Nucleosome-binding by TP53, TP63, and TP73 is determined by the composition, accessibility, and helical orientation of their binding sites.

The p53 family of transcription factors plays key roles in driving development and combating cancer by regulating gene expression. TP53, TP63, and TP73-the three members of the p53 family-regulate gene expression by binding to their DNA binding sites, many of which are situated within nucleosomes. To thoroughly examine the nucleosome-binding abilities of the p53 family, we used Pioneer-seq, a technique that assesses a transcription factor's binding affinity to its DNA binding sites at all possible positions within the nucleosome core particle. Using Pioneer-seq, we analyzed the binding affinity of TP53, TP63, and TP73 to 10 p53-family binding sites across the nucleosome core particle. We found that the affinity of TP53, TP63, and TP73 for nucleosomes was largely determined by the positioning of p53-family binding sites within nucleosomes; p53-family members bind strongly to the more accessible edges of nucleosomes but weakly to the less accessible centers of nucleosomes. We also found that the DNA-helical orientation of p53-family binding sites within nucleosomal DNA impacted the nucleosome-binding affinity of p53-family members. The composition of their binding sites also impacted each p53-family member's nucleosome-binding affinities only when the binding site was located in an accessible location. Taken together, our results show that the accessibility, composition, and helical orientation of p53-family binding sites collectively determine the nucleosome-binding affinities of TP53, TP63, and TP73. These findings help explain the rules underlying p53-family-nucleosome binding and thus provide requisite insight into how we may better control gene-expression changes involved in development and tumor suppression.