ISWI Is an ATP-Dependent Nucleosome Remodeling Factor Davide F.V Corona, Gernot Längst, Cedric R Clapier, Edgar J Bonte, Simona Ferrari, John W Tamkun, Peter B Becker  Molecular Cell  Volume 3, Issue 2, Pages 239-245 (February 1999) DOI: 10.1016/S1097-2765(00)80314-7

Figure 1 ISWI Is a Nucleosome-Stimulated ATPase (A) ATPase assays contained either of the following: 1.3 pmol of rISWI (160 ng), an equivalent volume of the control protein preparation, or 0.6 pmol of CHRAC. The substrates of the reaction were either 3.75 pmol (360 ng) of a 145 bp fragment (D) or the equivalent amount of nucleosomal DNA (N). The asterisk points to the signal derived from free phosphate generated during the 30 min assay. (B) A comparison of the ATPase activity of similar amounts of rISWI-wt and the K159R mutant derivative of rISWI. In order to monitor any residual ATPase activity of the K159 mutant, the reaction was run for 60 min. (C) The ISWI ATPase is not stimulated by free DNA. The following DNA substrates (100 ng) were included into standard ATPase assays (30 min reactions) containing recombinant rISWI or purified CHRAC: nucleosomal DNA (N), a 3 kb supercoiled plasmid (SC), a 146 bp PCR fragment (PCR), a synthetic four-way junction (4WJ), phage lambda DNA (λ, approx. 50 kb), polyA+ RNA (RNA). The ATPase activity is displayed as the percentage of ATP hydrolyzed during the assay. (D) Competition of histone N-terminal “tails” with nucleosomes. ATPase assays as in (B) contained either only buffer (B), 1 pmol of nucleosomes (N), or nucleosomes plus 50 pmol of individual Drosophila histone N termini (as indicated) fused to GST, or as a control, the GST moiety alone (GST). Molecular Cell 1999 3, 239-245DOI: (10.1016/S1097-2765(00)80314-7)

Figure 2 ISWI Is a Nucleosome Remodeling Factor (A) Nucleosome remodeling in the NURF assay. The nucleosome remodeling reaction is described in the text. The reactions contained ATP (throughout), GAGA factor as indicated, and the following remodeling factors: 0.15 pmol CHRAC, 0.13 pmol rISWI, rISWI-K159R, or 4 μl crude embryo extract, and a chromatin substrate equivalent of roughly 1 pmol nucleosomes. DNA fragments of diagnostic lengths: Arrow, mononucleosome; filled circle, dinucleosome; asterisk, atypical particle indicative of remodeling. The upper panel indicates the status around the GAGA boxes of the hsp26 promoter, while the lower panel shows the control hybridization of the same membrane with a vector-derived DNA probe. (B) TTF-1-dependent nucleosome remodeling at the rDNA promoter. TTF-1 binding to the rDNA promoter was assayed in the context of chromatin-bound remodeling machines (panel 1) or after endogenous remodeling factors had been removed by Sarkosyl treatment (panel 2), in the presence of either CHRAC or rISWI or rISWI-K159R as indicated. TTF-1 binding (box to the left) and aligned nucleosomes (ellipsoids) were visualized by indirect end labeling. (C) TTF-I-dependent nucleosome rearrangements on chromatin reconstituted from pure DNA and histones using polyglutamic acid as a histone carrier. Nucleosome remodeling was assayed analogous to (B) in the absence or presence of TTF-I, CHRAC, rISWI, and rISWI-K159R as indicated. Molecular Cell 1999 3, 239-245DOI: (10.1016/S1097-2765(00)80314-7)

Figure 3 rISWI Functions as a Chromatin Assembly Factor Nucleosomes were assembled from 200 ng purified DNA and histones using dNAP1 as a histone carrier (panel 1) and in the presence of 0.7 pmol of rISWI, 0.7 pmol of rISWI-K159R, or 0.2 pmol of CHRAC as indicated. The resulting nucleoprotein complex was digested with Micrococcal nuclease, the DNA fragments were purified and analyzed by agarose gel electrophoresis and ethidium bromide staining. Molecular Cell 1999 3, 239-245DOI: (10.1016/S1097-2765(00)80314-7)