CLASSIC PROTOCOL A look back: finding chromatin’s footprints Researchers have been aware of the existence and magnesium, could be partially digested of chromatin for well over a hundred years, with by endogenous nucleases to yield a pattern of Albrecht Kossel first identifying this darkly staining repeating units6, apparently resulting from the nuclear material in 1884 (ref. 1). Subsequent research nuclease resistance of chromatin-associated soon had identified chromatin as a combination of DNA. Similar work with nucleases had been various histone proteins and DNA in a roughly equal performed by chromatin pioneers such as Kensel methods weight ratio, although its actual structural details van Holde and Gary Felsenfeld, but due to the would remain enigmatic for nearly a century to come. design of their experiments, a repeating pattern Roger Kornberg first arrived at the MRC Laboratory had not been observed. of Molecular Biology in Cambridge in 1973, with the Kornberg and Noll were intrigued, but initial .com/nature e goal of working with Aaron Klug, an expert in X-ray efforts by Noll to purify the endogenous nuclease crystallography and a colleague of Francis Crick. were thwarted by low yields of enzyme, and he .natur Having become acquainted with Crick’s recent studies began investigating various commercially available w of chromosomal structure2, Kornberg approached Klug nucleases. Micrococcal nuclease worked beautifully, about the possibility of applying physical techniques specifically digesting the DNA between individual to the detailed analysis of chromatin. “He was very nucleosomes to yield clean patterns of bands http://ww 7 enthusiastic,” recalls Kornberg, “and he knew, as it separated by 205–base pair intervals —the length turned out, an immense amount about it—more than of DNA predicted to be nucleosomally associated in oup r I ever could have imagined… [but] he warned me Kornberg’s model. The efficiency and consistency of G that it would be, in his words, ‘a messy problem’.” the cleavage confirmed that most chromosomal DNA Messy indeed—early studies of histones was bound up in nucleosomal units, and micrococcal appeared to demonstrate that the purified proteins nuclease digestion rapidly became the technique of lishing b aggregate freely and randomly, and a major review choice for chromatin analysis. “Even a few months Pu published on the topic in 1973 asserted that the after the paper appeared in Nature… it was very hard evidence strongly disfavored a unitary structure for to get micrococcal nuclease,” recalls Noll. “There was chromatin3. Kornberg had noticed several problems this meeting in 1977 at Cold Spring Harbor, and half Nature with contemporary histone studies, however—in of it was this kind of work with micrococcal nuclease– 5 particular, he was concerned about the harsh digested chromatin fragments.” 200 conditions under which histones were purified. Noll would briefly continue his chromatin work, © “My key recognition was that these were actually using nuclease digestion to reveal the chromatin denatured molecules, and that’s why the interactions shift that occurs at active promoters8, before apparently made very little sense,” he says. “All of the making the move into developmental biology. For histones stuck to one another every which way, and Kornberg, however, this was only the beginning of there was no pattern or sensible set of interactions a long and fruitful career in chromatin research, from which one could hope to deduce anything about and even now, he praises the power of nuclease the structure.” digestion. In fact, his group recently used the Kornberg performed a new series of histone technique to demonstrate the surprising finding that experiments, and incorporated his findings with nucleosomes are fully removed from sites of active a reinterpretation of existing data to develop a transcription9, work that Kornberg describes as “a new model, wherein chromatin is composed of denouement for the entire [chromatin] story”—proof ‘nucleosomes’⎯repeating units of chromosomal DNA that micrococcal nuclease still remains useful for wrapped around a core octamer of histone proteins4,5. clarifying chromatin controversies. It was an intriguing—and controversial—concept, Michael Eisenstein and promptly caught the attention of newly arrived 1. Kossel, A. Z. Physiol. Chem. 8, 511–515 (1884). MRC postdoc Markus Noll. “Kornberg’s model wasn’t 2. Crick, F. Nature 234, 25–27 (1971). really completely supported by experimental facts—it 3. Huberman, J.A. Annu. Rev. Biochem. 42, 355–378 was still quite a bit speculative,” says Noll, “but he (1973). 4. Kornberg, R.D. & Thomas, J.O. Science 184, 865–868 really had put together the facts that he thought (1974). were credible into a beautiful model.” 5. Kornberg, R.D. Science 184, 868–871 (1974). Likewise, Kornberg was not fully certain of his 6. Hewish, D.R. & Burgoyne, L.A. Biochem. Biophys. Res. theory, until he became aware of recent data from Commun. 52, 504–510 (1973). 7. Noll, M. Nature 251, 249–251 (1974). a pair of Australian researchers who had found 8. Levy, A. & Noll, M. Nature 289, 198–203 (1981). that nuclear DNA, when incubated with calcium 9. Boeger, H. et al. Mol. Cell 11, 1587–1598 (2003). 718 | VOL.2 NO.9 | SEPTEMBER 2005 | NATURE METHODS.