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Single-molecule imaging of DNA curtains reveals intrinsic energy landscapes for nucleosome deposition

Nature Structural & Molecular Biology volume 16, pages 1056–1062 (2009)Cite this article

Abstract

Here we use single-molecule imaging to determine coarse-grained intrinsic energy landscapes for nucleosome deposition on model DNA substrates. Our results reveal distributions that are correlated with recent in silico predictions, reinforcing the hypothesis that DNA contains some intrinsic positioning information. We also show that cis-regulatory sequences in human DNA coincide with peaks in the intrinsic landscape, whereas valleys correspond to nonregulatory regions, and we present evidence arguing that nucleosome deposition in vertebrates is influenced by factors that are not accounted for by current theory. Finally, we demonstrate that intrinsic landscapes of nucleosomes containing the centromere-specific variant CenH3 are correlated with patterns observed for canonical nucleosomes, arguing that CenH3 does not alter sequence preferences of centromeric nucleosomes. However, the nonhistone protein Scm3 alters the intrinsic landscape of CenH3-containing nucleosomes, enabling them to overcome the otherwise exclusionary effects of poly(dA-dT) tracts, which are enriched in centromeric DNA.

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Figure 1: Visualizing fluorescently tagged recombinant nucleosomes on DNA curtains.
Figure 2: Predicted and observed nucleosome distribution patterns on λ bacteriophage DNA.
Figure 3: Predicted and observed nucleosome distribution patterns on human β-globin DNA.
Figure 4: Thermodynamic landscapes for centromeric nucleosomes.

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Acknowledgements

We thank R. Axel, H. Bussemaker, R. Gonzalez, A. Grishok, R. Mann, and members of the Greene laboratory for critically reading the manuscript. We thank B. Cairns (University of Utah and the Howard Hughes Medical Institute (HHMI)) for S. cerevisiae H2A, H2B, H3 and H4 expression plasmids. We thank C. Wu and H. Xiao (National Institutes of Health) for the Cse4 and Scm3 expression plasmids and for sharing unpublished results. We thank J. Widom (Northwestern University) for the plasmid bearing the 601 nucleosome-binding sequence, and for communicating unpublished results. We thank L. Kaplan for cloning Flag-H4 and purifying Scm3, J. Gorman for assisting with data analysis and Y. Kwon (Yale University) for assistance with the salt dialysis protocol. This work was supported by a Basil O'Connor Starter Scholar Award from the March of Dimes and a grant from the US National Institutes of Health (GM082848) to E.C.G., and was partially funded by the Initiatives in Science and Engineering grant program through Columbia University, by the Nanoscale Science and Engineering Initiative of the National Science Foundation under NSF Award Number CHE-0641523 and by the New York State Office of Science, Technology, and Academic Research (NYSTAR).

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Authors and Affiliations

  1. Department of Biochemistry and Molecular Biophysics, Columbia University, New York, New York, USA

    Mari-Liis Visnapuu & Eric C Greene

  2. Howard Hughes Medical Institute, New York, New York, USA

    Eric C Greene

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  1. Mari-Liis Visnapuu
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M.-L.V. performed all experiments; M.-L.V. and E.C.G. designed the experiments, analyzed and interpreted the data and wrote the manuscript.

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Correspondence to Eric C Greene.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–10 (PDF 2900 kb)

Supplementary Video 1

Nanofabricated DNA curtains. (MOV 1934 kb)

Supplementary Video 2

Visualizing DNA curtains and fluorescent nucleosomes. (MOV 2763 kb)

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Visnapuu, ML., Greene, E. Single-molecule imaging of DNA curtains reveals intrinsic energy landscapes for nucleosome deposition. Nat Struct Mol Biol 16, 1056–1062 (2009). https://doi.org/10.1038/nsmb.1655

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