19]. To investigate the putative roles of the subregions of ING2 in DNA damage responses, several truncated ING2 proteins were generated. As shown in Fig. 1A and B, ING2 DPHD, DLZL and DL+P mutants encoded 1–220, 63– 280 and 136–280 amino acids, respectively. Since we have previously demonstrated that ING2 can facilitate the removal of bulky photolesions or DNA adducts caused by UV light in melanoma cells and knockdown of ING2 renders cells resistant to DNA repair [13], a host-cellreactivation assay with luciferase reporter system was performed to investigate the role of different domains in ING2mediated DNA repair by co-transfecting a UV-damaged pRL-CMV-luciferase plasmid with vector, wt ING2, DPHD, DLZL or DL+P into MMRU melanoma cells. The luciferase activity was used as an indicator of the extent of DNA repair. The ING2 DPHD mutant, similar to the wt ING2, had a 2.8fold increase in DNA repair compared with control (P < 0.01, t-test), while the enhancement in DNA repair was abolished in DLZL or DL+P-transfected cells (Fig. 1C), indicating that LZL domain is an important component for ING2 to exert its role in DNA repair in melanoma cells. 3.2. Leucine zipper-like domain is essential for ING2-mediated apoptosis induced by UV radiation Gozani et al. reported that PHD–phosphoinositide interactions directly regulated nuclear responses to DNA damage and silencing of ING2 or abrogation of PHD domain reduced the responses to cell death stimuli [14]. On the other hand, ING1b mediated UV-induced apoptosis by binding to PCNA through the N-terminal PIP domain [9]. Recently, we also showed that ING2 cooperated with p53 to regulate apoptosis via the activation of both the mitochondrial/intrinsic and death-receptor/extrinsic apoptotic pathways in melanoma cells [18]. In this study, we confirmed that overexpression of wt ING2 significantly induced UV-mediated apoptosis in MMRU melanoma cells as suggested by increased sub-G1 population in flow cytometry analysis (Fig. 2A). We also found that full-length ING2 and its DPHD mutant induced significantly higher rate of apoptosis (54.6% and 59.8%, respectively)
Y. Wang et al. / FEBS Letters 580 (2006) 3787–3793
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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 · FLAGDPHD, 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.
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(P < 0.001, t-test) compared with cells transfected with vector (24.4%), DLZL (27.2%), or DL+P (27.9%) (P < 0.001, t-test) after 400 J/m2 UVB irradiation (Fig. 2A). Cells were stained with Hoechst 33258, which binds to DNA, to further confirm that ING2-mediated cell death after UVB irradiation was the result of apoptosis, not necrosis. Cells undergoing apoptosis are characterized by chromatin condensation, DNA fragmentation, and formation of apoptotic bodies [23]. Consistently, there were significantly more apoptotic cells in ING2 or DPHD transfected MMRU cells (50.5% and 49.6%, respectively) after UV irradiation compared to vector (18.7%), DLZL (15.5%) or DL+P transfected cells (17.3%) (P < 0.001, t-test) (Fig. 2B).
Y. Wang et al. / FEBS Letters 580 (2006) 3787–3793
3.3. Leucine zipper-like domain of ING2 plays a critical role in the process of histone H4 hyperacetylation and chromatin relaxation upon UV irradiation Only recently has the role of histone modification in cell signaling or facilitating DNA repair begun to be investigated [24]. The covalent acetylation or deacetylation of lysine residues occurring within the N-terminal histone tails is believed to alter the chromatin structure and its accessibility to protein complexes involved in transcription regulation, DNA repair or other cellular events [25,26]. p53 has been demonstrated to play an important role in modulating histone modifications after 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.
