Article Figures & Data
Figures
- Fig. 1.
Generation of histone octamers. Histone octamers were reconstituted from appropriate combinations of full-length and taillessXenopus histones (as indicated below the figure), purified by gel filtration, and separated in a 15% sodium dodecyl sulfate gel which was stained with Coomassie blue. The mobility of the histones is indicated on the left of the figure. Tailless histones, of which only the globular part contributes to the nucleosome, are indicated with the prefix “g” (e.g., gH4 indicates a tailless H4).
- Fig. 2.
The H4 tail is required for CHRAC-mediated nucleosome sliding. (Top) End-positioned nucleosomes reconstituted from histone octamers either containing all four full-length histones (intact) or with one tail missing (e.g., the H3 tail in the gH3 sample) were incubated with increasing concentrations of CHRAC, in which the CHRAC-to-nucleosome ratio was from 1:120 to 1:20. All reactions contained ATP, except for the one presented in the last panel. The reaction mixture was separated by native polyacrylamide gel electrophoresis. The position of traces of free DNA is indicated (DNA). (Bottom) Centrally positioned nucleosomes were mobilized with ISWI, and the ISWI-to-nucleosome ratio was from 1:20 to 1:10. Nucleosome sliding was analyzed as described in the legend for the top panel.
- Fig. 3.
ISWI ATPase is activated by the histone H4 tail. (A) ATPase assays were performed under conditions where ISWI generates regular nucleosome ladders and contained DNA, the recombinant histones indicated, and purified yNAP-1 and ISWI. The asterisk indicates the signal derived from free phosphate during the 1-h incubation. (B) Quantitation of ISWI ATPase as described for panel A in the presence of either DNA alone (lane 1) or the indicated histone octamers. The ATPase activity is displayed as the percentage of ATP hydrolyzed during the assay. The bars represent the average of three independent experiments, and the variability is indicated by the error bars.
- Fig. 4.
The H4 tail is required for ISWI to generate regular chromatin. (A) Two-dimensional supercoiling assay. Nucleosomes were reconstituted from the indicated mixtures of recombinant histones on circular plasmids using NAP-1 as a chaperone in the absence (−) or presence (+) of ISWI, and the resulting superhelicity was relaxed with topoisomerase I. The topoisomer distribution in the purified DNA was visualized by two-dimensional gel electrophoresis. (B) MNase digestion. Histone octamers of the indicated type were assembled into chromatin in a NAP-1 chromatin assembly system and subjected to MNase digestion. The resulting DNA fragments were purified and visualized by agarose gel electrophoresis and ethidium bromide staining. ISWI-generated regularity of nucleosomal arrays can be evaluated from a comparison of the patterns without (− ISWI) and with (+ ISWI) ISWI. The marker is a 123-bp DNA ladder.
- Fig. 5.
Recombinant ISWI binds to nucleosomes. ISWI (5 to 75 fmol) was incubated with mononucleosomes reconstituted with either four wild-type recombinant histones (intact) or three wild-type histones and one lacking the N-terminal tail (e.g., gH3 for globular H3) on a 248-bp radioactively labeled DNA fragment. Resulting complexes were separated by native polyacrylamide gel electrophoresis and visualized by autoradiography. Nucleosome-ISWI complexes are marked by arrows.
