p53-induced Regulation of Transcription in the Chromatin Context

Division Of Basic Sciences - Nci

Investigators

Linked publications & trials

Abstract

p53 regulates a wide spectrum of genes. The absolute level of the p53-induced response varies drastically, which is important for biological functioning. In particular, the p53-induced activation of the cell cycle arrest genes (CCA-genes) is stronger and occurs much faster than activation of the apoptotic genes (Apo-genes). Since p53 can bind nucleosomal DNA, we sought to understand if the two groups of p53 sites differ in their accessibility when embedded in nucleosomes. To this aim, we analyzed the sequence-dependent bending anisotropy of human genomic DNA containing p53 sites. We calculated rotational positioning patterns predicting that most of the CCA-sites are exposed on the nucleosome surface. This is consistent with experimentally observed positioning of human nucleosomes in the vicinity of the CCA-sites. Remarkably, the sequence-dependent DNA anisotropy of both the p53 sites and flanking DNA work in concert producing strong positioning signals. By contrast, both the predicted and observed rotational settings of the Apo-sites in nucleosomes suggest that many of these sites are buried inside, thus preventing immediate p53 recognition and delaying gene induction. We also measured the p53 binding to its cognate sites embedded in the in strongly positioned '601' nucleosome in vitro. Our data suggest that the p53 affinity to DNA strongly correlates with the rotational positioning of its RE in nucleosome, in agreement with our computational analysis. The exposed configurations of the p53 sites in nucleosome (like CCA-sites) demonstrate significantly stronger affinity to p53 compared to the buried configurations (similar to the Apo-sites). Thus, the difference in nucleosomal organization of the two sets of p53 response elements appears to be a key factor affecting the strength of p53-DNA binding and kinetics of induction of the p53 target genes. Recently, we performed a comprehensive analysis of the published p53 cistromes and identified thousands binding sites in normal and cancer cells. Our analysis revealed two distinct epigenetic features underlying p53-DNA interactions in vivo. First, we found that p53 binding sites are associated with transcriptionally active histone marks (H3K4me3 and H3K36me3) in normal-cell chromatin, but with repressive histone marks (H3K27me3) in cancer-cell chromatin. Second, p53 binding sites in cancer cells are characterized by a lower level of DNA methylation than their counterparts in normal cells, probably related to global hypomethylation in cancers. In addition, regardless of the cell type, p53 sites are highly enriched in the endogenous retroviral elements of the ERV1 family, highlighting the importance of this repeat family in shaping the transcriptional network of p53. Moreover, the p53 sites exhibit an unusual combination of chromatin patterns: high nucleosome occupancy and, at the same time, high sensitivity to DNase I. Our results suggest that p53 can access its target sites in a chromatin environment that is non-permissive to most DNA-binding factors, which may allow p53 to act as a pioneer transcription factor in the context of chromatin. In addition, we analyzed the nucleosome organization in the vicinity of Alu repeats and the p53 REs occurring in these repeats. Our observations indicate dramatic shortening of nucleosome spacing in breast cancer (BRC), in particular, at the 5'-ends of Alu elements incorporated in genes. As we demonstrated earlier, the shortening of inter-nucleosome distances by 5 bp is associated with the increased level of transcription. We anticipate that focusing on these observations, we will be able to reveal important determinants of transcription reprogramming at various stages of the BRC transformation. Recently, we studied p53 binding to nucleosomes with modified histones, mimicking epigenetic acetylation of the histone N-tails. We developed an in vitro model of the epigenetic acetylation of the lysines K5 and K7 of histone H2A. To this aim, we reconstituted well-positioned nucleosomes using Xenopus histone octamers with wild type (WT) and H2A tail-less histones (H2A-del). Analyzing p53 binding to the WT and H2A-del nucleosomes, we observed the following difference between the two. As mentioned above, the p53 affinity to nucleosomal DNA strongly correlates with the rotational setting of its RE in WT nucleosome. This correlation holds for H2A-del nucleosomes as well. In addition, for the H2A-del nucleosomes, the relationship between the p53 affinity to RE and its proximity to the nucleosome end is monotonic (which is intuitively obvious because the closer is RE to the nucleosome end, the more flexible it is, and hence, the more accessible for p53 binding.) However, for the WT nucleosomes the above relationship is non-monotonic. This is a novel effect, apparently coupled with the presence of histone tails. Based on these results, we conclude that the histone H2A tails produce a 'shielding' effect modulating the p53-DNA binding affinity. Importantly, this 'shielding' effect is local and very selective, because it changes the p53 affinity to the REs in the vicinity of the position where the H2A tail interacts with DNA in x-ray structure, rather than to the whole nucleosome. It remains to be seen whether the H4 N-tail produces a similar effect, and it will be the subject of our future studies. Potentially, this observation may be relevant to other TFs as well and thus, may have far-reaching implications for epigenetic regulation of TF binding to chromatin.