Accessing Nucleosomal DNA

ongoing

Transcription factors (TFs) regulate gene expression and govern cell identity. In chromatinised genomes TF binding sites are frequently encapsulated in nucleosomes, which severely restrict access. While specialised pioneer factors have been proposed to bind to these sterically hindered sites, the exact mechanism by which these factors access nucleosomes remains unknown. A number of biochemical models have been put forward that would allow DNA sequence read-out, these include: (i) TFs compatible with the nucleosome architecture, (ii) alternative, partial, DNA motifs that render TFs nucleosome compatible, (iii) exposure of DNA binding sites through histone-nucleosome breathing dynamics, or (iv) remodelling of the histone core by TF binding. Despite being a fundamental question in genome regulation, no structural rationale is currently available for any of these proposed mechanisms. In unpublished work, we developed a novel biochemical assay allowing to simultaneously investigate all DNA registers on a nucleosome for TF access. This tool enabled us to solve the 3.8 Å structure OCT4/SOX2 bound to a nucleosome revealing the unexpected binding modes these TFs employ to engage nucleosomal binding sites (unpublished). Building on this novel biochemical workflow, we propose to solve additional structures of TFs compatible with the nucleosome-architecture and of those TFs suspected to bind to the ends of nucleosomal DNA. By dissecting multiple structures with diverse DNA binding domains in vitro and in cells, we wish to understand general principles of how DNA sequence motifs are being read-out at different sites on a nucleosome. The key questions we wish to address are: How does the location of a TF motif on a nucleosome impact affinity and/or the binding mechanism? Can TF binding remodel the nucleosome architecture? How do arrays of nucleosomes impact TF accessibility to DNA?