Single Molecule Probing of P53's Ability to Dynamically Regulate Chromatin Structure

The tumor suppressor p53 protein is a transcriptional activator that binds to its response elements (REs) on target promoters and activates expression of a large number of genes involved in tumor suppression. Previous ChIP-seq studies indicate that p53 binds to its REs embedded in regions densely populated with nucleosomes. However, it is unknown if nucleosomes help or hinder binding of p53 to its response elements to regulate transcription.To decipher the interaction of p53 with nucleosomes, we have utilized a combination of Next Generation Proteomics, bioinformatics, bulk biochemical and real-time single molecule FRET assays. Our proteomic assays show that p53 interacts with peptides that have strong homology to histones H2A, H2B, and H4. Biochemical assays indicate that p53 can stably interact with histone peptides in the absence of DNA. Genomic maps of p53 REs and nucleosome positions further reveal that p53 REs cluster specifically within 2 regions of the nucleosome. Furthermore these clustered p53 REs are adjacent to histone regions identified in our proteomic studies, suggesting localization of two physiologically relevant binding platforms for p53 on the nucleosome.We have also established a single molecule FRET assay to characterize dynamic structural changes in nucleosomes with and without p53 REs. Interestingly, our studies reveal that p53 can bind to our biochemically defined nucleosomal surface and dynamically alter the structure of nucleosomal DNA independently of the presence of a p53 RE. Intriguingly, p53 has a higher affinity for its RE when incorporated into nucleosomes assembled with native DNA compared to REs on naked DNA. Thus, our studies indicate that histone/p53 contacts may enhance p53 directed transcription by creating a stable platform for p53 promoter recruitment.