1. Volume 6, Issue 5, Pages 1049-1058 (November 2000)
ATP-Driven Chromatin Remodeling Activity and Histone Acetyltransferases Act Sequentially during Transactivation by RAR/RXR In Vitro 
F.Jeffrey Dilworth, Catherine Fromental-Ramain, Ken Yamamoto, Pierre Chambon 
Molecular Cell 
Volume 6, Issue 5, Pages (November 2000)
DOI: /S (00)

2. Figure 1 Characterization of In Vitro Assembled Chromatin
(A) Histone H1 increases nucleosome repeat length and resistance to micrococcal nuclease (MNase) digestion. Chromatin (DR5)5β2G templates (160 ng) with (+H1) or without (−H1) histone H1 were digested with 6.5 U MNase (55 μl, 24°C) for the time indicated in seconds (see Dilworth et al. 1999). Following deproteinization and separation on a 1.2% agarose gel in 0.5× TBE, DNA was stained with ethidium bromide.
(B) Incorporation of Histone H1 into “crude” chromatin (DR5)5β2G templates does not render transactivation Acetyl CoA dependent. Transcription was performed using HeLa cell NE and purified RARα/RXRα heterodimers on “crude” chromatin (DR5)5β2G templates with or without H1. TIF2, p300, tRA/BMS649, and Acetyl CoA were added as indicated. S1 nuclease digestion of RNA transcripts originating from the (DR5)5β2G and internal “control” pG1 templates generated 179 and 60 nt fragments, respectively (Dilworth et al. 1999).
(C) Histone H1 remains associated with chromatin template after size-exclusion chromatography. “Purified” chromatin (DR5)5β2G template (160 ng) assembled in the presence of H1 was digested with 0.35 U of MNase and analyzed.
(D) SWI/SNF endogenous to the chromatin assembly extract is removed during size-exclusion chromatography. “Crude” or “purified” chromatin (DR5)5β2G templates (70 ng) with or without H1 were applied to a nitrocellulose membrane (slot blot apparatus) and probed for dSWI/SNF using antibodies directed against the BRM subunit.
(E) Purification of human SWI/SNF. Human SWI/SNF complexes were purified from HeLa cells expressing Flag-tagged Ini1. Purified SWI/SNF was separated on a 10% SDS–PAGE gel before staining with Coomassie blue or Western blot using antibodies recognizing either SNF2β or Flag-Ini1.
(F) Purification of human ISWI complexes. Human complexes containing SNF2h (ISWI) were immunoprecipitated from HeLa NE using a SNF2h monoclonal antibody. Complexes were eluted using an excess of the peptide used to generate the antibody. Western blot analysis was performed using antibodies against WCRF180 (Bochar et al. 2000) or a second epitope of SNF2h.
(G) Chromatin purification removes HAT activity associated with the “crude” preparation. “Crude” or “purified” chromatin (DR5)5β2G preparation (30 ng) with or without H1 were incubated with free core histones and [14C] Acetyl CoA at 30°C. Ovalbumin and p300 (1 pmol of each) were used as negative and positive controls, respectively.
Molecular Cell 2000 6, DOI: ( /S (00) )

3. Figure 5
ATP-Dependent Remodeling of (DR5)5β2G Chromatin Templates in the Presence of RAR/RXR Heterodimers Is Restricted to the Promoter Region
(A) The structures of the (DR5)5β2G reporter template (inserted into the pBluescribe plasmid) is represented in a linear diagram. The positioning of oligonucleotides used to generate 32P-labeled probes for Southern blot analysis are illustrated below the diagram.
(B) “Purified” (DR5)5β2G chromatin templates (1 nM) were incubated as indicated in the presence or absence of RARα/RXRα (5 nM), and/or ATP (0.1 mM) at 27°C. After 30 min, chromatin templates were digested with MNase (1 U, volume 60 μl, 24°C) for the time indicated in seconds. Following deproteinization and separation on a 1.2% agarose gel in 0.5× TBE, DNA was transfered to a nylon membrane and the chromatin structure at specific regions was examined using 32P-labeled oligonucleotides described in (A).
Molecular Cell 2000 6, DOI: ( /S (00) )

