High-resolution mapping of changes in histone-DNA contacts of nucleosomes remodeled by ISW2 - PubMed (original) (raw)
High-resolution mapping of changes in histone-DNA contacts of nucleosomes remodeled by ISW2
Stefan R Kassabov et al. Mol Cell Biol. 2002 Nov.
Abstract
The imitation switch (ISWI) complex from yeast containing the Isw2 and Itc1 proteins was shown to preferentially slide mononucleosomes with as little as 23 bp of linker DNA from the end to the center of DNA. The contacts of unique residues in the histone fold regions of H4, H2B, and H2A with DNA were determined with base pair resolution before and after chromatin remodeling by a site-specific photochemical cross-linking approach. The path of DNA and the conformation of the histone octamer in the nucleosome remodeled or slid by ISW2 were not altered, because after adjustment for the new translational position, the DNA contacts at specific sites in the histone octamer had not been changed. Maintenance of the canonical nucleosome structure after sliding was also demonstrated by DNA photoaffinity labeling of histone proteins at specific sites within the DNA template. In addition, nucleosomal DNA does not become more accessible during ISW2 remodeling, as assayed by restriction endonuclease cutting. ISW2 was also shown to have the novel capability of counteracting transcriptional activators by sliding nucleosomes through Gal4-VP16 bound initially to linker DNA and displacing the activator from DNA.
Figures
FIG. 3.
Mapping the changes in the position of the H2A/H2B histone fold with DNA before and after ISW2 remodeling. (A) DNA mapping results with histone octamers containing mutant H2A Ala 45 or H2B Ser 53 changed to Cys are shown. Samples were prepared and processed as described in the legend to Fig. 2B. The locations of the cut sites are indicated on the left and right sides. The modified histone octamer used in each sample is indicated at the top of the lane. ISW2, Gal4-VP16, and ATP were added as indicated. (B) Summary of the DNA cleavage results with histone octamers modified at residues 45 of H2A and 53 of H2B with correlation to the dyad axis mapping results for histone octamers modified at residue 47 of H4. The vertical arrows above and below the DNA sequence indicate the cut sites obtained with modified H2A or H2B. The horizontal arrows show where the predicted cut sites would be based on the previously determined dyad axis for either the unremodeled or remodeled nucleosome. (C) Gal4-VP16 is bound quantitatively to the183-bp nucleosome before and after remodeling. Small portions of the H2B C53 mapping reactions analyzed in panel A, lanes 1 to 4, were loaded onto a 4% native polyacrylamide gel to assess the extent of Gal4-VP16 and ISW2 binding to the nucleosome. Identical results were obtained with the other two modified nucleosomes (data not shown).
FIG. 1.
Evidence for sliding of mononucleosomes to the center of DNA by ISW2. (A) Nucleosomes were reconstituted on radiolabeled DNAs of 183, 210, and 243 bp and separated on a native 5% polyacrylamide gel. The longer DNAs when assembled into nucleosomes bound in different translational positions that were resolved and are indicated as N1 to N4 on the right. ISW2 and ATP were added as indicated at the top of the lanes. (B) The ATPase activity of ISW2 is stimulated by nucleosomes with as little as 183 bp of DNA. Mononucleosomes were reconstituted with recombinant histone octamer and DNAs of 183, 210, and 243 bp and used as substrate in ATPase assays of ISW2. Three different concentrations of ISW2 were used: 21, 7, and 2.3 nM.
FIG. 2.
High-resolution mapping of the 183-bp nucleosome before and after ISW2 remodeling. (A) The locations of the site-specific modification of the nucleosome at histone H4 residue 47, histone H2A residue 45, and histone H2B residue 53 are shown on the nucleosome. The nucleosome structure is shown with the specific residues highlighted by open circles and with the nucleosome shown in two orientations rotated 180° around the DNA superhelical axis (front and back views). (B) Site-directed mapping results with histone octamers containing histone H4 with Cys 47 coupled to an aryl azide. DNA radiolabeled at the 5′ end of either the upper or lower strand was used to map the location of the DNA cleavage site induced by the cross-linking of histone H4. The A and G sequencing ladders of the same DNA with unphosphorylated primers (labeled A and G) were also loaded for comparison. A lighter exposure of the sequencing ladders is shown to compensate for the higher level of signal in these lanes. The samples contained ATP (300 μM), ISW2 (2 nM), and Gal4-VP16 (50 nM), as indicated at the top of each lane. (C) Cut sites on the upper and lower strands of the 183-bp DNA are shown by arrows above and below the DNA sequence. The cut sites corresponding to each translational position, referred to as A, B, and C, are shown separately. The asterisk indicates the dyad axis of each, as mapped by the DNA cut sites, and the position of the nucleosome is shown as a shaded oval over the DNA sequence. The thickness of the arrow represents the relative efficiency of cutting, and the _Eco_RV restriction endonuclease site is underlined in the DNA sequence and labeled.
FIG.4.
DNA photoaffinity labeling of histones from bp 12 to 66. (A) Five DNA photoaffinity probes were constructed from the 183-bp GUB DNA with photoreactive and 32P-labeled nucleotides incorporated at the depicted sites. The underlined C and U are the photoreactive nucleotides DB-dCMP and DB-dUMP, and the asterisk over the p is the 32P-labeled phosphodiester. The relative positions of these modifications in the nucleosome are shown before (dyads at bp 84 and 104) and after (dyad at bp 93) ISW2 remodeling. (B) The nucleosomes were assembled with 183-bp modified DNA probes containing photoreactive nucleotides at bp 12, 19, 42, 52/54, and 66 in the top strand. ISW2, ATP, and γ-S-ATP were added as indicated above each lane. The photoaffinity-labeled samples were processed as described in Materials and Methods and visualized by phosphorimaging.
FIG. 5.
Gal4-VP16 does not block nucleosome sliding by ISW2. Gal4VP16 was prebound to nucleosomes for 30 min at 30°C in reaction mixtures containing 300 μM ATP, 4.6 pmol of Gal4-VP16, and 3.4 pmol of nucleosomes, and remodeling was started by addition of 0.2 pmol of ISW2 for 1 to 15 min (lanes 4 to 13). After remodeling, Gal4-VP16 was competed with 25 pmol of a short DNA fragment containing single Gal4 binding site for 5 min at 30°C (lanes 3 and 10 to 13). To stop remodeling during competition, the reaction mixture was supplemented with 10 mM γ-S-ATP and 3 μg of salmon sperm DNA. The reactions were analyzed by gel shift on 5% polyacrylamide native PAGE (acrylamide/bisacrylamide ratio, 60/1) gel containing 0.2× TBE at 4°C. In lane 10, the salmon sperm DNA and γ-S-ATP were added before the addition of ISW2.
FIG.6.
ISW2 does not make the nucleosomal DNA more accessible to the restriction endonuclease _Eco_RV. Sucrose gradient-purified nucleosomes (10 nM) were subjected to cleavage with _Eco_RV (200 U) in the absence (lane 1) or presence of various amounts of ISW2 (2 to 150 nM, lanes 3 to 7) or SWI/SNF (50 nM, lane 2) and ATP (800 μm) for 40 min at 30°C. Free DNA was digested to completion under those conditions in less than 0.5 min (data not shown). After deproteinizing, the cleaved DNA was resolved from the intact DNA on a 4% native polyacrylamide gel, and the signal in the bands was quantified by phosphorimaging.
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