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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/ or corepressor complexes [14]. We also found that ING2 is involved in histone acetylation and chromatin relaxation to mediate the repair of UV-damaged DNA [19]. To explore if deletion of ING2 LZL domain eliminates the role of ING2 in histone acetylation and chromatin relaxation, we examined the level of histone H4 acetylation and performed the micrococcal nuclease digestion assay in MMRU cells after transfection with mutant ING2 constructs and irradiation with 200 J/m2 UVC. Consistent with the DNA repair experiments, full-length ING2 as well as DPHD mutant, enhanced histone H4 acetylation by 1.8-fold compared to control cells after UV irradiation, while overexpression of ING2 DLZL or DL+P mutants did not affect histone H4 acetylation (Fig. 3A). Moreover, chromatin was more sensitive to the micrococcal nuclease (1 U treatment) in cells overexpressing wt ING2 or DPHD mutants (86% and 85% of the DNA fragments <2 kb, respectively) upon UVC irradiation compared to cells transfected with vector (15%), DLZL (22.5%) or DL+P (23.2%) (Fig. 3B). 3.4. Leucine zipper-like domain is required for ING2-mediated association between p53 and p300 Our previous data demonstrated that ING2-mediated DNA repair and apoptosis after UV irradiation require functional p53 [18,19]. Nagashima et al. and Pedeux et al. reported that ING2 regulates cell proliferation and replicative senescence by acetylation-mediated stabilization of p53 [13,29]. In this study, we investigated the role of LZL domain of ING2 in p53 stability and the interactions among ING2, p53 and p300. We found that deletion of LZL domain is required for the physical binding between ING2 and p53 and the association between p53 and p300 (Fig. 4A). However, LZL domain is not required for interaction between ING2 and p300 (Fig. 4B). Furthermore, we confirmed that overexpression of ING2 increased p53 level, while ING2 DLZL mutant failed to induce p53 (Fig. 4C).
4. Discussion We have previously demonstrated that ING2 cannot only enhance UV-induced apoptosis, but also increase the repair of UV damaged DNA by promoting global histone acetylation and chromatin relaxation [18,19]. In this study, we reported that the LZL motif, instead of the PHD region, plays a critical role for the interaction between ING2 and certain HAT or HDAC proteins to regulate histone H4 acetylation, chromatin
decondensation, and NER. We have previously shown that PHD region of ING1b plays an essential role in NER as the PHD deletion mutant is deficient in repair of UV-damaged DNA [17]. Due to the structural similarity between ING1b and ING2, we sought to investigate whether PHD of ING2 is also involved in the DNA repair process. Our data indicated that unlike ING1b, the PHD region of ING2 is dispensable, while the LZL domain is critical for NER (Fig. 1C). The reason for different domains of ING family members required for the DNA repair process is unclear. It is possible that distinct domain of each ING member is required for the interactions with specific HAT and/or HDAC complexes to induce histone acetylation and chromatin relaxation. We also observed that LZL motif, but not PHD domain, is essential for ING2 to enhance UV-medicated apoptosis in melanoma MMRU cells (Fig. 2). Gozani et al. previously showed that mutation in the PHD region of ING2 abolished ING2-induced apoptosis in human fibrosarcoma HT1080 cells [14]. This discrepancy may be due to cell type-specific responses. We has recently demonstrated that ING2 not only activated p53-dependent pathway, but also activated the Fas pathway to induce apoptosis in MMRU cells [18], while ING2 did not mediate Fas ligand-induced apoptosis in HT1080 cells [12]. This cell type-specific response was further addressed by knockdown of ING2 which did not render MMRU cells resistant to apoptosis, while RNAi-treated HT1080 cells were highly resistant to apoptosis [14]. Moreover, another ING family member, ING3, promoted UV-induced apoptosis only through Fas pathway in MMRU cells [30], despite that ING3 modulates p53-dependent apoptosis in RKO cells [31]. ING2 has been known to enhance the p300-dependent acetylation of p53, which stabilizes p53 and plays an important role in the cellular responses to DNA damage agents [13,29]. Our results indicated that LZL deletion mutant abrogated ING2-mediated association between p53 and p300 (Fig. 4A). 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 heptad repeats of leucines with an average of six repeats and a minimum of only four heptad repeats to make up a functional leucine zipper [32]. Although the N-terminal sequence of ING2 does not stringently match this motif, this leucine zipper-like (LZL) region has been suggested to help target particular HAT or HADC complexes to chromatin considerably more dynamic [20]. Therefore, it will be important to clarify how LZL domain of ING2 binds to p53 and targets it for HAT or HADC complexes (e.g p300, mSin3A), since ING2
