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Issue 10, 2021
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High-throughput AFM analysis reveals unwrapping pathways of H3 and CENP-A nucleosomes

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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 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 in contrast to force-induced unwrapping, we find only a weak sequence-dependent asymmetry. Notably, centromeric CENP-A nucleosomes do not unwrap anti-cooperatively, in stark contrast to H3 nucleosomes. Finally, our results reconcile previous 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 and to facilitate future high-throughput AFM studies that involve heterogeneous nucleoprotein complexes.

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

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Supplementary files

Article information


Submitted
02 Dec 2020
Accepted
30 Jan 2021
First published
08 Mar 2021

This article is Open Access

Nanoscale, 2021,13, 5435-5447
Article type
Paper

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

S. F. Konrad, W. Vanderlinden, W. Frederickx, T. Brouns, B. H. Menze, S. De Feyter and J. Lipfert, Nanoscale, 2021, 13, 5435
DOI: 10.1039/D0NR08564B

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