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. 2008 Aug;19(8):3308-22.
doi: 10.1091/mbc.e08-02-0123. Epub 2008 May 28.

Chromatin remodeling complexes interact dynamically with a glucocorticoid receptor-regulated promoter

Thomas A Johnson  1 , Cem Elbi, Bhavin S Parekh, Gordon L Hager, Sam John
Affiliations

Chromatin remodeling complexes interact dynamically with a glucocorticoid receptor-regulated promoter

Thomas A Johnson et al. Mol Biol Cell. 2008 Aug.

Abstract

Brahma (BRM) and Brahma-related gene 1 (BRG1) are the ATP-dependent catalytic subunits of the SWI/SNF family of chromatin-remodeling complexes. These complexes are involved in essential processes such as cell cycle, growth, differentiation, and cancer. Using imaging approaches in a cell line that harbors tandem repeats of stably integrated copies of the steroid responsive MMTV-LTR (mouse mammary tumor virus-long terminal repeat), we show that BRG1 and BRM are recruited to the MMTV promoter in a hormone-dependent manner. The recruitment of BRG1 and BRM resulted in chromatin remodeling and decondensation of the MMTV repeat as demonstrated by an increase in the restriction enzyme accessibility and in the size of DNA fluorescence in situ hybridization (FISH) signals. This chromatin remodeling event was concomitant with an increased occupancy of RNA polymerase II and transcriptional activation at the MMTV promoter. The expression of ATPase-deficient forms of BRG1 (BRG1-K-R) or BRM (BRM-K-R) inhibited the remodeling of local and higher order MMTV chromatin structure and resulted in the attenuation of transcription. In vivo photobleaching experiments provided direct evidence that BRG1, BRG1-K-R, and BRM chromatin-remodeling complexes have distinct kinetic properties on the MMTV array, and they dynamically associate with and dissociate from MMTV chromatin in a manner dependent on hormone and a functional ATPase domain. Our data provide a kinetic and mechanistic basis for the BRG1 and BRM chromatin-remodeling complexes in regulating gene expression at a steroid hormone inducible promoter.