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has been found in both HAT and HADC complexes [3]. On the other hand, the PCR domain is a novel predicted motif located after LZL domain of ING2. It is also found in other family members and may play a critical role in binding to HAT/HDAC complexes since it linked ING1b to the Sin3/ HDAC complex through direct interaction with SAP30 [22]. The results from this study demonstrated no significant difference in the biological functions between the cells transfected with DLZL and DL+P constructs, suggesting deletion of LZL motif alone is enough to eliminate ING2 function as a regulator of apoptosis and DNA repair. However, there is no direct evidence showing that the putative PCR domain is not required for ING2 function. One possibility is that PCR region may mediate the direct interaction between ING2 and p300 or other HAT/HDAC proteins. Further experiments are required to test this hypothesis. Taken together, ING2 may possibly function as a scaffold protein that mediates the interaction between p53 and p300, thereby inducing the acetylation level of p53 and p53-dependent chromatin remodeling, apoptosis and DNA repair process. LZL domain plays a key role for ING2 to exert its function in DNA repair and apoptosis, possibly by mediating protein–protein interaction or DNA–protein interaction to regulate gene transcription and/or target particular HAT and HDAC complexes to chromatin. Acknowledgments: We thank Dr. C.C. Harris for providing pcDNA3ING2 plasmid and Dr. R. Byers for the MMRU cell line. This work was supported by grants from the Canadian Institutes of Health Research and the National Cancer Institute of Canada to G.L. G.L. is a recipient of the Research Scientist Award from the National Cancer Institute of Cancer with funds provided by the Canadian Cancer Society.
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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
Abstract The plant homodomain (PHD) of ING2 was shown to regulate p53-dependent apoptosis through phosphoinositides signaling. However, the role of a predicted leucine zipper-like (LZL) motif in N-terminus of ING2 is unclear. Here, we show that LZL motif is critical for the proper functions of ING2 in DNA repair, apoptosis and chromatin remodeling after UV irradiation. Deletion of LZL domain also abrogated the association between ING2 and p53, but not between ING2 and p300, suggesting that ING2 modulates p53-dependent chromatin remodeling, apoptosis and DNA repair by functioning as a scaffold protein to mediate the interaction between p53 and p300. 2006 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. Keywords: ING2; p53; Leucine zipper-like domain; DNA repair; Apoptosis; Histone acetylation
1. Introduction The ING (inhibitor of growth) family consists of five genes that encode at least seven proteins which have been known as the regulators of transcription, apoptosis, cell cycle control, DNA repair, senescence and angiogenesis [1,2]. It is also notable that ING proteins both physically and functionally form complexes with several factors possessing intrinsic histone acetyltransferase (HAT) and histone deacetylase complex (HDAC) activities, thus linking the function of ING tumor suppressors to chromatin remodeling [1–3]. These proteins share a conserved plant homeodomain (PHD) in the C-terminus, which is a sequence encoding a specialized form of zinc finger. This PHD zinc finger has been widely discovered in nuclear DNA-binding proteins with known or suspected roles in regulating chromatin organization or gene expression [4]. The ING proteins also possess the strong nuclear localization sequences (NLS). Three intrinsic nucleolar translocation sequences (NTS) are located within the NLS of ING1 and ING2, which has been shown to target ING1 to the nucleolus
[5]. Several other motifs have also been predicted in ING proteins including the potential chromatin regulatory (PCR) domain which may play a critical role in binding to HAT/ HDAC complexes [6]. However, previous studies on either the founding member ING1b or its homology ING2 has predominantly focused on the PHD zinc finger since it has been shown to be involved in chromatin remodeling and modulate p53 function as a transcription factor and is also conserved in three yeast and mouse ING homologues [7,8]. So far, the function of the N-terminal region of the ING proteins remains largely unclear. Only the ING1b has been known to bind to the proliferating cell nuclear antigen (PCNA) through a specific Nterminal sequence named the PCNA-interacting protein (PIP) domain [9], which is found in proteins involved in growth inhibition, 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 induction of G1-phase cell cycle arrest and apoptosis [13] and interacts with phosphoinositides, Ptdlns(3)P and Ptdlns(5)P through PHD zinc finger that function in DNA damage-initiated stress signaling [14]. In addition, we have recently demonstrated that ING2, like its homologue ING1b [15–17], significantly promotes UV-induced apoptosis [18] and enhances nucleotide excision repair (NER) in melanoma cells in a p53-dependent manner by rapidly inducing histone H4 acetylation, chromatin relaxation, and the recruitment of the damage recognition factor XPA to the DNA photolesions [19]. A leucine zipper-like (LZL) motif consisting of leucine residues spanning every seven amino acids has been predicted in the N-terminal of ING2 [20], while no work has been done to demonstrate its role in ING2 function yet. To further characterize the roles of the specific domains of ING2, especially LZL domain, we constructed a panel of truncated ING2 mutants to examine the putative role of sub-regions in ING2mediated histone acetylation, chromatin assembly, DNA repair and apoptosis. Here we report that the LZL domain, 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.
