SWI-SNF-mediated nucleosome remodeling: role of histone octamer mobility in the persistence of the remodeled state - PubMed (original) (raw)
SWI-SNF-mediated nucleosome remodeling: role of histone octamer mobility in the persistence of the remodeled state
M Jaskelioff et al. Mol Cell Biol. 2000 May.
Abstract
SWI-SNF is an ATP-dependent chromatin remodeling complex that disrupts DNA-histone interactions. Several studies of SWI-SNF activity on mononucleosome substrates have suggested that remodeling leads to novel, accessible nucleosomes which persist in the absence of continuous ATP hydrolysis. In contrast, we have reported that SWI-SNF-dependent remodeling of nucleosomal arrays is rapidly reversed after removal of ATP. One possibility is that these contrasting results are due to the different assays used; alternatively, the lability of the SWI-SNF-remodeled state might be different on mononucleosomes versus nucleosomal arrays. To investigate these possibilities, we use a coupled SWI-SNF remodeling-restriction enzyme assay to directly compare the remodeling of mononucleosome and nucleosomal array substrates. We find that SWI-SNF action causes a mobilization of histone octamers for both the mononucleosome and nucleosomal array substrates, and these changes in nucleosome positioning persist in the absence of continued ATP hydrolysis or SWI-SNF binding. In the case of mononucleosomes, the histone octamers accumulate at the DNA ends even in the presence of continued ATP hydrolysis. On nucleosomal arrays, SWI-SNF and ATP lead to a more dynamic state where nucleosomes appear to be constantly redistributed and restriction enzyme sites throughout the array have increased accessibility. This random positioning of nucleosomes within the array persists after removal of ATP, but inactivation of SWI-SNF is accompanied by an increased occlusion of many restriction enzyme sites. Our results also indicate that remodeling of mononucleosomes or nucleosomal arrays does not lead to an accumulation of novel nucleosomes that maintain an accessible state in the absence of continuous ATP hydrolysis.
Figures
FIG. 1
Remodeled nucleosomes do not accumulate in the context of linear nucleosomal arrays. (A) Schematic representation of the nucleosomal DNA templates used for the coupled restriction enzyme–SWI-SNF remodeling assay. Each template is composed of head-to-tail repeats of a 5S rDNA nucleosome positioning sequence from L. variegatus. The first, second, or sixth nucleosome is tagged by a unique _Sal_I/_Hin_cII restriction site. (B) The nucleosomal arrays were incubated with _Hin_cII (□), SWI-SNF (○, ▵), or both (◊). After 1 h, _Hin_cII (○) or _Hin_cII and apyrase (▵) were added to the reaction to test for accumulation of remodeled template or for ATP dependence, respectively. Cleavage rates were quantified as described in Materials and Methods. Similar results were obtained in three separate experiments.
FIG. 2
Remodeled nucleosomes do not accumulate on closed circular nucleosomal arrays. (A) Nucleosomal arrays were reconstituted on negatively supercoiled closed circular plasmids bearing a unique _Sal_I/_Hin_cII site on a single 5S nucleosome positioning sequence. (B) The closed circular arrays were subjected to _Hin_cII digestion for 30 min followed by a 30-min incubation with (lane 2) or without (lane 1) SWI-SNF. Alternatively, the arrays were incubated for 30 min with SWI-SNF, followed by removal of ATP with apyrase and digestion with _Hin_cII for 30 min (lane 3). (C) A supercoiling assay was performed on the closed circular nucleosomal arrays (lane 1) in order to detect remodeling as changes in plasmid linking number. Addition of calf thymus topoisomerase I resulted in the appearance of approximately 15 bands after deproteination and agarose gel electrophoresis (lane 2). Incubation of the arrays with SWI-SNF and ATP for 30 min had no effect on topology (lane 3). Addition of SWI-SNF, ATP, and topoisomerase I resulted in a redistribution of the topoisomers (lane 4). Removal of ATP by addition of apyrase after 30 min of incubation in the presence of SWI-SNF plus topoisomerase did not affect the distribution of topoisomers (lane 5). Incubation of the arrays with SWI-SNF and ATP for 30 min followed by apyrase and then topoisomerase resulted in a topoisomer distribution similar to that in lane 2 (lane 6). Quantification of the autoradiograph indicates that for lanes 2 and 6, the predominant band corresponds to a linking number of 8; for lanes 4 and 5, the predominant band corresponds to a linking number of 6. Note that apyrase treatment of the circular array results in an increase in the nicked plasmid form (B, lane 3; C, lanes 5 and 6). Apyrase-induced plasmid nicking does not affect _Hin_cII activity or the relative distribution of topoisomers upon topoisomerase I treatment of the array (data not shown).
FIG. 3
SWI-SNF remodeling of isolated mononucleosomes leads to persistent DNA accessibility. (A) Purified mononucleosomes, obtained following _Pst_I digestion of a 6-mer nucleosomal array, were treated with SWI-SNF, _Sal_I, and/or apyrase as indicated. (B) Mononucleosomes assembled on a 154-bp DNA fragment were used as substrates in the same assays as panel A. Similar results were obtained in two additional, independent experiments.
