TAp73 isoforms antagonize Notch signalling in ... - Wiley Online Library

Journal of Neurochemistry, 2006, 99, 989–999

doi:10.1111/j.1471-4159.2006.04142.x

TAp73 isoforms antagonize Notch signalling in SH-SY5Y neuroblastomas and in primary neurones Claudie Hooper,* Mahvash Tavassoli, J. Paul Chapple,* Dafe Uwanogho,* Richard Goodyear,à Gerry Melino,§,¶ Simon Lovestone* and Richard Killick* *King’s College London, MRC Centre for Neurodegenerative Research, Institute of Psychiatry, London, UK Cancer Gene Therapy Group, King’s College London, The Rayne Institute, London, UK àSchool of Biological Sciences, Sussex University, Falmer, East Sussex, UK §Biochemistry Laboratory, Instituto Dermopatico Immacolata, c/o Department of Experimental Medicine, University of Roma Tor Vergata, Italy ¶Medical Research Council, Toxicology Unit, Leicester, UK

Abstract p73, like Notch, has been implicated in neurodevelopment and in the maintenance of the mature central nervous system. In this study, by the use of reporter-gene assays, we demonstrate that C-promoter binding factor-1 (CBF-1)-dependent gene transcription driven by the Notch-1 intracellular domain (N1ICD) is potently antagonized by exogenously expressed transactivating (TA) p73 splice variants in SH-SY5Y neuroblastomas and in primary neurones. Time course analysis indicated that the inhibitory effects of TAp73 are direct and are not mediated via the product of a downstream target gene. We found that endogenous TAp73 stabilized by either c-Abl or cisplatin treatment also potently antagonized N1ICD/CBF-1dependent gene transcription. Furthermore, western blotting revealed that exogenous TAp73 suppressed endogenous hairy and enhancer of split-1 (HES-1) protein levels and antagonized the increase in HES-1 protein induced by exogenous N1ICD expression. Evidence of a direct physical

The p73 protein, identified by Caput and colleagues (Kaghad et al. 1997), is a transcription factor that shares a high sequence identity with the transactivation (TA) domain, DNA binding domain (DBD) and oligomerization domain (OD) of the tumour suppressor p53 and the third family member p63 (Irwin and Kaelin 2001; Yang et al. 2002). There are multiple C-terminal splice variants of p73, including p73a, b, c, d, e, j, f and F, which also exist as N-terminally truncated (DN) forms. DN isoforms lack part of the TA domain and act in a dominant negative fashion over TA isoforms (Grob et al. 2001; Ishimoto et al. 2002). Activation of the p53 family proteins involves either homo-

interaction between N1ICD and TAp73a was demonstrated by co-immunoprecipitation. Using Notch deletion constructs, we demonstrate that TAp73a binds the N1ICD in a region C-terminal of aa 2094. Interestingly, DNp73a and TAp73aR292H also co-purified with N1ICD, but neither inhibited N1ICD/CBF-1dependent transcription. This suggests that an intact transactivation (TA) domain and the ability to bind DNA are necessary for TAp73 to antagonize Notch signalling. Finally we found that TAp73a reversed the N1ICD-mediated repression of retinoic acid-induced differentiation of SH-SY5Y neuroblastomas, providing functional evidence for an inhibitory effect of TAp73a on notch signalling. Collectively, these findings may have ramifications for neurodevelopment, neurodegeneration and oncogenesis. Keywords: development, neurodegeneration, Notch, oncogenesis, p73, p53. J. Neurochem. (2006) 99, 989–999.

Received May 19, 2006; revised manuscript received July 12, 2006; accepted July 21, 2006. Address correspondence and reprint requests to Richard Killick, King’s College London, MRC Centre for Neurodegenerative Research, Institute of Psychiatry, De Crespigny Park, Denmark Hill, London, SE5 8AF, UK. E-mail: [email protected] Abbreviations used: CBF, C-promoter binding factor; DIC, days in culture; EGFF, enhanced green fluorescent protein; FBS, fetal bovine serum; HA, haemagglutinin; HEK293a, human embryonic kidney 293a cells; HERP, HES-related protein; HES, hairy and enhancer of split; DN, N-terminally truncated; N1ICD, Notch-1 intracellular domain; RA, retinoic acid; TA, transactivation domain; TACE, tumour necrosis factor a converting enzyme.

