Octamer transfer and creation of stably remodeled nucleosomes by human SWI-SNF and its isolated ATPases - PubMed (original) (raw)

Octamer transfer and creation of stably remodeled nucleosomes by human SWI-SNF and its isolated ATPases

M L Phelan et al. Mol Cell Biol. 2000 Sep.

Abstract

Chromatin remodeling complexes help regulate the structure of chromatin to facilitate transcription. The multisubunit human (h) SWI-SNF complex has been shown to remodel mono- and polynucleosome templates in an ATP-dependent manner. The isolated hSWI-SNF ATPase subunits BRG1 and hBRM also have these activities. The intact complex has been shown to produce a stable remodeled dimer of mononucleosomes as a product. Here we show that the hSWI-SNF ATPases alone can also produce this product. In addition, we show that hSWI-SNF and its ATPases have the ability to transfer histone octamers from donor nucleosomes to acceptor DNA. These two reactions are characterized and compared. Our results are consistent with both products of SWI-SNF action being formed as alternative outcomes of a single remodeling mechanism. The ability of the isolated ATPase subunits to catalyze these reactions suggests that these subunits play a key role in determining the mechanistic capabilities of the SWI-SNF family of remodeling complexes.

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Figures

FIG. 1

BRG1 and hBRM are capable of interconverting mononucleosome cores between a rotationally phased monomeric state and a stably remodeled dimeric state. (A) Creation of remodeled dimer product by hSWI-SNF and isolated ATPases. SWI-SNF (hS/S; 1 nM), 4 nM BRG1, or 12 nM hBRM was incubated with 390 pM labeled TPT cores and 2 nM unlabeled HeLa cell mononucleosomes in 200-μl remodeling reactions under standard conditions (see Materials and Methods), but with 30 mM KCl, 3.5 mM MgCl2, and 0.5 mM ATP, at 30°C for 40 min. The reactions were stopped by addition of KCl to 240 mM, and the products were separated on a 10 to 30% glycerol gradient by centrifugation for 19.5 h. Samples (2 μl) of the reaction products loaded on the gradients (Load) were analyzed by the gel shift method (lanes 1, 4, and 7). Peak gradient fractions (5-μl volumes) containing mononucleosome cores (mono) (peak 1; lanes 2, 5, and 8) and the novel dimer species (peak 2; lanes 3, 6, and 9) were also analyzed by the gel shift method. Percentages indicate the distances of these fractions from the top of the gradient, which is indicative of relative S values. (B) Comparison of the DNase I digestion patterns of mononucleosome dimers formed by BRG1, hBRM, and the hSWI-SNF complex. Aliquots of peak gradient fractions from the gel shown in panel A (0.075 ng of labeled DNA) were incubated with 0.25 ng of unlabeled HeLa cell mononucleosomes/μl under standard conditions, but with 36 mM KCl, 3.5 mM MgCl2, and 0.5 mM ATP, at 30°C for 30 min; this was followed by equilibration to room temperature for 5 min and digestion with 80 U of DNase I/ml for 2 min. DNase digestion was stopped with 200 μl of phenol, and DNA was isolated and separated by denaturing PAGE (lanes 1 to 6). Positions where cutting is increased (closed arrows) or decreased (open arrows) in the dimer species (lanes 2, 4, and 6) are indicated. For comparison, the DNase I patterns of unseparated BRG1 and SWI-SNF remodeling reactions, performed in the presence (+) or absence (−) of ATP, are shown (lanes 7 to 10). (C) BRG1 reconverts remodeled dimer back to mononucleosome cores. Remodeled dimers (4 pM), generated by BRG1 and isolated by glycerol gradient centrifugation (Fig. 1A, lane 6), were incubated with 2.5 nM HeLa cell mononucleosomes and with 250 pM (lanes 1 and 2) or 500 pM (lanes 3 and 4) hSWI-SNF or 4 nM (lanes 5 and 6) or 8 nM (lanes 7 and 8) and BRG1 for 35 min at 30°C in 50-μl standard reaction volumes containing 44 mM KCl and 3.5 mM MgCl2, with (+) or without (−) 0.5 mM ATP. The reactions were stopped by increasing the KCl concentration to 240 mM, and the products were resolved by EMSA. The bare DNA in these lanes resulted from dissociation of the nucleosomes in the dimer-containing gradient fraction following storage at 4 to 8°C for several weeks.

