Article |
Address correspondence to Gary D. Kao, Hospital of the University of Pennsylvania, Department of Radiation Oncology, 3400 Spruce St. 2 Donner, Philadelphia, PA 19104. Tel.: (215) 573-5503. Fax: (215) 349-0090. E-mail: kao{at}xrt.upenn.edu
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Abstract |
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Key Words: HDAC4; 53BP1; DNA damage; irradiation; G2 checkpoint
* Abbreviations used in this paper: ATM, ataxia telangiectasia mutated; DNA-PK, DNA protein kinase; HDAC, histone deacetylase; IR, irradiation; NBS, Nijmegen breakage syndrome; RNAi, RNA interference; siRNA, short interfering RNA; TSA, trichostatin A.
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Introduction |
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Chromatin undergoes expansion and compaction in the course of many fundamental cellular processes, including gene expression, differentiation, and cell cycle progression. These alterations of the chromatin are largely mediated by histone acetylases and histone deacetylases (HDACs).*HDACs act on key acetylated lysine residues of core histones to induce chromatin compaction, which in turn results in gene silencing and heterochromatin formation (Taunton et al., 1996; Yang et al., 1996; Fischle et al., 1999; Grozinger et al., 1999). HDACs have been categorized into three classes that are based on sequence homology and domain organization. Class I HDACs (HDAC1, HDAC2, HDAC3, and HDAC8) are similar to the yeast deacetylase Rpd3 (Yang et al., 1996; Dangond et al., 1998; Emiliani et al., 1998; Buggy et al., 2000; Hu et al., 2000). Class II HDACs (HDAC4, HDAC5, HDAC6, and HDAC7) possess catalytic domains homologous to that of yeast Hda1 (Rundlett et al., 1996; Fischle et al., 1999; Grozinger et al., 1999; Miska et al., 1999; Verdel and Khochbin, 1999; Wang et al., 1999; Kao et al., 2000). Proteins similar to the yeast NAD1-dependent deacetylase Sir2 (Frye, 2000; Imai et al., 2000; Landry et al., 2000; Smith et al., 2000) compose the third class of HDACs. Class I and II HDACs have been found to function as corepressors recruited for transcriptional repression, whereas the class III HDACs are important for gene silencing at telomeres and HM (mating type) loci in yeast (Sherman et al., 1999).
Although HDACs are most prominently linked with transcriptional repression, there are indications that HDACs may play broader roles in regulating cellular processes that affect survival after exposure to DNA-damaging agents. For example, p53 has been found to be deacetylated and inactivated by human Sir2 in mammalian cells, leading to reduced apoptosis and increased survival after exposure to ionizing radiation or etoposide. Conversely, expression of catalytically inactive Sir2
leads to increased apoptosis and radiosensitization in mammalian cells (Luo et al., 2001; Vaziri et al., 2001). Effects of the class III HDACs may extend beyond effects on p53. In budding yeast, members of the SIR2 family of HDACs appear to be important components of the DNA damage pathway. Whereas SIR2 appears to be static, SIR3 and SIR4 are mobilized to sites of DNA breaks (Gottschling et al., 1990; Tsukamoto et al., 1997; Martin et al., 1999; Moazed, 2001). SIR2, SIR3, and SIR4 are required for the efficient recircularization of linear plasmids by nonhomologous end joining, and mutations in these genes resulted in increased sensitivity to
-radiation in fission yeast (Boulton and Jackson, 1998; Tsukamoto et al., 1997). A role in the DNA damage response has not yet been described for the class I and II HDACs, but is suggested by the observation that treatment with the class I and II HDAC inhibitor trichostatin A (TSA) leads to significant radiosensitization of human cells (Biade et al., 2001) (the class III Sir2 family deacetylases are resistant to TSA [Bernstein et al., 2000]). The mechanism of how TSA might lead to radiosensitization has likewise been unclear.
To assess for a role for class I and II HDACs in the DNA damage response in human cells, we examined several members for their ability to form foci in response to DNA damage. After exposure to double-strand DNA damage in a variety of human cells, we found that human HDAC4 is recruited to nuclear foci with kinetics similar to 53BP1. We found that HDAC4 and 53BP1 colocalize at foci and could be coimmunoprecipitated. HDAC4 foci formation was seen in a wide variety of cell lines, which included radiosensitive cell lines lacking the gene products of ATM (ataxia telangiectasia mutated), Nibrin, and DNA protein kinase (DNA-PK). HDAC4 foci disappeared after several hours in repair-proficient cells that were exposed to nonlethal doses of radiation. However, HDAC4 foci failed to resolve in radiosensitive cell lines or in normal cells that were exposed to a lethal dose of radiation. Silencing of HDAC4 resulted in decreased 53BP1 protein levels and did not markedly affect cell cycle distribution in unirradiated cells, but markedly abrogated the G2 checkpoint in radiosensitized cells after DNA damage. These results together suggest that HDAC4 is a critical component of the DNA damage response pathway and suggests an additional role for this protein beyond transcriptional silencing.