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Figures

Figure 1.
Figure 1.
SWI/SNF chromatin remodelers potentiate the hormone-dependent activation of the MMTV-LTR. (A) Schematic representation of BRG1 and BRM with conserved domains. (B) Location of lysine to arginine point mutations in the highly conserved ATPase domain of BRG1 and BRM. This mutation abolishes the ability of BRG1 and BRM to hydrolyze ATP and remodel chromatin. (C) BRG1 and BRM potentiate the transcriptional activity of MMTV in BRG1-, BRM-, and GR-deficient human adrenal carcinoma cells (SW13). SW13 cells were transfected with MMTV-LTR-Luciferase and pCMV β-galactosidase (internal control) along with pGR, pBRG1, pBRM, pBRG1-K-R, or pBRM-K-R expression vectors. The cells were treated with 100 nM dexamethasone or vehicle control for 4 h. Luciferase reporter gene activity was assayed and normalized to β-galactosidase reporter gene activity. The data shown is from two independent experiments.
Figure 2.
Figure 2.
BRG1 and BRM but not ISWI chromatin remodeling activities are recruited to the MMTV array in a hormone-dependent manner. Mouse mammary adenocarcinoma cells (3134) with a single, stably integrated MMTV-LTR array were treated with 100 nM dexamethasone for 30 min. Fixed cells were probed with specific antibodies to detect endogenous BRG1, BRM, or ISWI by indirect immunofluorescence. MMTV RNA was detected by a RNA FISH using a probe specific to the MMTV transcript. The arrows in E and M point to the immunofluorescence detected localization of BRG1 and BRM on the MMTV array. The overlays (yellow) in C, G, K, O, S, and W indicate colocalization of the immunofluorescence and RNA FISH signals. The arrows in C, G, K, O, S, and W point to the end positions of linescans. Linescan analyses in D, H, L, P, T, and X quantitatively show the ligand-dependent recruitment or lack thereof of the BRG1, BRM, or ISWI chromatin-remodeling complexes to the MMTV-LTR array. The linescan in L runs through the RNA FISH signal that is adjacent but not coincident with the BRM fluorescence intensity peak. In H, P, and X, fluorescence intensity peaks for BRG1 and BRM but not for ISWI coincided with MMTV RNA. Bar, 4 um. Treatment of cells with 100nM dexamethasone for 30 minutes increases the intensity (Y) as well as the size (Z) of the MMTV RNA FISH signal.
Figure 3.
Figure 3.
Transcription from the MMTV-LTR is regulated by the ATP-hydrolysis activity of BRG1 or BRM. 3134 mouse mammary adenocarcinoma cells with a stably integrated MMTV-LTR array were transfected with Flag-tagged forms of wild-type BRG1, BRM, and mutant BRG1-K-R, BRM-K-R (A–H). Cells were treated with 100 nM dexamethasone for 30 min and fixed. BRG1, BRG1-K-R, wild-type BRM, and BRM-K-R were detected by indirect immunofluorescence using an anti-Flag antibody and MMTV RNA was detected by RNA FISH using a probe specific to the MMTV transcript. Arrows (A, C, E, and G) point to the localization of MMTV transcript. Average integrated RNA FISH intensities from 35 randomly selected cells in each transfected category (A, C, E, and G) and untransfected category (B, D, F, and H) were measured as described in Materials and Methods and plotted as a bar histogram (I). Error bars, SE. (J–N) A stably expressed dominant negative form of BRG1 (BRG1-K-R) inhibits MMTV transcription. The 5555 mouse mammary adenocarcinoma cells with stably integrated MMTV-LTR array express a Flag-tagged dominant negative form of BRG1 (BRG1-K-R) under the control of a tetracycline-repressible system (J). 5555 cells were grown in the presence or absence of tetracycline to facilitate the expression of BRG1-K-R (K). Western blot analysis was performed using an anti-Flag antibody. 5555 cells were grown in the presence (L) or absence (M) of tetracycline. Cells were treated with 100 nM dexamethasone for 30 min, fixed, and processed for RNA FISH analysis using a probe specific for MMTV transcript. Arrows point to the localization of the MMTV transcript. DNA was stained by DAPI. Average integrated RNA FISH intensity from 35 randomly selected cells grown in the presence (−BRG1 K-R) or absence (+BRG1 K-R) of tet were measured as described in Materials and Methods and plotted as a bar histogram (N). Error bars, SE. siRNA-mediated depletion of BRG1 inhibits MMTV transcription (O–Q). 3134 cells with stably integrated MMTV-LTR array were transfected with a scrambled sequence (O) or with a siRNA pool targeted to BRG1 (P). Cells were treated with dexamethasone for 30 min, fixed, and processed for RNA FISH analysis using a probe specific for MMTV transcripts. Endogenous BRG1 was detected by indirect immunofluorescence using a BRG1-specific antibody. Arrows point to the localization of MMTV transcript. Average integrated RNA FISH intensity of 35 randomly selected cells from cells transfected with siRNAs to BRG1 or cells transfected with scrambled siRNAs were measured and plotted as a bar histogram (Q). Error bars, SE. Bar, 4 um.
