Chromatin Remodeling In Vivo Thomas G Fazzio, Toshio Tsukiyama  Molecular Cell  Volume 12, Issue 5, Pages 1333-1340 (November 2003) DOI: 10.1016/S1097-2765(03)00436-2

Figure 1 Association of Isw2 Complex with Chromatin upon Induction with Galactose (A) Diagram of the system. (B) Kinetics of Isw2p association with the POT1 locus as measured by chromatin immunoprecipitation. 50-, 250-, and 1250-fold dilutions of DNA isolated from input chromatin are shown for comparison. Shown is one of two independent experiments that gave similar results. (C) Quantitation of the chromatin immunoprecipitation data shown in (B), expressed as a percentage of the input. Molecular Cell 2003 12, 1333-1340DOI: (10.1016/S1097-2765(03)00436-2)

Figure 2 Nucleosomes Migrate Upstream toward the POT1 Promoter upon ISW2 Induction Indirect end labeling of chromatin samples digested with (A), DNaseI, (B), methidiumpropyl-EDTA-iron(II) (MPE-Fe2+) and (C), micrococcal nuclease (MNase) during a time course of ISW2 induction. Times after galactose addition are indicated. Δisw2 and wild-type (WT) samples are shown for comparison. Inferred nucleosome positions for isw2 and wild-type cells are shown to the left and right, respectively, of each panel. The band marked by an asterisk is a cleavage or hybridization artifact also observed when cells untreated with nuclease are subjected to the same DNA isolation, restriction digestion, and blotting procedure (Fazzio et al., 2001). Molecular Cell 2003 12, 1333-1340DOI: (10.1016/S1097-2765(03)00436-2)

Figure 3 High-Resolution Mapping of Chromatin Remodeling Intermediates Reveals Isw2-Dependent Nucleosome Sliding at the POT1 Locus (A) DNA samples from micrococcal nuclease-digested chromatin were subjected to primer extension analysis using a primer corresponding to base pairs +126 to +96 with respect to the POT1 initiation codon. Shown for reference is a 50 bp ladder, digestions of naked DNA (N), and dideoxy sequencing reactions (G, A, T, C), as well as Δisw2 and wild-type (WT) samples. Small blue dots mark small to moderate increases or decreases in nuclease sensitivity, while large red dots indicate larger changes. Asterisks indicate MNase cleavage sites that persist throughout the time course. These sites are located within stretches of AT-rich sequences, which are hypersensitive to MNase cleavage. (B) magnified view of the upstream region in (A). (C and D) Densitometry analysis of MNase cleavage sites during the chromatin remodeling time course. Peaks represent the intensity of bands, normalized for loading. Blue and red arrows mark the peaks noted in (A) and (B) by dots of the same color. Upward and downward pointing arrows indicate increasing and decreasing MNase sensitivity, respectively. Inferred nucleosome positions in Δisw2 and wild-type cells are indicated above and below each panel. Molecular Cell 2003 12, 1333-1340DOI: (10.1016/S1097-2765(03)00436-2)

Figure 4 Analysis of Chromatin Structure at the REC104 Locus Reveals Isw2-Dependent Nucleosome Sliding at a Second Class of Genes (A) Primer extension mapping of MNase cleavage sites at the REC104 locus during a time course of ISW2 induction. Labels are as in Figure 3A. The location of the Ume6p binding site (URS1) is indicated. (B) Densitometry analysis of MNase cleavage sites during the chromatin remodeling time course. Labels are as in Figures 3C and 3D. Molecular Cell 2003 12, 1333-1340DOI: (10.1016/S1097-2765(03)00436-2)

Figure 5 Isw2 Complex Slides Nucleosomes Immediately upon Association with Chromatin, without Disrupting Nucleosomal Integrity (A and B) Accessibility of chromatin to the BanI and NcoI restriction enzymes over a time course of ISW2 induction. Times after galactose addition are indicated. Locations of the BanI and NcoI recognition sites are indicated in Figure 2A. For each enzyme, one of two experiments with very similar results is shown. (C) Quantitation of the data in (A) and (B) reveal the kinetics of chromatin remodeling at the POT1 locus. (D and E) Sensitivity of chromatin at the POT1 (D) and GAL10 (E) loci to extensive MNase digestion during galactose-mediated induction of ISW2. Three doubling concentrations of MNase are shown for each time point. Wild-type (WT) and Δisw2 control cells were grown in raffinose medium and processed identically to the inducible strain. (F) Ethidium bromide stain of the digested DNA blotted in (D) and (E), indicating the bulk chromatin digestion pattern. Molecular Cell 2003 12, 1333-1340DOI: (10.1016/S1097-2765(03)00436-2)