Abbreviations: HAT, histone acetyltransferase; HDAC, histone deacetylase; LZL, leucine zipper-like; NER, nucleotide excision repair; PCNA, proliferating cell nuclear antigen; PCR, potential chromatin regulatory; PHD, plant homeodomain; PIP, PCNA-interacting protein; wt, wild-type
2. Materials and methods 2.1. Cell lines and cell culture MMRU melanoma cells were cultured in Dulbecco’s Modified Eagle Medium supplemented with 10% fetal bovine serum (Invitrogen, Mississauga, Ont., Canada), 100 U/ml penicillin, and 100 lg/ml streptomycin 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 2.2. Plasmids construction The pcDNA3-ING2 plasmid (a gift from Dr. C.C. Harris) [14] was digested with EcoRI and XbaI (New England Biolabs, Ipswich, MA), and subcloned into p3 · FLAG vector (Sigma, St. Louis, MO) between EcoRI and XbaI sites to get the p3 · FLAG-ING2 construct. The truncated 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 XbaI sites to get p3 · FLAG-DPHD, p3 · FLAG-DLZL and p3 · FLAG-DL+P constructs, respectively. The primers were: 5 0 -GGAATTCCGGTACCGAGCTCGGATCCA-3 0 (sense) and 5 0 -GCTCTAGAGCCTACACTTGGTTGCATAAGCA-3 0 (anti-sense) for DPHD, 5 0 -GGAATTCCATTGATGATGTCTACGA-3 0 (sense) and 5 0 -GCTCTAGAGCAGAATTCTACTACCTCGA-3 0 (anti-sense) for DLZL, and 5 0 -GGAATTCCGATAAAGCAAAGATGGA-3 0 (sense) and 5 0 GCTCTAGAGCAGAATTCTACTACCTCGA-3 0 (anti-sense) for DL+P. All the constructs were sequenced across the newly created junctions to confirm no reading frame shift induced by polymerase chain reaction. 2.3. Host-cell-reactivation assay DNA repair was determined with the host-cell-reactivation assay [21,22] The pRL-CMV plasmid (Promega, Madison, WI) was damaged with UVC at 400 J/m2 and co-transfected with gene of interest using Lipofectamine 2000 (Invitrogen). Forty hours after transfection, the luciferase activity was measured with the Renilla luciferase assay kit (Promega) on a 96-well plate format luminometer (Berthold Technologies, Bad Wildbad, Germany). To estimate repair efficiency, the results were normalized to values obtained from undamaged plasmids. 2.4. Western blotting Western analysis was performed as previously described [18,19]. The antibodies used in this study included anti-ING2, anti-p53, anti-b-actin (Santa Cruz Biotechnology, Santa Cruz, CA), anti-acetylated histone H4, anti-p300 (Upstate, Charlottesville, VA) and anti-FLAG (Applied Biological Materials Inc., Vancouver, BC, Canada) antibodies. Protein expression was quantified by densitometry using the Quantity One software (Bio-Rad, Mississauga, Ont., Canada). 2.5. Micrococcal nuclease (MNase) digestion Cells in 100-mm dishes were harvested by scraping in 12 ml lysis solution (10 mM Tris/HCl [pH 8], 10 mM MgCl2, 1 mM DTT) 30 min after UV irradiation. Then 0.3 ml of 2% NP-40 detergent was 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 was discarded and the nuclei pellet was resuspended in 200 ll MNase buffer (10 mM Tris/HCl [pH 8], 50 mM NaCl, 300 mM sucrose, 3 mM MgCl2). MNase (Sigma) was added to the nuclei samples to digest the DNA for 5 min at 37 C. The reaction was terminated with the stop solution (1% SDS, 20 mM EDTA). After centrifugation at 10,000 · g for 10 min, the aqueous phase was collected for DNA extraction with phenol:chloroform followed by precipitating with ethanol and analyzing on 1% agarose gel. 2.6. Hoechst staining Cells grown on coverslips in 6-well plates were transfected with wildtype (wt) or mutant ING2 for 24 h and then irradiated with UVB at 400 J/m2. After another 24 h, cells fixed in fixation buffer (2% formaldehyde, 0.5% Triton X-100 in PBS, pH 7.2) and incubated for 45 min at room temperature. The fixed cells were washed with 0.1% Triton X100/PBS and stained with the 2.5 lg/ml Hoechst 33258 in PBS at room temperature for 5 min. The cells were then washed with PBS, mounted onto the slides with mounting media (Fisher Scientific, Nepean, Ont., Canada) and visualized under a fluorescent microscope (Zeiss, Chester, VA) for apoptotic bodies. Images were taken with a cooled mono 12bit Retiga-Ex camera. 2.7. Flow cytometry 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 transfection. After another 24 h, cells were collected by trypsinization, pelleted by centrifugation at 200 · g for 3 min and resuspended in 1 ml of
