Journal:

Article Title: BAT3 and SET1A Form a Complex with CTCFL/BORIS To Modulate H3K4 Histone Dimethylation and Gene Expression ▿ †

doi: 10.1128/MCB.00568-08

Figure Lengend Snippet: BAT3 physically interacts with BORIS in HCT116 cells. (A) Yeast two-hybrid design. Diagram of 13 bait sequences of BORIS used to identify binding partners. Light hatched rectangles represent baits that captured binding partners (Table ​(Table1),1), while dark dotted rectangles represent baits with no binding partners (see methods in the supplemental material). (B) BAT3 binds to BORIS in vitro. HCT116 cell lysates were subjected to IP with an anti-BORIS antibody (see Fig. S1 and S2 in the supplemental material), followed by Western analysis with an anti-BAT3 antibody. IgG, immunoglobulin G. (C) HCT116 cell lysates were subjected to IP with an anti-BAT3 antibody, followed by Western analysis with an anti-BORIS antibody. (D) COS-7 cells were transfected with pCMV-GFP, pCMV-GFP-BORIS, and/or pCMV-BAT3 and subjected to IP with an anti-GFP antibody. Immunoprecipitates were resolved by SDS-polyacrylamide gel electrophoresis and blotted with an anti-BAT3 antibody. (E) BORIS binds to the BAT3 proline-rich region. HCT116 cell lysates were incubated with purified wild-type His-tagged BAT3 or His-tagged ΔP2-BAT3, which contains a deletion in the proline-rich region (aa 387 to 675 deleted). Lysates were subjected to IP with an anti-His antibody (Santa Cruz, Inc.), and the His tag IP products were resolved and immunoblotted with either an anti-BAT3 (top panel) or anti-BORIS (lower panel) antibody.

Article Snippet: HCT116 cell lysates were incubated with purified wild-type His-tagged BAT3 or His-tagged ΔP2-BAT3, which contained a deletion in the second proline-rich region (aa 387 to 675 deleted [see Fig. S4 in the supplemental material]), followed by the addition of an anti-His antibody (Santa Cruz, Inc.).

Techniques: Binding Assay, In Vitro, Western Blot, Transfection, Polyacrylamide Gel Electrophoresis, Incubation, Purification

Journal:

Article Title: BAT3 and SET1A Form a Complex with CTCFL/BORIS To Modulate H3K4 Histone Dimethylation and Gene Expression ▿ †

doi: 10.1128/MCB.00568-08

Figure Lengend Snippet: (A and B) BAT3 and BORIS bind to the promoter regions of BRCA1 and Myc and to the H19 DMR. HCT116 cells were fixed with 1% formaldehyde to cross-link protein-DNA interactions and sonicated, and fixed cells were subjected to IP with either an anti-BAT3 or an anti-BORIS antibody. DNA was eluted and purified before analysis by either PCR (A) or QPCR (B) with primers to either the promoters of BRCA1 or Myc or the H19 DMR. All ChIP experiments were done in triplicate, and error bars represent one standard deviation about the arithmetic mean. Fold enrichment for all ChIP experiments was normalized to the immunoglobulin G control IP. (C and D) BAT3 and BORIS bind to a CTCF-binding element. HCT116 cells were harvested, and nuclear cell extracts were used for EMSA with a 32P-labeled oligonucleotide containing a consensus CTCF-binding sequence. Lane 1 is probe alone and lanes 2 to 4 were incubated with cellular extract. Supershift assays were done by preincubating extracts with increasing concentrations (+ or ++) of either anti-BORIS (C) or anti-BAT3 (D) antibodies (lanes 3 and 4). Sections of fluorograms from native gels using a Typhoon phosphorimager are shown. Arrows indicate the supershifted complexes as well as the protein-CTCF-DNA complex and unbound (free) oligonucleotide probe.

Article Snippet: HCT116 cell lysates were incubated with purified wild-type His-tagged BAT3 or His-tagged ΔP2-BAT3, which contained a deletion in the second proline-rich region (aa 387 to 675 deleted [see Fig. S4 in the supplemental material]), followed by the addition of an anti-His antibody (Santa Cruz, Inc.).

Techniques: Sonication, Purification, Standard Deviation, Control, Binding Assay, Labeling, Sequencing, Incubation

Journal:

Article Title: BAT3 and SET1A Form a Complex with CTCFL/BORIS To Modulate H3K4 Histone Dimethylation and Gene Expression ▿ †

doi: 10.1128/MCB.00568-08

Figure Lengend Snippet: siRNA knockdown of BAT3 eliminates BAT3 binding to the promoter regions of BRCA1 and Myc, decreases gene expression, and alters H3K4 dimethylation. (A and B) BAT3 is not required for BORIS DNA binding. HCT116 cells were treated with either nonsense (control) or BAT3 siRNA, and ChIP analysis was done followed by QPCR to with primers to the Myc (A) or BRCA1 (B) promoters (see Fig. S5 in the supplemental material). (C) siRNA knockout of BAT3 decreases BRCA1 and MycR RNA levels. HCT116 cells were treated with nonsense (Cont) or BAT3 siRNA (in duplicate); RNA was isolated; and BRCA1 and Myc expression was determined by RT-PCR that was done in triplicate. Error bars represent one standard deviation about the arithmetic mean, and statistical significance was established by Student's t test. *, P < 0.05. (D) siRNA knockout of BAT3 decreases histone H3K4 dimethylation. HCT116 cells were treated with nonsense (Cont) or BAT3 siRNA and harvested for ChIP using either an H3K4 or H3K9 antibody followed by QPCR with primers to either the Myc (left panel) or BRCA1 (right panel) promoter. All ChIP experiments were done in triplicate, and error bars represent one standard deviation about the arithmetic mean. Statistical significance was established by Student's t test. *, P < 0.05 by t test.