FIG. 2

hSWI-SNF and ATPases BRG1 and hBRM can transfer histone octamers from nucleosomes to bare DNA. (A) hSWI-SNF, BRG1, and hBRM transfer histone octamers. Labeled bare TPT fragment (120 pM) was incubated for 35 min at 30°C with 24 nM bulk HeLa cell mononucleosomes (mono) in 12.5-μl standard reactions but with 30 mM KCl and 3.5 mM MgCl2 and with (+) or without (−) 0.5 mM ATP. The remodeler concentrations were 5.6 nM hSWI-SNF complex (lanes 4 and 5), 8 nM BRG1 (lanes 6 and 7), and 8 nM hBRM (lanes 8 and 9). Reactions were stopped by addition of 2 μg of plasmid DNA, and the products were separated by EMSA. The product marked with an asterisk is created by SWI-SNF and bare DNA in the absence of nucleosomes. Preliminary results suggest that its formation, which is stimulated by ATP (see, e.g., Fig. 5A), is due to protein binding (J. Guyon and R. E. Kingston, unpublished observations). However, since its formation is actually repressed by the presence of donor nucleosomes, this product was not further analyzed here. (B) The transfer product has the same gradient mobility as a nucleosome core. A scaled-up transfer reaction mixture contained 590 pM bare TPT, 280 ng of HeLa cell polynucleosomes (∼11 nM in nucleosomes), and 4 nM SWI-SNF in a 200-μl volume, and the reaction was performed under standard reaction conditions but with 30 mM KCl, 3.5 mM MgCl2, 1 mM ATP, and no glycerol. After 1 h, the reaction was stopped by addition of 22 μg of a 8-kb plasmid (pKS-BRG1), and the products were purified on a 10 to 30% GGB-glycerol gradient. Fractions were then separated by EMSA. (C) DNase I accessibility of DNA before and after octamer transfer. Gradient-isolated cores (fraction no. 12, lane 4) or bare DNA (fraction no. 18, lane 2) and control cores (lane 3) or DNA (lane 1) (0.3 ng each) were brought up to a 100-μl volume by addition of 10% GGB, adjusted to 5 mM MgCl2 and 40-μg/ml BSA, and digested for 2 min at room temp with DNase (0.03 U for bare DNA; 0.3 U for nucleosomal DNA). The DNase reaction was stopped by addition of EDTA to 15 mM prior to DNA purification and denaturing PAGE. Arrows indicate the 10-bp periodicity of DNase cuts indicative of a rotationally phased nucleosome core.

FIG. 3

Remodeling activities coelute with monomeric BRG1. (A) Coelution of octamer transfer and remodeled-dimer activities with monomeric BRG1. BRG1 incubated in a solution containing 1 M urea, 1 M NaCl, and 95 mM KCl was centrifuged through a 10 to 40% glycerol gradient (see Materials and Methods). Fractions were analyzed by silver staining following sodium dodecyl sulfate-PAGE (top panel; 16.7 μl of each peak fraction). The relative position of myosin (200 kDa) centrifuged under identical conditions is indicated (below), as are the positions of molecular mass markers in the gel (left; in kilodaltons). The ∼150-kDa band comigrates with a BRG1 breakdown product identified by Western blotting (data not shown). The BRG1-containing fractions were analyzed for their ability to convert mononucleosome cores to a dimeric form (middle panel; EMSA) and to transfer octamers from unlabeled genomic HeLa mononucleosomes to labeled TPT DNA (bottom panel; EMSA). These reactions were performed under standard conditions, except that the final reaction conditions were 33 mM KCl, 0.3% NP-40, and 11 mM (dimer formation) or 33 mM (octamer transfer) urea. Dimer formation reactions contained 2% gradient fraction and 240 pM TPT mononucleosomes, while octamer transfer reactions contained 20% gradient fraction, 120 pM bare TPT, and 24 nM HeLa cell mononucleosomes (mono). The concentration of BRG1 in these reactions was estimated, from the silver stained gel, to range from 0.5 to 3 nM (dimer formation) or from 5 to 25 nM (octamer transfer). (B) Remodeling activity correlates with BRG1 in a quantitative assay. BRG1 incubated in a solution containing 1 M urea, 930 mM NaCl, and 65 mM KCl was centrifuged through a 10 to 40% glycerol gradient (see Materials and Methods) and analyzed by silver staining following sodium dodecyl sulfate-PAGE (top panel; 20 μl of each fraction). The remodeling activity of these fractions was assayed by measuring changes in the rate of restriction enzyme cutting of a nucleosomal array (shown; see Materials and Methods for details). The rate of cutting in the presence of 15 μl of each gradient fraction (or a 1:1 mixture of fractions 19 and 20) in a total of 25 μl is shown in the bottom panel. The concentration of BRG1 in these reactions was estimated to range from 1 to 5 nM.