Y. Wang et al. / FEBS Letters 580 (2006) 3787–3793 hypotonic fluorochrome buffer (0.1% Triton X-100, 0.1% sodium citrate, 25 lg/ml RNase A, 50 lg/ml PI). After incubation at 4 C for 30 min, the samples were then analyzed by a Coulter EPICS XLMCL flow cytometer (Beckman Coulter, Miami, FL) to determine the percentage of subdiploid DNA. 2.8. Immunoprecipitation Cells were transfected with wt or mutant ING2 and harvested 48 h post transfection. Cell lysates were prepared as described [18,19]. Lysates (300 lg of proteins) were pre-cleared with 60 ll of protein A or G agarose (50% slurry), incubated for 3 h at 4 C with 3 lg of monoclonal anti-p53 or p300 and then precipitated with fresh protein A or G beads (60 ll of 50% slurry). Mouse IgG (Santa Cruz Biotechnology) was used as a negative control. The beads were then pelleted by centrifugation at 10,000 · g for 1 min, washed thrice with PBS and boiled in 2 · Tris–glycine SDS sample buffer for Western blot analysis using anti-p300 and anti-FLAG antibodies.
3. Results 3.1. The leucine zipper-like domain is required for ING2mediated repair of UV-damaged DNA in melanoma cells The novel tumor suppressor ING2 has been shown to regulate p53-mediated cell proliferation, apoptosis and DNA repair [13,18,19]. To investigate the putative roles of the subregions of ING2 in DNA damage responses, several truncated ING2 proteins were generated. As shown in Fig. 1A and B, ING2 DPHD, DLZL and DL+P mutants encoded 1–220, 63– 280 and 136–280 amino acids, respectively. Since we have previously demonstrated that ING2 can facilitate the removal of bulky photolesions or DNA adducts caused by UV light in melanoma cells and knockdown of ING2 renders cells resistant to DNA repair [13], a host-cellreactivation assay with luciferase reporter system was performed to investigate the role of different domains in ING2mediated DNA repair by co-transfecting a UV-damaged pRL-CMV-luciferase plasmid with vector, wt ING2, DPHD, DLZL or DL+P into MMRU melanoma cells. The luciferase activity was used as an indicator of the extent of DNA repair. The ING2 DPHD mutant, similar to the wt ING2, had a 2.8fold increase in DNA repair compared with control (P < 0.01, t-test), while the enhancement in DNA repair was abolished in DLZL or DL+P-transfected cells (Fig. 1C), indicating that LZL domain is an important component for ING2 to exert its role in DNA repair in melanoma cells. 3.2. Leucine zipper-like domain is essential for ING2-mediated apoptosis induced by UV radiation Gozani et al. reported that PHD–phosphoinositide interactions directly regulated nuclear responses to DNA damage and silencing of ING2 or abrogation of PHD domain reduced the responses to cell death stimuli [14]. On the other hand, ING1b mediated UV-induced apoptosis by binding to PCNA through the N-terminal PIP domain [9]. Recently, we also showed that ING2 cooperated with p53 to regulate apoptosis via the activation of both the mitochondrial/intrinsic and death-receptor/extrinsic apoptotic pathways in melanoma cells [18]. In this study, we confirmed that overexpression of wt ING2 significantly induced UV-mediated apoptosis in MMRU melanoma cells as suggested by increased sub-G1 population in flow cytometry analysis (Fig. 2A). We also found that full-length ING2 and its DPHD mutant induced significantly higher rate of apoptosis (54.6% and 59.8%, respectively)
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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 · FLAGDPHD, 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.