Article Snippet: HCT116 cell lysates were incubated with purified wild-type His-tagged BAT3 or His-tagged ΔP2-BAT3, which contained a deletion in the second proline-rich region (aa 387 to 675 deleted [see Fig. S4 in the supplemental material]), followed by the addition of an anti-His antibody (Santa Cruz, Inc.).

Techniques: Knockdown, Binding Assay, Gene Expression, Control, Knock-Out, Isolation, Expressing, Reverse Transcription Polymerase Chain Reaction, Standard Deviation

Journal:

Article Title: BAT3 and SET1A Form a Complex with CTCFL/BORIS To Modulate H3K4 Histone Dimethylation and Gene Expression ▿ †

doi: 10.1128/MCB.00568-08

Figure Lengend Snippet: SET1A and ASH2 bind to the Myc and BRCA1 promoters and the H19 DMR, and shRNA knockdown of SET1A decreases both H3K4 dimethylation and gene expression. (A) SET1A and ASH2 bind to the promoter regions of Myc and BRCA1 as well as the H19 DMR. HCT116 cells were harvested and subjected to IP with either an anti-BORIS, an anti-SET1A (Santa Cruz, Inc.), or an anti-ASH2 (Santa Cruz, Inc.) antibody. DNA was eluted and purified before analysis with primers to BRCA1, Myc, or the H19 DMR. All ChIP experiments were done in triplicate, and error bars represent one standard deviation about the arithmetic mean. (B) HCT116 cells were transfected with either vector only (Cont) or a SET1A shRNA and then harvested, separated by SDS-polyacrylamide gel electrophoresis, transferred onto nitrocellulose, and immunoblotted using an anti-SET1A antibody (Bethyl Laboratories). (C and D) shRNA knockdown of SET1A decreases SET1A and ASH2, but not BORIS or BAT3, binding to the Myc (C) or BRCA1 (D) promoter. HCT116 cells were treated with nonsense (Cont) or SET1A shRNA, and ChIP was done with an anti-BORIS, anti-BAT3, anti-SET1A, or anti-ASH2 antibody followed by QPCR (as described above). (E) shRNA knockdown of SET1A decreases BRCA1, Myc, and H19 gene expression. HCT116 cells were treated with nonsense (Cont) or SET1A shRNA (in duplicate), RNA was isolated, and BRCA1, Myc, and H19 RNA levels were determined by RT-PCR that was done in triplicate. Error bars represent one standard deviation about the arithmetic mean. *, P < 0.05 by t test. (F) shRNA knockdown of SET1A decreases promoter histone H3K4 dimethylation. HCT116 cells were treated with nonsense (Cont) or SET1A shRNA, harvested for ChIP with either an H3K4 or H3K9 antibody, and analyzed by QPCR with primers to either the Myc or BRCA1 promoter. All ChIP experiments were done in triplicate, and error bars represent one standard deviation about the arithmetic mean. Statistical significance was established by Student's t test. *, P < 0.05.

Article Snippet: HCT116 cell lysates were incubated with purified wild-type His-tagged BAT3 or His-tagged ΔP2-BAT3, which contained a deletion in the second proline-rich region (aa 387 to 675 deleted [see Fig. S4 in the supplemental material]), followed by the addition of an anti-His antibody (Santa Cruz, Inc.).

Techniques: shRNA, Knockdown, Gene Expression, Purification, Standard Deviation, Transfection, Plasmid Preparation, Polyacrylamide Gel Electrophoresis, Binding Assay, Isolation, Reverse Transcription Polymerase Chain Reaction

Journal:

Article Title: BAT3 and SET1A Form a Complex with CTCFL/BORIS To Modulate H3K4 Histone Dimethylation and Gene Expression ▿ †

doi: 10.1128/MCB.00568-08

Figure Lengend Snippet: shRNA knockdown of BORIS eliminates BORIS, SET1A, and ASH2 DNA binding, decreases Myc and BRCA1 expression, and reduces H3K4 dimethylation. (A and B) shRNA knockdown of BORIS decreases SET1A and ASH2 binding to the Myc and BRCA1 promoters. HCT116 cells were transfected with either nonsense (Cont) or BORIS shRNA, and ChIP was done with an anti-BORIS, anti-SET1A, or anti-ASH2 antibody. DNA was eluted and purified before analysis with primers to either the Myc (A) or BRCA1 (B) promoter. (C) shRNA knockdown of BORIS decreased Myc and BRCA1 expression. HCT116 cells were transfected with either nonsense (Cont) or BORIS shRNA (in duplicate), RNA was isolated, and Myc and BRCA1 expression was determined by RT-PCR in triplicate. Error bars represent one standard deviation about the arithmetic mean. (D) shRNA knockdown of BORIS decreases promoter histone H3K4 dimethylation. HCT116 cells were treated with nonsense (Cont) or BORIS shRNA, harvested for ChIP with either an H3K4 or H3K9 antibody, and analyzed by QPCR. All ChIP experiments were done in triplicate, and error bars represent one standard deviation about the arithmetic mean. Statistical significance was established by Student's t test. *, P < 0.05.

Article Snippet: HCT116 cell lysates were incubated with purified wild-type His-tagged BAT3 or His-tagged ΔP2-BAT3, which contained a deletion in the second proline-rich region (aa 387 to 675 deleted [see Fig. S4 in the supplemental material]), followed by the addition of an anti-His antibody (Santa Cruz, Inc.).

Techniques: shRNA, Knockdown, Binding Assay, Expressing, Transfection, Purification, Isolation, Reverse Transcription Polymerase Chain Reaction, Standard Deviation