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

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 155 bp, 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.

Nucleosome-Positioning Sequence Repeats Impact Chromatin Silencing in Yeast Minichromosomes

“…The remaining lanes contain samples digested with 0, 1, 2, 4, and 8 units/ml of MNase. The MNase cutting pattern was prominent with increasing enzyme concentrations as expected and consistent with MNase preferentially cutting at A-T-rich nucleotides in the nucleosome linkers in vitro (Nikitina et al 2013). The strongest MNase cutting sites are shown by boldface arrows ( Figure 3C).…”

“…The observed cutting pattern of the clone-601 nucleosome sequence is also schematized in Figure 3E. The dyad axis position at nucleotide 131 is according to nucleosome structural studies (Makde et al 2010;Nikitina et al 2013).…”

“…Here we examined if placing a clone-601 DNA sequence in combination with linker DNA of variable lengths between the silencer and the reporter would impede or facilitate silencing of the URA3 reporter gene. The clone-601 DNA sequence that directs positioning of the 147 bp nucleosome core (Nikitina et al 2013) is referred to as the "nucleosome-positioning sequence." We placed a 167 bp DNA fragment (601-16731) containing one nucleosome-positioning sequence between the E-silencer and the URA3 reporter gene (as shown in Figure 1, A-C, and schematic representations) and between the I-silencer and the URA3 reporter gene in different orientations ( Figure 1, D and E, and schematic representations).…”

Genome‐Wide Analysis of Nucleosome Positions, Occupancy, and Accessibility in Yeast: Nucleosome Mapping, High‐Resolution Histone ChIP, and NCAM

“… Exonuclease III is used to help eliminate the A/T cleavage bias of MNase and has been shown to reduce nucleosome footprints to the crystallographic footprint ( Nikitina et al 2013 ). If scaling the protocol for a higher starting cell number, chromatin may be digested with higher MNase concentration or for a longer time and will likely require optimization.…”

The proto-chromatosome: A fundamental subunit of chromatin?

“…2). 1,19 In this situation, it should be possible to produce less digested mono-nucleosomes using MNase and to remove the residual linker DNA at a much faster rate, before internal digestion of nucleosomes by MNase becomes significant. Since ExoIII is a single-strand 3 0 ->5 0 -exonuclease, mung bean nuclease was initially included to destroy the other strand.…”