Oncogene (2006) 25, 1–7 & 2006 Nature Publishing Group All rights reserved 0950-9232/06 $30.00 www.nature.com/onc ORIGINAL ARTICLE tumor suppressor regulates the levels of huntingtin expression

Z Feng1,7, S Jin1,2,7, A Zupnick3, J Hoh4, E de Stanchina5, S Lowe5, C Prives3 and AJ Levine1,6

1Cancer Institute of New Jersey, University of Medicine and Dentistry of New Jersey, New Brunswick, NJ, USA; 2Department of Pharmacology, University of Medicine and Dentistry of New Jersey, Piscataway, NJ, USA; 3Department of Biological Sciences, Columbia University, New York, NY, USA; 4Department of Epidemiology and Public Health, Yale University, New Haven, CT, USA; 5Cold Spring Harbor Laboratories, Cold Spring Harbor, NY, USA and 6School of Natural Sciences, Institute for Advance Study, Princeton, NJ, USA

The p53 protein is a transcription factor that integrates the laterstages of the disease, extends to a varietyof various cellular stress signals. The accumulation of the brain regions (Huntington’s Disease Collaborative mutant huntingtin protein with an expanded polygluta- Research Group, 1993; Li and Li, 2004). This disease mine tract plays a central role in the pathology of human is caused by expansion of a polymorphic trinucleotide Huntington’s disease. We found that the huntingtin gene repeat (CAG)n located in the coding region of the contains multiple putative p53-responsive elements and huntingtin gene, which translates as a polyglutamine p53 binds to these elements both in vivo and in vitro. p53 repeat in the protein product. Accumulation of cyto- activation in cultured human cells, either by a tempera- plasmic and nuclearaggregates of the mutant huntingtin ture-sensitive mutant p53 protein or by gamma-irradiation protein with an expanded is thought (c-irradiation), increases huntingtin mRNA and protein to play a central role in the pathology of Huntington’s expression. Similarly, murine huntingtin also contains disease (DiFiglia et al., 1997). The huntingtin gene is multiple putative p53-responsive elements and its expres- expressed in most tissues and it is required for normal sion is induced by p53 activation in cultured cells. development (Nasir et al., 1995). The normal function of Moreover, c-irradiation, which activates p53, increases the huntingtin protein is not fully understood. In huntingtin in the and cortex of addition to its possible functions in the cell nucleus, mouse brain, the major pathological sites for Huntington’s recent studies indicate that normal huntingtin protein disease, in p53 þ / þ but not the isogenic p53À/À mice. is required for fast axonal trafficking (Trushina et al., These results demonstrate that p53 protein can regulate 2004). Reduction of normal huntingtin expression, huntingtin expression at transcriptional level, and suggest or expression of the polyglutamine track expanded that a p53 stress response could be a modulator of the mutant huntingtin , which interact with the process of Huntington’s disease. normal huntingtin proteins, impairs vesicular and mito- Oncogene (2006) 25, 1–7. doi:10.1038/sj.onc.1209021; chondrial trafficking in mammalian , leading to published online 7 November2005 mitochondrial dysfunction and toxicity (Trushina et al., 2004). Although the regulation of huntingtin gene Keywords: p53; hungtingtin; Huntington’s disease; expression has not been well studied, both genetic and p53-responsive element; transcription regulation; gene environmental stress factors could affect huntingtin gene expression expression (Dixon et al., 2004), and that in turn could impact upon the onset and prognosis of Huntington’s disease (Georgiou et al., 1999; Anca et al., 2004). p53 is a tumorsuppressorgenethat is mutated in Introduction more than 50% of human cancer (Levine, 1997; Vogelstein et al., 2000). It encodes a transcription factor Huntington’s disease is a progressive neurodegenerative that plays a central role in integrating various stress disorder characterized by selective loss of neurons signals, in particular, genotoxic stresses such as DNA (Huntington’s Disease Collaborative Research Group, damage, hypoxia, and oncogene activation (Levine, 1993). The degeneration preferentially occurs in the 1997; Vogelstein et al., 2000; Jin and Levine, 2001). striatum and the deep layers of the cortex and, during These stresses trigger signal transduction pathways leading to the activation of the p53 protein, which in turn binds to the p53-responsive elements (p53 REs), Correspondence: Dr AJ Levine, School of Natural Sciences, Institute in its downstream target and alters their rates for advanced study, Einstein Drive, Princeton, NJ 8540, USA. of transcription. The transcriptional activation of E-mail: [email protected] 7These two authors contributed equally to this work downstream target genes appears to account for most Received 17 May 2005; revised 7 July 2005; accepted 7 July 2005; of the mechanism by which p53 regulates its down- published online 7 November2005 stream cellular programs, including cell cycle arrest and p53 regulates huntingtin Z Feng et al 2 . The consensus p53-responsive elements contain a repeat of PuPuPuC(A/T) (T/A)GPy- PyPy (Pu, purine; Py, pyramidine) with a spacer ranging from 0 to 14 (el-Deiry et al., 1992; Hoh et al., 2002). Recently a novel algorithm (p53MH) was developed fordetecting the p53-responsive genes (Hoh et al., 2002). A genome wide search for p53 target genes in human and mouse was carried out using this algorithm. Interestingly, the huntingtin gene was found to contain a numberof putative p53 binding sites scattered throughout the gene (Table 1, Supplementary Information; and Figure 1a). Since p53 is a central integrator for stress signals that is involved in regulating cell death and senescence, and a mutant form of the huntingtin is responsible for an age related neurodegen- erative disease, Huntington’s disease, we investigated the regulation of the huntingtin gene by p53. Here we demonstrate that the p53 protein interacts with the putative p53-responsive elements in the huntingtin gene both in vivo and in vitro. Moreover, in both cultured cells and mouse brain tissues, p53 mediates the regulation of the huntingtin gene that is triggered after DNA damage. These results demonstrate that the p53 protein regulates the huntingtin gene.

