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CENP-A octamers do not confer a reduction in nucleosome height by AFM

Nature Structural & Molecular Biology volume 21, pages 2–3 (2014)Cite this article

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CENP-A is a histone H3 variant required for accurate segregation of chromosomes during mitosis. Over the past 20 years, workers have unveiled several structural and mechanical features encoded within CENP-A nucleosomes1,2,3,4,5,6,7 that enable the nucleosomes to serve as an epigenetic platform for centromere assembly. In a recent study, Miell et al.8 present evidence that, when measured by atomic force microscopy (AFM), in vitro–reconstituted octamers containing CENP-A have reduced heights compared to those of nucleosome octamers containing H3. These data led the authors to propose that previous AFM images—in which native CENP-A nucleosomes purified from Drosophila or human cells4,5,6 were shown to possess shorter heights relative to those of native H3 nucleosomes—could have resulted from octameric nucleosomes. The authors propose that CENP-A octamers might be inherently smaller, owing to CENP-A2–histone H42 compaction2,3 within the core of the octamer. The data by Miell et al.8 were surprising. They contradict seminal AFM analysis of in vitro–reconstituted human CENP-A octameric nucleosomes, for which ∼580 nm3 octameric volumes were carefully measured over a decade ago9. They also contradict the recently solved crystal structure of the CENP-A octameric nucleosome10 in which, with the exceptions of looser entry and exit DNA, subtle alterations in loop 1 and the unstructured C-terminal six amino acids, a near-perfect atomic correspondence exists between the cores of CENP-A and H3 octameric nucleosomes in vitro. It is puzzling that subtle differences between CENP-A and H3 octameric nucleosomes10 could translate into differences in height8 that would be detected by AFM performed under native conditions.

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Figure 1: AFM analysis of recombinant CENP-A and H3 octameric nucleosomes shows no appreciable difference in size.

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Acknowledgements

We thank A. Straight (Stanford University) and J. Ottesen (Ohio State University) for the gift of recombinant human CENP-A and H3 octamers; R. Allshire (University of Edinburgh) for protocols; S. Henikoff (Fred Hutchinson Cancer Research Center) and J. Gerton (Stowers Institute for Medical Research) for the gift of recombinant yeast CSE4 octamers; C. Wu (US National Institutes of Health and Janelia Farm, Howard Hughes Medical Institute) for the gift of the Widom 601 plasmid; R. Gill and M. Bui for technical assistance; T. Misteli, G. Hager and S. Grewal for critical comments and K. Takeyasu for discussions on the AFM analysis presented in ref. 9.

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  1. National Cancer Institute, National Institutes of Health, Bethesda, Maryland, USA

    Marcin P Walkiewicz & Yamini Dalal

  2. National Institute for BioImaging and BioEngineering, National Institutes of Health, Bethesda, Maryland, USA

    Emilios K Dimitriadis

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Correspondence to Yamini Dalal.

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Supplementary information

Supplementary Figures and Text

Supplementary Figures 1 and 2, and Supplementary Note (PDF 4333 kb)

Supplementary Data Set 1

Raw AFM measurements demonstrate no appreciable difference between CENP-A and H3 octamers. (XLS 96 kb)

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Walkiewicz, M., Dimitriadis, E. & Dalal, Y. CENP-A octamers do not confer a reduction in nucleosome height by AFM. Nat Struct Mol Biol 21, 2–3 (2014). https://doi.org/10.1038/nsmb.2742

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