Combined Micrococcal Nuclease and Exonuclease III Digestion Reveals Precise Positions of the Nucleosome Core/Linker Junctions: Implications for High-Resolution Nucleosome Mapping

Combined Micrococcal Nuclease and Exonuclease III Digestion Reveals Precise Positions of the... Micrococcal nuclease (MNase) is extensively used in genome-wide mapping of nucleosomes but its preference for AT-rich DNA leads to errors in establishing precise positions of nucleosomes. Here, we show that the MNase digestion of nucleosomes assembled on a strong nucleosome positioning sequence, Widom's clone 601, releases nucleosome cores whose sizes are strongly affected by the linker DNA sequence. Our experiments produced nucleosomal DNA sizes varying between 147 and 155bp, with positions of the MNase cuts reflecting positions of the A⋅T pairs rather than the nucleosome core/linker junctions determined by X-ray crystallography. Extent of chromatosomal DNA protection by linker histone H1 also depends on the linker DNA sequence. Remarkably, we found that a combined treatment with MNase and exonuclease III (exoIII) overcomes MNase sequence preference producing nucleosomal DNA trimmed symmetrically and precisely at the core/linker junctions regardless of the underlying DNA sequence. We propose that combined MNase/exoIII digestion can be applied to in situ chromatin for unbiased genome-wide mapping of nucleosome positions that is not influenced by DNA sequences at the core/linker junctions. The same approach can be also used for the precise mapping of the extent of linker DNA protection by H1 and other protein factors associated with nucleosome linkers. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Journal of Molecular Biology Elsevier

Combined Micrococcal Nuclease and Exonuclease III Digestion Reveals Precise Positions of the Nucleosome Core/Linker Junctions: Implications for High-Resolution Nucleosome Mapping

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Publisher
Elsevier
Copyright
Copyright © 2013 Elsevier Ltd
ISSN
0022-2836
D.O.I.
10.1016/j.jmb.2013.02.026
Publisher site
See Article on Publisher Site

Abstract

Micrococcal nuclease (MNase) is extensively used in genome-wide mapping of nucleosomes but its preference for AT-rich DNA leads to errors in establishing precise positions of nucleosomes. Here, we show that the MNase digestion of nucleosomes assembled on a strong nucleosome positioning sequence, Widom's clone 601, releases nucleosome cores whose sizes are strongly affected by the linker DNA sequence. Our experiments produced nucleosomal DNA sizes varying between 147 and 155bp, with positions of the MNase cuts reflecting positions of the A⋅T pairs rather than the nucleosome core/linker junctions determined by X-ray crystallography. Extent of chromatosomal DNA protection by linker histone H1 also depends on the linker DNA sequence. Remarkably, we found that a combined treatment with MNase and exonuclease III (exoIII) overcomes MNase sequence preference producing nucleosomal DNA trimmed symmetrically and precisely at the core/linker junctions regardless of the underlying DNA sequence. We propose that combined MNase/exoIII digestion can be applied to in situ chromatin for unbiased genome-wide mapping of nucleosome positions that is not influenced by DNA sequences at the core/linker junctions. The same approach can be also used for the precise mapping of the extent of linker DNA protection by H1 and other protein factors associated with nucleosome linkers.

Journal

Journal of Molecular BiologyElsevier

Published: Jun 12, 2013

References

  • The DNA-encoded nucleosome organization of a eukaryotic genome
    Kaplan, N.; Moore, I.K.; Fondufe-Mittendorf, Y.; Gossett, A.J.; Tillo, D.; Field, Y.
  • Determinants of nucleosome organization in primary human cells
    Valouev, A.; Johnson, S.M.; Boyd, S.D.; Smith, C.L.; Fire, A.Z.; Sidow, A.
  • Nucleosome organization in the Drosophila genome
    Mavrich, T.N.; Jiang, C.; Ioshikhes, I.P.; Li, X.; Venters, B.J.; Zanton, S.J.
  • Short nucleosome repeats impose rotational modulations on chromatin fibre folding
    Correll, S.J.; Schubert, M.H.; Grigoryev, S.A.
  • Micrococcal nuclease does not substantially bias nucleosome mapping
    Allan, J.; Fraser, R.M.; Owen-Hughes, T.; Keszenman-Pereyra, D.
  • New DNA sequence rules for high affinity binding to histone octamer and sequence-directed nucleosome positioning
    Lowary, P.T.; Widom, J.
  • Nucleosomal locations of dominant DNA sequence motifs for histone–DNA interactions and nucleosome positioning
    Thastrom, A.; Bingham, L.M.; Widom, J.
  • Structure of RCC1 chromatin factor bound to the nucleosome core particle
    Makde, R.D.; England, J.R.; Yennawar, H.P.; Tan, S.
  • Sequence and position-dependence of the equilibrium accessibility of nucleosomal DNA target sites
    Anderson, J.D.; Widom, J.
  • Crystal structure of the human centromeric nucleosome containing CENP-A
    Tachiwana, H.; Kagawa, W.; Shiga, T.; Osakabe, A.; Miya, Y.; Saito, K.
  • A map of nucleosome positions in yeast at base-pair resolution
    Brogaard, K.; Xi, L.; Wang, J.P.; Widom, J.

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