Gain-of-function p53 mutants have widespread genomic locations partially overlapping with p63

Elena Martynova,Diletta Dolfini,Roberto Mantovani,Silvia Pozzi,Giulio Pavesi,Federico Zambelli,Carol Imbriano,Valentina Basile

doi:10.18632/oncotarget.447

Elena Martynova, Diletta Dolfini + Show 6 more

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https://doi.org/10.18632/oncotarget.447

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Abstract

p53 and p63 are transcription factors -TFs- playing master roles in the DNA-damage response and in the development and maintenance of pluristratified epithelia, respectively. p53 mutations are common in epithelial tumors and HaCaT keratinocytes harbor two p53 alleles -H179Y and R282Q- with gain-of-function (GOF) activity. Indeed, functional inactivation of mutp53 affects the growth rate of HaCaT. We investigated the strategy of mutp53, by performing ChIP-Seq experiments of mutp53 and p63 and analyzed the transcriptome after mutp53 inactivation. Mutp53 bind to 7135 locations in vivo, with a robust overlap with p63. De novo motifs discovery recovered a p53/p63RE with high information content in sites bound by p63 and mutp53/p63, but not by mutp53 alone: these sites are rather enriched in elements of other TFs. The HaCaT p63 locations are only partially overlapping with those of normal keratinocytes; importantly, and enriched in mutp53 sites which delineate a functionally different group of target genes. Our data favour a model whereby mutp53 GOF mutants act both by tethering growth-controlling TFs and highjacking p63 to new locations.

Highlights

P53/p63/p73 are a family of transcription factors -TFs- that share a conserved DNA-binding domain and a similar DNA target sequence in promoters and enhancers [1,2,3,4]. p63 is involved in the development and maintenance of the skin and of pluristratified epithelia
Clones were selected, pooled and mRNA and protein levels of mutp53 and p63 controlled by qRT-PCR and Western blot: Fig. 1A shows that mutp53 mRNA levels are substantially decreased, the protein levels essentially abolished (Fig. 1B). p63 mRNA was modestly decreased
The most notable difference, was the growth rate, as curves were flatter in HaCaT deprived of mutp53 (Fig. 1F)

Summary

Introduction

P53/p63/p73 are a family of transcription factors -TFs- that share a conserved DNA-binding domain and a similar DNA target sequence in promoters and enhancers [1,2,3,4]. p63 is involved in the development and maintenance of the skin and of pluristratified epithelia. HaCaT cells are immortalized, non tumorigenic keratinocytes with mutant p53 alleles, R282Q and H179Y [6], which are typical UVB signatures [7]. In this system, the p53 pathway is functional in response to UVB irradiation, in terms of cell cycle block and induction of apoptosis [8, 9]. The issue of the interplay between mutp and p63/p73 is quite relevant, because genetic experiments suggest a complex role of p63 isoforms in transformation [4], and p53 missense mutants, including those produced by HaCaT alleles, have an increased affinity for p63/p73 [12-14, reviewed in 15]. We decided to explore mutp in vivo DNA-binding and functional activities, and relate it to p63 locations in HaCaT cells

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