Defining p53 pioneering capabilities with competitive nucleosome binding assays

Abstract

Accurate gene expression requires the targeting of transcription factors (TFs) to regulatory sequences often occluded within nucleosomes. The ability to target a transcription factor binding site (TFBS) within a nucleosome has been the defining characteristic for a special class of TFs known as pioneer factors. Recent studies suggest p53 functions as a pioneer factor that can target its TFBS within nucleosomes, but it remains unclear how p53 binds to nucleosomal DNA. To comprehensively examine p53 nucleosome binding we competitively bound p53 to multiple in vitro formed nucleosomes containing a high or low-affinity p53 TFBS located at differing translational and rotational positions within the nucleosome. Stable p53-nucleosome complexes were isolated and quantified using next generation sequencing. Our results demonstrate p53 binding is limited to nucleosome edges with significant binding inhibition occurring within 50-bp of the nucleosome dyad. Binding site affinity only affects p53 binding for TFBS located outside the nucleosome core at the same nucleosomal positions. Furthermore, p53 has strong non-specific nucleosome binding facilitating its interaction with chromatin. Our in vitro findings were confirmed by examining p53 induced binding in a cell line model, showing induced binding at nucleosome edges flanked by a nucleosome free region. Overall, our results suggest that the pioneering capabilities of p53 are driven by non-specific nucleosome binding with specific binding at nucleosome edges.