Reconstitution of Chromatin by Stepwise Salt Dialysis

[Abstract] Chromatin, rather than plain DNA, is the natural substrate of the molecular machines that mediate DNA-directed processes in the nucleus. Chromatin can be reconstituted in vitro by using different methodologies. The salt dialysis method yields chromatin that consists of purified histones and DNA. This biochemically pure chromatin is well-suited for a wide range of applications. Here, we describe simple and straightforward protocols for the reconstitution of chromatin by stepwise salt dialysis and the analysis of the chromatin by the micrococcal nuclease (MNase) digestion assay. Chromatin that is reconstituted with this method can be used for efficient homology-directed repair (HDR)-mediated gene edited with the CRISPR-Cas9 system as well as for biochemical studies of chromatin dynamics and function.

as octamers (which exist as octamers at high salt concentrations and as H2A-H2B dimers and H3-H4 tetramers at low salt concentrations) rather than individual core histones because the separate histones need to be combined in the correct stoichiometry and then purified as octamers before use (Khuong et al., 2017). Protocols for the purification of core histone octamers from different sources are described elsewhere (Laybourn and Kadonaga, 1991;Bulger and Kadonaga, 1994;Fyodorov and Levenstein, 2002;Peterson and Hansen, 2008;Khuong et al., 2017). Histones prepared by these methods have been extensively used for the efficient assembly of chromatin in vitro. We also tested a sample of commercially available native human histone octamers (catalog number 52065; BPS Bioscience, San Diego, CA) and found that they are suitable for reconstituting chromatin by the salt dialysis method.
2. DNA. To achieve the efficient reconstitution of chromatin, the quality of the DNA is extremely important. For the protocol described here, the DNA can be easily prepared by using the HiSpeed Plasmid Maxi Kit (catalog number 12662; QIAGEN, Hilden, Germany) or by carrying out two successive CsCl density gradient centrifugation steps. We typically use plasmid DNA prepared by these methods for reconstituting chromatin donor templates for HDR-mediated gene editing (Cruz-Becerra and Kadonaga, 2020). In addition, for mononucleosome gel shift assays, we use this salt dialysis protocol to reconstitute mononucleosomes with DNA fragments (≥ 147 bp) that are prepared 4. MNase digestion assay. In this assay, the partial digestion of chromatin with different concentrations of MNase (which cleaves the linker DNA between nucleosomes) reveals the formation of arrays of nucleosomes on the DNA template. After deproteinization, the resulting DNA fragments that are derived from the oligonucleosomes show a repeating ladder pattern on an agarose gel. The detection of a periodic array of oligonucleosomes, such as tetra-and pentanucleosomes, is an indication of high quality chromatin. 3 www.bio-protocol.org/e3977   In summary, we describe a protocol for the reconstitution of chromatin by simple stepwise salt dialysis.
This protocol can be completed in about 12 h (total time for chromatin reconstitution and analysis), and can be used with core histones from different sources and DNA prepared by standard laboratory procedures. In addition to the reconstitution of nucleosomes onto circular plasmid DNA, this method can be used for the preparation of mononucleosomes or polynucleosomes with linear DNA of different lengths (Chavez et al., 2019).

A. Reconstitution of chromatin
Here we describe the protocol for a standard chromatin reconstitution experiment. In this protocol, it is assumed that the optimal amounts of histones and DNA have been previously established.
To determine the optimal histone:DNA ratio with new preparations of histones and/or DNA, this

Load the histone-DNA mix from
Step A1 into a dialysis chamber.
The dialysis chamber consists of the sample reservoir, the dialysis membrane, and a sealing ring ( Figure 2). Assemble the dialysis chamber as follows: a. Cut a microcentrifuge tube (please see Note 8) such that the lid of the tube (i.e., the sample reservoir) is surrounded by a ring (i.e., the sealing ring).
b. Detach the sample reservoir from the sealing ring by cutting the linker between them. Then pull the two pieces apart. 6 www.bio-protocol.org/e3977  With the chromatin reconstitution procedure described in Section A, we generally recover greater than 90% of the starting amount of DNA (Figure 3). For many applications, the nucleosomal DNA concentrations can be estimated (within 10% of the method described below and shown in Figure   3) by the A260nm readings (using the extinction coefficient for pure double-stranded DNA) on a NanoDrop One C spectrophotometer (model ND-ONEC-W; Thermo Fisher Scientific, Waltham, MA). This is probably due to the low A260nm absorbance of the histones because of their low content of aromatic amino acid residues. Alternatively, the nucleosomal DNA concentrations can be determined by removal of the histones and quantification of the resulting DNA samples, as described below. In contrast, the use of a Qubit Fluorometer is not recommended because it does not provide accurate quantification of the nucleosomal DNA in the chromatin sample, probably because the binding of fluorescent dye molecules to nucleosomal DNA is less efficient than their binding to free DNA. m. Subject the DNA to electrophoresis on a 1.0% agarose-TBE gel at 3.7 V/cm until the Orange G dye reaches the bottom of the gel. The running time will vary according to the gel dimensions. As an example, a gel that is 5 cm × 6 cm × 0.5 cm (width × length × height) will 9 www.bio-protocol.org/e3977  The arrows indicate the DNA bands that correspond to mono-, di-, tri-, tetra-, and pentanucleosomes. 10 www.bio-protocol.org/e3977  b. Subject the DNA to electrophoresis on a 1.3% agarose-TBE gel at 3.3 V/cm until the Orange G dye reaches the bottom of the gel (this will take about 2.5 h with a gel that is 11 cm, 14 cm, and 0.6 cm in width, length, and height, respectively).
5. Visualize the DNA by EB staining.
a. Stain the gel for 30 min in EB staining solution.
b. Remove the excess EB with water (typically, two 10 min rinses in water).
c. Visualize the DNA with a UV transilluminator. 8. Chromatin can be reconstituted in reaction volumes of 150 µl and 75 µl with similar results. We use dialysis chambers prepared from 1.7 ml or 0.65 ml low binding tubes for 150 µl or 75 µl reconstitution reactions, respectively. Do not use smaller reaction volumes with these dialysis chambers. 9. As an alternate option, the Slide-A-Lyzer MINI dialysis units from Thermo Fisher Scientific (catalog number: 69550, Thermo Fisher Scientific, Waltham, MA) could be used instead of the dialysis chambers described here. 10. We recommend that the conductivity of the reconstituted chromatin sample is measured to determine the completion of dialysis. The conductivity of the reconstituted chromatin at the end of dialysis should be within 10% of the conductivity of TE Buffer containing 0.05 M NaCl.