PT  - JOURNAL ARTICLE
AU  - Sebastian F. Konrad
AU  - Willem Vanderlinden
AU  - Wout Frederickx
AU  - Tine Brouns
AU  - Björn Menze
AU  - Steven De Feyter
AU  - Jan Lipfert
TI  - High-throughput AFM analysis reveals unwrapping pathways of H3 and CENP-A nucleosomes
AID  - 10.1101/2020.04.09.034090
DP  - 2020 Jan 01
TA  - bioRxiv
PG  - 2020.04.09.034090
4099  - http://biorxiv.org/content/early/2020/04/10/2020.04.09.034090.short
4100  - http://biorxiv.org/content/early/2020/04/10/2020.04.09.034090.full
AB  - 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 α-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 StatementThe authors have declared no competing interest.