Rapid Periodic Binding and Displacement of the Glucocorticoid Receptor during Chromatin Remodeling  Akhilesh K Nagaich, Dawn A Walker, Ron Wolford, Gordon L Hager  Molecular Cell  Volume 14, Issue 2, Pages 163-174 (April 2004) DOI: 10.1016/S1097-2765(04)00178-9

Figure 1 Periodic Binding Detected by UV Laser Crosslinking (A) Protocol for detection of real-time protein/DNA interactions. Aliquots are subjected to a single pulse of UV laser irradiation as the reaction proceeds, using the fourth harmonic (266 nm) from a Nd:YAG laser. Individual aliquots are then processed to detect covalent crosslinking. All remodeling reactions are initiated by addition of biotinylated MMTV chromatin (Experimental Procedures) bound to strepavidin-coated paramagnetic beads and ATP to a master mix of GR, hSWI/SNF, and either nonspecific competitor chromatin or DNA (molar ratio 1:1:10). Aliquots (30 μl) in siliconized microcentrifuge tubes are crosslinked every 30 s with a single 5 ns pulse of laser UV. Crosslinked chromatin is washed with high salt buffer (Experimental Procedures) to remove noncrosslinked proteins, digested with micrococcal nuclease to liberate mononucleosomes, and then blotted on nitrocellulose membrane to immobilize DNA. The membrane is then incubated with antibodies for specific proteins, and the extent of bound protein was determined by chemiluminescent detection. (B) Coomassie staining (lanes 1 and 2) and silver staining (lanes 3 and 4) is shown for SDS-PAGE of chromatin reconstituted on beads, both before and after the washing procedure (Fletcher et al., 2000). Lane 5 presents the silver staining profile for a typical hSWI/SNF preparation. (C) The extent of GR/DNA crosslinking is shown during a 20 min remodeling reaction using equimolar concentrations of GR and hSWI/SNF. (D) A quantitative profile for GR (-■-) and Brg1 (-♢-) crosslinking to DNA. (a) indicates the repeat period for GR interactions, and (b) indicates the approximate resident time for GR. The inset panel shows the crosslinking profile for a reaction carried out with a 10-fold reduction in the hSWI/SNF conc. Molecular Cell 2004 14, 163-174DOI: (10.1016/S1097-2765(04)00178-9)

Figure 2 Periodicity Requires ATP, SWI/SNF, and Chromatin (A) A reaction was performed without ATP, and the extent of GR (-■-) and Brg1 (-♢-) crosslinking was determined. Interactions of both GR and Brg1 with DNA are invariant with time under these conditions. (B) The periodic binding of GR requires the continuous action of hSWI/SNF. In a reaction carried out without added hSWI/SNF, the extent of GR (-■-) crosslinking increases slightly with time, with no evidence of cyclic binding. (C) The periodic binding of Brg1 requires the presence of GR. In a reaction carried out with hSWI/SNF, but without added GR, the extent of Brg1 (-♢-) crosslinking to DNA is invariant over time. (D) The interaction of GR and Brg1 with deproteinized DNA. In a complete reaction with GR, hSWI/SNF, and ATP, the Brg1 (-♢-) interaction with DNA comes rapidly to equilibrium and changes little over time. GR binding (-■-) requires approximately 6 min under the reaction conditions but also remains invariant after reaching equilibrium. Molecular Cell 2004 14, 163-174DOI: (10.1016/S1097-2765(04)00178-9)

Figure 3 A Periodic, Internal Nucleosome Rearrangement Detected by UV Laser Crosslinking (A) Reactions were performed in the presence of GR, hSWI/SNF, and ATP as described in Figure 1. The extent of DNA crosslinking for each of the four core histones was determined during the course of a 20 min reaction (Experimental Procedures). For histones H3 (-•-) and H4 (-○-), the extent of crosslinking is invariant with reaction time. For histones H2A (-▾-) and H2B (-▿-), however, crosslinking efficiency varies dramatically, with a periodicity of approximately 5 min. For each binding cycle, the reorganization of H2B with respect to DNA precedes the alteration in DNA contacts observed for H2A. In the absence of ATP (B), no reorganization of histone/DNA contacts is detected. Molecular Cell 2004 14, 163-174DOI: (10.1016/S1097-2765(04)00178-9)

