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Updated: Jan 19, 2026
Genome-wide Mapping of Protein-DNA Interactions with ChEC-seq in Saccharomyces cerevisiae
Published on: June 3, 2017
Quantitative MNase-seq accurately maps nucleosome occupancy levels
Răzvan V Chereji1, Terri D Bryson2, Steven Henikoff3
1Division of Developmental Biology, Eunice Kennedy Shriver National Institute for Child Health and Human Development, National Institutes of Health, Bethesda, MD, 20892, USA.
Abstract:
Micrococcal nuclease (MNase) is widely used to map nucleosomes. However, its aggressive endo-/exo-nuclease activities make MNase-seq unreliable for determining nucleosome occupancies, because cleavages within linker regions produce oligo- and mono-nucleosomes, whereas cleavages within nucleosomes destroy them. Here, we introduce a theoretical framework for predicting nucleosome occupancies and an experimental protocol with appropriate spike-in normalization that confirms our theory and provides accurate occupancy levels over an MNase digestion time course. As with human cells, we observe no overall differences in nucleosome occupancies between Drosophila euchromatin and heterochromatin, which implies that heterochromatic compaction does not reduce MNase accessibility of linker DNA.
Insights
Micrococcal nuclease sequencing (MNase-seq) can be unreliable for measuring nucleosome occupancy due to its nuclease activity. This study presents a new theoretical framework and experimental method for accurate nucleosome occupancy determination.
Area of Science:
- Molecular Biology
- Genomics
- Epigenetics
Background:
- Micrococcal nuclease (MNase) is a standard tool for mapping nucleosomes in DNA.
- However, MNase's potent nuclease activity can lead to inaccurate nucleosome occupancy measurements.
- This inaccuracy stems from both inter-nucleosome (linker) and intra-nucleosome DNA cleavage.
Purpose of the Study:
- To develop a reliable method for quantifying nucleosome occupancy.
- To introduce a theoretical framework for predicting nucleosome occupancy.
- To validate the method using experimental data and spike-in normalization.
Main Methods:
- Development of a theoretical model for nucleosome occupancy prediction.
- Establishment of an experimental protocol using MNase digestion.
- Implementation of spike-in normalization for accurate quantification.
- Analysis of nucleosome occupancy across a digestion time course.
Main Results:
- The study introduces a validated theoretical framework for predicting nucleosome occupancy.
- An experimental protocol with spike-in normalization provides accurate occupancy levels.
- No significant difference in nucleosome occupancy was found between euchromatin and heterochromatin in Drosophila.
- This suggests heterochromatin compaction does not impede MNase accessibility of linker DNA.
Conclusions:
- The developed method offers accurate nucleosome occupancy quantification.
- Heterochromatin accessibility to MNase is not reduced by compaction.
- This finding challenges assumptions about the impact of chromatin structure on nuclease accessibility.
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