Journal: Molecular and Cellular Biology

Article Title: Mammalian SWI/SNF-A Subunit BAF250/ARID1 Is an E3 Ubiquitin Ligase That Targets Histone H2B ▿

doi: 10.1128/MCB.00540-09

Figure Lengend Snippet: Association of Cul2 with BAF250b. (A) BAF250b associates specifically with Cul2 and not Cul5. T7 immunoprecipitates of transiently transfected T7-BAF250b with Myc-Cul2 (lanes 1, 2, 5, and 6) or Myc-Cul5 (lanes 3, 4, 7, and 8) were washed in a buffer containing 0.3 M KCl and analyzed by immunoblotting with an antibody to T7 (α-T7) or Myc. (B) Bacterially expressed Cul2 interacts with BAF250bCTD. GST pulldown assays were performed as for Fig. ​Fig.1C1C with His-Cul2 expressed in E. coli. (C) Cul2 association with the BAF250b C terminus is BC box dependent. Lysates from cells transiently transfected with T7-BAF250bNTD, T7-BAF250bCTD, or T7-BAF250bCTDBC* and with Myc-Cul2 were immunoprecipitated with T7 antibody, washed in a buffer containing 0.3 M KCl, and analyzed by immunoblotting with an antibody to T7 or Myc. (D) Cul2 association with BAF250b N terminus is ARID dependent. Lysates from cells transiently transfected with T7-BAF250bNTD or the T7-BAF250b N terminus with the ARID deletion (ΔA) and Myc-Cul2 were immunoprecipitated with T7 antibody, washed in a buffer containing 0.3 M KCl, and analyzed by immunoblotting with an antibody to T7 or Myc. (E) Endogenous Cul2 association with BAF250b is BC box dependent. T7 immunoprecipitates of transiently transfected T7-BAF250b, T7-BAF250b with ARID deleted (ΔA), the T7-BAF250b BC box mutant (BC*), or T7-BAF250b with ARID deleted and the BC box mutated (ΔA and BC*) were washed in a buffer containing 0.3 M KCl and analyzed by immunoblotting with an antibody to T7 or endogenous Cul2. (F) A schematic summarizing the interactions between BAF250b constructs and Elo C and Cul2. n.d., not determined. (G) Association of endogenous BAF250a/b and Cul2. BAF250a and BAF250b were immunoprecipitated from HEK293 cells and probed for Cul2 and BAF250. The bands corresponding to BAF250a and BAF250b are indicated by ▪ and ▪▪, respectively. (H) Endogenous Roc1 is enriched in immunopurified Flag-BAF250b complex. Flag-BAF250b immunoprecipitated from induced (+) or uninduced (−) 16e cell nuclear extracts was washed in a buffer containing 0.3 M (lanes 3 and 4) or 0.5 M (lanes 5 and 6) KCl and eluted with a Flag peptide. Eluted material was analyzed by immunoblotting for endogenous Roc1 or Flag-BAF250b.

Article Snippet: Thirty microliters of Flag-BAF250b complexes (purified from induced 16e cells [16e+]) or the negative-control eluant (purified from uninduced 16e cells [16e−]) was dialyzed into ubiquitination buffer (50 mM Tris-HCl [pH 7.9], 5 mM MgCl 2 , 2 mM NaF, 0.6 mM dithiothreitol [DTT]) at 4°C for 1 h. After adjusting for changes in volume during dialysis, equivalent amounts of 16e−, Flag-BAF250b complexes, or recombinant Flag-tagged Ring1b were incubated with 2 mM ATP, 0.1 μg E1 (Boston Biochem), 0.5 μg E2 (UbcH5c; Boston Biochem), 1 μg hemagglutinin antigen-tagged ubiquitin (HA-Ub) (Boston Biochem), and 5 to 10 μg of in vitro -reconstituted oligonucleosomes (wild type or H2BK120A) in 30- to 40-μl reaction volumes for 30 min at 37°C.

