High-throughput AFM analysis reveals unwrapping pathways of H3 and CENP-A nucleosomes
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 introd...
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Veröffentlicht in: | Nanoscale 2021-03, Vol.13 (1), p.5435-5447 |
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Sprache: | eng |
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Zusammenfassung: | 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.
The thermal unwrapping pathways of canonical H3 and centromeric CENP-A nucleosomes are elucidated in unprecedented detail by high-throughput analysis of AFM images. |
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ISSN: | 2040-3364 2040-3372 |
DOI: | 10.1039/d0nr08564b |