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. 2005 Sep;25(18):7953-65.
doi: 10.1128/MCB.25.18.7953-7965.2005.

The BRG1- and hBRM-associated factor BAF57 induces apoptosis by stimulating expression of the cylindromatosis tumor suppressor gene

Li Wang  1 , Robert A Baiocchi, Sharmistha Pal, George Mosialos, Michael Caligiuri, Saïd Sif
Affiliations

The BRG1- and hBRM-associated factor BAF57 induces apoptosis by stimulating expression of the cylindromatosis tumor suppressor gene

Li Wang et al. Mol Cell Biol. 2005 Sep.

Abstract

Mutation of BRG1, hBRM, and their associated factors, INI1 and BAF57, in primary human tumors has suggested that inactivation of human SWI/SNF (hSWI/SNF) complexes may be involved in neoplastic transformation. BT549 is an invasive human breast carcinoma cell line that lacks expression of BAF57, a key hSWI/SNF subunit that mediates interaction with transcriptional activators and corepressors. In this study we investigated the role of BAF57 in suppressing tumorigenesis by establishing BT549 stable cell lines that expresses full-length BAF57 protein. BT549 clones expressing BAF57 demonstrated marked phenotypic changes, slow growth kinetics, and restoration of contact inhibition. Altered growth was found to be due in part to cell cycle arrest and induction of apoptosis. Furthermore, microarray analysis revealed that BAF57-mediated cell death was associated with up-regulation of proapoptotic genes including the tumor suppressor familial cylindromatosis (CYLD), which was found to be a direct target of BAF57 as determined by chromatin immunoprecipitation analysis. Increased expression of CYLD in BT549 cells induced apoptosis, while its suppression by small interfering RNA inhibited cell death in BAF57 expressing BT549 cells. These findings demonstrate the importance of BAF57 in cell growth regulation and provide a novel link between hSWI/SNF chromatin remodelers and apoptosis.

