1. Volume 12, Issue 4, Pages 983-990 (October 2003)
Targeting Activity Is Required for SWI/SNF Function In Vivo and Is Accomplished through Two Partially Redundant Activator-Interaction Domains 
Philippe Prochasson, Kristen E Neely, Ahmed H Hassan, Bing Li, Jerry L Workman 
Molecular Cell 
Volume 12, Issue 4, Pages (October 2003)
DOI: /S (03)

2. Figure 1
The Snf5 N-Terminal Domain and Residues 329–657 of Swi1 Interact with Activator
(A) Schematic of the Snf5 protein and the different constructs used. The two highly glutamine-rich domains (Q), the three proline-rich regions (P), a highly charged middle region (hatched), and the two conserved repeats Rep1 and Rep2 domains (solid line) are indicated. Solid lines below the Snf5 protein scheme represent the smaller constructs that were expressed for the GST pull-down experiments. The letters indicated on the right correspond to the following pieces of Snf5 used: A, full-length Snf5 (1–905 aa); B, Snf5N 1–334 aa; C, Snf5M 335–700 aa; D, Snf5C 701–905 aa; E, Snf5MC1 335–905 aa; F, Snf5NM 1–700 aa. GST pull-down assays were performed using the GST fusion proteins indicated and individually expressed the 35S-labeled Snf5 proteins indicated on the right. Fifty percent of each Input and supernatant (S) and 100% of the proteins associated with the beads (B) were loaded on a 10% SDS-PAGE. The radiolabeled proteins were visualized by autoradiography.
(B) Schematic of the Swi1 protein and the different constructs used. The asparagine-rich N terminus domain (N), the glutamine-rich domain (Q), the ARID (AT rich interacting domain, conserved among Swi1 homologs proteins in Drosophila and humans) domain, and the two highly conserved domains C1 and C2 among Swi1 homologs proteins are indicated. Solid lines below the Swi1 protein scheme represent the smaller constructs used for the GST pull-down analysis. The letters indicated on the right correspond to the following pieces of Swi1 used: A, Swi1 1–328 aa; B, Swi1 329–657 aa; C, Swi1 658–985 aa; D, Swi1 985–1314 aa. GST pull-down assays were performed as describe above. The GST fusion proteins and the individually expressed 35S-labeled Swi1 proteins are indicated.
Molecular Cell  , DOI: ( /S (03) )

3. Figure 2
Deletion of Residues 329–657 of Swi1 and the Snf5 N-Terminal Domain Impairs SWI/SNF Complex Activator Interaction
Schematic of the Swi1 and Snf5 mutants used. Swi1ΔB corresponds to the Swi1 protein deleted of amino acids 329–657; Snf5M corresponds to amino acids 335–700 of Snf5 protein. Dashed lines represent the region removed from the Swi1 and the Snf5 proteins. Yeast whole-cell extracts from strains expressing different combination of Swi1 and Snf5 wild-type (WT), mutant (ΔB, M), or no protein (−), as indicated, were incubated with either GST-Vp16, GST-Gcn4 fusion protein, or GST alone bound to glutathione Sepharose beads. The input whole-cell extracts and the bead fractions were assayed for the presence of Swi3 by Western blotting.
Molecular Cell  , DOI: ( /S (03) )

4. Figure 3
The Acidic Activator-Interacting-Deficient SWI/SNF Complex Is Intact
The SWI/SNF complexes were purified as described in the Experimental Procedures from the following yeast cells containing the wild-type SWI/SNF complex (WT) (Swi1- and Snf5-wild-type), the SWI/SNF activator-interacting mutant complex (M) (Swi1ΔB, Snf5M), and the SWI/SNF complex lacking Swi1p and Snf5p (−) (swi1Δ, snf5Δ). The fractions peak of the MonoQ purification were loaded onto a Superose 6 column as described, and the Superose 6 fractions were assayed for the presence of Swi3 by Western blotting.
Molecular Cell  , DOI: ( /S (03) )

