p53-induced Regulation of Transcription in the Chromatin Context

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As a transcription factor, p53 is unique in regulating a wide spectrum of genes. The absolute level of the p53-induced response also 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 site in nucleosome, in agreement with the computational analysis described above. 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 initiated genome-wide analysis of the nucleosome repositioning in glioblastoma multiforme (GBM) cells (in collaboration with V. Teif, Essex University, UK). Our aim is to compare the nucleosome occupancy (and thus, the DNA accessibility to TFs) in the GBM tissues and in healthy brain tissues from GBM patients. In particular, we are interested in the nucleosome rearrangement in the vicinity of CTCF binding sites (many of which are involved in stabilization of chromatin loops), in Alu repeats that are abundant around the CTCF sites, and the p53 REs frequently occurring in these repeats. We anticipate that focusing on these three elements, we will be able to reveal important determinants of transcription reprogramming at various stages of GBM transformation. Unfortunately, there was a delay in our research due to restricted access to the lab space. Hopefully, this study will be completed by the end of 2020.