Single Molecule Imaging of p53's Dynamic Interaction with Chromatin

Abstract

P53 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 genome-wide studies showed that p53 binds to its REs in regions densely populated with nucleosomes. However, it is unknown how p53 accesses its target REs in the context of chromatin to regulate transcription.To better decipher the interaction of p53 with chromatin targets, we utilized a combination of in vivo live cell and in vitro single molecule imaging along with bioinformatics and Next Generation Proteomic screens. Our live cell imaging studies indicate a dynamic interaction between p53 and chromatin that varies in distinct sub-nuclear compartments. In vitro, p53 binds a mononucleosome on a native p53 target gene with nearly identical residence times as our live cell measurements. Strikingly, p53 has a higher affinity for its RE when incorporated into nucleosomes compared to an RE on naked DNA, suggesting a novel role for p53 as a pioneer factor. In addition, p53 transiently interacts and alters the structure of nucleosomal DNA, presumably to facilitate access of additional p53 bound chromatin modifiers.Bioinformatic analysis reveal that p53 REs cluster specifically within 2 regions of the nucleosome. Interestingly, p53 interacts with peptides that have strong homology to histones H2A, H2B, and H4 in our next generation proteomic screens. Furthermore based upon crystal structures, these histone peptides are adjacent to the clustered p53 REs, strongly suggesting localization of two physiologically relevant binding platforms for p53 on the nucleosome. Collectively, our studies indicate that p53/histone contacts combined with p53's interaction with REs may enhance p53 directed transcription by creating a stable multivalent platform for p53's recruitment to target promoters.