2006 The Authors Journal Compilation 2006 International Society for Neurochemistry, J. Neurochem. (2006) 99, 989–999

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or hetero-dimerization with either themselves or other family members. The dimers then combine to form active tetramers, which activate gene transcription by directly binding to specific DNA sequences within the promoters of target genes. p73 plays a pivotal role during development (Yang et al. 2000), in particular during the development of the nervous system (Meyer et al. 2002; Pan et al. 2003; Rentzsch et al. 2003; Abraham et al. 2004), and in neuronal maintenance and survival in the mature CNS (Pozniak et al. 2002; Walsh et al. 2004). Notch signalling regulates many aspects of invertebrate and vertebrate development, ranging from somite formation (Jiang et al. 1998) to epithelial–mesenchymal interactions (Weinmaster et al. 1991) and the determination of neural stem-cell fate (Yoon and Gaiano 2005). Notch signalling is also active in adult tissues, including the brain where it is involved in synaptic plasticity and memory (Costa et al. 2003; Wang et al. 2004). There are four mammalian Notch receptors (Notch 1–4), which undergo a series of sequential proteolytic cleavages. The first cleavage (S1) occurs in the Golgi by a furin-like convertase generating two fragments that remain non-covalently attached to form the mature Notch receptor at the cell surface. Ligands of the Jagged and Delta families, expressed on neighbouring cells, bind to the Notch receptor leading to the second cleavage (S2) by tumour necrosis factor a converting enzyme (TACE), which results in the shedding of the extracellular domain. The second cleavage is followed by a constitutive cleavage (S3) within the transmembrane domain of Notch by c-secretase. This cleavage releases the Notch intracellular domain (NICD), which migrates to the nucleus (Schroeter et al. 1998; Struhl and Adachi 1998) where it interacts with the C-promoter binding factor-1 (CBF-1), the mammalian homologue of Suppressor of hairless (Drosophila melanogaster) and Lag-1 (Caenorhabditis elegans), a transcription factor of the CSL family, also known as RBP-Jj (Tamura et al. 1995). In the absence of NICD CBF-1 forms a complex with repressor proteins, resulting in the suppression of gene transcription. Interaction with NICD displaces the repressor proteins and allows the entry of transcriptional activators such as p300/ Creb Binding Protein (CBP) and Mastermind, which convert the CBF-1 repressor complex into an activator of gene transcription (Kadesch 2000). Notch/CSL target genes include the hairy and enhancer of split (HES) family and the HES-related proteins (HERP/Hey) that encode basic helix-loop-helix transcription factors, which in turn suppress differentiation by antagonizing the expression of downstream lineage-specifying genes. Associations between Notch and the p53 family have been previously noted (Laws and Osborne 2004; Yang et al. 2004). p53 negatively regulates Notch-1 activation during Tcell development (Laws and Osborne 2004), and p63 exerts an inhibitory effect on the involvement of Notch in the balance between keratinocyte self-renewal and differentiation

(Nguyen et al. 2006). Furthermore, TAp73 over-expression promotes neuronal differentiation and neurite growth (De Laurenzi et al. 2000), as well as oligodendrocyte differentiation (Billon et al. 2004), two processes regulated, at least in part, by Notch (Wang et al. 1998; Berezovska et al. 1999; Park et al. 2005). These findings suggested to us that p73 might be a negative regulator of Notch signalling, particularly within nervous tissues. Materials and methods Cell lines, antibodies and plasmid constructs SH-SY5Y neuroblastoma cells and Saos-2 cells (a p53–/– human osteosarcoma cell line) were obtained from the European Collection of Cell Cultires (ECACC) (Health Protection Agency, Porton Down, Salisbury, Wiltshire, UK). Human embryonic kidney 293a cells (HEK293a; an adherent clone of HEK293 cells) were purchased from Quantum Biotechnologies (Montreal, QC, Canada). Hemagglutinin (HA)-tagged tetracycline-inducible TAp73c Saos-2 cells have been previously described (Melino et al. 2004). Anti-HA monoclonal agarose conjugate was from Sigma-Aldrich (Gillingham, Dorset, UK). Mouse anti-HA was from Roche (Welwyn Garden City, UK). Rabbit anti-p73 was a kind gift of Dr A. Emre Sayan (MRC, Leicester, UK). Mouse anti-myc was from Cell Signaling Technology (Herts, UK). Rabbit anti-HES-1 was from Chemicon (Hampshire, UK). Rabbit antitotal tau was from Dakocytomation (Cambridgeshire, UK). Goat anti-Notch-1, rabbit anti-p300, goat anti-mouse IgG, goat anti-rabbit IgG and donkey anti-goat IgG were from Santa Cruz Biotechnology (Santa Cruz, CA, USA). The myc-His tagged human N1ICD, DTAD-N1ICD and DRA-N1ICD constructs were generous gifts from Dr T. Kadesch (University of Pennsylvania, Philadelphia, PA, USA). All the cDNA expression constructs encoding the p73 isoforms have been previously described (Grob et al. 2001). TAp63a was a generous gift from Dr K. Engeland (Leipzig, Germany). p53 was from Dr B. Vogelstein (John Hopkins University, Baltimore, MD, USA). The Notch-CBF-1 reporter, 4xwtCBF-1-Luc, which contains four tandem repeats of the consensus CBF-1 DNA binding sequence, GTGGGAA, and the mutated control 4xmut-CBF–Luc, with four tandem repeats of CTTGGAA, were generous gifts from Dr G. Weinmaster (UCLA Medical School, Los Angeles, CA, USA). The reporters containing the region from )2500 to +43 bp of the HES-1 promoter (HES-1-Luc) and from )800 to +73 bp of HES-5 (HES-5-Luc) were from Dr R. Kageyama (Kyoto, Japan). The reporter containing from )2839 to +87 bp of the Hey-1 promoter was from Professor M. Gessler (Universitat Wurzburg, Germany). BAX-Luc and p21waf-Luc were from Dr T. Soussi (Institut Curie, Paris, France). 14xp53-Luc containing 14 tandemly arranged repeats of the p53 responsive element (TGCCTGGACTTGCCTGG) was from Stratagene (Amsterdam, The Netherlands). p300 was from Dr H. Lu (Oregon Health & Science University, Portland, OR, USA). p300 siRNA was from Dharmacon (Northumberland, UK). C-Abl was a gift from Dr R. van Etten (Boston, USA). Cell culture All cells were maintained in 5% CO2 in air in a humidified incubator at 37C. HEK293a and Saos-2 cells were cultured in low glucose Dulbecco’s modified Eagle’s medium (DMEM) – all culture media