Results

Human huntingtin gene contains putative p53-responsive elements The tumor suppressor p53 protein is a transcription factor that specifically recognizes a degenerate DNA responsive element loosely defined by PuPuPuC(A/T) (T/A)GPyPyPy (N)0–14 PuPuPuC(A/T) (T/A)GPyPyPy, wherePu stands forpurine,Py stands forpyramidine Figure 1 The Putative human huntingtin p53 REs interact with p53 in in vivo and in vitro.(a) The potential p53 REs in human and N stands forany nucleotide (el-Deiry et al., 1992). huntingtin gene. Numberindicates the nucleotide position relative Hoh et al. (2002) developed a novel algorithm, which to the transcription initiation site. Pu, purine and Py, pyramidine. employs a weighted matrix to score the likelihood of p53 (b) Chromatin (ChIP) assay of interactions binding, and a program for genome-wide scanning for between p53 and chromatin regions (as indicated) containing p53 REs in H1299/V138 cells and the parental p53 null H1299 cells. putative p53-responsive elements. The H1299/V138 and H1299 cells were cultured at 321C for18 h before was scanned forputative p53 sites and the genes that collecting. DO-1 p53 antibody (lanes 4 and 5), no antibody (lane 2) contain high-score putative p53 RE sites were identified ornonspecific mouse IgG (lane 3) wereused forChIP assay. 1/20 and compiled (Hoh et al., 2002). Interestingly, hunting- DNA of ChIP was used forPCR as Input DNA (lane 1). MDM2 tin was one of the genes that contained high-score promoter p53 RE region was used as a positive control. (c) Gel mobility shift and supershift analysis. Reaction mixtures containing putative p53 RE sites (Supplementary Information, 32P-labeled GADD45, human huntingtin intron 2, or intron 3 Table 1). As shown in Figure 1a there are three potential oligonucleotides were incubated with 40, 60, or 80 ng of bacterial p53 RE sites, including one in the promoter region, one His-p53 protein (lanes 1–3, 7–9, and 13–15). Unlabeled DNA in intron 2, and one in intron 3. (50 Â ) of the corresponding sequence was added as a specific competitor(lanes 4, 10, and 16). p53 specific antibody PAb1801 was added to supershift p53-DNA complexes (lanes 5, 11, and 17). p53 binds to human huntingtin p53-responsive elements in Mutant GADD45 oligonucleotide was used to indicate nonspecific binding (lane 19). Migration positions of free probe, p53-oligo, and vivo and in vitro 1801-p53-oligo complexes are shown. The ability of these sites to interact with the p53 protein was examined both in vivo and in vitro. First, a chromatin immunoprecipitation (ChIP) assay was per- p53 (Pochampally et al., 1999). By shifting the culture formed in a human cell line, H1299/V138, to test the in temperature from 37 to 321C, p53 is stabilized, binds to vivo interactions between p53 and these three potential p53 REs and activates transcription of its downstream p53-responsive elements. H1299/V138 was established genes. As shown in Figure 1b, the H1299/V138 cells by stably transfecting a temperature-sensitive mutant were shifted from 37 to 321C and cultured for another form of p53 (alanine 138 to valine) into H1299, a human 18 h, and the ChIP assay performed with an anti-p53 lung cancercell line that does not contain endogenous antibody, DO-1, detected the interactions between p53