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Results |
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HDAC4 foci induced by IR persist in radiosensitive cells
We next examined in a range of human cell lines the genetic determinants that specified HDAC4 foci formation. We found that HDAC4 foci formation did not depend on DNA damage response genes, i.e., ATM (ataxia telangiectasia mutated), Nibrin, or DNA-PK, as cell lines defective for these genes formed foci with similar kinetics as HeLa cells (Fig. 3). However, the repair-deficient cell lines differed from HeLa (and other repair-proficient) cells in the persistence of HDAC4 foci after exposure to low doses of IR. For example, HDAC4 foci readily formed 1 h after radiation in the ATM-deficient FT169 cell line, as well as in its isogenic derivatives Y25 (in which ATM is restored by expression of a full-length cDNA) and PEB (expressing empty vector and hence remaining ATM deficient). 24 h after IR, HDAC4 foci were significantly reduced only in the ATM-positive Y25 cells (Fig. 3, AD). A similar difference in the resolution of HDAC4 foci was observed between DNA-PKdeficient MO59J cells and DNA-PKpositive MO59K cells. Although MO59K cells did not completely resolve their HDAC4 foci, the average number of foci was less than in the MO59J cells (Fig. 3, EG). We believe that MO59K cells did not efficiently resolve HDAC4 foci because of their inherent radiosensitivity relative to HeLa cells (Wang et al., 1997; unpublished data), which efficiently resolves foci at low doses of IR (Fig. 3 J). Lastly, we examined HDAC4 foci formation in the radiosensitive Nijmegen breakage syndrome (NBS) mutant cell lines and found that they too retained high levels of foci 24 h after IR (Fig. 3, H and I). We found that foci formation by HDAC4 in HeLa cells was unimpeded by TSA. However, the resolution of HDAC4 foci in HeLa cells was partially inhibited by TSA (Fig. 3, H and K).
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Silencing of HDAC4 expression via RNAi results in decreased cell viability
We further studied the effects of silencing HDAC4 on cell cycle distribution and cellular viability after DNA damage. Treatment with HDAC4 siRNA did not appear to have a major effect in altering the cell cycle distribution of unirradiated HeLa cells, compared with cells treated with control siRNA (Fig. 6, A and B) or untreated cells (unpublished data). IR of HeLa cells treated with control siRNA resulted in the expected accumulation of cells in G2, characteristic of the IR-induced checkpoint. In contrast, treatment with HDAC4 siRNA markedly decreased the proportion of cells with G2/M DNA content but was accompanied by an increase in cells with DNA content less than 2N (sub-G1) (Fig. 6, A and B). This reduction in G2/M cells was not due to an S-phase delay because HDAC4 siRNA did not affect normal S-phase progression, as determined by FACS® analysis and BrdU incorporation (unpublished data). Flow cytometry based on DNA content alone does not distinguish between cells in G2 and mitosis. We therefore performed immunofluorescence on irradiated cells that were transfected with control and HDAC4 siRNA. Control cells were blocked in G2 after IR, as determined by high cyclin B1 levels in the cytoplasm, CENP-F distributed uniformly in the nucleus, and uncondensed chromatin (Fig. 6, C and D), as described previously (Liao et al., 1995; Kao et al., 2001). In contrast, few of the HDAC4 siRNAtreated cells appeared to be in G2. Although many of these cells still retained cyclin B1 and CENP-F staining, the cyclin B1 was often not uniformly cytoplasmic, the CENP-F staining of the nuclei was frequently uneven, and many of nuclei were condensed or fragmented. Many of these condensed cells (>70%), in fact, showed high levels of phosphohistone H3 staining, along with cyclin B1, suggesting progression into mitosis (see Fig. S3, available at http://www.jcb.org/cgi/content/full/jcb.200209065/DC1). These observations together suggest that cells silenced for HDAC4 do not maintain a uniform G2 delay after IR. One notes, however, that as 53BP1 has been established to mediate the damage-induced G2 checkpoint, the abrogation of this checkpoint by silencing HDAC4 may be mediated, at least in part, through decreasing 53BP1 protein (DiTullio et al., 2002; Fernandez-Capetillo et al., 2002; Wang et al., 2002).
