LAUSR

Nucleosomes are the basic units of chromatin and critical to the storage and expression of eukaryotic genomes. Chromatin accessibility and gene readout are heavily regulated by epigenetic marks of which post-translational modifications of histones play a key role. However, the mode of action and the structural implications on the single-molecule level of nucleosomes is still often poorly understood. Here, we apply a high-throughput AFM imaging and analysis pipeline to investigate the conformational landscape of the nucleosome variants H3K36me3, H3S10phos and H4K5/8/12/16ac. Our data set of >25,000 nucleosomes reveals nucleosomal unwrapping steps corresponding to 5 bp DNA. We find that H3K36me3 nucleosomes unwrap significantly more than wild type nucleosomes and additionally unwrap stochastically from both sides similar to CENP-A nucleosomes and in contrast to the highly anti-cooperative unwrapping of wild type nucleosomes. Nucleosomes with H3S10phos or H4K5/8/12/16ac modifications show unwrapping populations similar to wild type nucleosomes and also retain the same level of anti-cooperativity. Our findings help putting the mode of action of these modifications into context: While H3K36me3 likely partially acts by directly affecting nucleosome structure on the single-molecule level, H3S10phos and H4K5/8/12/16ac must predominantly act through higher-order processes. Our analysis pipeline is readily applicable to other nucleosome variants and will facilitate future high-resolution studies of the conformational landscape of nucleoprotein complexes.