Quantitative Measurement of Nucleosome Occupancy and DNA Accessibility

Abstract

Micrococcal nuclease (MNase) is typically used to digest free DNA in order to map nucleosome positions genome-wide. Unfortunately, MNase has a strong DNA sequence specificity, and different levels of digestion release from chromatin different ensembles of nucleosomes [1,2]. Mild digestions release from chromatin the more accessible nucleosomes, while extensive digestions destroy the former ones and release the less accessible nucleosomes. Recently, we have developed a new nucleosome mapping method that does not use MNase, and eliminates the biases introduced by this nuclease [3]. Nevertheless, MNase-seq remains the most used method of mapping nucleosomes, although its results are not quantitative and are strongly affected by the level of digestion. Using a rigorous mathematical treatment of all the steps involved in DNA digestion by nucleases, we have developed a quantitative framework of obtaining the nucleosome occupancy and DNA accessibility genome-wide, at the single-nucleosome level. We show that the traditional way of analyzing MNase-seq experiments is flawed, and the coverage obtained by stacking the undigested DNA fragments obtained at any level of digestion is not proportional to the real nucleosome occupancy. Therefore, the MNase-seq coverage is an inaccurate measure of the fraction of cells containing a nucleosome at a given position. Using a series of digestions, we determine the real fraction of cells containing a nucleosome at each position, DNA accessibility, and the rates of MNase digestion corresponding to each position in the genome of Drosophila melanogaster. [1] Chereji RV et al. - Nucleic Acids Res 44, 1036 (2016) [2] Chereji RV et al. - Mol. Cell 65, 565 (2017) [3] Chereji RV et al. - Genome Biol. 19, 19 (2018)