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bioRxiv · 10.1101/2020.04.09.034090

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

Konrad, S. F.·Vanderlinden, W.·Frederickx, W.·Brouns, T.·Menze, B.·De Feyter, S.·Lipfert, J.
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Abstract

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 -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.

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Konrad, S. F., Vanderlinden, W., Frederickx, W., Brouns, T., Menze, B., De Feyter, S., Lipfert, J.. 2020-04-10. High-throughput AFM analysis reveals unwrapping pathways of H3 and CENP-A nucleosomes. https://doi.org/10.1101/2020.04.09.034090

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