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, 19 (2), 1460-9

Cyclin E Associates With BAF155 and BRG1, Components of the Mammalian SWI-SNF Complex, and Alters the Ability of BRG1 to Induce Growth Arrest

Affiliations

Cyclin E Associates With BAF155 and BRG1, Components of the Mammalian SWI-SNF Complex, and Alters the Ability of BRG1 to Induce Growth Arrest

F Shanahan et al. Mol Cell Biol.

Abstract

SWI-SNF complexes have been implicated in transcriptional regulation by chromatin remodeling. We have identified an interaction between two components of the mammalian SWI-SNF complex and cyclin E, an essential cell cycle regulatory protein required for G1/S transition. BRG1 and BAF155, mammalian homologs of yeast SWI2 and SWI3, respectively, are found in cyclin E complexes and are phosphorylated by cyclin E-associated kinase activity. In this report, we show that overexpression of BRG1 causes growth arrest and induction of senescence-associated beta-galactosidase activity, which can be overcome by cyclin E. Our results suggest that cyclin E may modulate the activity of the SWI-SNF apparatus to maintain the chromatin in a transcriptionally permissive state.

Figures

FIG. 1
FIG. 1
Association of cyclin E with cellular proteins. (A) Immunoprecipitations were performed with cyclin E monoclonal antibody (HE172), cyclin A (cycA) (BF683), or a negative control antibody (PAb419) as indicated from [35S]methionine metabolically labeled ML-1 cell lysates. (B) Cyclin E (HE172)- or control antibody (PAb419)-cross-linked beads were used to immunoprecipitate protein complexes from 1 mg of ML-1 extract. Polypeptides were separated by SDS-PAGE and analyzed by immunoblotting with antibiotics as indicated. One hundred micrograms of ML-1 extract was included as a positive control. (C) Immunoprecipitations were performed with cyclin E monoclonal antibody (HE172), cyclin A (BF683), or negative control antibody (PAb419) as indicated from ML-1 cells. Immunocomplexes were subjected to an in vitro kinase assay with 5 μCi of [γ-32P]ATP before separation by SDS-PAGE and autoradiography. (D) Immunoprecipitations were performed as described above from ML-1 cells metabolically labelled with 2 mCi of 32Pir/ml for 30 min. The identities of associated molecules are as indicated; asterisks denote unknown proteins.
FIG. 2
FIG. 2
Cyclin E associates with BAF155. (A) Peptide sequences obtained from mass spectroscopic sequencing of p150. (B) Schematic representation of BAF155. Alignments of peptides to BAF155 are shown by solid bars. The shaded regions share sequence homology with yeast SWI3 (57). (C) A SalI-NotI fragment encoding full-length BAF155 in pSPORT (Invitrogen) was in vitro translated (ivt) with T7 RNA polymerase with the TNT kit (Promega). V8 partial proteolytic mapping of in vitro-translated BAF155 protein and p150 derived from cyclin E immunoprecipitation (cycE ip) was performed according to standard protocols (46). (D) One milligram of ML-1 cell lysate was immunoprecipitated with cyclin E monoclonal antibodies from the HE series (46). PAb419 was included as a negative control. Immunoprecipitates (IP) were analyzed by immunoblotting with antibodies against BAF155 and cdk2. (E) One milligram of ML-1 (lanes 1 to 4) or U2OS (lanes 5 to 9) cell lysate was immunoprecipitated with monoclonal antibodies against cyclin E (HE172), cyclin A (BF683), and cyclin B (GNS1) and polyclonal peptide antisera against cyclins D1, D2, and D3 as indicated. PAb419 and normal rabbit serum (NR) were included as negative controls. Immunoprecipitates were separated by SDS-PAGE and immunoblotted with antibodies against BAF155. cdc2, cdk2, or cdk6.
FIG. 3
FIG. 3
BRG1 is associated with the cyclin E-BAF155 complex. (A) V8 partial proteolytic mapping of in vitro-translated (ivt) BRG1 protein and p200 derived from cyclin E (cyc E) and BAF155 immunoprecipitations (ip). (B) Immunoblot analysis of cyclin E (HE172, HE67) or control (PAb419) immunoprecipitations from ML-1 cells with antibodies to BRG1 and Ini1 as indicated. One hundred micrograms of cell lysate was loaded as a positive control. (C) Immunoblot analysis of cyclin E (HE172) or control (PAb419) immunoprecipitations from ML-1 cells with antibodies to hBRM. One hundred micrograms of cell lysate was loaded as a positive control.
FIG. 4
FIG. 4
BRG1 is required for the interaction between cyclin E and BAF155. (A) Immunoblot analysis of cyclin E (HE172) or control (PAb419) immunoprecipitations from cell lines as indicated, probed with antibodies against BAF155 or cyclin E. Lysates were also probed directly for BAF155 expression (lower panel). (B) SW13 cells were transiently transfected with pHook-1 (Invitrogen) alone (lanes 3 and 4) or together with pBJ5.BRG1 (lanes 5 and 6), pBJ5.BRG1-K798R (lanes 7 and 8), and pBJ5.BRG1 plus pCMV-cyclin E (lanes 9 and 10) as indicated. Transfected cells were harvested with the Capture-Tec kit (Invitrogen) 72 h after transfection. Cyclin E (HE172) or control (PAb419) immunoprecipitations were performed from 100 μg of lysate and probed with antibodies to BAF155 (upper panel), BRG1 (middle panel), and cyclin E (lower panel). Untransfected SW13 (lanes 1 and 2) and ML-1 (lanes 11 and 12) cells were included as controls.
FIG. 5
FIG. 5
Association of cyclin E with SWI-SNF complex does not require the presence of pRb family member proteins. (A) Immunoblot analysis of cyclin E (HE172 [lane 1]) or control (PAb419 [lane 2]) immunoprecipitations (IP) from SAOS-2 cells. In lanes 3 to 6, lysates were precleared prior to immunoprecipitation with antibodies to p107 and p130 (lanes 4 and 6) or normal rabbit (NR) serum (lanes 3 and 5). ML-1 lysate was run as a positive control in lane 7. Blots were probed with antibodies against BAF155. (B) Extracts (100 μg) from lysates precleared with normal rabbit serum or p107 and p130 were immunoblotted with antibodies as indicated.
FIG. 6
FIG. 6
BRG1 induces growth arrest. SW13 cells were transfected as described in Materials and Methods with 6 μg of total plasmid DNA, including 2 μg of pBJ5.BRG1. pBSK was used as the carrier. Cells were grown in the presence of puromycin for 10 days. Cells were then either fixed and stained with 2% crystal violet–20% methanol for 5 h (original magnification, ×40 [A and B]), fixed and stained with normal rabbit serum and counterstained with DAPI (original magnification, ×40) [C and D]), fixed and stained with BRG1 antibody (J1) (25) and counterstained with DAPI (original magnification, ×40 [E and F]), labelled with BrdU (original magnification, ×20 [G]), or stained for SA β-galactosidase activity as previously described (8) (original magnification, ×40 [H]).

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