Oncogene p53 regulates huntingtin Z Feng et al 3 and the chromatin regions containing the putative p53 REs in huntingtin protomer region, intron 2 and intron 3, respectively (Figure 1b, lane 4). These interactions are comparable to that between p53 and the MDM2 gene p53 RE located in the promoter region. As negative controls, these chromatin fragments could not be co-immunopre- cipitated with the DO-1 antibody in the parental H1299 cells cultured at 321C, which does not contain any endogenous p53 protein (Figure 1b, lane 5); and a nonspecific mouse IgG antibody could not precipitate these fragments in H1299/V138 cells shifted to 321C (Figure 1b, lane 3). These results clearly show that p53 interacts with all these three putative p53 REs in the cells. To further confirm the direct interactions between p53 and these putative p53 REs, gel mobility shift and supershift analyses were performed. The oligonucleo- tides corresponding to the putative p53 REs and their endogenous flanking sequences in intron 2 and intron 3 of the human huntingtin gene were 32P-labeled and incubated with the p53 protein. As shown in Figure 1c, these two oligonucleotides bind to the p53 protein in a dose-dependent manner(lanes 6–9 and lanes 12–15, respectively), and the interactions between 32P-labelled oligonucleotides and p53 were disrupted by an excess amount of nonradio-labelled oligonucleotides (lanes 10, Figure 2 p53 activation increases huntingtin gene expression in cultured human cells. (a) Quantitative real-time PCR analysis of 16). Moreover, the identities of the p53-oligonucleotide huntingtin mRNA levels after temperature shift from 37 to 321C complexes were further confirmed by mobility supershift forindicated amount of time in H1299/V138 cells and in H1299 assay with a p53 antibody, Pab1801 (lanes 11 and 17). cells. The levels of huntingtin mRNA were normalized against the Taken together, these results indicate that p53 specifi- levels of actin. (b) Western blot analysis of huntingtin protein in cally interacts with these putative p53 REs, although the H1299/V138 cells and in H1299 cells aftergrowingat 32 1C for indicated amount of time. The protein levels of actin were affinities between these elements and p53 are somewhat determined as control. (c) Western blot analysis of huntingtin lower than that between p53 and the well-characterized protein in HCT116 p53 þ / þ cells and HCT116 p53À/À cells after p53 RE in GADD45 gene (compare lanes 6–11, lanes g-irradiation (IR). HCT116 p53 þ / þ and HCT116 p53À/À cells at 12–17, to lanes 1–5). 50% confluence were treated with g-irradiation (5 Gy) and cultured for indicated amount of time before harvesting. Proteins were extracted and Western blot analysis was performed to determine the levels of huntingtin protein. The protein levels of GAPDH were Activation of p53 in cultured cells increases huntingtin determined as control. The data represent three independent mRNA and protein levels experiments, and the error bar represents standard deviation. To analyse the functional significance of the interactions between p53 and the p53 REs in the huntingtin gene, the ability of p53 to regulate the expression of the logical stress induces huntingtin gene expression, huntingtin gene was examined. p53 activity was induced we measured huntingtin mRNA and protein levels in by shifting the culture temperature of the H1299/V138 HCT116 cells upon gamma(g)-irradiation. HCT116 cells from 37 to 321C, and the mRNA levels of p53 þ / þ is a human colon carcinoma cell line, which huntingtin in these cells were measured by real-time contains a wild-type p53 gene, whereas the HCT116 PCR. As shown in Figure 2a (left panel), upon p53 p53À/À is a p53 knockout cell line derived from activation, the huntingtin mRNA levels increased in a HCT116 p53 þ / þ by homologus recombination (Bunz time dependent manner. By 24 h, huntingtin mRNA et al., 1998). As shown in Figure 2c, g-irradiation (5 Gy), increased by B2.8-fold as normalized against actin which induces double-stranded DNA breaks and mRNA. As a control, exactly the same temperature shift activates p53, increased the huntingtin protein levels had little effect on huntingtin expression in the parental by B2–3-fold in HCT116 p53 þ / þ cells. It is apparent H1299 cells (Figure 2a, right panel), which lacks that the huntingtin upregulation is p53-dependent, since endogenous p53 protein. Similarly, the huntingtin g-irradiation had no effect on huntingtin expression protein levels increased by B3-fold in H1299/V138 cells in HCT116 p53À/À cells (Figure 2c, right panel). The upon temperature shift to 321C, while little changes in mRNA levels of huntingtin were also determined by huntingtin protein levels were observed in the parental real-time PCR analysis and g-irradiation increased H1299 cells upon the same treatment (Figure 2b). huntingtin mRNA levels by B2-fold in HCT116 DNA damage is among the most important physio- p53 þ / þ cells but had little effect in HCT116 p53À/À logical signal that activates the p53 protein (Levine, cells (data not shown). The same p53-dependent 1997; Vogelstein et al., 2000; Jin and Levine, 2001). To upregulation of huntingtin gene expression after determine whether activation of p53 by this physio- g-irradiation was also observed in human lung epithelial