4. Figure 2
Requirements for Activation of Transcription on Purified Chromatin Templates
(A) Transcriptional flow chart.
(B) Ligand-dependent activation of transcription on purified chromatin templates required coactivator acetyltransferase and ATP-dependent chromatin remodeling activities. Transcription was performed using a HeLa NE on “crude” or “purified” chromatin (DR5)5β2G templates assembled with or without H1. RARα/RXRα, tRA/BMS649, p300, TIF2, hSWI/SNF, ATP, and/or Acetyl CoA were included as indicated.
(C) Acetyl CoA and ATP are required for transactivation on “purified” chromatin. Transcription was performed on H1-containing “purified” (DR5)5β2G chromatin using HeLa NE, RARα/RXRα, tRA/BMS649 ligands, p300, TIF2, hSWI/SNF, ATP, and Acetyl CoA, as indicated.
(D) TIF2 and p300 stimulate activation of transcription. Transcription was performed on H1-containing “purified” (DR5)5β2G chromatin templates using a HeLa NE, RARα/RXRα, ATP, hSWI/SNF, and Acetyl CoA, p300, TIF2, and tRA/BMS649, as indicated .
Molecular Cell 2000 6, DOI: ( /S (00) )

5. Figure 7
ATP-Driven Chromatin Remodeling Activities and Coactivators Act Sequentially to Mediate the Ligand-Dependent Stimulatory Effect of RARα/RXRα Heterodimers
(A) Transcriptional timeline and schematic diagram indicating the “normal” time of addition of the different components of the in vitro transcription system. Transcription was performed on H1-containing “purified” (DR5)5β2G chromatin templates using HeLa NE, ATP, hSWI/SNF, Acetyl CoA, p300, TIF2, RARα/RXRα, and/or tRA/BMS649.
(B) Effect of varying the time of addition of RARα/RXRα heterodimers and tRA/BMS649.
(C) Effect of varying the time of addition of p300/TIF2 and hSWI/SNF.
(D) Effect of varying the time of Acetyl CoA addition.
(E) Effect of varying the time of ATP addition. Additions were as indicated at either the same time as the chromatin template (−40 min relative to transcription initiation), or 20 min later (−20 min), the same time as (−10 min) or 5 min after (−5 min) HeLa NE, 0.5 min before the rNTPs (−0.5 min), or not at all (none or −). Experiments displayed in (B)–(E) were repeated at least three times with different chromatin preparations and yielded similar results.
Molecular Cell 2000 6, DOI: ( /S (00) )

6. Figure 3
TIF2, p300, and SWI/SNF Act Synergistically to Stimulate Transcription by Enhancing the Formation of Productive Initiation Complexes
Transcription was performed on H1-containing “purified” (DR5)5β2G chromatin templates. RARα/RXRα, tRA/BMS649, p300, TIF2, hSWI/SNF, ATP, and/or Acetyl CoA were as indicated. Sarkosyl (0.5% final) was added 1 min after rNTPs. Relative transcription is that observed in the absence of sarkosyl (multiple rounds) and is expressed relative to that achieved when all components of the system were present. Rounds of transcription were calculated by dividing the intensity of the signal in the absence of sarkosyl (multiple rounds) by that in the presence of sarkosyl (single round) and are rounded off to a whole number. Similar results were obtained in several experiments carried out with different chromatin preparations and sarkosyl concentrations.
Molecular Cell 2000 6, DOI: ( /S (00) )

7. Figure 4
ATP and ISWI-Containing Complexes Are Required for Efficient Binding of RARα/RXRα Heterodimers to Their Response Elements on Chromatin Templates
(A) H1-containing “purified” (DR5)5β2G chromatin templates were incubated as indicated in the presence or absence of RARα/RXRα, tRA, BMS649, TIF2, p300, hSWI/SNF, ATP, and/or Acetyl CoA for 30 min at 27°C, and then processed for DNase I footprinting.
(B) Same as (A) except that hISWI was added, where indicated.
Molecular Cell 2000 6, DOI: ( /S (00) )

8. Figure 6
RAR/RXR Heterodimers Target Histone Acetyltransferase Activity to (DR5)5β2G Promoter Region in a Ligand-Dependent Manner
(A) “Purified” chromatin (DR5)5β2G (200 pM) and pSG5 (200 pM) templates were incubated in the presence of RARα/RXRα (1 nM), hSWI/SNF (2 ng/μl), ATP (0.1 mM), Acetyl CoA (2 μM), tRA and BMS649 (1 μM each), and/or TIF2 and p300 (500 pM each) as indicated. ChIP assays were then performed. The same blot was successively hybridized with probe C, probe B, and pSG5 probe.
(B) Chromatin immunoprecipitations were performed as in (A) except that, where indicated, RARα/RXRα were added to the chromatin assembly reaction after 3.5 hr and left to incubate for 30 min at 27°C to permit binding to DR5 elements before purification of the chromatin template. Each pair of panels correspond to a single blot, which was first hybridized with probe B. Data displayed in (A) and (B) are representative of three independent experiments.
Molecular Cell 2000 6, DOI: ( /S (00) )