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and supplements were obtained from Invitrogen (Paisley, UK) unless otherwise stated – containing 10% (v/v) fetal bovine serum (FBS) (Autogen Bioclear, Wiltshire, UK), 2 mM L-glutamine, 100 IU penicillin and 100 mg/mL streptomycin. SH-SY5Y neuroblastomas were grown in a 50/50 mix of F12/EMEM supplemented with 15% (v/v) FBS, 2 mM L-glutamine, 100 IU penicillin and 100 mg/mL streptomycin. SH-SY5Y neuroblastomas were differentiated by the addition of retinoic acid 1 lM (Sigma-Aldrich) to culture medium supplemented as above, but containing 0.5% (v/v) FBS. Saos-2 cells stably transfected with an HA-tagged tetracycline inducible TAp73c construct were grown in RPMI media supplemented with 10% (v/v) tetracycline-free FBS, geneticin 500 lg/mL (Sigma-Aldrich), hygromycin B 0.25 mg/mL (Sigma-Aldrich), 2 mM L-glutamine, 100 IU penicillin and 100 mg/mL streptomycin. TAp73c expression was induced by the addition of 2.5 lg/mL of doxycycline to the culture medium. Rat primary neurones were prepared and cultured in Neurobasal media plus B27 supplement as previously described (Theuns et al. 2003). Luciferase assays Cells were transfected with 400 ng of firefly luciferase-based reporter DNA (4x-wtCBF-1-luc, 4x-mutCBF-Luc, Hey-1-Luc, HES-1-Luc, HES-5-Luc, 14xp53-Luc, BAX-Luc or p21waf-Luc) and 800 ng of each plasmid containing the cDNA constructs being examined using FuGene 6 according to the manufacturer’s instructions (Roche). To control for transfection efficiency, 50 ng of phTKRenilla luciferase (Promega, Southampton, UK) was also included in transfections. Empty vector DNA was included where necessary to maintain constant DNA concentrations. The medium was removed from the cells 24 h post transfection and the firefly and Renilla luciferase activities were sequentially measured using DualGlo reagents (Promega) in a Wallac Trilux 1450 Luminometer (Perkin Elmer, Buckinghamshire, UK). Firefly values were divided by the Renilla value from the same well to control for non-specific effects. Data for each set of four replica transfections was averaged, the control in each set normalized to 1 and data presented as fold increases over control. All experiments were performed in triplicate. Preparation of cell lysates, immunoprecipitation and western blotting Cell lysates were harvested in RIPA buffer [150 mM NaCl, 50 mM Tris, 1% NP40, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate (SDS), 1 mM benzamidine, 4 lg/mL leupeptin, 0.1% bmercaptoethanol, pH 7.4] either 24 h after transfection with the appropriate cDNA (1 lg) using FuGene 6 or after an 8-h treatment with cisplatin (25 lM) in the case of Saos-2 cells. For immunoprecipitations HEK293a cells were transfected with N1ICD, myc-His tagged DTAD-N1ICD or myc-His tagged DRA-N1ICD (4 lg of each) either independently or in combination with either HA-tagged TAp73a or DNp73 (4 lg of each) using FuGene 6. Cells were harvested in RIPA buffer 24 h after transfection and then an anti-HA antibody conjugated to agarose (25 lL) was incubated with the lysates for 2 h at 4C with rotation to capture interacting proteins. Western blotting was performed by standard protocols using mouse anti-HA (1 : 2000), rabbit anti-p73 (1 : 1000), goat anti-Notch-1 (1 : 1000), mouse anti-myc (1 : 5000) or rabbit anti-HES-1 (1 : 1000) followed by incubation with the corresponding secondary antibody conjugated to horseradish peroxidase (HRP; 1 : 2000).

Proteins were visualized using enhanced chemiluminescence reagents (Amersham Pharmacia, Buckinghamshire, UK). To ensure equal protein loading, membranes were re-probed with mouse antib-actin (1 : 1000) where appropriate. All experiments were performed in triplicate. Figures shown are representative images from a single experiment. Nucleofection SH-SY5Y neuroblastomas were nucleofected following Amaxa’s optimization protocol. For experiments 1 million cells were nucleofected with 5 lg of N1ICD and/or TAp73a using solution V and programme A-23 for high cell viability. Empty vector DNA was included where necessary to maintain constant DNA concentrations. Confocal microscopy SH-SY5Y neuroblastomas were nucleofected with N1ICD and/or TAp73a in duplicate as described above. The following day the SHSY5Y cells were treated with retinoic acid (1 lM). Cells were subsequently fixed in ice-cold methanol after either 2 or 6 days in culture and then stained according to standard protocols. Briefly, cells were incubated with rabbit anti-total tau (1 : 500), mouse anti-HA (1 : 250) or goat anti-Notch-1 (1 : 250) before being incubated with the appropriate fluorescent secondary antibody (1 : 200). Nuclei were counter-stained with Hoescht 33342. Immunofluorescence was visualized and captured using a Zeiss LSM510 meta-confocal microscope (Zeiss, Hertfordshire, UK). Images were processed using LSM5 image examiner (Zeiss). All experiments were performed in triplicate. Figures shown are representative of a single experiment.