FIG. 4
SWI-SNF action leads to increased protection of DNA ends. (A) Schematic of the 216-bp mononucleosome substrate. (B) The ends of the DNA fragment are more protected after a 30-min incubation with SWI-SNF. The graph represents the percentage of cleaved nucleosomal DNA after restriction enzyme digestion without SWI-SNF (−) or after a 30-min incubation with SWI-SNF followed by removal of ATP with apyrase (+). Error bars represent the standard deviation from at least three experiments. (C) Difference in percent nucleosomal DNA cleaved by the restriction enzyme in the absence (−) and presence (+) of SWI-SNF (see panel B). (D) Time course of _Bam_HI DNA cleavage. Mononucleosomes were preincubated for 30 min in the absence of SWI-SNF (□), in the presence of SWI-SNF without ATP (⧫), or in the presence of SWI-SNF and ATP (●, ▴). Reactions containing SWI-SNF and ATP were then incubated with (▴) or without (●) apyrase, _Bam_HI was added to all reactions, and the amount of cleavage was determined throughout a 50-min time course.
FIG. 5
SWI-SNF action alters the translational positioning of a mononucleosome. Mononucleosomes reconstituted onto a 416-bp DNA fragment that contains two copies of a 5S nucleosome positioning sequence were electrophoresed on a 4% native polyacrylamide gel for 8 h. Arrows denote eight electrophoretically distinct particles that reflect different nucleosome translational positions. Mononucleosomes were incubated for 30 min at 37°C in the absence of SWI-SNF (lane 1), in the presence of SWI-SNF but in the absence of ATP (lane 2), and in the presence of both SWI-SNF and ATP (lane 3). Prior to loading of samples, all three reactions received a 100-fold molar excess of unlabeled chicken oligonucleosomes to compete for binding of SWI-SNF to the mononucleosome substrate. Note that SWI-SNF and ATP shifts the distribution of mononucleosome particles to the faster-migrating species without leading to an increase in the amount of free, naked DNA. Similar results were obtained in at least three additional experiments.
FIG. 6
SWI-SNF-dependent remodeling changes nucleosome positions within reconstituted arrays. (A) Schematic representation of the DNA template showing positions of restriction endonuclease sites unique to the central nucleosome (open bars). _Eco_RI sites (filled bars) are located in the linker region between nucleosomes in every 5S repeat. (B) Change in the accessibility of restriction endonuclease sites at the nucleosome dyad (HincII) and at the linker region (NcoI and BamHI) following SWI-SNF reaction. The bars represent percentages of uncut arrays after restriction endonuclease cleavage of reconstituted arrays in the presence of ATP and/or SWI-SNF as indicated. In apyrase (APY) experiments, the SWI-SNF reaction was stopped with apyrase and restriction enzyme was then added. Error bars represent the standard deviation from at least three experiments. (C) SWI-SNF reaction results in a decrease in accessibility of linker regions throughout nucleosomal arrays. The _Eco_RI digestion of reconstituted nucleosomal arrays was carried out as in panel B; DNA was isolated and analyzed in a 4% acrylamide gel. Gel was stained with Vistra Green (Amersham) and scanned with a Molecular Dynamics Storm scanner. The limit digestion product (208-bp 5S DNA) is marked with an arrow. The 232-bp product represents the _Hin_cII/_Sal_I-marked nucleosome. Larger products represent partial digestion products. Lane M contains a 100-bp DNA ladder (New England Biolabs).
FIG. 7
Persistent randomization of nucleosome positions within reconstituted arrays as a result of SWI-SNF-dependent remodeling. (A) MNase digestion of reconstituted 11-mer arrays. [α-32P]dATP-labeled reconstituted nucleosomal arrays were digested with increasing amounts of MNase in the presence of SWI-SNF and either in the presence or absence of ATP before or after addition of apyrase (APY). DNA was isolated and analyzed on a 1% agarose gel. Lane M, 32P-labeled 1-kb DNA ladder (Gibco BRL). Note that 5S array DNA also exhibits a repeating pattern of MNase cleavages that is the inverse pattern of nucleosomal arrays. (B) SWI-SNF-dependent remodeling does not change the sedimentation properties of nucleosomal arrays. Shown are 10 to 40% linear glycerol gradient profiles of naked DNA and reconstituted nucleosomal arrays either before or after SWI-SNF-dependent remodeling. 32P-labeled reconstituted arrays were incubated in the presence of ATP and/or SWI-SNF for 30 min, loaded on top of the gradient, and centrifuged in an SW28 rotor (Beckman) at 33,000 × g for 16 h. Fractions of 0.4 ml were collected from the top of the gradient and counted by scintillation. (C) Nucleosomes within remodeled arrays protect ∼150 bp of DNA. Nucleosomal arrays were incubated in the presence of SWI-SNF with or without ATP, the reaction was stopped with apyrase, and remodeled arrays were digested with a high concentration of MNase. DNA was isolated, fractionated in a 4% acrylamide gel, and visualized by staining with Vistra Green. The position of the 146-bp nucleosomal DNA is marked with an arrow. Lane M, 100-bp DNA ladder.
References
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