Figure 4 The Effect of ATP Depletion during the Binding and Displacement Reaction Remodeling reactions were carried out as described in Figure 1. Reactions were split at the times indicated ([A] 4 min, [B] 5 min, [C] 6 min), and hexokinase/glucose and γ-S-ATP were added to half of the reaction (see Experimental Procedures). Time-dependent GR crosslinking was determined with (-○-) or without (-■-) ATP depletion. Brg1 crosslinking efficiencies (-▴-) were also determined but are presented only for the control reaction in (A). Molecular Cell 2004 14, 163-174DOI: (10.1016/S1097-2765(04)00178-9)

Figure 5 hSWI/SNF Interactions Are Localized to the Nucleosome B Region of the MMTV Template in the Presence of GR Nucleosome positions on the reconstituted template (Fletcher et al., 2000) are depicted. Formaldehyde-based chromatin immunoprecipitation was used to localize Brg1 crosslinking to specific nucleosomes, using primers for each core position (example shown for nucleosome 2) (see Experimental Procedures for primer locations) ([A] nucleosome C, [B] nucleosome B, (C) nucleosome A, [D] nucleosome 1, [E] nucleosome 2, [F] nucleosome 3). MMTV nucleosomal arrays assembled with wild-type DNA and DNA mutated at all six GREs were incubated in the presence (rows 3, 4, 7, and 8) or absence of GR (rows 1, 2, 5, and 6) followed by the addition of hSWI/SNF (rows 2, 4, 6, and 8) and competitor chromatin as indicated (columns b, d, f, and h). After 1% formaldehyde crosslinking, washing, MNase digestion, and immunoprecipitation with anti-Brg1 antisera, crosslinking was reversed using standard ChIP protocol (Experimental Procedures). Immunoprecipitated DNA was slot blotted and probed with radiolabeled DNA specific to the different nucleosomes. Graphical representations of the average values and the standard deviation of data from three independent experiments are shown in (G)–(L). (M) Site-specific Brg1 interactions induced by laser crosslinking were determined using oligonucleotide probes (20 bp) complementary to the dyad regions of specific nucleosomes (Experimental Procedures). Reconstituted MMTV chromatin was incubated with GR and hSWI/SNF (1:1.5 molar ratio) in the presence of HeLa oligonucleosomes as competitor chromatin and crosslinked with five pulses of laser UV. After micrococcal nuclease digestion, mononucleosomes were immunoprecipitated with Brg1 antibody. DNA was isolated, quantitated by fluorimetry, and blotted on Zeta Probe GT membrane (Experimental Procedures). DNA (1, 10, and 50 ng) was hybridized with nucleosome-specific radiolabeled oligonucleotide probes (lanes 1–6). Molecular Cell 2004 14, 163-174DOI: (10.1016/S1097-2765(04)00178-9)

Figure 6 Remodeling-Coupled Restriction Enzyme Accessibility Assay Shows that GR Targets Remodeling Activity to the Correct Position on MMTV Chromatin (A) The in vivo remodeling region covers all of the B nucleosome and the 3′ half of the C nucleosome. Complete chromatin-remodeling reactions were carried out with or without competitor oligonucleosomes in the presence of SacI or BsrB1 (with or without added ATP) to test for localization of the remodeling event. The fragments were end labeled with α-32P-dGTP, released by EcoR1 digestion, and analyzed on agarose gels. Representative results from 40 min reactions are shown in (B) for SacI and (C) for BsrB1. (D and E) Fractional cleavage of the chromatin was then determined by quantitating the digested and undigested bands. Digestion in the presence and absence of ATP was determined, and ATP-dependent digestion is expressed as F(x) (Fragoso et al., 1998; Fletcher et al., 2002) (Experimental Procedures). Molecular Cell 2004 14, 163-174DOI: (10.1016/S1097-2765(04)00178-9)

Figure 7 Proposed Mechanism for the Periodic Interaction of GR and hSWI/SNF with the Template during Chromatin Remodeling (1) GR initially binds weakly to GREs located on the B-C nucleosomal region. (2) Binding of GR targets hSWI/SNF to the B nucleosome. (3 and 4) During the subsequent nucleosome remodeling, histones H2B, then H2A, undergo extensive reorganization. This reorganization is accompanied by transient binding of GR to GREs. (5) hSWI/SNF leaves the template, and the remodeled state collapses, leading to (6) a rapid loss of GR from the template. (7) Chromatin reverts to the ground state, poised for next cycle of GR directed hSWI/SNF action. Molecular Cell 2004 14, 163-174DOI: (10.1016/S1097-2765(04)00178-9)