Figure 3.
Figure 3.
Transcription from the MMTV-LTR is regulated by the ATP-hydrolysis activity of BRG1 or BRM. 3134 mouse mammary adenocarcinoma cells with a stably integrated MMTV-LTR array were transfected with Flag-tagged forms of wild-type BRG1, BRM, and mutant BRG1-K-R, BRM-K-R (A–H). Cells were treated with 100 nM dexamethasone for 30 min and fixed. BRG1, BRG1-K-R, wild-type BRM, and BRM-K-R were detected by indirect immunofluorescence using an anti-Flag antibody and MMTV RNA was detected by RNA FISH using a probe specific to the MMTV transcript. Arrows (A, C, E, and G) point to the localization of MMTV transcript. Average integrated RNA FISH intensities from 35 randomly selected cells in each transfected category (A, C, E, and G) and untransfected category (B, D, F, and H) were measured as described in Materials and Methods and plotted as a bar histogram (I). Error bars, SE. (J–N) A stably expressed dominant negative form of BRG1 (BRG1-K-R) inhibits MMTV transcription. The 5555 mouse mammary adenocarcinoma cells with stably integrated MMTV-LTR array express a Flag-tagged dominant negative form of BRG1 (BRG1-K-R) under the control of a tetracycline-repressible system (J). 5555 cells were grown in the presence or absence of tetracycline to facilitate the expression of BRG1-K-R (K). Western blot analysis was performed using an anti-Flag antibody. 5555 cells were grown in the presence (L) or absence (M) of tetracycline. Cells were treated with 100 nM dexamethasone for 30 min, fixed, and processed for RNA FISH analysis using a probe specific for MMTV transcript. Arrows point to the localization of the MMTV transcript. DNA was stained by DAPI. Average integrated RNA FISH intensity from 35 randomly selected cells grown in the presence (−BRG1 K-R) or absence (+BRG1 K-R) of tet were measured as described in Materials and Methods and plotted as a bar histogram (N). Error bars, SE. siRNA-mediated depletion of BRG1 inhibits MMTV transcription (O–Q). 3134 cells with stably integrated MMTV-LTR array were transfected with a scrambled sequence (O) or with a siRNA pool targeted to BRG1 (P). Cells were treated with dexamethasone for 30 min, fixed, and processed for RNA FISH analysis using a probe specific for MMTV transcripts. Endogenous BRG1 was detected by indirect immunofluorescence using a BRG1-specific antibody. Arrows point to the localization of MMTV transcript. Average integrated RNA FISH intensity of 35 randomly selected cells from cells transfected with siRNAs to BRG1 or cells transfected with scrambled siRNAs were measured and plotted as a bar histogram (Q). Error bars, SE. Bar, 4 um.
Figure 4.
Figure 4.
BRG1 is required for the hormone-dependent remodeling of MMTV chromatin and associated loading of RNA pol II at the MMTV promoter. 5555 and 3617 cells were untreated or treated with 100 nM dexamethasone for 30 min and tetracycline as indicated. Nuclei were isolated and digested with SacI and DpnII restriction enzymes. Digestion products were detected by linear amplification using a radiolabeled primer specific to the MMTV promoter region. Percent cleavage and the fractional change in the accessibility of MMTV promoter to restriction enzymes are indicators of nuclease hypersensitivity and chromatin remodeling of MMTV promoter region. (A) Intensity of SacI digestion product is divided by the sum of the intensities of SacI and DpnII digestion products and presented as percent cleavage at the bottom of each lane. (B) Bar graph shows dexamethasone- or dexamethasone-, GR-, and BRG1-K-R– induced change in percent cleavage in SacI hypersensitivity between 5555 and 3617 cells from A. (C) Expression of BRG1-K-R reduced the loading of RNA pol II to MMTV promoter. 5555 and 3617 cells were untreated or treated with 100 nM dexamethasone for 30 min and tetracycline as indicated. Chromatin was immunoprecipitated using an antibody specific for RNA pol II or no antibody (control). Immunoprecipitated and input DNA were amplified using primers specific to the MMTV promoter. (D) Expression of BRG1-K-R reduced the loading of RNA pol II to the dexamethasone-induced Rgs2 locus. 5555 cells were untreated or treated with 100 nM dexamethasone for 30 min and tetracycline as indicated. cDNA was prepared from RNAs isolated from the indicated conditions. Chromatin was immunoprecipitated using an antibody specific for RNA pol II or no antibody (control). Immunoprecipitated and input DNA or cDNA were amplified using primers specific to the Rgs2 coding region.
