There is a remarkable degree of specificity in the establishment and maintenance of cellular identity, given that our cells have the same DNA but vastly different gene expression profiles. The basis of this ‘epigenetic’ regulation is a complex network of gene activation and repression events that takes place in the context of our chromatinized genomes - DNA wrapped around histones that make up nucleosomes. While we know specific gene activation is driven by DNA-binding proteins called transcription factors, our knowledge of how they engage naked DNA vastly outstrips our insights into how they interact with chromatinized DNA in vivo. This prompts the critical question: how do transcription factors read or interpret chromatin?
We previously developed an in vitro genomics pipeline to enable the first structural characterization of transcription factors binding to nucleosomal DNA (Michael/Grand/Isbel et al., 2020, Science), which we now apply to p53. This reveals that p53 engages motifs at key points along the nucleosome, which we provide evidence for in stem cells by profiling binding with Cut and Run. Cellular characterization using proteomics and precision mutants with functional genomics measurements (i.e ChIPseq/ATACseq/RNAseq) reveals that p53 utilizes a repressive cofactor protein called Trim24 to interpret chromatin. We reveal the mechanistic basis of this activity: Trim24 utilizes a histone binding domain to engage unmethylated lysine 4 of histone H3 at closed chromatin, while it is blocked at open chromatin by lysine 4 methylation.
These findings demonstrate how chromatin restricts the ability of p53 to activate genes that are initially off, so-called ‘pioneering activity’. Additionally, as we demonstrate that the majority of p53 targets in human tissues are in closed chromatin, suggesting a means to specifically amplify its activity at repressive chromatin by targeting Trim24 in cancer. Notably, these results underscore the considerably variability in transcription factors ability to navigate chromatin.