Results

TAp73a inhibits N1ICD-dependent transcription Given the connections between TAp73 and the Notch signalling pathway we asked whether TAp73 might directly modulate Notch signalling at the level of NICD/CBF-1dependent gene transcription. To investigate this we used a CBF-1 luciferase reporter gene, 4xwt-CBF–Luc, and its mutated control, 4xmut-CBF–Luc. These reporters were transfected into SH-SY5Y neuroblastomas singly and in combination with DNA constructs encoding the intracellular domain of Notch-1 (N1ICD) and HA-tagged full-length p73 (TAp73a) as indicated in Fig. 1(a). Singly transfected N1ICD activated 4xwt-CBF-1-Luc by approximately 80-fold in SHSY5Y neuroblastomas, whilst singly transfected TAp73a had a negligible effect on reporter activity (Fig. 1a). In combination, TAp73a consistently antagonized reporter activation resulting from N1ICD by over 60% (a 30-fold reduction in activation). Furthermore, neither N1ICD nor TAp73a affected transcription from the control reporter, 4xmut-CBF–Luc (Fig. 1b), which provides evidence that the response is specific. We explored the antagonistic effects of TAp73a on NICD/ CBF-1-dependent transcription further by performing a dose–response study of the effect of TAp73a cDNA on the N1ICD-induced activation of the 4xwt-CBF-1-Luc reporter



Fig. 1 TAp73a inhibits Notch-1 intracellular domain (N1ICD)/ C-promoter binding factor-1 (CBF-1)-dependent transcription. Panels (a) and (b) show that TAp73a inhibits the N1ICD activation of a CBF-1 reporter. (a) Either the CBF-1 reporter (4xwt-CBF-Luc) or (b) the mutated control reporter (4xmut-CBF-Luc) was transfected into SHSY5Y neuroblastomas with empty vector, N1ICD or TAp73a cDNA alone, or in combination as indicated. Cells were lysed and luciferase activities were determined 24 h post transfection. Data are presented as -fold increases in luciferase activity over control (as is the case for all other reporter experiments unless otherwise indicated). (c) The dose–response of TAp73a antagonism of N1ICD/CBF-1-dependent transcription. SH-SY5Y neuroblastomas were transfected with CBFLuc with empty vector, N1ICD or TAp73a alone, or with a constant quantity of N1ICD and increasing doses of TAp73a cDNA. The luciferase activity was determined 24 h post transfection. (d) Western blot analysis of N1ICD expression in the presence and absence of TAp73a. SH-SY5Y cells were transfected with empty vector, N1ICD or N1ICD

and TAp73a in combination. In all three transfections a constant quantity of an enhanced green fluorescent protein (EGFP) expression construct was also included. Cell lysates were harvested, blotted and probed with antibodies to the C-terminus of Notch-1 (top panel), bactin (middle panel) or EGFP (bottom panel) 24 h post transfection. (e) Time course of TAp73c expression in an inducible Saos-2 cell line. Cells were treated with 2.5 lg/mL doxycycline and at the time points indicated lysates were prepared and the levels of TAp73c protein were determined by western blotting with an anti-haemagglutinin (anti-HA) antibody. (f) Doxycyline treatment of TAp73c inducible Saos-2 cells leads to the suppression of N1ICD-induced transcription from CBF-Luc. Inducible TAp73c Saos-2 cells were transfected with CBF-Luc and either empty vector or N1ICD in the presence of doxycyline (2.5 lg/ mL). At the indicated time points cells were lysed and luciferase activities were determined. (g) Doxycyline treatment of TAp73c inducible Saos-2 cells activates 14xp53-Luc. Details are the same as for panel (f), but performed with 14xp53-Luc.



(from here on referred to as CBF–Luc). When transfecting 200 ng of N1ICD cDNA per well, as little as 50 ng of TAp73a cDNA was found to inhibit the activation of the reporter in SH-SY5Y cells (Fig. 1c). A maximal effect was achieved with 300 ng of TAp73a cDNA, which reduced reporter activity by more than 75%. None of these doses of TAp73a affected reporter activity in the absence of exogenously expressed N1ICD (data not shown), and neither N1ICD nor TAp73a (at all doses) affected transcription from the control reporter, 4xmut-CBF–Luc (data not shown). To ensure that the reduction observed in Notch signalling was neither a result of lowered transfection efficiency nor a result of a reduction in protein expression, possibly caused by TAp73a-induced cell-cycle arrest (Melino et al. 2002), SH-SY5Y neuroblastomas were transfected with N1ICD alone and in conjunction with TAp73a, and N1ICD protein levels were examined by western blotting. The presence of TAp73a neither affected N1ICD protein levels nor the expression of a non-specific control for protein expression, enhanced green fluorescent protein (EGFP) (Fig. 1d). Furthermore, the antagonistic effects of TAp73a on N1ICD/CBF1-dependent transcription were not attributable to increased apoptosis, as demonstrated using anti-caspase-3 and Hoechst 33342 staining (data not shown). In order to determine if the inhibition of N1ICD/CBF-1dependent transcription by TAp73a was direct, a time course was performed using an inducible TAp73c Saos-2 cell line. Cells were treated with 2.5 lg/mL doxycycline and at various time points after treatment the levels of induced TAp73c protein were determined by western blotting. HAtagged TAp73c was undetectable in lysates from untreated cells, but became detectable 3 h after doxycycline treatment and the expression of TAp73c increased with time (Fig. 1e). Reporter assays demonstrated that doxycycline-induced TAp73c antagonized N1ICD-dependent CBF-Luc activity after 2 h (Fig. 1f). Activation was antagonized further as the duration of doxycycline treatment increased. In a parallel experiment, TAp73c was found to induce the activation of 14xp53-Luc after 2 h and luciferase activity increased with time (Fig. 1g). The lack of delay between the p73-induced inhibition of N1ICD-dependent transcription and p73-induced activation of 14xp53-Luc, suggests that the inhibitory effects of TAp73c on N1ICD are direct and are not attributable to the expression of a p73 target gene. Again the antagonistic effects of TAp73 on N1ICD-dependent transcription over the time course examined were not attributable to apoptosis as demonstrated by anti-caspase-3 and Hoechst 33342 staining (data not shown). However, Saos-2 cells are sensitive to TAp73c-induced apoptosis (Melino et al. 2004). TAp73 isoforms, but not DNp73, antagonize N1ICD Considering that TAp73a and TAp73c (as demonstrated using Saos-2 cells harbouring a tetracycline-inducible HAtagged TAp73c construct) both inhibited Notch signalling,

