Genome-wide impact of the BRG1 SWI/SNF chromatin remodeler on the transforming growth factor beta transcriptional program

Abstract

The transcription factors Smad2 and Smad3 mediate a large set of gene responses induced by the cytokine transforming growth factor beta (TGFbeta), but the extent to which their function depends on chromatin remodeling remains to be defined. We observed interactions between these two Smads and BRG1, BAF250b, BAF170, and BAF155, which are core components of the SWI/SNF chromatin-remodeling complex. Smad2 and Smad3 have similar affinity for these components in vitro, and their interactions are primarily mediated by BRG1. In vivo, however, BRG1 predominantly interacts with Smad3, and this interaction is enhanced by TGFbeta stimulation. Our results suggest that BRG1 is incorporated into transcriptional complexes that are formed by activated Smads in the nucleus, on target promoters. Using BRG1-deficient cell systems, we defined the BRG1 dependence of the TGFbeta transcriptional program genome-wide. Most TGFbeta gene responses in human epithelial cells are dependent on BRG1 function. Remarkably, BRG1 is not required for the TGFbeta-mediated induction of SMAD7 and SNON, which encode key mediators of negative feedback in this pathway. Our results provide a genome-wide scope of the participation of BRG1 in TGFbeta action and suggest a widespread yet differential involvement of BRG1 SWI/SNF remodeler in the transcriptional response of many genes to this cytokine.

FIGURE 1. Interaction of Smad2 and Smad3 with a BRG1 SWI/SNF complex

A, HeLa cell extracts were subjected to affinity purification with the indicated baits. Eluted proteins were resolved on SDS-PAGE and stained with Coomassie Blue. Bands were excised and identified by mass spectrometry analysis. Red letters indicate SWI/SNF components. B, the interaction between BRG1 and Smad2/3 was verified by immunoprecipitation (IP) of TGFβ-treated or untreated HaCaT cells with anti-Smad2/3 antibody or rabbit IgG, followed by Western immunoblotting (IB) with anti-BRG1 or anti-smad2/3 antibodies. C and D, HEK293 cells were transfected with vectors encoding FLAG-tagged BRG1 or FLAG-tagged BAF170, and HA-tagged Smad proteins, in the presence of either constitutive active (C) TGFβ type I receptor (HA-TβRI(AAD) construct) or kinase-dead (D) receptor (HA-TβRI(KR) construct), as shown. Whole cell lysates were immunoprecipitated and subsequently immunoblotted with antibodies as shown. Protein expression was monitored by immunoblot analysis of total cell extracts (input). E, scheme of the Smad3 and BRG1 domain structures; brackets indicate the interacting domains.

FIGURE 2. Differential sensitivity of TGFβ gene responses to BRG1 depletion

A and B, BRG1 was depleted in HaCaT cells using a combination of two siRNA oligonucleotides targeting BRG1, as shown by immunoblot analysis (inset). Control and BRG1 knockdown HaCaT cells were incubated with 100 pM TGFβ for 2 h, and then the total RNA was analyzed using Affymetrix HG-U133-plus 2.0 GeneChip. Blue bars depict the fold change in the mRNA signal of 97 genes whose expression in control cells was increased or decreased by at least 2-fold (p < 0.05) in response to TGFβ. The fold change of the same genes in BRG1-depleted cells is indicated with red bars. C, all 97 genes were ranked according to the percentage of reduction of their response to TGFβ in BRG1-depleted cells. The genes whose response was verified by qRT-PCR are labeled in red. IB, immunoblotting.

FIGURE 3. BRG1 dependence of selected TGFβ gene responses

A and B, control (-) and BRG1 knockdown HaCaT cells with two independent siRNAs (bars 1 and 2) were incubated with TGFβ for 2 h. Total RNA was subjected to qRT-PCR with appropriate primers. C, for each gene, the fold change in mRNA levels in response to TGFβ was calculated based on data in A and B. The data are the mean values ± S.D. from at least three independent experiments.

FIGURE 4. BRG1-dependent gene responses in stable BRG1 knockdown cells

Stable BRG1 knockdown HaCaT cells were constructed using two different shRNAs. Knockdown and control cells were treated with TGFβ for different lengths of time. A, BRG1, phospho-Smad2, Smad2, and Smad3 protein levels in control and BRG1 knockdown HaCaT cells were detected by immunoblotting (IB) with the indicated antibodies. B, BRG1 levels after 7 h of treatment with TGFβ as determined by Western immunoblotting. Smad2/3 was used as loading control. C, the interaction between BRG1 or BAF170 and Smad2/3 was tested by immunoprecipitation (IP) of TGFβ-treated HaCaT control or BRG1 knockdown cells with anti-Smad2/3 antibodies or rabbit IgG, followed by Western immunoblotting (IB) with anti-BRG1, anti-BAF170, or anti-Smad2/3 antibodies. D, mRNA levels of selected genes at different time points after TGFβ treatment were measured by qRT-PCR. The fold change in mRNA is calculated and plotted against the indicated time course. The data are the mean values ± S.D. of triplicate determinations. The experiments were repeated at least twice, with similar results each time.

FIGURE 5. Differential recruitment of BRG1 to TGFβ/Smad target gene promoters

A, HaCaT cells were left untreated (-) or treated (+) with TGFβ for 2 h, and ChIP assays were performed with the indicated antibodies and PCR primers specific for the promoter regions of the indicated target genes, and β-actin as a control. B, HaCaT control (ctr) and stable BRG1 knockdown (KD) cells were subjected to assays as described under A. Acetyl H4, acetylated histone 4; H3, histone 3; POLII, RNA polymerase II.

FIGURE 6. Rescue of TGFβ gene responses in lung cancer cells with restored BRG1 expression

A, NCI-H522 cells were infected with retrovirus encoding TβR-II or control virus. The infected cells were selected, and the rescue of TGFβ-induced Smad2 phosphorylation was determined by Western immunoblotting (IB) using the indicated antibodies. B, NCI-H522 cells where transduced with a TβRII retrovirus vector alone or this vector and BRG1 vector. BRG1 mRNA expression and its lack of response to TGFβ were verified by qRT-PCR. C, proliferation of NCI-H522, NCI-H522 (TβRII), NCI-H522 (BRG1), or NCI-H522 (TβRII/BRG1) was measured by counting cell numbers 3 days after culture with or without TGFβ addition. The data are the averages ± S.D. of triplicate determinations. D, NCI-H522 (empty vector), NCI-H522 (BRG1), NCI-H522 (TβRII), and NCI-H522 (TβRII/BRG1) cells were incubated with or without 100 pM TGFβ for 2 h, and total RNA was subjected HG-U133-plus 2.0 microarray analysis. Upper panel, fold change in mRNA signal in response to TGFβ in the NCI-H522 (TβRII) and NCI-H522 (TβRII/BRG1) cell samples. Lower panel, ±TGFβ heat map of the signals corresponding to these genes in the three cell samples.

FIGURE 7. BRG1-dependent and -independent gene responses

A, the heat map plot shows 14 TGFβ gene responses in NCI-H522 (BRG1/TβRII) cells that are shared in other human epithelial cell lines; cell lines responsive to TGFβ responses in cells shared with HaCaT, HPL1, MDA-MB-231, and MCF10A cells. B, NCI-H522 cells transduced with the indicated vectors were incubated with or without TGFβ for 2 h, and the expression level of six genes of interest was analyzed by qRT-PCR. The data are the averages ± S.D. of triplicate determinations.