Identification of genomic DNA sequences bound by mutant p53 protein (Gly245→Ser) in vivo

Abstract

Mutant p53 proteins were shown to exert complex DNAinteractions in vitro, like binding to MAR-DNA, but so far it is unknown whether such interactions also occur in vivo. Therefore we analysed the binding of mutant (mut) p53 (Gly 245 ?Ser) in Onda 11 glioma cells to cellular DNA in vivo, using p53-speci®c chromatin immunoprecipitation (CHIP) after in vivo cross-linking of mut p53 to genomic DNA with cisplatin. We identi®ed genomic DNA fragments to which mut p53 (Gly 245 ?Ser) could be cross-linked in vivo. Puri®ed recombinant mut p53 (Gly 245 ?Ser) was able to bind speci®cally to such elements in PCR-EMSA in vitro, supporting the idea that this mut p53 protein interacts with genomic DNA in vivo. The genomic DNA fragments identi®ed are vastly di erent in sequence, but display as a common feature a high likelihood to adopt a non B-DNA conformation. Therefore we propose that structural determinants within these DNA elements are important for their interaction with mut p53 (Gly 245 ?Ser) in vivo. Oncogene (2000) 19, 4178 ± 4183.

Mutant p53: an oncogenic transcription factor

“…Despite rapidly accumulating evidence, the knowledge of the molecular details of the transcriptional activity of mutant p53 is still very scarce; (c) recent work by Di Agostino et al Mutant p53: an oncogenic transcription factor S Strano et al mutant p53 is recruited in vivo to such regulatory regions through the physical interaction with the transcription factor NF-Y (Figure 3). Altogether, these results show that the repertoire of the genes regulated by p53 gain-offunction mutants can be dictated by the specific interaction partner; (d) original work from Deppert's lab has shown that mutant p53 tightly associates with the nuclear matrix in vivo, and with high affinity with nuclear matrix attachment region DNA in vitro Koga and Deppert, 2000;Gohler et al, 2005;Kim and Deppert, 2006). These findings suggest that mutant p53 interacting with key structural components of the nucleus exerts its gain-of-function activity through the perturbation of the nuclear structure and function.…”

Interactions of mutant p53 with DNA: guilt by association

“…A distinctly different mode of mutp53:DNA interaction relies on the ability of mutp53 proteins to bind directly and preferentially DNA in a non-linear conformation (reviewed by Kim and Deppert, 2004). This mode, termed DNA structure-selective binding (p53-DSSB) (reviewed by Kim and Deppert, 2004), is persistently exhibited by mutp53 proteins in vitro with artificially constructed DNA structures (Go¨hler et al, 2005;Walter et al, 2005) and is operative in vivo as demonstrated with structurally flexible genomic sequences (Koga and Deppert, 2000;Walter et al, 2005). From such sequences, MAR/SAR sequences and trinucleotide (CTG CAG) repeats had been identified as genomic targets bound by mutp53 in the p53-DSSB mode (reviewed by Kim and Deppert, 2004).…”

“…Such a scenario, previously suggested for the p53-SSDB-dependent DNA looping (Stenger et al, 1994), might have a much broader application and underlie distinct modes of DNA binding not only of wtp53, but even more so of mutp53 proteins. Furthermore, considering that the binding of mutp53 proteins to structurally flexible DNA (Wei ker et al, 1992;Mu¨ller et al, 1996) or unusual DNA structures (Koga and Deppert, 2000;Go¨hler et al, 2005) is mediated jointly by the DNA binding activities of the p53-DBD and the p53-CTD, and that the core domains of mutp53 proteins exhibit considerable structural diversity (Campomenosi et al, 2001;Ang et al, 2006;Joerger et al, 2006); see also Joerger and Fersht, 2007, this issue), it can be expected that the patterns of mutp53-DSDB will vary greatly among mutp53 proteins in terms of their different affinity toward certain types of DNA structures.…”

“…Until now, all attempts to derive a linear consensus sequence for mutp53 binding sites have been largely futile, as genomic sequences associated with mutp53 proteins are notoriously heterogeneous and elude their stratification by sequence homology. However, despite the lack of sequence similarity, the sequences to which mutp53 proteins bind preferentially do exhibit some common features, such as repetitiveness and the propensity to adopt alternative DNA conformations (Wei ker et al, 1992;Mu¨ller et al, 1996;Koga and Deppert, 2000;Walter et al, 2005). It appears that a certain specificity of the interactions of mutp53 proteins with DNA can be realized by versatile modes that either involve intrinsic DNA binding activities of the mutp53 proteins themselves (direct binding), or can be mediated by the interaction of mutp53 proteins with DNA binding proteins (passive targeting).…”

Mutant p53 is constitutively phosphorylated at Serine 15 in UV-induced mouse skin tumors: involvement of ERK1/2 MAP kinase

“…Certain mutants of p53 have been shown to promote cell growth (Dittmer et al, 1993), transactivate promoters of epidermal growth factor receptor and basic fibroblast growth factor proteins (Ueba et al, 1994;Ludes-Meyers et al, 1996;Sheikh et al, 1997), and potentiate the expression of vascular endothelial growth factor (Kieser et al, 1994). Although tumor-derived mutants are generally defective in binding to promoters containing wild-type p53 response elements (Ko and Prives, 1996), they are nonetheless capable of binding to nonspecific DNA sequences (Koga and Deppert, 2000). Structural requirements for acquiring the 'gain of function' phenotype by mutant p53 proteins consist of retention of the intact transactivation and nonsequence-specific DNA-binding domains (Lin et al, 1995;Lanyi et al, 1998) as well as p53 oligomerization (Lanyi et al, 1998).…”