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Post-translational modifications (PTM) of histones include acetylation, methylation, ubiquitination, phosphorylation, ADP-ribosylation and sumoylation, which play important roles in regulating transcription, chromatin assembly, DNA repair, recombination and DNA replication (1-2). Histone modifications also serve as epigenetic marks that can be inherited through cell division to maintain lineage specificity (3). Therefore, determination of PTM functions has been a central focus of the chromatin field for the past two decades (4).
Chemically modified histone peptides are commonly utilized to identify PTM recognition modules or used as substrates for various enzymatic reactions in vitro. Although this type of assay has led to many major discoveries in the field, some intrinsic shortcomings limit their broad applications. First, short histone peptides may only cover a partial functional surface of histones; second, without DNA, free histone peptides may not recapitulate the native conformation of intact nucleosomes; third, technical limitations of peptide synthesis may prevent the desired combination of PTM when multiple histones are involved or a great distance between modifications is needed. Therefore, using chromatin templates that carry specific PTM becomes increasingly desirable for biochemical analysis of histone modifications. In this chapter, we describe two sets of protocols for reconstituting designer nucleosomes that contain specifically modified histones. We first present a small scale reconstitution method in which radiolabeled DNA templates are used and resulting nucleosomes are suitable for electro-mobility shift assays (EMSA) or chromatin remodeling reactions (Figure 1 and subheading 3.1, 3.2 and 3.3). We then discuss a generic method to prepare modified nucleosomes on a large scale for functional and structural studies. Histone modifications can be introduced either at the level of individual histones through chemical approaches (Figure 1, subheading 3.2) (5-7) or at the level of nucleosomes via a broad range of site specific histone modifying enzymes (Figure 1, subheading 3.3 and 3.4) (8-9). With proper pairings of these two strategies, one can expect to generate a single nucleosome containing various combinations of histone modifications for studying cross-talk between PTMs. Due to space limitation, in the cases where procedures described here were adapted from previously established protocols (5, 10-12), we will primarily emphasize the modifications which we have made and the critical parameters that are important for successful subsequent steps.
The protocol in this session is based on a previous method with minor modifications (5).
These nucleosomes can be directly used as substrates in EMSA assays or chromatin remodeling assays as described in another chapter by Tom Owen-Hughes.
|TEBS2.0 (2M NaCl)||1hr|
|1×TEB||overnight (at 4°C)|
1We prepare each individual recombinant histone based on the general protocol described by the Luger lab (10). We typically use 3-liter culture for wild type histones and 1.5 liter for mutants, which yield about 100-500mg of histones. We found that applying the solubilized inclusion bodies through a single ion-exchange column (5ml Hi-Trap SP (GE)) results in adequate purity for the procedures described here.
2The concentrations of histones are determined by OD276 using the theoretical extinction coefficients calculated on the ExPASy Proteomics Server. For mutant histones, concentrations are further confirmed by running on a SDS-PAGE gel and comparing to a standard curve of a histone with known concentration.
3For histones containing native cysteines, these residues should first be mutated to alanine. The resulting “wild type” histones should be tested in available in vitro and/or in vivo functional assays to ensure that the C-to-A mutation itself introduces minimal interference within the intended experiments. The commonly used histone H3C110A does not result in any noticeable phenotype in all tested assays thus far. Your favorite lysine(s) can then be substituted with cysteine(s) in this “wild type” background. Multiple cysteines can be introduced in one histone (such as H3Kc9 and H3Kc36); however, only one type of methylation analog can be installed at these residues using the current protocol. We have tested various combinations of double lysine mutant histones, and found that the recipes described here are sufficient for completion of such double reactions.
4If chemical approach can generate all intended histone modification combinations, non-biotinylated DNA templates can be used. Proceed to nucleosome reconstitution (Step 4-6). In this case, these nucleosomes can be directly gel-purified starting from Step 17 in subheading 3.3. 32P also can be incorporated into DNA using a body-labeling method in which PCR reactions using the same primer sets are carried out in the presence of 32P-labeled nucleotides.
5We measure radioactivity incorporation (cpm) by counting 1μl of purified DNA on a scintillation counter. We then determine the DNA concentration (ng/μl) by running 1 μl of DNA on a 2% Agarose gel with known concentration DNA markers. This allows us to deduce the ratio of cpm/ng for the labeled DNA and estimate the amount of DNA during subsequent steps by monitoring the radioactivity (cpm).
6Theoretically histone and DNA should be in a 1:1 molar ratio of nucleosomes binding sites to octamers or a 1:1.3 mass ratio of histone octamers to DNA. We typically perform a titration test with 1.25 fold increments. Once the optimal histone/DNA ratio for any given histone octamer is determined, it can be consistently used for the same preparation with different DNA probes.
7The histone modifying enzymes that are suitable for the assays described in this chapter should be highly active and possess nucleosomal activity. Preliminary tests are normally performed to determine the necessary amount of enzymes and the completeness of the modification reactions. If 80-90% of a target population is modified, these nucleosomal templates are generally satisfactory for most other assays.
8The generic histone acetyltransferase complexes used here are purified from yeast using the TAP method (13). We typically use Ada2-TAP, use of which yields a mixture of three HAT complexes: SAGA, SLIK and ADA, to provide the histone H3 specific HAT, and the NuA4 (Epl1-TAP) complex to provide the histone H4 HAT.
9A pBlueScript vector carrying a single copy 601sequence flanked by several restriction sites (Figure 2, top left panel) is digested with KpnI and BamHI, the small fragment is ligated to the same vector digested with KpnI and BglII to produce the 2× 601 plasmid. Since the junction site of BamHI and BglII can no longer be cut by either enzyme, the two steps in Figure 2 can be multiplied several times to generate more repeats. We found that for the single sequence that is less than 300bp in length, a 16× version of construct can be routinely obtained.
10A previous established procedure for a large scale plasmid preparation (10) is adopted with minor modifications. We normally start with 2 liters of 2×TY culture. After 18-22 hrs incubation at 37°C, about 20 grams of cells can be harvested, which yield about 10 mg of plasmid DNA.
11The simple procedure described here only removes free co-factors. Most modifying enzymes remain in the final nucleosome fraction, which should be factored into the data interpretation as appropriate.
12The following procedures are based on a previous protocol (12). Spot 5 μl of acetylated nucleosomes on a piece of negatively charged P81 phosphocellulose filter. Air dry filters for 5mins. Wash filters with 50 ml of HAT wash buffer at RT for 5 mins and then repeat two more times. Filters are briefly rinsed with 30 ml acetone and then air dried for 5 min. Place the filters into scintillation vials containing 4 ml of scintillation fluid (for filters) and measure the radioactivity using a scintillation counter.