Techniques: Transfection, Western Blot, Immunoprecipitation, Mutagenesis, Construct

Journal: Molecular and Cellular Biology

Article Title: Mammalian SWI/SNF-A Subunit BAF250/ARID1 Is an E3 Ubiquitin Ligase That Targets Histone H2B ▿

doi: 10.1128/MCB.00540-09

Figure Lengend Snippet: BAF250 contains a BC box and associates with Elo C. (A) Schematic diagram of BAF250b (GenBank accession no. AF521671). 16e, amino acids 419 to 2165; NTD, 419 to 1547; CTD, 1518 to 2165. Putative BC boxes identified by MAST scanning of human BAF250a/b and Drosophila Osa are shown. Amino acid numbering in the second column corresponds to the GenBank accession numbers listed in parenthesis. Sequences of consensus and putative BC boxes are listed in the third column, with key residues highlighted in bold. (B) The BAF250a/b C-terminal domain associates with Elo C. Upper two panels, cells were transiently transfected with Flag-Elo C alone (lanes 1, 3, and 5) or together with T7-BAF250aCTD (amino acids 1635 to 2285, GenBank accession no. O14497) (lanes 2, 4, and 6). Lysates were immunoprecipitated (IP) with α-T7 antibody, washed in a buffer containing 0.3 M KCl (lanes 3 and 4) or 0.5 M KCl (lanes 5 and 6), and analyzed by immunoblotting with an antibody to T7 or Flag. Lower two panels, the same experiment was performed with T7-BAF250bCTD. (C) Bacterially expressed Elo C interacts with BAF250b CTD. Polyhistidine (His)-tagged Elo C was expressed in E. coli, and the lysate was mixed with purified GST-BAF250bCTD (HR2) (lane 3) or control GST-TAFH (lane 2). Immunoblotting (WB) with an antibody to the poly-His tag shows specific binding of Elo C to BAF250bCTD. HR2 represents the second conserved region in BAF250bCTD, which contains the BC box (residues 1786 to 2165, boxed in gray in Fig. ​Fig.1A).1A). (D) BAF250bCTD interaction with Elo C is BC box dependent. Flag antibody immunoprecipitates of transiently transfected Flag-Elo C with T7-BAF250bNTD, T7-BAF250bCTD, or T7-BAF250bCTD with the BC box mutation (BC*) were washed in a buffer containing 0.3 M KCl and immunoblotted with an antibody to T7 or Flag. *, nonspecific band. (E) BAF250b interacts with Elo C in a BC box-dependent manner. T7 antibody immunoprecipitates of transiently transfected vector, T7-BAF250b, or T7-BAF250bBC* with Flag-Elo C were washed in a buffer containing 0.3 M KCl and immunoblotted with an antibody to T7 or Flag.

Article Snippet: Thirty microliters of Flag-BAF250b complexes (purified from induced 16e cells [16e+]) or the negative-control eluant (purified from uninduced 16e cells [16e−]) was dialyzed into ubiquitination buffer (50 mM Tris-HCl [pH 7.9], 5 mM MgCl 2 , 2 mM NaF, 0.6 mM dithiothreitol [DTT]) at 4°C for 1 h. After adjusting for changes in volume during dialysis, equivalent amounts of 16e−, Flag-BAF250b complexes, or recombinant Flag-tagged Ring1b were incubated with 2 mM ATP, 0.1 μg E1 (Boston Biochem), 0.5 μg E2 (UbcH5c; Boston Biochem), 1 μg hemagglutinin antigen-tagged ubiquitin (HA-Ub) (Boston Biochem), and 5 to 10 μg of in vitro -reconstituted oligonucleosomes (wild type or H2BK120A) in 30- to 40-μl reaction volumes for 30 min at 37°C.