we examined the effects of several other p73 isoforms (TAp73b, c, d, e, DNp73a and a transcriptionally inactive mutant form of p73a, TAp73aR292H, harbouring an arginine to histidine subsitution at amino acid 292 within the DNA binding domain) on N1ICD/CBF-1-dependent gene transcription. Schematics of the p73 isoforms and the two alternate p73 gene promoter start sites are shown in Fig. 2(a and b), respectively. Initially we tested the transcriptional effects of the isoforms on Bax-Luc and p21waf1-Luc in SH-SY5Y neuroblastomas. Consistent with previous reports (Jost et al. 1997; Kaghad et al. 1997; De Laurenzi et al. 1998; Flores et al. 2002) TAp73a, b, c, d and e activated the reporters to varying degrees, whereas DNp73a and TAp73aR292H did not activate either reporter (Fig. 2c). The N1ICD-mediated activation of the CBF-Luc reporter was antagonized by all of the TAp73 isoforms, but neither by DNp73 nor TAp73aR292H, in SH-SY5Y cells (Fig. 2d), in Saos-2 cells (Fig. 2e) and in HEK293a cells (data not shown). The effects of TAp73a, TAp73aR292H and DNp73 on N1ICD/CBF-1-dependent transcription in rat primary cortical neurones were also examined (Fig. 2f). Despite the low transfection efficiency of primary neurones N1ICD activated CBF–Luc, but the activation was lower than in the cell lines examined. TAp73a again antagonized N1ICD, whereas DNp73 and TAp73aR292H exerted no inhibitory effects. We proceeded to examine whether the related family members p53 and TAp63a might also modulate N1ICD-dependent transcription. Indeed when co-transfected with N1ICD, both p53 and TAp63a antagonized N1ICD-induced reporter activity by > 80% in SH-SY5Y neuroblastomas (Fig. 2g) and in HEK293a cells (data not shown). Endogenous TAp73 antagonizes N1ICD/CBF-1dependent transcription Next we examined whether endogenous TAp73 could antagonize Notch signalling. To do this we either transfected the p53–/– cell line, Saos-2, with the non-receptor tyrosine kinase c-Abl or treated the cells with 25 lM cisplatin for 8 h, both of which have been shown to stabilize endogenous p73 (Agami et al. 1999; Gong et al. 1999). Western blotting demonstrated that either exogenously expressed c-Abl or treatment with cisplatin both markedly increased endogenous TAp73 protein levels (Fig. 3a). Both treatments also reduced the N1ICD-induced activation of CBF–Luc to a similar extent as exogenously expressed TAp73a in Saos-2 cells (Fig. 3b), but had no effect on the control reporter (data not shown). TAp73 isoforms antagonize the transcription of the Notch target genes Hey-1, HES-1 and HES-5 The effect of p73 on the Notch-induced activation of reporters containing luciferase under the transcriptional control of the Hey-1, HES-1 and HES-5 promoters was subsequently investigated. The reporters, Hey-1-Luc, HES-1Luc and HES-5-Luc, were transfected into SH-SY5Y



Fig. 2 Transactivation (TA) domain, but not N-terminally truncated (DN), isoforms of p73 antagonize Notch-1 intracellular domain (N1ICD)/ C-promoter binding factor (CBF-1) transcriptional activity. (a) Schematic representation of the p53 family showing the alternatively spliced isoforms of p73. TAD, transactivation domain; DBD, DNA-binding domain; OD, oligomerization domain. (b) Schematic representation of the p73 gene showing alternative promoter sites. The 14 exons are numbered. (c) SH-SY5Y neuroblastomas were transfected with either BAX-Luc or p21-Luc with empty vector, p73a, b, c, d, e, DNp73a or TAp73aR292H as indicated. Cells were lysed and luciferase activities were determined 24 h post transfection. (d) The N1ICD-induced acti-

vation of CBF-Luc was antagonized by all TAp73 isoforms, but neither by DNp73 nor TAp73aR292H in SH-SY5Y cells. The CBF-1 reporter was transfected into SH-SY5Y cells with empty vector, N1ICD, TAp73a, b, c, d, e, DNp73a or TAp73aR292H alone, or in combination as indicated. Cells were lysed and luciferase activities were determined 24 h post transfection. (e) Details are the same as for panel (d), but performed in Saos-2 cells. (f) Primary mouse neurones were transfected as in panel (c), but only the TAp73a and DNp73a isoforms were compared. (g) SH-SY5Y cells were transfected as in panel (c), but the effects of p53 and TAp63a on the N1ICD-induced transcription from CBF-Luc was investigated.