Oncogene p53 regulates huntingtin Z Feng et al 4 H460 cells containing wild-type p53 while not in p53 and age-matched isogenic p53 knockout mice were null human lung epithelial H1299 cells (data not shown). exposed to 6 Gy of g-irradiation. Various parts of the Taken together, these results demonstrated that activa- mouse brain tissues were dissected out and the effect of tion of p53 indeed upregulates the expression the p53 activation on huntingtin gene expression was huntingtin gene in cultured cells. examined in cortex, cerebellum, and striatum tissues by real-time PCR. The p53 activation in these tissues p53 mediates the upregulation of huntingtin upon DNA upon g-irradiation was confirmed by measuring mRNA damage by g-irradiation in mouse striatum and cortex levels of the p21 gene, a known p53 downstream target Although huntingtin gene is expressed in most tissue gene. As shown in Figure 4b (left panel), p21 mRNA increased by B2-fold in cortex and cerebellum, and types, the majortargets affected by the mutant B (trinucleotide expanded) huntingtin protein are striatum 4-fold in striatum tissues in wild-type mice. The p21 and deep layers of cortex neurons (Huntington’s Disease mRNA increase was clearly mediated by p53, since no Collaborative Research Group, 1993; Sharp and Ross, increase in p21 mRNA levels were observed in p53 1996; Li and Li, 2004). Apparently, accumulation of the knockout mice (Figure 4b, right panel). The expression mutant huntingtin protein aggregates contributes to patterns of the huntingtin gene were determined in the degeneration in the pathological regions, which same mice brain tissues. As shown in Figure 4a, huntingtin mRNA levels increased by B2.1-fold in causes most symptoms in Huntington’s disease. There- B fore, it is of interest to examine the effect of p53 wild-type mice cortex tissues, 2.7-fold in striatum 24 h activation on huntingtin gene expression in these tissues after g-irradiation, while huntingtin mRNA levels in animals. As shown in Supplementary Table 1, similar changed little in tissues of the p53À/À mice. The to the human huntingtin gene, the mouse huntingtin gene p53-dependent activation of huntingtin is similarin also contains multiple high-score putative p53 REs. Thus, magnitude compared to that of the p21 gene in these the ability of p53 in regulating huntingtin gene was tested tissues. However, the p53-dependent regulation of the in murine cells, Vm10 cells, the fibroblast cells expressing huntingtin gene was not observed in cerebellum tissue a temperature-sensitive mutant p53 protein (alanine 135 (Figure 4a, left panel), which may be due to a tissue- to valine) (Wu and Levine, 1994). As shown in Figure 3a, specific difference in transcriptional regulation. Consis- upon a temperature shift from 39 to 321C, huntingtin tent with the increase of the huntingtin mRNA, the huntingtin protein levels increased by B2.4-fold in the mRNA levels increased in a time dependent manner and B reached B5-fold increased levels by 24 h, as measured by cortex and 3.2-fold in striatum tissues of the wild-type Northern blot analysis with a murine huntingtin specific mice 24 h postirradiation, as detected by Western blot probe. Similarly, the huntingtin protein levels increased in analysis with an anti-huntingtin antibody (Figure 4c), these cells and by 24 h they reached B4-fold increased whereas there were little change in huntingtin protein levels (Figure 3b). These results demonstrated that p53 levels in the age- and sex-matched isogenic p53 knock- induces huntingtin gene expression in cultured murine out mice (Figure 4d). The p53-dependent upregulation cells, as p53 does in human cells. of huntingtin gene by g-irradiation and its correlation To directly test whether a similar regulation occurs in with p21 transactivation in the striatum and cortex mouse brain tissues, both wild-type mice and the sex- strongly suggest that p53 can regulate huntingtin gene transcription in these tissues.