Techniques: Transfection, Immunoprecipitation, Western Blot, Purification, Binding Assay, Mutagenesis, Plasmid Preparation

Journal: Molecular and Cellular Biology

Article Title: Mammalian SWI/SNF-A Subunit BAF250/ARID1 Is an E3 Ubiquitin Ligase That Targets Histone H2B ▿

doi: 10.1128/MCB.00540-09

Figure Lengend Snippet: BC* degradation by the proteasome is Cul2 dependent. (A) Stability of BAF250b and BC* protein. HeLa cells transiently transfected with T7-BAF250bWT (lanes 1 to 3) or T7-BAF250bBC* (lanes 4 to 6) were treated with vehicle (−) or cycloheximide (CHX) for the times indicated. Whole-cell extracts were immunoblotted for T7-250b and endogenous BRG1. (B) BC* degradation is exacerbated by overexpression of Cul2. HeLa cells were transiently transfected with T7-BAF250b (lanes 1 to 6) or T7-BAF250bBC* (lanes 7 to 12) and Myc-Cul2 (lanes 1 to 12). Cells were treated with MG132 or CHX for the times indicated. Whole-cell extracts were immunoblotted for T7, Myc, or endogenous BRG1. (C) The degradation of BAF250bBC* is exacerbated by Cul2 but not Cul5. HeLa cells were transiently transfected with T7-BAF250bBC* (lanes 1 to 12) and Myc-Cul5 (lanes 1 to 6) or Myc-Cul2 (lanes 7 to 12). Cells were treated with MG132 or CHX for the times indicated. Whole-cell extracts were immunoblotted for T7, Myc, or endogenous Elo A (loading control).

Article Snippet: Thirty microliters of Flag-BAF250b complexes (purified from induced 16e cells [16e+]) or the negative-control eluant (purified from uninduced 16e cells [16e−]) was dialyzed into ubiquitination buffer (50 mM Tris-HCl [pH 7.9], 5 mM MgCl 2 , 2 mM NaF, 0.6 mM dithiothreitol [DTT]) at 4°C for 1 h. After adjusting for changes in volume during dialysis, equivalent amounts of 16e−, Flag-BAF250b complexes, or recombinant Flag-tagged Ring1b were incubated with 2 mM ATP, 0.1 μg E1 (Boston Biochem), 0.5 μg E2 (UbcH5c; Boston Biochem), 1 μg hemagglutinin antigen-tagged ubiquitin (HA-Ub) (Boston Biochem), and 5 to 10 μg of in vitro -reconstituted oligonucleosomes (wild type or H2BK120A) in 30- to 40-μl reaction volumes for 30 min at 37°C.

Techniques: Transfection, Over Expression

Journal: Molecular and Cellular Biology

Article Title: Mammalian SWI/SNF-A Subunit BAF250/ARID1 Is an E3 Ubiquitin Ligase That Targets Histone H2B ▿

doi: 10.1128/MCB.00540-09

Figure Lengend Snippet: BAF250b is ubiquitinated and degraded by the proteasome. (A) BAF250bCTD BC* mutants are degraded during immunoprecipitation. T7 antibody immunoprecipitates of transiently transfected T7-BAF250b, T7-BAF250b with ARID deleted (ΔA), the T7-BAF250b BC box mutant (BC*), or T7-BAF250b with ARID deleted and the BC box mutated (ΔA and BC*), with Myc-Cul2, were washed in a buffer containing 0.5 M KCl and 0.1% NP-40 and analyzed by immunoblotting with an antibody to T7 or Myc. The film exposure for the input panels was shorter than that for the IP panels. (B) MG132 and NEM stabilize BAF250b mutant proteins in the presence of Myc-Cul2. HeLa cells were transiently transfected with T7-BAF250b, T7-BAF250b with ARID deleted (ΔA), the T7-BAF250b BC box mutant (BC*), or T7-BAF250b with ARID deleted and the BC box mutated (ΔA, BC*) and with Myc-Cul2, treated with 5 μM MG132, and harvested in the presence of NEM. BAF250b proteins were immunoprecipitated with an antibody to T7 and probed for Myc-Cul2. (C) BAF250bCTDBC* is autoubiquitinated. T7 antibody immunoprecipitates of cells transiently transfected with vector (−) (lanes 1, 5, and 9), T7-BAF250bCTDWT (+) (lanes 2, 6, and 10), T7-BAF250bCTDBC* (lanes 3, 7, and 11), or T7-BAF250bCTDBC** (lanes 4, 8, and 12) and Myc-Cul2 (lanes 1 to 12) were washed in a buffer containing 0.3 M KCl and 0.5% NP-40 and immunoblotted for ubiquitin (endogenous), Myc, and T7.