Figure 5.
Figure 5.
Large-scale chromatin decondensation is regulated by BRG1 and BRM chromatin-remodeling complexes. 3134 cells were transfected with Flag-tagged forms of wild-type BRG1 (A and B), mutant BRG1-K-R (C), wild-type BRM (D), and mutant BRM-K-R (E). Cells were treated with 100 nM dexamethasone for 90 min, fixed, and processed for DNA FISH analysis. BRG1, BRG1-K-R, BRM, and BRM-K-R were detected by indirect immunofluorescence using an anti-Flag antibody and MMTV DNA was detected by DNA FISH using a probe specific to the entire MMTV-LTR array. The average DNA FISH signal areas obtained from 35 randomly selected cells in the transfected (A–E) and untransfected (F–J) populations were measured and plotted as a bar histogram (K). Error bars, SE. The inset rectangle shows an enlarged image of the DNA FISH signal. Expression of BRG1-K-R and BRM-K-R prevent hormone-induced decondensation of MMTV chromatin. Bar, 4 um.
Figure 6.
Figure 6.
Chromatin remodeling complexes have distinct kinetic properties and dynamically associate with MMTV-LTR array in a ligand and ATPase-dependent manner. (A–C) Qualitative FRAP analysis of BRG1, mutant BRG1-K-R and BRM in 1365.1 cells. 1365.1 mouse fibroblast cells were transfected with YFP-BRG1, YFP-BRG1-K-R, or GFP-BRM and treated with 100 nM dexamethasone for 30 min. BRG1 (A), BRM (B), or BRG1-K-R (C) bound to the MMTV-LTR array was imaged before and during recovery after photobleaching of the array for 120 ms. Images were acquired at the indicated times after the end of the bleach pulse. The MMTV-LTR array and the area of the bleached region is indicated by a red rectangle and shown as an enlarged pseudocolor image in the bottom panels. (D) Quantitative FRAP analysis of YFP-BRG1 or GFP-BRM in the nucleoplasm (control) or bound to the MMTV-LTR array after treatment with dexamethasone for 30 min (Dex). BRG1 and BRM bound to the MMTV-LTR array showed slower recovery kinetics after ligand treatment. (E) Quantitative FRAP analysis of YFP-BRG1, YFP-BRG1-K-R, or GFP-BRM bound to the MMTV-LTR array after treatment with dexamethasone for 30 min. The recovery kinetics of mutant BRG1-K-R bound to the MMTV array was slower than the wild-type BRG1 or wild-type BRM bound to MMTV-LTR array. All quantitative data values in the FRAP studies represent averages ± SE from at least 25 cells imaged in three independent experiments. Bar, 4 um.
Figure 6.
Figure 6.
Chromatin remodeling complexes have distinct kinetic properties and dynamically associate with MMTV-LTR array in a ligand and ATPase-dependent manner. (A–C) Qualitative FRAP analysis of BRG1, mutant BRG1-K-R and BRM in 1365.1 cells. 1365.1 mouse fibroblast cells were transfected with YFP-BRG1, YFP-BRG1-K-R, or GFP-BRM and treated with 100 nM dexamethasone for 30 min. BRG1 (A), BRM (B), or BRG1-K-R (C) bound to the MMTV-LTR array was imaged before and during recovery after photobleaching of the array for 120 ms. Images were acquired at the indicated times after the end of the bleach pulse. The MMTV-LTR array and the area of the bleached region is indicated by a red rectangle and shown as an enlarged pseudocolor image in the bottom panels. (D) Quantitative FRAP analysis of YFP-BRG1 or GFP-BRM in the nucleoplasm (control) or bound to the MMTV-LTR array after treatment with dexamethasone for 30 min (Dex). BRG1 and BRM bound to the MMTV-LTR array showed slower recovery kinetics after ligand treatment. (E) Quantitative FRAP analysis of YFP-BRG1, YFP-BRG1-K-R, or GFP-BRM bound to the MMTV-LTR array after treatment with dexamethasone for 30 min. The recovery kinetics of mutant BRG1-K-R bound to the MMTV array was slower than the wild-type BRG1 or wild-type BRM bound to MMTV-LTR array. All quantitative data values in the FRAP studies represent averages ± SE from at least 25 cells imaged in three independent experiments. Bar, 4 um.
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