5. Figure 4
The Wild-Type and the Activator Mutant SWI/SNF Complexes Show Identical Remodeling Activity Using Restriction Enzyme Accessibility Assays
(A) 5′ end-labeled GUB template was mock reconstituted (Naked DNA) or reconstituted into mononucleosome cores (Mononucleosome) and incubated with the same amount of indicated SWI/SNF complex (WT, wild-type; or Mut, activator mutant) in the presence or absence of ATP for 1 hr at 30°C. Then, SalI, XhoI, or PvuII restriction enzymes were added to the sample and incubated 30 more minutes at 30°C. After ethanol precipitation, DNA was dissolved in formamide loading buffer, heat denatured, and resolved on an 8% acrylamide-8 M urea sequencing gel. The gel was dried and exposed to Phosphorimager to be quantified. The quantifications of three independent experiments are indicated as percentage of cleavage corrected for the background cleavage obtained in the absence of ATP. The SalI, XhoI, and PvuII generate labeled cut fragments of, respectively, 70, 112, and 130 bp after cleavage of the 183 bp template.
(B) Western blotting probed for the presence of the Swp61 SWI/SNF subunit showing that equal amounts of wild-type and activator mutant purified SWI/SNF complexes were used for the experiments shown in Figures 4A and 5. The wild-type and activator mutant SWI/SNF complexes were purified over a cation exchange column (SP sepharose) followed by an anion exchange (Mono Q HR5/5 column), and then were Flag-immunoprecipitated.
Molecular Cell  , DOI: ( /S (03) )

6. Figure 5
Equal Remodeling Activity of Wild-Type and Activator Mutant SWI/SNF Complex Using DNase I Digestion Assay
5′ end-labeled GUB template was mock reconstituted (Naked DNA) or reconstituted into mononucleosome cores (Mononucleosome), and incubated with the same amount of indicated SWI/SNF complex (WT, wild-type; or Mut, activator mutant) in the presence or absence of ATP for 1 hr at 30°C. Then, the remodeling reaction mixtures were treated with 0.4 or 0.04 unit of DNase I for 1 min at RT, for nucleosome template or naked DNA, respectively. After stopping the digestion, the DNA was ethanol precipitated, then dissolved in formamide loading buffer, heat denatured, and resolved on an 8% acrylamide-8 M urea sequencing gel.
Molecular Cell  , DOI: ( /S (03) )

7. Figure 6
The Acidic Activator-Interacting-Deficient SWI/SNF Mutant Shows Reduced Growth under Conditions Requiring Activated Transcription
Yeast strain YPP33 (swi1::HIS3 snf5::HIS3) was transformed with vector alone (−) or plasmids encoding either wild-type (+) or activator mutant (ΔB or M) Swi1p or Snf5p. The resulting transformants were plated on either CSM media (−Trp−Ura) with dextrose (Control), or with raffinose (+Raffinose), or with galactose (+Galactose) or on CSM dextrose media (−Trp−Ura) with 1 μg/ml of sulfometuron methyl (+SMM) or without inositol (−Inositol). Scans of plates are representative of at least three individual experiments.
Molecular Cell  , DOI: ( /S (03) )

8. Figure 7
Expression of SUC2 mRNA in Derepression Condition Is Strongly Affected in the SWI/SNF Activator Mutant Strain Compared to the Wild-Type Strain
Total RNA (16 μg per lane) from W303 (wild-type control strain), WT (corresponding to the swi1::HIS3 snf5::HIS3 strain bearing centromeric expression plasmids for wild-type Swi1 and Snf5), and Mut strain (swi1::HIS3 snf5::HIS3 bearing centromeric expression plasmids for Swi1ΔB and Snf5M activator mutant) was electrophoresed through a 1.2% agarose gel and transferred to a charged Nylon membrane. Northern blots were hybridized with full-length SUC2 and SCR1 probes and exposed to autoradiographic film.
Molecular Cell  , DOI: ( /S (03) )