Mechanism of chromatin remodeling

Abstract

Results from biochemical and structural studies of the RSC chromatin-remodeling complex prompt a proposal for the remodeling mechanism: RSC binding to the nucleosome releases the DNA from the histone surface and initiates DNA translocation (through one or a small number of DNA base pairs); ATP binding completes translocation, and ATP hydrolysis resets the system. Binding energy thus plays a central role in the remodeling process. RSC may disrupt histone-DNA contacts by affecting histone octamer conformation and through extensive interaction with the DNA. Bulging of the DNA from the octamer surface is possible, and twisting is unavoidable, but neither is the basis of remodeling.

Fig. 1.

Effects of RSC- and ATPγS binding upon DNase I digestion of nucleosomal DNA. (A) DNase I digestion patterns of naked DNA and nucleosomes, under the conditions indicated above the lanes. Digestion was with 0.33 units of DNase I (ProMega) for 15 sec (lanes 1 and 2) or with 0.6 units for 40 sec (lanes 3–8). Positions of size markers produced by digestion of naked DNA with either Msp I or Alu I are indicated by DNA length on the right. (B) Profile of radioactivity in lane 4 of the region indicated by the bracket on the right of (A). Arrows identify cutting sites exposed by RSC–nucleosome interaction. (C) DNase I digestion pattern in the vicinity of the hypersensitive site indicated by the arrow in (A), under the conditions indicated above the lanes. In this case, the nucleosomal DNA was labeled at the end opposite to that in (A). Amounts of enzyme and times of digestion were the same as in (A).

Fig. 2.

Effect of RSC-binding upon exo III digestion of nucleosomal DNA. Digestion of nucleosomes (2.5 ng DNA) was performed with 40 u exo III (New England Biolabs) for 5 min under the conditions indicated above the lanes (0.3 μg RSC, 0.5 mM ATP or ATPγS). Position of size marker produced by digestion of naked DNA with Msp I is indicated by DNA length on the right. Product of limit exo III digestion, approximately half the length of the starting nucleosomal DNA, is indicated by an asterisk.

Fig. 3.

Dissociation of H2A-H2B dimers from nucleosomes and reversal in the presence of acetate. Nucleosomes (1 ng DNA) were incubated with naked DNA (1 μg pUC19) for 5 min and analyzed by gel electrophoresis. For lanes to the left of the center line, incubation was in the presence of potassium acetate at the concentrations indicated; for lanes to the right of the center line, incubation was with naked DNA alone, followed by the addition of potassium acetate at the concentrations indicated. The bands in the gel correspond to the nucleosome and tetramer particle. The relative intensities are plotted as black diamonds and red squares below.

Fig. 4.

Proposed mechanism of chromatin remodeling. Schematic of nucleosome at left depicts histone octamer as gray disk and DNA as solid curve. Schematic of RSC–nucleosome complex in center depicts RSC as yellow volume surrounding the nucleosome, with Sth1 region in red, with positively charged surface for interaction with the nucleosome, and with DNA largely dissociated from surface of the histone octamer. Schematic of RSC–nucleosome at right shows DNA translocated in the presence of ATP.