Discussion

Hungtington’s disease is one of the most prevalent neurodegenerative diseases caused by expansion of polyglutamine tract in disease-associated huntingtin protein. Although the huntingtin protein was discovered over 10 years ago, its function, regulation, and the mechanisms in pathogenesis are still largely unknown. However, accumulation of cytoplasmic and nuclear aggregates of the mutant huntingtin protein in patho- logical sites of the brain is believed to play a central role in the pathology of Huntington’s disease (DiFiglia et al., Figure 3 p53 activation increases huntingtin gene expression in 1997); and it has been suggested that both genetic and cultured murine cells. Vm10 cells were grown to 50% confluence at 391C and transferred to a 321C incubatorand culturedfor environmental stress factors could affect huntingtin gene indicated amount of time. Total RNA and proteins were extracted expression, which in turn could impact upon the onset from each sample. Northern blot analysis (a) orWesternblot and prognosis of Huntington’s disease (Georgiou et al., analysis (b) were performed to determine the levels of huntingtin 1999; Anca et al., 2004; Dixon et al., 2004). mRNA and protein. The mRNA levels of GAPDH and the protein levels of RAN were also determined as control. The data represent The tumor suppressor gene p53 encodes a transcrip- three independent experiments, and the error bar represents tion factor that represents a central integrator of various standard deviation. stress signals, such as cellular DNA damage, hypoxia,