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Lastly, we examined whether abrogation of the G2 delay occurs when HDACs other than HDAC4 are silenced. Silencing of HDAC2 and HDAC6 did not interfere with the cell's ability to arrest in G2 in response to DNA damage (Fig. 7, A and B). Only silencing of HDAC4 appreciably diminished the proportion of cells delayed in G2 after IR.
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Discussion |
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These data suggest that HDAC4 foci may facilitate recruitment of, or stabilize, repair factors such as 53BP1, which in turn may help maintain damage-induced cell cycle checkpoints. The interaction between HDAC4 and 53BP1 is established here by the findings that the two proteins colocalize and coimmunoprecipitate, as well as the unexpected findings that siRNA-mediated silencing of HDAC4 leads to reduced levels of 53BP1 protein and vice-versa. Thus, the stability of each protein appears to depend on the stability of the other, or on being associated in a common complex. Our findings that silencing of HDAC4 expression abrogates the damage-induced G2 delay and increases radiosensitivity support the notion that the checkpoint determines the ability of the cell to survive radiation damage. Along with 53BP1, localization of HDAC4 to unrepaired damage may signal the cell to stall at a checkpoint to allow the repair to be completed, while possibly serving as a marker to facilitate repair. These results, therefore, may be consistent with the model proposed by Fernandez-Capetillo et al. (2002) in which 53BP1 and other factors capable of signal transduction may accumulate into a chromatin microenvironment within megabases of an actual DNA double-strand break. This localized concentration of protein complexes may then generate the amplification of signal sufficient to invoke the G2 checkpoint.
These findings do not exclude roles for HDAC4 in mediating other cellular functions. HDAC4 is an established component of complexes mediating transcriptional repression (Guenther et al., 2001; Fischle et al., 2002). Although transcription is globally decreased during mitosis (Spencer et al., 2000; Pflumm, 2002), it appears unlikely that the role of HDAC4 in the DNA damage response is restricted to transcriptional repression. Radiation, at the doses used in this study, does not globally repress transcription (Maity et al., 1995), and drugs that globally inhibit transcription, such as -amanitin, do not reverse the effects of silencing HDAC4 (unpublished data). The radiation dose dependency of the number of HDAC4 and 53BP1 foci does not exclude, but does reduce, the likelihood that foci are mere storage sites for these proteins.
The recruitment of HDACs to sites of DNA damage may be at least partially evolutionarily conserved. Members of the SIR2 family of HDACs have been implicated in the DNA response in budding yeast, contributing either directly or indirectly to the repair of double-strand breaks (Astrom et al., 1999; Bennett et al., 2001). Whereas SIR2 has been most prominently implicated in the silencing of chromatin at the mating type loci and at telomeres, SIR3 is recruited to sites of DNA damage to form foci that are visible at the cytological level. It has been proposed that the accumulation of some of these deacetylases may induce a repressed chromatin state to facilitate repair or protect unrepaired broken DNA ends (Tsukamoto et al., 1997; Martin et al., 1999). Although HDAC4 does not belong to the same family of deacetylases, we propose that HDAC4 may perform an analogous function in mammalian cells. As a component of protein complexes that mediate transcriptional repression by inducing heterochromatin formation, HDAC4 likely serves a similar role in processing chromatin in the DNA damage response. Recruitment of HDAC4 to foci after DNA damage might reflect its role in silencing the chromatin near the site of damage. The silenced chromatin would prevent processes such as DNA replication and transcription from passing through damaged DNA. In addition, the silenced chromatin might also reduce nonspecific end joining of broken DNA ends or unwanted recombination events.
In linking a HDAC with the DNA damage response to agents inducing double-strand breaks, the results presented here may also have clinical implications for treating patients with cancer. Inhibitors of HDAC inhibitors have entered clinical trials with reports of efficacy in certain tumors (Piekarz et al., 2001; Sandor et al., 2002). These protocols have largely entailed administering the HDAC inhibitor as a single agent. With further development and confirmation, the observations reported here suggest that it may be fruitful to pursue strategies to block HDAC function in combination with standard treatments, such as radiation and chemotherapy, to maximize the killing of cancer cells.
