The TP53 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 TP53 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 TP53 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 TP53-family binding sites across the nucleosome core particle. We found that the affinity of TP53, TP63, and TP73 for nucleosomes was primarily determined by the positioning of TP53-family binding sites within nucleosomes
TP53-family members bind strongly to the more accessible edges of nucleosomes but weakly to the less accessible centers of nucleosomes. Our results further show that the DNA-helical orientation of TP53-family binding sites within nucleosomal DNA impacts the nucleosome-binding affinity of TP53-family members, with binding site composition impacting each TP53-family member's affinity only when the binding site location was accessible. Taken together, our results show that the accessibility, composition, and helical orientation of TP53-family binding sites collectively determine the nucleosome-binding affinities of TP53, TP63, and TP73. These findings help explain the rules underlying TP53-family-nucleosome binding and thus provide requisite insight into how we may better control gene-expression changes involved in development and tumor suppression.