High-throughput AFM analysis reveals unwrapping pathways of H3 and CENP-A nucleosomes

• Published in: bioRxiv
• Main Authors: Konrad, Sebastian F, Vanderlinden, Willem, Frederickx, Wout, Brouns, Tine, Menze, Bjoern, De Feyter, Steven, Lipfert, Jan
• Format: Paper
• Language: English
• Published: Cold Spring Harbor Cold Spring Harbor Laboratory Press 10.04.2020
• Subjects: Atomic force microscopy; Chromatin; Genomes; Image processing; Nucleosomes
• DOI: 10.1101/2020.04.09.034090

Nucleosomes, the fundamental units of chromatin, regulate readout and expression of eukaryotic genomes. Single-molecule experiments have revealed force-induced transient nucleosome accessibility, but a high-resolution unwrapping landscape in the absence of external forces is currently lacking. Here, we introduce a high-throughput pipeline for the analysis of nucleosome conformations based on atomic force microscopy and automated, multi-parameter image analysis. Our data set of ~10,000 nucleosomes reveals multiple unwrapping states corresponding to steps of 5 bp DNA. For canonical H3 nucleosomes, we observe that dissociation from one side impedes unwrapping from the other side, but unlike to force-induced unwrapping, we find only a weak sequence-dependent asymmetry. Centromeric CENP-A nucleosomes do not unwrap anti-cooperatively, in stark contrast to H3 nucleosomes, likely due to their shorter N-terminal alpha-helix. Finally, our results reconcile previously conflicting findings about the differences in height between H3 and CENP-A nucleosomes. We expect our approach to enable critical insights into epigenetic regulation of nucleosome structure and stability. Competing Interest Statement The authors have declared no competing interest.