Fig. 3 Endogenous p73 antagonizes Notch-1 intracellular domain (N1ICD)/ C-promoter binding factor (CBF-1)-dependent transcription. Panels (a) and (b) show that endogenous p73 proteins antagonize the N1ICD/CBF-1-dependent transcription in p53–/– Saos-2 cells. (a) Western analysis of Saos-2 cells either transfected with c-Abl cDNA or treated with 25 lM cisplatin for 8 h. Saos-2 cell lysates were collected and endogenous p73 proteins detected using an anti-p73 antibody. (b) Saos-2 cells were transfected with CBF-Luc with either empty vector or N1ICD in the presence of c-Abl cDNA, cisplatin or TAp73a cDNA as indicated. Cells were lysed and luciferase activity was measured 24 h post transfection.

neuroblastomas. N1ICD increased the activity of Hey-1-Luc by 7-fold (Fig. 4a), the activity of HES-1-Luc by 12-fold (Fig. 4b) and the activity of HES-5-Luc by 14-fold (Fig. 4c). As seen with the CBF-Luc reporter, all TAp73 isoforms as well as p53 and TAp63a antagonized the N1ICDinduced Hey-1-Luc, HES-1-Luc and HES-5-Luc activity, whereas DNp73 and TAp73aR292H had no effect on N1ICDdependent transcription. The induction of TAp73c expression in the inducible Saos-2 cell line by doxycyline treatment was also found to inhibit N1ICD-induced Hey-1, HES-1 and HES5 reporter activity (data not shown). Furthermore, western blotting revealed that TAp73a suppressed endogenous HES1 protein expression in SH-SY5Y neuroblastomas, and antagonized the increase in HES-1 expression induced by N1ICD (Fig. 4d). TAp73a and N1ICD co-precipitate, which is indicative of a direct physical interaction Given that TAp73 inhibits N1ICD-dependent transcription, we performed co-immunoprecipitation experiments to determine if the two proteins directly interact. HEK293a cells were cotransfected with myc-His tagged N1ICD and either TAp73aHA or DNp73a-HA either singly or in combination (Fig. 5a). A monoclonal anti-HA antibody/agarose conjungate was used to immunoprecipitate the HA-tagged proteins from cell lysates. Immunoprecipitates were then probed with the antiNotch-1 antibody following western blotting (Fig. 5a, upper panel). To demonstrate that TAp73a was equally expressed,

Fig. 4 TAp73 isoforms inhibit the Notch-1 intracellular domain (N1ICD)-induced transcription of Hey-1, hairy and enhancer of split -1 (HES-1) and HES-5. SH-SY5Y neuroblastomas were transfected with Hey-1-Luc (a), HES-1-Luc (b) or HES-5-Luc (c) with empty vector, N1ICD, the p73 isoforms, p53 or TAp63a alone, or in combination as indicated. Cells were lysed and luciferase activity was determined 24 h post transfection. (d) Western analysis of SH-SY5Y neuroblastomas transfected with empty vector, N1ICD or TAp73a alone, or N1ICD and TAp73a in combination. Lysates were collected and probed for endogenous HES-1 protein.



Fig. 5 p73a and DNp73 bind a region C-terminal of aa 2093 in the Notch-1 intracellular domain (N1ICD). (a) TAp73a and DNp73a both coprecipitate with N1ICD. Human embryonic kidney 293a (HEK293a) cells were transfected with N1ICD (lane 1), TAp73a-haemagglutinin (HA) (lane 2), DNp73-HA (lane 3), with N1ICD and TAp73a-HA (lane 4) or with N1ICD and DNp73a-HA (lane 5) in combination. Cells were lysed and the HA-tagged p73 proteins were immunoprecipitated using an HA-antibody/agarose conjugate 24 h post transfection. Precipitated proteins were probed using an anti-Notch antibody (upper panel). To ensure the presence of the HA-tagged p73 isoforms in the immunoprecipitates, membranes were reprobed with an anti-HA antibody (lower panel). (b) Schematic diagram showing N1ICD and the deletion constructs, DRA-N1ICD and DTAD-N1ICD. Top, the intact S3 cleaved N1ICD in which the major recognized domains are depicted; the RAM23 (RAM) domain; the seven Ankyrin repeats; three nuclear localization signals (NLS), red rectangles; TAD, the transactivation domain, within which lies the serine/threonine-rich region (STR); the opposite paired (OPA) sequence; and the PEST (proline, glutamate, serine, threonine-rich) sequence. Below are depicted DTAD-N1ICD, which terminates at aa residue 2093 and DRA-N1ICD, which starts at aa 2094 and ends at 2556. (c) A region of the N1ICD C-terminal of aa 2093 binds p73. HEK293a cells were transfected with DRA-N1ICD (lane 1), DTAD-N1ICD (lane 2), TAp73a-HA (lane 3), DRA-N1ICD and TAp73a-HA (lane 4) or DTAD-N1ICD and TAp73a-HA (lane 5) in combination and immunoprecipitated as detailed in panel (a). Membranes were probed using an anti-myc antibody (upper panel) then an anti-HA antibody (lower panel). DRA-N1ICD but not DTAD-N1ICD co-precipitated with TAp73a (lane 4).