Article Snippet: Thirty microliters of Flag-BAF250b complexes (purified from induced 16e cells [16e+]) or the negative-control eluant (purified from uninduced 16e cells [16e−]) was dialyzed into ubiquitination buffer (50 mM Tris-HCl [pH 7.9], 5 mM MgCl 2 , 2 mM NaF, 0.6 mM dithiothreitol [DTT]) at 4°C for 1 h. After adjusting for changes in volume during dialysis, equivalent amounts of 16e−, Flag-BAF250b complexes, or recombinant Flag-tagged Ring1b were incubated with 2 mM ATP, 0.1 μg E1 (Boston Biochem), 0.5 μg E2 (UbcH5c; Boston Biochem), 1 μg hemagglutinin antigen-tagged ubiquitin (HA-Ub) (Boston Biochem), and 5 to 10 μg of in vitro -reconstituted oligonucleosomes (wild type or H2BK120A) in 30- to 40-μl reaction volumes for 30 min at 37°C.

Techniques: Immunoprecipitation, Transfection, Mutagenesis, Western Blot, Plasmid Preparation

Journal: Molecular and Cellular Biology

Article Title: Mammalian SWI/SNF-A Subunit BAF250/ARID1 Is an E3 Ubiquitin Ligase That Targets Histone H2B ▿

doi: 10.1128/MCB.00540-09

Figure Lengend Snippet: The BAF250b complex contains an E3 ligase activity that ubiquitinates histone H2B at K120. (A) Immunopurification of Flag-BAF250b complex. A silver-stained gel of SWI/SNF purified from a HeLa cell line, 16e, induced to express Flag-BAF250b is shown (lane 2). Purification from uninduced (−) 16e cells is shown as a negative control (lane 1). The SWI/SNF subunits identified by mass spectrometry are indicated. (B) Identification of SWI/SNF subunits by immunoblotting. Endogenous BRG1, BAF170, BAF155, and E3 ligase subunit Cul2 were detected in Flag-BAF250b-immunopurified material eluted with Flag peptide from induced cells (lane 4). An antibody to Flag was used to detect Flag-BAF250b. (C) The BAF250b complex monoubiquitinates histone(s) in a ubiquitination assay. An in vitro nucleosome ubiquitination assay mixture was reconstituted with the Flag-BAF250b complex (lanes 2 to 4), control Flag-eluted material (lane 1), or recombinant Ring1b (lane 5) and HA-ubiquitin (lanes 1 to 5). The products were immunoblotted (WB) with an antibody to HA and H2B. Ten microliters of eluted material correlates roughly with each lane in the silver-stained gel shown in panel A. Oligonucleosomes were omitted from the reaction in lane 4. Arrow, ubiquitinated H2A/H2B; *, HA-Ub-conjugated E1 and E2. (D) Bacterially expressed BAF250bCTD interacts with histone H2B. GST-BAF250bCTD (HR2) (lane 2) and control GST-TAFH (lane 3) proteins were expressed and purified from E. coli and incubated with histones extracted from HeLa cells. Immunoblotting (WB) with an antibody to H2B shows specific binding of H2B to BAF250bCTD. The lower panel shows the upper portion of the same gel stained with Coomassie blue. (E) The BAF250b complex monoubiquitinates histone H2B on K120. An in vitro nucleosome ubiquitination assay was performed as for panel C with the Flag-BAF250b complex (lanes 2 and 4), control material (lane 1), or recombinant Ring1b (lanes 3 and 5). The products were immunoblotted with an antibody to HA and H2B. The nucleosomes used were reconstituted in vitro with wild-type H2B (lanes 1 to 3) or H2B with lysine-120 mutated to alanine (H2BK120A) (lanes 4 and 5). Arrow, ubiquitinated H2A/H2B; *, HA-Ub-conjugated E1 and E2.