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Materials and methods |
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Cell lines
All cells were grown in DME medium supplemented with 20% FBS at 37°C with 5% CO2. The ATM-deficient FT169A (ATM-), ATM-restored YZ5 cells (ATM+, consisting of FT169A transfected with and stably expressing full-length ATM), and ATM-deficient vector-control PEB cells (ATM control, consisting of FT169A cells transfected with the parental vector only) were provided by Y. Shiloh (Tel Aviv University, Ramat Aviv, Israel). Nibrin-deficient human cells were obtained from the American Type Culture Collection. Cells grown on coverslips were irradiated with cesium-137 rays from a JL Shepherd and Associates 8114R panoramic irradiator at a dose rate of 1.35 Gy/min. UV IR was delivered in a single pulse (50 J/m2) using a Stratalinker UV source (Stratagene). Before UV IR, the culture medium was removed, and the medium was replaced immediately after IR. All cells were returned to the incubator for recovery and harvested at the indicated times. Etoposide was used at 20 µg/ml for 20 min. To block cells in mitosis, cells were exposed to 0.04 µg/ml of nocodazole for 15 h.
RNAi
RNAi was performed with siRNA that was commercially synthesized (Dharmacon) and used as described in protocols provided by the manufacturer. Cells were treated with siRNA to a final concentration of 10 µM. siRNA against HDAC6 was applied twice on consecutive days, whereas all other siRNAs were applied once and harvested as described for each experiment. Paired siRNA sequences targeting each protein were as follows: HDAC4, GACGGGCCAGUGGUCACUG and CAGUGACCACUGGCCCGUC; 53BP1, CACACAGAUUGAGGAUACG and CGUAUCCUCAAUCUGUGUG; HDAC2, GCCUCAUAGAAUCCGCAUG and CAUGCGGAUUCUAUGAGGC; HDAC6, CCAGCCAGCGAAGAAGUAG and CUACUUCUUCGCUGCCUGG. Control siRNA consisted of the unannealed single-strand RNA and siRNA targeted against luciferase (both of which did not affect levels of endogenous proteins).
Assays
HDAC assays were performed as previously described (Huang et al., 2000). In brief, [3H]acetylated histones purified from HeLa cells (25,000 cpm/10 µg) were incubated with enzymes at 37°C for 15 min. The reaction was stopped by the addition of concentrated HCl, extracted with 1 ml of ethylacetate, and the amount of radioactivity released into the organic layer was quantitated using a scintillation counter. In experiments in which HDAC4 deacetylase activity was inhibited, HeLa cells were pretreated with 1 µM TSA. For immunoprecipitations, for each sample, 0.1 µg of antibody or 10 µl of preimmune rabbit serum was incubated with cell lysate from 1.0 x 106 cells and incubated in the presence of ethidium bromide (10 µg/ml) to exclude nonspecific proteinDNA associations (Lai and Herr, 1992). Cell preparation, image acquisition and processing, and FACS® analysis were as previously described (Kao et al., 1997, 2001). Endogenous mRNA was isolated using Trizol reagent (GIBCO BRL), as per the manufacturer's instructions, and assessed via RT-PCR. The Titan One Tube RT-PCR System (Roche) was used with the following primers: HDAC4, CAAGAACAAGGAGAAGGGCAAAG and GGACTCTGGTCAAGGGAACTG; 53BP1, AGGTGGGTGTTCTTTGGCTTCC and TTGGTGTTGAGGCTTGTGGTGATAC; glyceraldehyde-3-phosphate dehydrogenase, CAACTTTGGTATCGTGGAAGGACTC and AGGGATGATGTT-CTGGAGAGCC (specific details regarding the PCR parameters used are available from the authors).
Plating efficiency was defined as the proportion of cells that remained viable 8 h after trypsinization and replating in fresh media. Cell viability was assessed by trypan exclusion. Clonogenic survival assays were performed as previously described (Biade et al., 2001), except that cells were counted and plated 48 h after treatment with siRNA and colonies of at least 50 cells were counted 14 d after plating. Statistical analyses were performed with SPSS for Windows Release 10 and Microsoft Excel (Office 2000).
Online supplemental material
Additional data (Figs. S1S4) regarding antibody specificity, Rad51 localization after IR, and phosphorylated histone H3 (Pi-Histone H3) labeling of siRNA-treated cells are available as supplemental material (http://www.jcb.org/cgi/content/full/jcb.200209065/DC1).
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Acknowledgments |
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G.D. Kao is a recipient of an award from the WW Smith Charitable Trust. T.J. Yen, R.J. Muschel, and W.G. McKenna are supported by PO1-CA75138-05. T.J. Yen was also supported by grants from the National Institutes of Health, PO1 core grant CA06927, and an Appropriation from the Commonwealth of Pennsylvania.
Submitted: 12 September 2002
Revised: 28 January 2003
Accepted: 5 February 2003
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