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(P < 0.001, t-test) compared with cells transfected with vector (24.4%), DLZL (27.2%), or DL+P (27.9%) (P < 0.001, t-test) after 400 J/m2 UVB irradiation (Fig. 2A). Cells were stained with Hoechst 33258, which binds to DNA, to further confirm that ING2-mediated cell death after UVB irradiation was the result of apoptosis, not necrosis. Cells undergoing apoptosis are characterized by chromatin condensation, DNA fragmentation, and formation of apoptotic bodies [23]. Consistently, there were significantly more apoptotic cells in ING2 or DPHD transfected MMRU cells (50.5% and 49.6%, respectively) after UV irradiation compared to vector (18.7%), DLZL (15.5%) or DL+P transfected cells (17.3%) (P < 0.001, t-test) (Fig. 2B).
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3.3. Leucine zipper-like domain of ING2 plays a critical role in the process of histone H4 hyperacetylation and chromatin relaxation upon UV irradiation Only recently has the role of histone modification in cell signaling or facilitating DNA repair begun to be investigated [24]. The covalent acetylation or deacetylation of lysine residues occurring within the N-terminal histone tails is believed to alter the chromatin structure and its accessibility to protein complexes involved in transcription regulation, DNA repair or other cellular events [25,26]. p53 has been demonstrated to play an important role in modulating histone modifications after 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.
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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/ or corepressor complexes [14]. We also found that ING2 is involved in histone acetylation and chromatin relaxation to mediate the repair of UV-damaged DNA [19]. To explore if deletion of ING2 LZL domain eliminates the role of ING2 in histone acetylation and chromatin relaxation, we examined the level of histone H4 acetylation and performed the micrococcal nuclease digestion assay in MMRU cells after transfection with mutant ING2 constructs and irradiation with 200 J/m2 UVC. Consistent with the DNA repair experiments, full-length ING2 as well as DPHD mutant, enhanced histone H4 acetylation by 1.8-fold compared to control cells after UV irradiation, while overexpression of ING2 DLZL or DL+P mutants did not affect histone H4 acetylation (Fig. 3A). Moreover, chromatin was more sensitive to the micrococcal nuclease (1 U treatment) in cells overexpressing wt ING2 or DPHD mutants (86% and 85% of the DNA fragments <2 kb, respectively) upon UVC irradiation compared to cells transfected with vector (15%), DLZL (22.5%) or DL+P (23.2%) (Fig. 3B). 3.4. Leucine zipper-like domain is required for ING2-mediated association between p53 and p300 Our previous data demonstrated that ING2-mediated DNA repair and apoptosis after UV irradiation require functional p53 [18,19]. Nagashima et al. and Pedeux et al. reported that ING2 regulates cell proliferation and replicative senescence by acetylation-mediated stabilization of p53 [13,29]. In this study, we investigated the role of LZL domain of ING2 in p53 stability and the interactions among ING2, p53 and p300. We found that deletion of LZL domain is required for the physical binding between ING2 and p53 and the association between p53 and p300 (Fig. 4A). However, LZL domain is not required for interaction between ING2 and p300 (Fig. 4B). Furthermore, we confirmed that overexpression of ING2 increased p53 level, while ING2 DLZL mutant failed to induce p53 (Fig. 4C).