blots were re-probed with a monoclonal anti-HA antibody (Fig. 5a, lower panel). Endogenous Notch-1 is expressed at low levels in HEK293a cells and no endogenous N1ICD was detected in immunoprecipitates from cells transfected with TAp73a only (Fig. 5a, lanes 2 and 3). Exogenous N1ICD was not detected in immunoprecipitates in the absence of TAp73a (Fig. 5a, lane 1), but was detected when co-expressed with TAp73a (Fig. 5a, lane 4), DNp73a-HA (Fig. 5a, lane 5) or TAp73aR292H (data not shown). Confocal microscopy revealed that exogenously expressed N1ICD, TAp73a-HA, DNp73a-HA and TAp73aR292H were all found in the nucleus in both HEK293a cells and in SH-SY5Y neuroblastomas (data not shown). Collectively, these findings indicate that TAp73a, DNp73a and TAp73aR292H all bind N1ICD either directly or as part of a larger protein complex. Moreover, the TA domain of p73, although not directly involved in binding, is required for the antagonism of N1ICD/CBF-1-dependent transcription, as is the ability of TAp73 to bind DNA. To investigate which regions of N1ICD interact with p73, immunoprecipitations were performed using myc-His tagged DTAD-N1ICD and DRA-N1ICD, which are N1ICD deletion constructs. DTAD-N1ICD runs from aa 1760 to aa 2093 and lacks the transactivation, the opposite paired (OPA) and the proline, glutamine, serine, threonine rich region (PEST) domains. DRA-N1ICD extends from aa 2094–aa 2556 and lacks the RAM23 (RAM) domain and the Ankyrin repeats (Fig. 5b). These N1ICD deletion constructs were transfected alone and in combination with TAp73a-HA, and immunoprecipitations were performed using the anti-HA agarose conjugate. DRA-N1ICD (Fig. 5c, lane 4), but not DTADN1ICD (Fig. 5c, lane 5), was found to co-precipitate with TAp73a-HA, which implies that neither the RAM domain nor the Ankyrin repeats are involved in binding with TAp73, but that a region C-terminal of aa 2094 either within the TA domain or beyond is required. TAp73a represses the N1ICD-mediated inhibition of retinoic acid-induced differentiation of SH-SY5Y neuroblastomas To assess whether TAp73 inhibits Notch activity at a functional level, the effects of TAp73 on the N1ICD-mediated suppression of retinoic acid (RA)-induced differentiation of SH-SY5Y neuroblastomas was investigated. SH-SY5Y cells cultured in normal medium in the absence of RA exhibited a rounded morphology and low tau immunoreactivity (Fig. 6a). RA evoked the differentiation of SH-SY5Y neuroblastomas after 6 days in culture as evidenced by the presence of long neurites and the increased expression of tau (Fig. 6b). However, SH-SY5Y cells nucleofected with N1ICD were resistant to RA-induced differentiation, and cells maintained a more rounded morphology with less pronounced tau immunoreactivity (Fig. 6c). SH-SY5Y cells nucleofected with TAp73a then treated with RA possessed a differentiated phenotype similar to that observed with RA



Fig. 6 TAp73a represses the Notch-1 intracellular domain (N1ICD)mediated inhibition of retinoic acid (RA)-induced differentiation of SHSY5Y neuroblastomas. (a) SH-SY5Y cells cultured in normal medium in the absence of RA exhibited a rounded morphology and weak tau immunoreactivity after 6 days in culture as evidenced using anti-total tau. In all cases nuclei were counterstained with Hoechst 3342. (b) RA evoked the differentiation of SH-SY5Y neuroblastomas after 6 days in culture (DIC) as demonstrated using anti-total tau. (c) Conversely, SHSY5Y cells nucleofected with N1ICD then treated with RA maintained a

more rounded morphology with weak tau immunoreactivity. (d) SHSY5Y cells nucleofected with TAp73a then treated with RA possessed a differentiated phenotype exemplified by strong tau immunoreactivity. (e) Following co-transfection with TAp73a and N1ICD, SH-SY5Y cells exhibited a neuronal-like morphology and exhibited extensive tau immunoreactivity. N1ICD exhibits a diffuse nuclear staining pattern in SHSY5Y neuroblastomas (f), as does TAp73a (g) when expressed independently (f and g) and in combination (h) after 2 days in culture. The scale bar in panel (h) represents 20 lm.

treatment alone (Fig. 6d). Moreover, TAp73a when conucleofected with N1ICD reversed the N1ICD-dependent inhibition of RA-induced differentiation (Fig. 6e). Following co-transfection with TAp73a and N1ICD, SH-SY5Y cells exhibited a neuronal-like morphology and exhibited extensive tau immunoreactivity. Immune staining demonstrated that approximately 50% of the SH-SY5Y cells expressed TAp73a and/or N1ICD 2 days after nucleofection (Figs 6f–h). By 6 days post-nucleofection, the expression of TAp73a and N1ICD was undetectable by immune-staining, which was attributed to the transient nature of the transfection (data not shown). Collectively, these findings provide functional evidence that TAp73 antagonizes Notch signalling.

not antagonize N1ICD. The most parsimonious explanation for these observations is that the transcriptionally active isoforms of TAp73 antagonize N1ICD/CBF-dependent transcription via their ability to increase the expression of an inhibitory factor. However, time course analyses indicated that the effects of TAp73 upon N1ICD are direct, as no lag between the N1ICD activation of CBF-Luc and the inhibition of N1ICD-dependent transcription was observed. In support of this TAp73a and N1ICD appear to directly interact, as demonstrated by immunoprecipitiation. Interestingly, DNp73a and TAp73aR292H were also found to co-purify with N1ICD, but did not inhibit N1ICD-dependent transcription. This suggests that an intact TA domain and the ability to bind DNA are required for TAp73 to antagonize Notch signalling. Thus, either TAp73 binds N1ICD directly and disrupts the activation of the CBF-1 repressor complex, or TAp73 binds N1ICD and recruits other protein(s), possibly via the TA domain, into the N1ICD/CBF-1/TAp73 complex, which in turn serves to inhibit N1ICD-dependent transcription. Both p73 and N1ICD interact with the histone acetylase p300 (Zeng et al. 2000; Oswald et al. 2001), raising the possibility that TAp73-induced inhibition of N1ICD signalling might be mediated by this common binding partner. However p300 was undetectable in TAp73a/N1ICD immuno-precipitates, as evidenced by western blotting (data not shown).

