BPS2026 – A novel cryo-EM method for deciphering nucleosome-protein complexes

Nucleosomes are the structural units in which DNA is packaged within chromatin. Each nucleosome disk is made up of an octamer of core histone proteins and approximately 150 base pairs of DNA which wrap around this octamer like a spool. Nucleosomes are commonly considered to be repressive structures, as they limit the accessibility of DNA, but they also act as interaction hubs for nuclear complexes performing DNA replication, DNA damage repair, and transcription. Thus, the perturbation of these interactions can lead to disease, including cancers. Proteomics studies have identified canonical nucleosome interactors; however, there exists gaps in knowledge in regards to the interactome of variant histones, which play an important role in maintaining genomic integrity and cell identity. Variant histones are distinct from their canonical counterparts in their chemical properties, resulting from differences in their amino acid sequences and thus participate in varying protein interaction networks. To address this gap in knowledge, we will utilize cryo-electron microscopy (cryo-EM) and proteomics, allowing us to observe and identify complexes formed between variant nucleosomes and nuclear proteins. Cryo-EM experiments will involve the use of monomeric streptavidin affinity grids, which will allow for tight binding to biotinylated nucleosomes. We will perform on-grid pull-downs of nuclear lysates, which will be complemented by traditional pull-downs and proteomics assays on the same samples. With mutations found in both nucleosome interactors and histones known to cause various cancers, it is imperative that the structures of these complexes are better understood for the potential identification of novel therapeutic targets and the development of treatments for these diseases.