Figure 6.

Direct association between CARM1 and BRG1. (A) CARM1 binds to purified BRG1 in GST pull-down assays, whereas no interaction was observed between CARM1 and ³⁵S-Met-labeled BAF155 or BAF 170 (right). Coomassie staining of GST, GST-CARM1, and GST-PRMT1 (left, arrowhead). BRG1 was visualized by Western blotting and 10% of fraction applied for binding was loaded as input. (B) Coimmunoprecipitation of CARM1 with BRG1 and BAF155 in SW13 cells. Nuclear extracts of SW13 cells transfected with indicated plasmids were immunoprecipitated with CARM1 antibody. The CARM1 immunoprecipitates were analyzed by Western blotting using HA/BRG1 or HA/BRG155 antibodies. The 1/10 of nuclear extracts from transfected cells were loaded as Input and probed with α-BRG1/BAF155 or α-CARM1 antibodies. (C) In the SWI/SNF complex, only BRG1 is recognized by CARM1 in a Far-Western assay. A total of 1.0 μg of SWI/SNF (lane 1) and 0.1 μg of BRG1 (lane 2) was loaded on 8% SDS-PAGE, transferred to PVDF membrane, and incubated with ³⁵S-Met-labeled CARM1. The membrane was washed three times with buffer (20 mM Tris at pH 7.6, 10% glycerol, 100 mM NaCl, 1 mM EDTA, 0.1% Tween 20) and exposed to X-ray film. (D) Stimulation of ATPase activity by two BRG1-interacting fragments of CARM1. GST-CARM1 fragments were expressed in E. Coli and purified as described in the Materials and Methods. Recombinant BRG1 is purified from Flag-BRG1 expressing baculovirus-infected Sf9 cells and 1/10 of the total rBRG1 applied to the GST-CARM1 affinity column is shown as input. BRG1 is detected by Western blotting. (Bottom) A total of 0.3 μg of GST-CARM1 or fragments was applied in BRG1 ATPase assay (final concentration of BRG1 is 5 nM). (E) Mapping of CARM1-interacting regions in BRG1. Comparable amounts of ³⁵S-labeled BRG1 fragments and purified GST-CARM1 proteins were used in each binding reaction as described (DiRenzo et al. 2000). When compared with the total input, strong (≥20%, +++), moderate (10%, ++), weak (5%, +), and no (-) binding are indicated. (HSA) Helicase domain with SANT association; (TCH) conserved in transcription and CHROMO domain helicases; (DEXDc) homology to DEAD-like RNA helicases superfamily; (HELICc) helicase superfamily carboxy-terminal domain; (BROMO) bromo domain. (F) Transactivation of CARM1 in ER signaling requires BRG1. BRG1 and BRM-deficient adrenal carcinoma SW13 cells were plated in 24-well dishes and transfected with indicated plasmids using FuGene (Roche). All experiments were normalized to internal β-gal controls and carried out in triplicates. The date represented the fold of induction by estrodial (1 μM). Similar conditions were used in GAL4-VP16 transfection assays.