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.