Oncogene p53 regulates huntingtin Z Feng et al 5

Figure 4 Huntingtin gene expression is upregulated by g-irradiation in a p53-dependent manner in mouse cortex and striatum. p53 þ / þ or p53À/À mice were exposed to g-irradiation (IR, 6 Gy) and killed at 24 h after irradiation. Total RNA and proteins were extracted and from the cerebellum, cortex, and striatum. The levels of huntingtin mRNA and p21 mRNA were determined by quantitative real-time PCR and normalized against the levels of GAPDH. The levels of huntingtin protein were analyzed by Western blot assay and normalized against the levels of GAPDH. (a) huntingtin mRNA levels in p53 þ / þ and p53À/À mice; (b) p21 mRNA levels in p53 þ / þ and p53À/À mice. (c) and (d), Western blot analysis with anti-huntingtin antibody (left panels) and quantification of huntingtin protein levels (right panels) in p53 þ / þ and p53À/À mice, respectively. The data represent three independent experiments, and the error bar represents standard deviation. and oncogene activation (Levine, 1997; Vogelstein et al., activation by DNA damage increases huntingtin ex- 2000; Jin and Levine, 2001). In this study, we have pression in the striatum and cortex tissues of mouse, the identified multiple potential p53-REs in both mouse and two majorpathological sites forHuntington’s disease. human huntingtin genes. The interactions between As an important signal integrator, p53 responds to a human huntingtin p53 REs and p53 have been tested number of stress signals. It is expected that a variety of and they are capable of interacting with p53 protein environmental factors that can alter p53 activity could both in vitro and in cells. In both cultured murine and then increase the mutant huntingtin protein expression human cells, p53 activation is capable of increasing levels. An accumulation of mutant huntingtin is huntingtin gene expression. Moreover, p53 mediates the deleterious, therefore the activation of p53 would lead increasing of huntingtin gene expression in mouse to increased expression of mutant huntingtin, thus tissues after g-irradiation. This study revealed a novel increase toxicity and alter the time of onset and the regulation of the huntingtin gene expression at tran- developmental course of this disease. Since mutant scriptional level by the tumor suppressor p53. Although huntingtin can accumulate in the nucleus, where it may we only tested the expression regulation of the normal then bind to p53 (Steffan et al., 2000), it is not clear huntingtin gene by p53, it is expected that the mutant whether an altered response to p53 might be observed in huntingtin gene is regulated in a similar way by p53 neurons of Huntington’s disease. This will be of interest since the trinucleotide repeat expansion occurs in the to determine and could be performed readily in the cell coding region while not in the regulatory regions or p53 lines used forthis study. In addition, experiments and REs in the huntingtin gene. It is notable that p53 epidemiological analyses should now be carried out to