Discussion

Our findings demonstrate that exogenous and endogenous TAp73 antagonizes N1ICD/CBF-1-dependent gene transcription, and at a functional level TAp73 reverses the N1ICD-dependent inhibition of SH-SY5Y neuroblastoma differentiation. All the TA-containing p73 isoforms examined, TAp73a, b, c, d and e, inhibited N1ICD/CBF-1dependent transcription, whereas a dominant negative isoform, DNp73a, which lacks the TA domain, and the TAp73aR292H mutant, which is unable to bind DNA, did



Furthermore, neither exogenously expressed p300 nor small interfering RNA raised against p300 had any measurable effects on the ability of TAp73a to antagonize the N1ICDinduced activation of CBF-Luc (data not shown). This indicates that the effect of TAp73a on N1ICD/CBF-1dependent transcription is independent of p300. The p73/Notch interaction requires a region of the N1ICD C-terminal of aa residue 2093. This is the same region with which other repressors of Notch signalling, such as Dishevelled (Axelrod et al. 1996) and Numb (Zhong et al. 1996), interact. The segment of p73 that binds to N1ICD lies in a region common to all the TAp73 isoforms examined. It neither lies in a portion of the TA domain absent from DNp73a nor in a region C-terminal of the oligomerization domain, because TAp73d, which lacks exons 11–14, inhibits N1ICD/CBF-1-dependent transcription. Thus it is likely to be part of the ‘core’ region of p73, encompassing the DNA binding domain and the oligomerization domain or flanking sequences involved in N1ICD binding. Consistent with this, p53 and TAp63a, which share these domains, also exerted an inhibitory effect on Notch signalling; the former finding is in accord with a previous report (Oswald et al. 2001). Notch and p73 are both implicated in CNS development and maintenance. TAp73 over-expression promotes neuronal differentiation and neurite growth (De Laurenzi et al. 2000), as well as oligodendrocyte differentiation (Billon et al. 2004), which could be explained by the inhibition of Notch signalling. Interestingly, TAp73 has been shown to upregulate Jagged-1 and -2 transcription (Sasaki et al. 2002). Thus, TAp73 might serve to suppress Notch signalling in the ‘receiving’ cell, but enhance Notch activity in neighbouring cells, thereby facilitating the process of lateral inhibition and cell fate decisions. Recent studies using Notch-1 antisense mice (Wang et al. 2004) and mice carrying a heterozygous null mutation of the Notch-1 gene (Costa et al. 2003) have highlighted a role for Notch-1 in spatial learning, long-term potentiation and memory formation. A conditional presenilin double knockout mouse also displays impairments in synaptic plasticity and memory (Saura et al. 2004), which might be attributable to a reduction in Notch signalling. It is feasible then that the antagonism of Notch by p73 might play a role in the suppression of long-term memory formation under physiological conditions. Moreover, the pathologically induced stabilization of p73 proteins, which occurs following amyloid-b (Ab) treatment through the activation of c-Abl (Alvarez et al. 2004), might contribute to long-term memory impairment, a characteristic of Alzheimer’s disease. In support of this, p73 is aberrantly expressed in Alzheimer’s where it is found in the nuclei of hippocampal neurones in contrast to the cytoplasmic distribution seen in normal brain (Wilson et al. 2004). The inhibition of N1ICD transcriptional activity by TAp73 may also play a positive role in Notch-dependent oncogen-

esis. In human acute lymphoblastic T-cell leukaemia, a chromosomal translocation gives rise to a constitutively activated version of Notch-1 (Ellisen et al. 1991). TAp73 is a known tumour suppressor, which acts by inducing G1/S cellcycle arrest and/or apoptosis in a variety of cancerous cells (Melino et al. 2002). Thus, antagonism of Notch signalling by TAp73 may confer a novel mode by which this protein, and indeed other family members, combat neoplastic transformation. In summary, our findings demonstrate that p73 physically interacts with N1ICD and acts to inhibit N1ICD-dependent gene transcription, as demonstrated by reporter assays and western blotting for HES-1 protein. TAp73 was also shown to inhibit the N1ICD-dependent repression of SH-SY5Y differentiation, providing functional evidence for an antagonistic effect of TAp73 on N1ICD-induced gene transcription. Further investigations of the interactions between p73 and N1ICD might increase our understanding of neurodevelopment, neurodegeneration and oncogenesis. Acknowledgements This work was supported by the Wellcome Trust, UK, the BBSRC, UK & Telethon, AIRC, EU (2003-Zhivotoski, 2004-Blandino, 2005-Vandenabeele), FIRB-2001, and MIUR, to GM. We also thank C. Towlson for his assistance with nucleofections and G. Cocks for his help with SH-SY5Y cell differentiation.

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