For nucleosome mapping, a major improvement in resolution has been provided by the development of chemical mapping methodologies in which a cysteine is engineered into one of the core histones, enabling localized generation of hydroxyl radicals and precise cleavage of the nearby DNA backbone (Brogaard et al. 2012). Briefly, yeast are engineered to carry a mutant histone H4S47C, and after cell lysis, chromatin is treated with N-(1,10-phenanthroline-5-yl) iodoacetamide, which reacts with the H4 cysteine. Treatment of this adduct with copper and hydrogen peroxide results in generation of short-lived hydroxyl radicals, which diffuse very short distances before reacting with the DNA backbone or other molecules. Given the location of this H4 residue near the nucleosomal dyad axis, predominant cleavage sites occur at the +1 and +6 (and the −1 and −6) positions relative to the dyad. Deconvolution of the mix of cleavage sites then yields a map of precise locations of nucleosome dyads.
