Identification of Accessible Chromatin Regions with MNase-seq

Background

Accessible chromatin regions (ACRs) of eukaryotes generally imply the functional genome of the species or the collection of cis-regulatory elements (CREs) (Lu et al., 2019), such as regulatory elements located in promoters and enhancers (Yan et al., 2019). By identifying ACRs, important non-coding CREs and their roles in the regulation of gene expression, species growth and development, environmental adaptation, and natural evolution can be explored. There are several methods to identify ACRs, the most common of which are DNase-seq, ATAC-seq, and MNase-seq (Tsompana and Buck, 2014; Klein and Hainer, 2020). Unlike the enzymes DNase I used in DNase-seq and Tn5 used in ATAC-seq, MNase has both endonuclease and exonuclease activities, which digest and degrade naked DNA, leaving only sequences bound by repressors such as nucleosomes or DNA-binding proteins. The process is highly sensitive to the degree of digestion (i.e., MNase dose or concentration) and can be used to study nucleosome occupancy and digestion sensitivity, indirectly obtaining ACRs. Compared with DNase-seq and ATAC-seq, MNase-seq can identify some ACRs that cannot be identified by the other methods (Zhao et al., 2020). For example, in Arabidopsis thaliana, 20% more ARCs were identified by MNase-sensitive sites, obtained by sequencing 20–100 bp fragments, than by DNase-seq or ATAC-seq reads coverage. Meanwhile, MNase-seq can be used to estimate and contrast histone and non-histone DNA-binding components (Chereji et al., 2017) and identify both open and closed chromatin regions (Vera et al., 2014). However, the identification of plant ACRs with this method has also the disadvantage of demanding more sequencing material and higher sequencing depth, which requires weighing the pros and cons.

There are multiple strategies for identifying ACRs by MNase-seq. One is to select and sequence small DNA fragments (generally significantly smaller than the length of a mononucleosome, such as <80 bp or <100 bp), which may be the binding sites of some non-histone DNA-binding proteins (e.g., TF), directly based on MNase light digestion and define enrichment location of these small fragments as ACRs (Zhao et al., 2020). An alternative strategy is to define ACRs by processing with MNase digestion concentration gradients and sequencing DNA fragments that show a length close to that of mononucleosomes, to detect loci with changes in susceptibility to digestion (Rodgers-Melnick et al., 2016). Another strategy is to use MNase-seq and then combine it with ChIP-seq of histone subunit H3 or H4 for capture, to indirectly calculate the ACRs (Cook et al., 2017). Using these strategies, ACRs have been identified, and cis-acting elements have been found in human and mammalian species, Drosophila, yeast, Arabidopsis, maize, rice, and wheat (Fang et al., 2016; Mieczkowski et al., 2016; Rodgers-Melnick et al., 2016; Brahma and Henikoff, 2019; Schwartz et al., 2019; Jordan et al., 2020; Zhao et al., 2020). There may be differences in the ACRs obtained by the above strategies or in different species. This is mainly due to different research objectives, with differences in the definition of accessible chromatin and the target ACRs (a typical example is the controversy about fragile nucleosomes). Here, we provide a detailed protocol for this MNase-seq method in the identification of ACRs using the strategy of differential nuclease sensitivity, illustrated by data from polyploid crop wheat, while optimized for plant cell nuclei extraction and polyploid genome-specific alignment (Figure 1).

Figure 1. Schematic overview of the protocol.The experimental flow is shown on the left and the data analysis flow isshown on the right. MSF: MNase-sensitive footprint; MRF: MNase-resistantfootprint; ACR: accessible chromatin region.