RSC Unravels the Nucleosome with the DNA

with the DNA. The stability of the nucleosome is reYahli Lorch, Mincheng Zhang, and Roger D. Kornberg* duced, but the gross structure of the nucleosome is Department of Structural Biology unaffected. This “activated” state of the nucleosome is Stanford School of Medicine dependent on the presence of the chromatin-remodelStanford, California 94305 ing complex but not on continuing ATP hydrolysis (Imbalzano et al., 1996; Cote et al., 1998; Lorch et al., 1998; Schnitzler et al., 1998). Summary Reduced stability of the activated nucleosome is manifested by “mobility” of the core histone octamer. The RSC and SWI/SNF chromatin-remodeling complexes activated nucleosome formed by RSC can transfer the were previously reported to generate a stably altered octamer to exogenous DNA, forming a new nucleosome nucleosome. We now describe the formation of hy(Lorch et al., 1999). SWI/SNF and also ISWI complexes brids between nucleosomes of different sizes, showpromote sliding of the octamer to adjacent positions ing that the stably altered structure is a noncovalent along the same DNA (Hamiche et al., 1999; Langst et dimer. A basis for dimer formation is suggested by an al., 1999). Sliding can occur without transfer and often effect of RSC on the supercoiling of closed, circular culminates in most stable positions of the octamer near arrays of nucleosomes. The effect may be explained the ends of linear DNA (Whitehouse et al., 1999). by the interaction of RSC with DNA at the ends of the Removal of the chromatin-remodeling complex from nucleosome, which could lead to the release 60–80 bp an activated nucleosome results in a persistently altered or more from the ends. DNA released in this way may state (Cote et al., 1998; Lorch et al., 1998; Schnitzler et be trapped in the stable dimer or lead to alternative al., 1998). The alteration is revealed by an increased fates such as histone octamer transfer to another DNA sedimentation rate and decreased electrophoretic moor sliding along the same DNA molecule. bility. The altered nucleosome retains a full complement of histones but with a diminished histone–DNA interacIntroduction tion, as shown by increased sensitivity to dissociation at elevated ionic strength. The coiling of DNA by histones in nucleosomes is a An activated nucleosome formed by a chromatinbarrier to the entry of proteins involved in transcription, remodeling complex can, therefore, meet three fates: replication, and other DNA transactions (Grunstein, histone octamer transfer, sliding, or conversion to a sta1990; Kornberg and Lorch, 1999; Struhl, 1999; Wyrick bly altered state. Here we investigate the perturbation of et al., 1999). The barrier is high due to the tight binding the nucleosome underlying these processes. We show of histones to DNA and consequent stability of the how the structure of the stably altered nucleosome and nucleosome. The barrier is breached at least in part a previously unexplained change in DNA supercoiling through the action of chromatin-remodeling complexes. of nucleosomal arrays may be related, with attendant Two families of such complexes containing Swi2/Snf2 insight into the mechanism of chromatin remodeling by or ISWI ATPases have been described, and evidence RSC and SWI/SNF complexes. for a third family based on the Mi-2 ATPase has been presented (Cairns, 1998; Aalfs and Kingston, 2000; Results Boyer et al., 2000; Vignali et al., 2000). Yeast SWI/SNF complex is implicated by microarray analysis in the tranFormation of the Persistently Altered Nucleosome scription of a few percent of all genes and is nonessential In previous studies, a nucleosome on a small DNA fragfor cell growth (Sudarsanam et al., 2000). A related comment was converted to an activated complex by incubaplex, termed RSC, contains several homologs of SWI/ tion with yeast RSC or human SWI/SNF in the presence SNF subunits, all of which are encoded by essential of ATP, followed by the removal of the chromatin-remodgenes (Cairns et al., 1996, 1998, 1999). RSC mutants eling complex by competition with excess nucleosomes have been identified with spt phenotypes, pointing to or DNA (Lorch et al., 1998; Schnitzler et al., 1998). Breaka role of the RSC complex in transcription as well (Cairns down of the activated complex yielded the original et al., 1998). nucleosome and a smaller amount of a persistently alYeast and human SWI/SNF complexes and yeast RSC tered form. We now find, in reactions performed with complex perturb the structure of nucleosomes in an RSC and a nucleosome on a slightly longer DNA fragATP-dependent manner in vitro. Nucleosomal DNA is ment (217 bp, compared with 154 bp in the original rendered more accessible to nuclease digestion, and experiments with RSC), that the altered nucleosome is DNA supercoiling of a closed circular array of nucleoobtained without the addition of competing nucleosomes is much diminished (Cote et al., 1994; Imbalzano somes or DNA. The altered nucleosome is released from et al., 1994; Cairns et al., 1996; Guyon et al., 1999; Jaskelthe complex with RSC over time (Figure 1A). Apparently, ioff et al., 2000). Despite the increase in accessibility the additional DNA protruding from the ends of the 217 and loss of supercoiling, the histones remain associated bp nucleosome facilitates the formation of or stablilizes the altered nucleosome. It thus accumulates despite conversion back to the original state, which is catalyzed * To whom correspondence should be addressed (e-mail: kornberg@