p53 chromatin epigenetic domain organization and p53 transcription

Epigenetic organization represents an important regulation mechanism of gene expression. In this work, we show that the mouse p53 gene is organized into two epigenetic domains. The first domain is fully unmethylated, associated with histone modifications in active genes, and organized in a nucleosome-free conformation that is deficient in H2a/H2b, whereas the second domain is fully methylated, associated with deacetylated histones, and organized in a nucleosomal structure. In mitotic cells, RNA polymerase is depleted in domain II, which is folded into a higher-order structure and is associated with H1 histone, whereas domain I conformation is preserved. Similar results were obtained for cells treated with inhibitors of associated regulatory factors. These results suggest that depletion of RNA polymerase II is the result of a physical barrier due to the folding of chromatin in domain II. The novel chromatin structure in the first domain during mitosis also suggests a mechanism for marking active genes in successive cell cycles.     

p53 binds single-stranded DNA ends through the C-terminal domain and internal DNA segments via the middle domain.

We have previously reported that wild-type p53 can bind single-stranded (ss) DNA ends and catalyze renaturation of ss complementary DNA molecules. Here we demonstrate that p53 can also bind to internal segments of ss DNA molecules via a binding site (internal DNA site) distinct from the binding site for DNA ends (DNA end site). Using p53 deletion mutants, the internal DNA site was mapped to the central region (residues 99-307), while the DNA end site was mapped to the C-terminal domain (residues 320-393) of the p53 protein. The internal DNA site can be activated by the binding of ss DNA ends to the DNA end site. The C-terminal domain alone was sufficient to catalyze DNA renaturation, although the central domain was also involved in promotion of renaturation by the full-length protein. Our results suggest that the interaction of the C-terminal tail of p53 with ss DNA ends generated by DNA damage in vivo may lead to activation of non-specific ss DNA binding by the central domain of p53.     

p53 gene expression and activation of p53-dependent transcription in melanoma cell lines

Malignant melanoma has poor prognosis because of its high metastatic potential and resistance to chemotherapy. A possible approach to more effective therapy is induction of p53-dependent apoptosis. This approach is promising, since the wild-type p53 is expressed in most melanomas. An attempt was made to estimate the functional activity of p53 in several malignant melanoma cell lines. Most lines were characterized by a high protein level and nuclear localization of p53. All cell lines expressing the wild-type p53 showed stabilization of p53, its translocation into the nucleus, and activation of several target genes in response to DNA-damaging agents, suggesting that p53 was functionally active. A high-molecular-weight protein localized in the cytoplasm and mimicking a p53 epitope was found in several cell lines. It was shown that the DO-1 epitope of this protein does not derive from p53, ruling out cytoplasmic retention of p53 in melanoma cell lines. A mechanism of camptothecin-induced stabilization of p53 by decreasing the level of the HDM2 mRNA was described for melanoma cells but not for normal melanocytes, which suggested a differential effect of camptothecin on tumor-derived and primary cells.     

How p53 binds DNA as a tetramer.

The p53 tumor suppressor protein is a tetramer that binds sequence-specifically to a DNA consensus sequence consisting of two consecutive half-sites, with each half-site being formed by two head-to-head quarter-sites (--><-- --><--). Each p53 subunit binds to one quarter-site, resulting in all four DNA quarter-sites being occupied by one p53 tetramer. The tetramerization domain forms a symmetric dimer of dimers, and two contrasting models have the two DNA-binding domains of each dimer bound to either consecutive or alternating quarter-sites. We show here that the two monomers within a dimer bind to a half-site (two consecutive quarter-sites), but not to separated (alternating) quarter-sites. Tetramers bind similarly, with the two dimers within each tetramer binding to pairs of half-sites. Although one dimer within the tetramer is sufficient for binding to one half-site in DNA, concurrent interaction of the second dimer with a second half-site in DNA drastically enhances binding affinity (at least 50-fold). This cooperative dimer-dimer interaction occurs independently of tetramerization and is a primary mechanism responsible for the stabilization of p53 DNA binding. Based on these findings, we present a model of p53 binding to the consensus sequence, with the tetramer binding DNA as a pair of clamps.     

The N-terminal interferon-binding domain (IBiD) homology domain of p300 binds to peptides with homology to the p53 transactivation domain

Two high affinity Ser-20-phospho-LXXLL p53-binding domains of p300 map to the C-terminal interferon-binding domain (IBiD) and N-terminal IBiD homology domain (IHD) regions. Purified fractions of a recombinant IHD miniprotein are active in a set of in vitro assays highlighting its affinity to the N-terminal LXXLL domain of p53 including (i) dose-dependent binding to Ser-20-phosphorylated p53 tetramers; (ii) DNA-stimulated binding to p53 tetramers; and (iii) inhibition of MDM2-mediated p53 ubiquitination. The active component of the IHD miniprotein was localized to a 75-amino-acid fragment corresponding to amino acids 401-475 on human p300. This minimal IHD miniprotein can function in vivo as a p53-binding polypeptide in assays including: (i) complex formation with VP16-LXXLL peptide motifs in the two-hybrid assay; (ii) action as a dominant negative inhibitor of p53 from p21 luciferase templates; and (iii) attenuation of endogenous p21 protein levels. Further, we show here that the IRF-1-dependent stabilization and reactivation of p53DeltaPRO protein (LXXLL+/PXXP-) can be neutralized by the minimal IHD miniprotein, suggesting that IHD can bind to the p53 LXXLL domain in vivo. Phage-peptide display to the IHD miniprotein gave rise to an LSQXTFSXLXXLL consensus binding site that displays significant homology to the LXXLL transactivation domain of p53. These data validate the IHD scaffold as an independent LXXLL peptide-binding domain within the p300 protein, complementing the known peptide-binding domains including IBiD, C/H1, and C/H3.