Perturbation of nucleosome core structure by the SWI/SNF complex persists after its detachment, enhancing subsequent transcription factor binding

Abstract

To investigate the mechanism of SWI/SNF action, we have analyzed the pathway by which SWI/SNF stimulates formation of transcription factor-bound nucleosome core complexes. We report here that the SWI/SNF complex binds directly to nucleosome cores and uses the energy of ATP hydrolysis to disrupt histone/DNA interactions, altering the preferred path of DNA bending around the histone octamer. This disruption occurs without dissociating the DNA from the surface of the histone octamer. ATP-dependent disruption of nucleosomal DNA by SWI/SNF generates an altered nucleosome core conformation that can persist for an extended period after detachment of the SWI/SNF complex. This disrupted conformation retains an enhanced affinity for the transcription factor GAL4-AH. Thus, ATP-dependent nucleosome core disruption and enhanced binding of the transcription factor can be temporally separated. These results indicate that SWI/SNF can act transiently in the remodeling of chromatin structure, even before interactions of transcription factors.

Figure 1

SWI/SNF complex binding to nucleosome cores or naked DNA. Purified SWI/SNF complex was incubated with a 176-bp 5S RNA gene probe either as reconstituted nucleosome cores (lanes 1–8) or as naked DNA (lanes 9–16). SWI/SNF was present at 0 nM in lanes 1 and 9, 3 nM in lanes 2 and 10, 10 nM in lanes 3 and 11, and 30 nM in lanes 4–8 and 12–16. Distamycin A was present during SWI/SNF binding in lanes 5 and 13 (0.1 μM), lanes 6 and 14 (1 μM), lanes 7 and 15 (10 μM), and lanes 8 and 16 (100 μM). All lanes contain 9 nM total nucleosomal or naked DNA (mainly HeLa unlabeled oligonucleosomes or DNA) and 1 mM Mg-ATP. Samples were run on 4% acrylamide gel (80:1, acrylamide/bis) in a Tris/glycine/EDTA buffer system.

Figure 2

Dissociation of the SWI/SNF complex leaves a persistently altered nucleosome core. (A) A 5S DNA probe as in Fig. 1 was reconstituted into nucleosome cores. SWI/SNF binding was analyzed by standard gel shift assay [4% acrylamide (29/1)/0.5× TBE]. Each reaction contains 12.5 nM total nucleosomes and 1 mM Mg-ATP, whereas 30 nM SWI/SNF was added in lanes 2 and 4. In lanes 3 and 4, after an initial 30-min incubation, 20-fold excess of cold HeLa oligonucleosomes were added followed by another 30-min incubation at 30°C before loading on the gel. (B) Binding reactions similar to A were performed and analyzed by DNase I footprint assay. 15 nM of SWI/SNF was added in lanes 2 and 4. Competitors and 2-fold more DNase I were used in lanes 3 and 4. Near stochiometric ratio of SWI to nucleosomes were used here and in the following experiments to insure almost complete binding of the nucleosomes in the initial reaction.

Figure 3

Temporal analysis of SWI/SNF action and enhanced activator binding. (A) SWI/SNF-treated nucleosome cores remain bound by GAL4-AH after SWI/SNF detachment. A 154-bp DNA probe bearing a single GAL4 site 32 bp from one end was reconstituted into nucleosome cores and incubated with 30 nM (lanes 2, 3, 6, 7, 10, and 11) and 100 nM (lanes 4, 5, 8, 9, 12, and 13) GAL4-AH dimers. All lanes contain 12.5 nM total nucleosomes and 1 mM ATP, and 10 nM SWI/SNF was included in the odd-numbered lanes. To study the temporal ATP requirement for SWI action, ATP was degraded by apyrase before SWI/SNF addition (lanes 2–5), after SWI/SNF and GAL4 incubation (lanes 6–9), or after SWI/SNF action but before GAL4 incubation (lanes 10–13). Finally, before loading the native gel, nucleosome cores were released from SWI by competition with 20-fold excess of cold oligonucleosomes for 30 min at 30°C. (B) SWI/SNF treatment of nucleosome cores allows GAL4-binding after SWI/SNF detachment. Gel shift assay of similar binding reactions as in A except for the time of SWI/SNF competition. In lanes 1–4 competitors were added after SWI/SNF action, at the same time as apyrase treatment, before GAL4 addition. In lanes 5–8 competitors were included from the beginning of the binding reaction, at the same time as SWI/SNF is added. (C) Footprinting assay of equivalent binding reactions. 30 nM GAL4-AH dimers was added in lanes 2–5 whereas 600 nM was used in lanes 7, 8, 11, and 12. SWI/SNF (40 nM) was added at the indicated time in lanes 3, 5, 8, and 12. Competitors were added at the indicated time in lanes 6–12. Two-fold more DNase I was used in lanes 6–12.

Figure 4

Perturbation of nucleosome core structure by SWI/SNF is stable for several hours after its detachment but eventually reverts back. Time-course experiment on the stability of the altered nucleosomal state induced by SWI/SNF. 5S nucleosome cores were incubated with 15 nM SWI/SNF complex for 30 min at 30°C in the presence of 1 mM ATP. Apyrase and 20-fold excess of both unlabeled HeLa oligonucleosomes and calf thymus DNA then were added. Reactions were put at 37°C for the indicated time before DNase I addition.

Figure 5

SWI/SNF alters the path of DNA bending around the histone octamer (A) The same core 5S DNA sequence as in Fig. 1 was end-labeled (lanes 2–4) or body-labeled (lanes 5–10), reconstituted into nucleosome cores, and digested with DNase I. Mock-reconstituted DNA is shown in lanes 2 and 5. 25 ng (12.5 nM) of total nucleosomes (probe plus donor oligonucleosomes) was incubated as before, followed by digestion with 0.05 unit (lanes 1, 5), 0.5 unit (lanes 3, 4), 1 unit (lane 8), 2 units (lane 9), or 10 units (lane 10) of DNase I; 1 mM Mg-ATP is present in lanes 4 and 7. (B) Body-labeled (lanes 1–5) and end-labeled (lanes 6–10) reconstituted probes were incubated in the presence or the absence of 2.5 nM SWI/SNF and/or ATP followed by digestion with 10 units (lanes 2–5) or 0.5 unit (lanes 7–10) of DNase I. Mock reconstitutions were digested with 1 unit (lane 1) and 0.05 unit (lane 6) of DNase I. (C) Reactions were performed as in B with the body-labeled reconstituted probe (using 10 units of DNase I) except that ATP is present in all lanes and 7.5 nM SWI/SNF is used in lane 4. Note the single 10-nucleotide digestion product kept in the gel.