4. Discussion We have previously demonstrated that ING2 cannot only enhance UV-induced apoptosis, but also increase the repair of UV damaged DNA by promoting global histone acetylation and chromatin relaxation [18,19]. In this study, we reported that the LZL motif, instead of the PHD region, plays a critical role for the interaction between ING2 and certain HAT or HDAC proteins to regulate histone H4 acetylation, chromatin
decondensation, and NER. We have previously shown that PHD region of ING1b plays an essential role in NER as the PHD deletion mutant is deficient in repair of UV-damaged DNA [17]. Due to the structural similarity between ING1b and ING2, we sought to investigate whether PHD of ING2 is also involved in the DNA repair process. Our data indicated that unlike ING1b, the PHD region of ING2 is dispensable, while the LZL domain is critical for NER (Fig. 1C). The reason for different domains of ING family members required for the DNA repair process is unclear. It is possible that distinct domain of each ING member is required for the interactions with specific HAT and/or HDAC complexes to induce histone acetylation and chromatin relaxation. We also observed that LZL motif, but not PHD domain, is essential for ING2 to enhance UV-medicated apoptosis in melanoma MMRU cells (Fig. 2). Gozani et al. previously showed that mutation in the PHD region of ING2 abolished ING2-induced apoptosis in human fibrosarcoma HT1080 cells [14]. This discrepancy may be due to cell type-specific responses. We has recently demonstrated that ING2 not only activated p53-dependent pathway, but also activated the Fas pathway to induce apoptosis in MMRU cells [18], while ING2 did not mediate Fas ligand-induced apoptosis in HT1080 cells [12]. This cell type-specific response was further addressed by knockdown of ING2 which did not render MMRU cells resistant to apoptosis, while RNAi-treated HT1080 cells were highly resistant to apoptosis [14]. Moreover, another ING family member, ING3, promoted UV-induced apoptosis only through Fas pathway in MMRU cells [30], despite that ING3 modulates p53-dependent apoptosis in RKO cells [31]. ING2 has been known to enhance the p300-dependent acetylation of p53, which stabilizes p53 and plays an important role in the cellular responses to DNA damage agents [13,29]. Our results indicated that LZL deletion mutant abrogated ING2-mediated association between p53 and p300 (Fig. 4A). 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 heptad repeats of leucines with an average of six repeats and a minimum of only four heptad repeats to make up a functional leucine zipper [32]. Although the N-terminal sequence of ING2 does not stringently match this motif, this leucine zipper-like (LZL) region has been suggested to help target particular HAT or HADC complexes to chromatin considerably more dynamic [20]. Therefore, it will be important to clarify how LZL domain of ING2 binds to p53 and targets it for HAT or HADC complexes (e.g p300, mSin3A), since ING2
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has been found in both HAT and HADC complexes [3]. On the other hand, the PCR domain is a novel predicted motif located after LZL domain of ING2. It is also found in other family members and may play a critical role in binding to HAT/HDAC complexes since it linked ING1b to the Sin3/ HDAC complex through direct interaction with SAP30 [22]. The results from this study demonstrated no significant difference in the biological functions between the cells transfected with DLZL and DL+P constructs, suggesting deletion of LZL motif alone is enough to eliminate ING2 function as a regulator of apoptosis and DNA repair. However, there is no direct evidence showing that the putative PCR domain is not required for ING2 function. One possibility is that PCR region may mediate the direct interaction between ING2 and p300 or other HAT/HDAC proteins. Further experiments are required to test this hypothesis. Taken together, ING2 may possibly function as a scaffold protein that mediates the interaction between p53 and p300, thereby inducing the acetylation level of p53 and p53-dependent chromatin remodeling, apoptosis and DNA repair process. LZL domain plays a key role for ING2 to exert its function in DNA repair and apoptosis, possibly by mediating protein–protein interaction or DNA–protein interaction to regulate gene transcription and/or target particular HAT and HDAC complexes to chromatin. Acknowledgments: We thank Dr. C.C. Harris for providing pcDNA3ING2 plasmid and Dr. R. Byers for the MMRU cell line. This work was supported by grants from the Canadian Institutes of Health Research and the National Cancer Institute of Canada to G.L. G.L. is a recipient of the Research Scientist Award from the National Cancer Institute of Cancer with funds provided by the Canadian Cancer Society.
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