Fig 2: Binding site composition modulates TP53 family–nucleosome binding affinity. (A) A TP53 sequence logo above binding sites: The high-affinity site is based on the TP53 sequence logo; the Mut1-high-affinity site, on the TP53 sequence logo but with one base mutated; the Mut2-high-affinity binding site, on the TP53 sequence logo but with two bases mutated; and the nonspecific STAT5A site. (B,D,F) TP53, TP63, or TP73 binding to the four binding sites in A at different nucleosomal positions, measured relative to TP53 family binding to nonspecific binding sites and represented by relative-supershift values (Equation 1). A nonspecific motif for STAT5A is shown for comparison in gray. (Edge) The right end of the 146 bp nucleosome core particle. Shading indicates SEM. n = 3. (C,E,G) Assessing the binding affinity between TP53, TP63, or TP73 and the +70 high-affinity nucleosome or the +70 mut1-high-affinity nucleosome, both of which are indicated in B. Bound nucleosome (%) was calculated via gel-shift assays featuring Cy5-labeled nucleosomes.

Fig 3: TP53 and TP63 enrichment relative to nucleosome positioning and binding-site affinity. (A) Average nucleosome occupancy at TP53 binding sites before activation with Nutlin in IMR-90 cells, as determined from MNase-seq data. These sites represent the locations where TP53 binds following its activation. (B) Average nucleosome occupancy at TP63 binding sites before expression in K562 cells, as determined from MNase-seq data. These sites correspond to the locations where TP63 binds upon expression. (C,D) Violin plots of TP53 and TP63 ChIP-seq peak enrichment scores, categorized by binding-site affinity and nucleosome position (lower/higher-affinity, near-dyad/outer nucleosome). Binding sites were identified using the MA0106.3 TP53 motif (JASPAR), and nucleosome positioning was determined from MNase-seq data.

Fig 4: TP53 family–nucleosome binding is collectively determined by the composition, accessibility, and helical orientation of TP53 family binding sites. (A) Outline of regression model predicting TP53 family–nucleosome binding based on binding-site FIMO scores, MNase-digestion scores, and solvent-accessible-surface-area Z-scores. The resulting equation illustrates the predictive relationship between these variables and TP53 family–nucleosome binding. (B–D) Correlating the relative-supershift values (Equation 1) of TP53, TP63, and TP73 to two TP53 family binding sites with either the MNase-digestion score of these binding sites, the solvent-accessible surface area of the CATGs of these binding sites, or the relative-supershift values of TP53, TP63, and TP73 to these binding sites predicted by the multiple regression model outlined in A. (E,F) Comparison of actual versus model-predicted TP53 relative-supershift values for high-affinity and Mut1-high-affinity binding sites.

Fig 5: Binding site helical orientation in nucleosomal DNA impacts TP53 family–nucleosome binding. (A) TP53 binding to a high-affinity (HA) TP53 family binding site at different nucleosomal positions, measured relative to nonspecific binding sites and represented by relative-supershift values (Equation 1). A nonspecific motif for STAT5A is shown for comparison in gray. (Edge) The right end of the 146 bp nucleosome core particle. Shading indicates SEM. n = 3. (B) Binding affinity comparison between +70 and +73 high-affinity nucleosomes—both of which are indicated in A. (C) Illustrative models of the +70 and +73 high-affinity nucleosomes. The TP53-relative-supershift value corresponding to each nucleosome is shown. Also shown are the solvent-accessible-surface area of the conserved CATG bases of the high-affinity binding site in each nucleosome and the number of atomic clashes between the TP53 tetramer and nucleosome. (D) TP53 family binding to the HA binding site at different nucleosomal positions is plotted in blue as “control-adjusted” relative-supershift values (i.e., relative-supershift values of nonspecific-binding-site-containing nucleosomes subtracted from relative-supershift values of HA-binding-site-containing nucleosomes). Solvent-accessible surface area (SAS) of the conserved CATG bases of the high-affinity binding site is plotted in purple. (Edge) The right end of the 146 bp nucleosome core particle. (E) Plot of TP53 relative-supershift values versus the number of atomic clashes between a TP53 dimer and the nucleosome at different nucleosomal positions.