FIG. 4

Octamer transfer requires an unstable intermediate of the hSWI-SNF remodeling reaction. (A) The remodeled dimer does not support transfer without SWI-SNF. Nonlabeled remodeled dimer was generated by incubating 100 nM HeLa cell mononucleosome cores with 3.9 nM SWI-SNF in a 200-μl volume under standard reaction conditions, but in the absence of glycerol and in the presence of 30 mM KCl and 3.5 mM MgCl2, for 2 h. The reaction was stopped by addition of KCl to 230 mM, and the products were separated on 180 mM–KCl–GGB–glycerol gradients as described in Materials and Methods. Octamer transfer reactions were performed with 80 pM labeled TPT bare DNA and 0.8 nM control mononucleosomes or gradient-purified remodeled dimers under standard conditions but with 0.1 mM ATP-MgCl2, 3 mM MgCl2, and 50 mM KCl. SWI-SNF was added at the onset of the reactions or either 10 or 2 min before the reactions were stopped (at 50 min) with 3 μg plasmid DNA. ∗, donor-independent nonnucleosomal band described in the legend to Fig. 2. (B) Transfer requires continuous ATP hydrolysis. Octamer transfer reactions with 4 nM HeLa cell cores and 120 pM labeled TPT bare DNA and 4.1 nM SWI-SNF were performed under standard conditions but with 2.5 mM MgCl2 and 0.1 mM ATP. Apyrase was added where indicated, and in the last two lanes the labeled TPT bare DNA was added after 30 min. All reactions were stopped with 3 μg of plasmid DNA at 55 min, and products were separated by EMSA. Percentages of cores formed, relative to total counts in the lane, are indicated.

FIG. 5

Effects of donor nucleosome and acceptor DNA type and concentration on transfer. (A) Donor and acceptor requirements for transfer. Transfer reactions (standard conditions but with 21 mM KCl) were performed with 0.12 nM labeled TPT DNA (left panel) or labeled bulk ∼150-bp DNA purified from HeLa cell mononucleosome cores (right panel), 3.8 nM hSWI-SNF, 2 mM MgCl2 with (+) or without (−) ATP, and 0.6 or 3 nM bulk HeLa cell mononucleosomes (Mono), Dinucleosomes (Di.), or TPT mononucleosomes (TPT) where indicated. At 30 min, 3 μg of plasmid DNA was added, and the reaction products were separated by EMSA. ∗, see the legend to Fig. 2. (B) Inhibition of transfer by high concentrations of bare DNA. Transfer reactions (standard conditions but with 3 mM MgCl2 and 34 mM KCl) were performed with 0.24 nM TPT cores (labeled [left panel] or unlabeled [right panel]), 3.8 nM SWI-SNF (where indicated), and the indicated amount of bare TPT DNA (80 pM of which was labeled in the panel on the right). Reactions were stopped with 3 μg of plasmid at 40 min, and products were separated by EMSA. (C) Quantitation of the results in panel B. For transfer from labeled cores (light bars), lane 4 (no added bare DNA) was set to background. For transfer from unlabeled cores (dark bars), lane 11 (no SWI-SNF) was set to background. The line represents the amount of remodeled dimer formed as a percentage of the amount formed in the absence of bare DNA (lane 4).

FIG. 6

Model of hSWI-SNF mechanism. (A) When the SWI-SNF ATPase(s) (BRG1 or hBRM alone, or in the SWI-SNF complex) binds two mononucleosome cores (left side) or one remodeled dimer (right side), the hydrolysis of ATP is hypothesized to generate a high-energy SWI-SNF nucleosome intermediate. The loosened histone-DNA contacts in this intermediate facilitate remodeling, allowing these transiently disrupted nucleosomes to stochastically reform normal mononucleosomes (reconversion; reaction I) or remodeled dimers (remodeling; reaction II). In addition, the weakened contacts may allow the histones in this disrupted complex to be pulled away by exogenous bare DNA, resulting in octamer transfer (reaction III). (B) Alternatively, transfer may occur in a concerted reaction when one SWI-SNF site is bound by a nucleosome and the other is bound by bare DNA.

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Octamer transfer and creation of stably remodeled nucleosomes by human SWI-SNF and its isolated ATPases - PubMed (original) (raw)