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Figures

FIG. 1.
FIG. 1.
Retrovirus-mediated expression of BAF57 in BT549 cells. (A) Whole-cell extracts (20 μg) from normal HMEC or transformed BT549, HCC1937, and MCF7 breast cancer cell lines were analyzed by Western blotting using anti-BAF57 antibody. As a control, MAD levels were also measured by reprobing the same blot with anti-MAD antibody. (B) Analysis of ectopically expressed BAF57 in stable BT-BAF57 cell lines. Approximately 20 μg of nuclear extract isolated from either BT549, BT549/pBABE (BT-pBABE), or BT549/BAF57 (BT-BAF57) cells and 100 ng of immunopurified flag-tagged human SWI/SNF (Fl-hSWI/SNF) were analyzed by Western blotting using anti-BAF57 antibody. Anti-MAD was used as a control to show equal loading. (C) Ectopically expressed BAF57 is incorporated into BRG1- and hBRM-based hSWI/SNF complexes. Nuclear extracts from BT549 and BT-BAF57 clones 3 and 6 were immunoprecipitated with either preimmune (PI) or immune (I) anti-BAF57 antibodies, and after extensive washing the retained proteins were analyzed by Western blotting using anti-BRG1, anti-hBRM, and anti-BAF155 antibodies.
FIG. 2.
FIG. 2.
Reexpression of BAF57 induces flat-cell morphology and inhibits transformation and cell proliferation. (A) An equal number (5 × 104) of BT549, BT-pBABE, BT-BAF57 clone 3, and BT-BAF57 clone 6 cells was seeded into 10-cm plates, and after 3 days pictures were taken at ×40 magnification. (B) Approximately 2 × 103 BT549, BT-pBABE, and BT-BAF57 clones 3 and 6 cells were grown for 14 days before anchorage-dependent colonies were visualized by crystal violet staining. This experiment was performed three times, and representative plates are shown. (C) Proliferation was measured by plating an equal number (5 × 104) of cells from the indicated cell lines and performing cell counts every 2 days for 6 days. The number of viable cells was determined by trypan blue exclusion. This experiment was repeated three times, and the data points represent the average counts from nine plates.
FIG. 3.
FIG. 3.
FACS analysis of wild-type and BAF57-expressing BT549 cell lines. An equal number (5 × 104) of BT549, BT-pBABE, BT-BAF57 clone 3, or BT-BAF57 clone 6 cells was seeded into 15-cm plates and allowed to grow for the indicated times. Next, cells were fixed, stained with propidium iodide, and analyzed on a FACSCalibur flow cytometer. The cell cycle profile of each cell line was determined at least three times, and representative histograms are shown.
FIG. 4.
FIG. 4.
Reexpression of BAF57 affects genes involved in cell cycle regulation and cell death. (A) Whole-cell extracts (20 μg) from BT549, BT-pBABE, and BT-BAF57 clone 3 cells were analyzed by Western blotting using antibodies specific for the indicated proteins. As a control, BAF57 levels were measured in BT549, BT-pBABE, and BT-BAF57 cell lines. (B) Genes affected by BAF57 reexpression were analyzed in BT-BAF57 clone 6 as described in panel A. (C) Total RNA from BT549 and BT-BAF57 clones 3 and 6 was used in RT-PCRs as described in Material and Methods. Different amounts of each RT reaction (0.2 and 2 μl) were PCR amplified in the presence of [α-32P]dCTP and gene-specific primers. Control (Ctrl) represents PCRs lacking 5′ primer. (D) Total RNA from untransfected (−) or transiently transfected (+) HMEC or BT549, HCC1937, and MCF7 cells was used to measure the levels of CYLD mRNA as described in panel C. Western blot analysis was performed to show that BAF57 was expressed at similar levels in the transiently transfected cells.
FIG. 5.
FIG. 5.
BAF57 is associated with the CYLD and CYCLIN E promoters. (A) Cross-linked chromatin from BT549, BT-BAF57 clone 3, and BT-BAF57 clone 6 cells was immunoprecipitated with either preimmune serum (PI) or anti-BAF57 antibodies (I), and the eluted genomic DNA was PCR amplified using promoter-specific primers for the indicated genes. Input represents 1/750 of total chromatin used in each reaction. (B) BRG1 and hBRM are differentially recruited to the CYLD promoter. Chromatin immunoprecipitation was conducted as described in panel A using either anti-BRG1 or anti-hBRM antibodies, and the eluted DNA (1, 2, or 5 μl) was PCR amplified using CYLD or suppressor of tumorigenicity 5 (ST5) promoter-specific primers. Mock, reaction without chromatin; No Ab, reaction without antibody.
FIG. 6.
FIG. 6.
CYLD induces apoptosis in BT549 cells. (A) An equal number (5 × 104) of cells from the BT-549, BT-CYLD, and BT-CYLD(1-932) cell lines was seeded into 10-cm plates, and pictures were taken at ×40 magnification. (B) Western blot analysis was conducted using 20 μg of whole-cell extract from the indicated stable cell lines. Both wild-type and mutant CYLD were detected using either anti-flag or anti-CYLD antibody. To show equal loading, the same blot was stripped and reprobed with anti-HDAC2 antibody. (C) Total RNA from BT549, BT-CYLD(1-932), and BT-CYLD was PCR-amplified using either BAX, BCL-2, or GAPDH gene-specific primers as described in the legend of Fig. 4C. (D) Approximately 5 × 104 cells from the BT549, BT-CYLD or BT-CYLD(1-932) cell lines were seeded into 10-cm plates and allowed to grow until plates became 80 to 90% confluent. Cells were then analyzed by FACS analysis. Cell cycle distribution of each cell line was measured at least three times, and representative histograms are shown.
FIG. 7.
FIG. 7.
Knock-down of CYLD by siRNA restores BT549 cell morphology and inhibits apoptosis. (A) An equal number (5 × 104) of BT-BAF57, BT-BAF57-NSsiRNA, and BT-BAF57-CYLDsiRNA cells was seeded into 10-cm plates, and pictures were taken at ×40 magnification. (B) To measure the mRNA levels of CYLD, BAX, and BCL-2, RT-PCR was conducted using total RNA from the indicated stable cell lines. As a control GAPDH levels were also analyzed. (C) To measure endogenous CYLD protein levels, Western blot analysis was conducted using whole-cell extracts from the indicated stable cell lines. As a control HDAC2 levels were also analyzed. (D) An equal number (5 × 104) of BT-BAF57, BT-BAF57-NSsiRNA, and BT-BAF57-CYLDsiRNA cells was seeded into 10-cm plates and analyzed by FACS analysis as described in the legend of Fig. 6D.
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