Oncogene p53 regulates huntingtin Z Feng et al 6 determine the impact of p53 activity (DNA damage, ForMdm2 promoterp53 RE site: 5 0-GGTTGACTCAGC hypoxia, chemical stresses, etc) upon the development of TTTTCCTCTTG-30 and 50-GGAAAATGCATGGTTTAAA Huntington’s disease in both Huntington’s disease TAGCC-30 (Jin et al., 2002). mouse models and in human patients. Electromobility shift assays Bacterially expressed 6 Â His-p53 protein was purified by Ni- Materials and methods NTA-agarose chromatography. Sequences of the linear, blunt oligonucleotides used were: GADD45, 50-GTAGCTGATATC GAATTCTCGAGCAGAACATGTCTAAGCATGCTGGGC Cells and cell culture 0 0 The human lung epithelial H1299 cells (American Type TCGAGAATTCCTGCAGCC-3 ; Huntingtin intron 2, 5 -CT Culture Collection, ATCC) were cultured in DMEM medium CGAGGCTGAGAGTTCATTATGCTTGTTCTACAGAA GAGCATGTTAAAAGGAGTTTTTGAGATCT-30; Huntingtin supplemented with 10% fetal bovine serum. H1299/V138 cells 0 (a generous gift from Jiandong Chen at H Lee Moffitt Cancer intron 3, 5 -CTCGAGCATGGTGCCATTTAGGGCCTGCT TCCAGTTAAGCTTGCTTCTCCACAGGCCTAAAGAT Center, Tampa, FL, USA), established by stably transfecting a 0 0 temperature-sensitive mutant form of p53 (alanine 138 to CT-3 ; mutant GADD45, 5 -GTAGCTGATATCGAATTCT valine) into H1299 cells (Pochampally et al., 1999), were CGAGCAGAAAATTTCTAAGAATTCTGGGCTCGAGA ATTCCTGCAGCG-30. DNA probes were labeled using cultured in DMEM medium supplemented with 10% fetal 32 bovine serum and 500 mg/ml G418. Human colon epithelial [g- P]ATP and T4 polynucleotide kinase. Reaction mixtures HCT116 p53 þ / þ , HCT116 p53À/À cells (generous gifts (20 ml) contained 20 mM HEPES (pH 7.9), 25 mM KCl, 0.1 mM from Bert Vogelstein at Johns Hopkins Medical Institutions, EDTA (pH 8.0), 2 mM MgCl2, 10% glycerol, 0.025% NP-40, 4mM spermidine, 2 mM DTT, 0.2 mg BSA, 25 Â mutant Baltimore, MD, USA) were cultured in McCoy’s 5A medium 32 supplemented with 10% fetal bovine serum (Bunz et al., 1998). GADD45 oligo, and 4 ng P-labeled oligonucleotide. After Vm10 cells, the murine fibroblast cells expressing a tempera- 30 min of incubation at room temperature, the reaction ture-sensitive mutant p53 protein (alanine 135 to valine), were mixtures were subjected to electrophoresis on a 4% poly- established in this lab as previously described (Wu and Levine, acrylamide gel (29:1 acrylamide/bisacrylamide) containing 1994), and were cultured in DMEM medium supplemented 0.5 Â Tris-borate-EDTA buffer at 165 V for 1.25 h at room with 10% fetal bovine serum and 500 mg/ml G418. temperature. The gel was dried and exposed to Kodax Biomax MR film. Mice and treatment C57BL/6 p53 knockout mice (4–6-week-old) (Lowe et al., Northern and Western blot analysis 1993) and age- and sex-matched C57BL/6 wild-type controls Standard Northern and Western blot analysis were used (Jackson Laboratories) were subject to 6 Gy of total body to analyse RNA and protein expression, respectively. irradiation with a 137Cs gamma source. Mice were killed at Anti-huntingtin (SC-8767), GAPDH (SC-20357) and RAN different time after irradiation and different tissues were (SC-1156) antibodies were purchused from Santa Cruz harvested for further experiments. Biotechnology (Santa Cruz, CA, USA). Anti-b-actin (A5441) was purchused from Sigma (Saint Louis, MO, USA). The Chromatin immunoprecipitation (ChIP) assay RNA and protein levels were quantified by digitalization of the ChIP assays were performed using Upstate ChIP Assay Kit X-ray film and analysed with Scion Image software (Scion (Lake Placid, NY, USA) according to the manufacturer’s Corporation, Maryland). instructions. In brief, cells were fixed with 1% formaldehyde for 10 min at room temperature. After washing with cold-PBS, Quantitative real-time PCR cells were lysed in SDS lysis buffer, and sonicated to shear Total RNA was prepared from cells or mouse brain tissues DNA to an average fragment size of 500 bp. Anti-p53 DO-1 with the RNeasy kit (Qiagen, Valencia, CA, USA) and treated antibody (SC-126, Santa Cruz, CA, USA) or normal mouse with the DNase I to remove residual genomic DNA. The IgG was added. Afterovernightincubation at 4 1C, immune cDNA was prepared with random primers using Taqman complexes were collected with salmon sperm DNA/protein A Reverse Transcription kit (Applied Biosystems, Foster agarose-50% sulrry (Upstate) for 1 h, and then extensively city, CA, USA). Real-time PCR was performed in triplicate washed. Samples were extracted with elution buffer (1% SDS, with Taqman PCR Mix (Applied Biosystems) for15 min 0.1 M NaHCO3), and heated at 651C overnight to reverse at 951C forinitial denaturing,followed by 40 cycles of crosslinks. DNA was purified and used for PCR. The primer 951C for30 s and 60 1C for30 s in the 7000 ABI sequence sets were designed to encompass the potential p53 RE sites in Detection System. Assay-on-demand forhuman huntingtin the human huntingtin gene. The sequences are as follows: (catalogue numberHs00169273_m1), human GAPDH Forpromoterp53 RE site: 5 0-GGGACTACAGGCATGCA (catalogue numberHs99999905_m1), mouse p21 (catalogue CCACTAC-30 and 50-AAAAATTAAGTTCCAGCGAGG numberMm00432448_m1), mouse GAPDH (catalogue TG-30 numberMm99999915_g1), mouse b-actin (catalogue number Forintron2 p53 RE site: 5 0-GCCTGGGTGACTGAGCG Mm00607939_s1) and assay-on-design formouse huntingtin AGAC-30 and 50-CTTGTTCTCCGTGCCTGCTGAT-30 (HD-EXN3-211888), were purchased from Applied Biosys- Forintron3 p53 RE site: 5 0-GGAAACAGCGATGAGCA tems. The expression of genes was normalized to expression of ATAAG-30 and 50-AATAGCTGTTAATGGTTTTCAC-30 housekeeping gene, GAPDH and b-actin.

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