Pioneer-seq maps TP53 family–nucleosome binding. (A) Outline of Pioneer-seq. A library of 217 bp nucleosome-positioning sequences, which are based on the Widom-601 nucleosome-positioning sequence and which have a TP53 family binding site in one of the 146 bp positions of the nucleosome core particle, is reconstituted into nucleosomes and then incubated with TP53, TP63, or TP73. Bound nucleosomes are separated via gel-shift assay, extracted, purified, and sequenced. (B–D) TP53, TP63, or TP73 binding to a CDKN1A-promoter TP53 family binding site at different nucleosomal positions. TP53 family binding to the CDKN1A-promoter binding site is measured relative to TP53 family binding to nonspecific (non-TP53 family) 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.
