Measuring occupancies of the nucleosome and nucleosome-interacting factors in vivo in Saccharomyces cerevisiae genome-wide

Abstract

Nucleosomes are the repeating units of chromatin. The presence of nucleosomes poses a major impediment to all DNA-dependent processes. As a result, access to DNA in chromatin is dynamically regulated by many factors, including ATP-dependent chromatin remodeling complexes. Digestion of chromatin by micrococcal nuclease (MNase) followed by chromatin immunoprecipitation (ChIP) and sequencing can be leveraged to determine nucleosome occupancy, positioning, and the ability of chromatin interacting factors to alter chromatin accessibility. Here we describe the procedure for performing MNase and MNase ChIP-seq in detail.

Figure 1. Schematics showing MNase-seq and ChIP MNase-seq processing.

MNase Digestion followed by chromatin immunoprecipitation and sequencing (MNase ChIP-seq) can be used to determine the occupancy, positioning, and accessibility of nucleosomes and factor-bound nucleosomes at a genome-wide scale. A) Schematic detailing the process of MNase ChIP-seq. Chromatin is digested by micrococcal nuclease (MNase) that digests accessible DNA, and the mostly-inaccessible nucleosomes are released into a soluble chromatin fraction that is collected after centrifugation. The MNase-protected DNA fragments (MPDFs; input DNA) representing nucleosomes can be purified. Additionally, the soluble chromatin can be subjected to ChIP followed by sequencing. The sequencing of the MPDFs from inputs and/or ChIP can be used to determine nucleosome occupancy, positioning, and accessibility after sequencing. The left-hand profile shown is a theoretical occupancy profile for an input, where the peaks correspond to the average occupancy of nucleosomes along a typical gene. The right-hand profile is a theoretical occupancy profile depicting the occupancy of a factor-bound nucleosome on a gene. B) Schematic showing the nucleosome accessibility for nucleosomes and factor-bound nucleosomes. Canonical nucleosomes are relatively inaccessible and show resistance to MNase digestion. Mono-nucleosome fragments from canonical nucleosomes typically yield fragments ~150 bp in length. Certain factors that bind to nucleosomes, such as chromatin remodelers, can act on nucleosomes to make their DNA more accessible. The DNA in the factor-bound nucleosomes could be digested to a greater extent leading to shorter than 150 bp. Thus, MNase ChIP-seq can be used to determine occupancies of chromatin-interacting factors at a nucleosomal resolution and also to determine differences between the accessibility of factor-bound nucleosomes from non-bound nucleosomes.

Figure 2. MNase digestion and library preparation

A) Purified DNA from MNase-digested chromatin is separated on an agarose gel. The digestion was done in replicates for three different MNase concentrations. Lanes 1-2 and 7-8; 3-4 and 9-10; and 5-6 and 11-12 represent digestion with low (45 units), medium (112.5 units), and high MNase concentrations (225 units), respectively. The digestion patterns are shown for the two different yeast strains. The bottom-most band represents mono-nucleosomes, and the next represents di-nucleosomes, and so on. The two strains show differences in MNase digestion patterns. M: 100 bp ladder (NEB). B) Gel pictures show the separation of DNA libraries from MNase-digested chromatin (lanes 1 and 2). The libraries show nucleosomal banding patterns. The gel picture on the left hand is before extracting the mononucleosomal band, and the right-hand gel shows after extracting the band for sequencing. M: 100 bp ladder (NEB).