Article Snippet: Thirty microliters of Flag-BAF250b complexes (purified from induced 16e cells [16e+]) or the negative-control eluant (purified from uninduced 16e cells [16e−]) was dialyzed into ubiquitination buffer (50 mM Tris-HCl [pH 7.9], 5 mM MgCl 2 , 2 mM NaF, 0.6 mM dithiothreitol [DTT]) at 4°C for 1 h. After adjusting for changes in volume during dialysis, equivalent amounts of 16e−, Flag-BAF250b complexes, or recombinant Flag-tagged Ring1b were incubated with 2 mM ATP, 0.1 μg E1 (Boston Biochem), 0.5 μg E2 (UbcH5c; Boston Biochem), 1 μg hemagglutinin antigen-tagged ubiquitin (HA-Ub) (Boston Biochem), and 5 to 10 μg of in vitro -reconstituted oligonucleosomes (wild type or H2BK120A) in 30- to 40-μl reaction volumes for 30 min at 37°C.

Techniques: Activity Assay, Immu-Puri, Staining, Purification, Negative Control, Mass Spectrometry, Western Blot, Ubiquitin Assay, In Vitro, Recombinant, Incubation, Binding Assay

Journal: Molecular and Cellular Biology

Article Title: Mammalian SWI/SNF-A Subunit BAF250/ARID1 Is an E3 Ubiquitin Ligase That Targets Histone H2B ▿

doi: 10.1128/MCB.00540-09

Figure Lengend Snippet: Global levels of H2B-Ub and H3K4me3 are decreased following knockdown of BAF250a and BAF250b by RNAi. (A) Knockdown of BAF250 by RNAi. Whole-cell extracts from HEK293 cells transiently transfected with siRNAs against luciferase (control), BAF250a, BAF250b, or both BAF250a and BAF250b (a+b) were immunoblotted with an antibody to BAF250b. *, nonspecific band used as a loading control. (B) The same extracts as for panel A were immunoblotted with an antibody to BAF250a. (C) Decreased global H2B-Ub levels upon BAF250 siRNA knockdown. Acid extracts from HEK293 cells depleted of BAF250a (lane 3), BAF250b (lane 2), or BAF250a+b (lane 4) were immunoblotted with an antibody to H2B. The band migrating above 20 kDa in the upper panel corresponds to the size of H2B-Ub. The upper and lower panels are from the same gel. The blot was separated for probing because of the difference in signal intensity between H2B-Ub and H2B. (D) The same extracts as for panel C were immunoblotted with an antibody specific to H3K4me3 (upper panel) and H3 (lower panel). (E) BAF250a is a positive regulator of the HoxA9 gene. Quantitative RT-PCR of HoxA9 mRNA levels in HEK293 cells transiently transfected with siRNAs targeting luciferase (control), BAF250a, BAF250b, or both BAF250a and BAF250b (a+b) is shown. Error bars indicate standard deviations.

Article Snippet: Thirty microliters of Flag-BAF250b complexes (purified from induced 16e cells [16e+]) or the negative-control eluant (purified from uninduced 16e cells [16e−]) was dialyzed into ubiquitination buffer (50 mM Tris-HCl [pH 7.9], 5 mM MgCl 2 , 2 mM NaF, 0.6 mM dithiothreitol [DTT]) at 4°C for 1 h. After adjusting for changes in volume during dialysis, equivalent amounts of 16e−, Flag-BAF250b complexes, or recombinant Flag-tagged Ring1b were incubated with 2 mM ATP, 0.1 μg E1 (Boston Biochem), 0.5 μg E2 (UbcH5c; Boston Biochem), 1 μg hemagglutinin antigen-tagged ubiquitin (HA-Ub) (Boston Biochem), and 5 to 10 μg of in vitro -reconstituted oligonucleosomes (wild type or H2BK120A) in 30- to 40-μl reaction volumes for 30 min at 37°C.

Techniques: Transfection, Luciferase, Quantitative RT-PCR