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 demon- strate that C-promoter binding factor-1 (CBF-1)-dependent gene transcription driven by the Notch-1 intracellular domain (N1 ICD ) is potently antagonized by exogenously expressed transactivating (TA) p73 splice variants in SH-SY5Y neurobl- astomas 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 N1 ICD /CBF-1- dependent 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 N1 ICD expression. Evidence of a direct physical interaction between N1 ICD and TAp73a was demonstrated by co-immunoprecipitation. Using Notch deletion constructs, we demonstrate that TAp73a binds the N1 ICD in a region C-ter- minal of aa 2094. Interestingly, DNp73a and TAp73a R292H also co-purified with N1 ICD , but neither inhibited N1 ICD /CBF-1- dependent transcription. This suggests that an intact trans- activation (TA) domain and the ability to bind DNA are necessary for TAp73 to antagonize Notch signalling. Finally we found that TAp73a reversed the N1 ICD -mediated repres- sion of retinoic acid-induced differentiation of SH-SY5Y neu- roblastomas, providing functional evidence for an inhibitory effect of TAp73a on notch signalling. Collectively, these find- ings may have ramifications for neurodevelopment, neurode- generation and oncogenesis. Keywords: development, neurodegeneration, Notch, onco- genesis, p73, p53. J. Neurochem. (2006) 99, 989–999. 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- 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: r.killick@iop.kcl.ac.uk 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; N1 ICD , Notch-1 intracellular domain; RA, reti- noic acid; TA, transactivation domain; TACE, tumour necrosis factor a converting enzyme. Journal of Neurochemistry , 2006, 99, 989–999 doi:10.1111/j.1471-4159.2006.04142.x Ó 2006 The Authors Journal Compilation Ó 2006 International Society for Neurochemistry, J. Neurochem. (2006) 99, 989–999 989