The nucleosome-binding affinities of p53, p63, and p73 are collectively 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. p53, p63, and p73, which are the three members of the p53 family, regulate gene expression changes by binding to their DNA binding sites, many of which are positioned within nucleosomes. To comprehensively examine the nucleosome binding abilities of the p53 family, we developed Pioneer seq. Pioneer seq is a novel technique that enables the comprehensive assessment of the binding affinity of a transcription factor to its DNA binding sites at all 147 base pair positions of the nucleosome core particle. Using Pioneer seq, we analyzed the binding affinity of p53, p63, and p73 to 10 p53 family binding sites across the nucleosome core particle. The nucleosomal positioning of p53 family binding sites had the largest impact on p53 family binding affinity. p53 family members bound strongly near the more accessible edges of nucleosomes but weakly near the less accessible centers of nucleosomes. The nucleosome binding affinity of each p53 family member was also impacted by the composition of their binding sites. That is, each p53 family member was able to bind nucleosomal DNA more strongly in the presence of high affinity p53 family binding sites than in the presence of low affinity p53 family binding sites at the same nucleosomal position. 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. Taken together, our results help explain the rules underlying p53 family nucleosome binding and thus provide requisite insight into how we may better control the expression of genes involved in development and tumor suppression.

p53家族转录因子通过调控基因表达，在驱动个体发育与抵御癌症的过程中发挥关键作用。p53、p63与p73作为该家族的三个成员，通过结合其DNA结合位点调控基因表达变化，其中多数结合位点定位于核小体（nucleosome）内部。为全面探究p53家族的核小体结合能力，我们开发了Pioneer seq技术。该技术可在核小体核心颗粒（nucleosome core particle）的全部147个碱基对（base pair, bp）位置上，全面评估转录因子与其DNA结合位点的结合亲和力。借助Pioneer seq技术，我们分析了p53、p63与p73对核小体核心颗粒上10个p53家族结合位点的结合亲和力。核小体上结合位点的位置对p53家族的结合亲和力影响最为显著：p53家族成员在核小体更易接触的边缘区域结合能力较强，而在核小体相对不易接触的中心区域结合能力较弱。每个p53家族成员的核小体结合亲和力还受其结合位点的序列组成影响：即在同一核小体位置上，当结合位点为高亲和力p53家族结合位点时，各家族成员对核小体DNA的结合能力强于对应低亲和力结合位点的情况。我们还发现，核小体DNA内p53家族结合位点的DNA螺旋取向，也会影响该家族成员的核小体结合亲和力。综上，本研究结果阐明了p53家族结合核小体的内在规律，为我们更好地调控发育与肿瘤抑制相关基因的表达提供了必要的理论依据。