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Chromatin remodeling is an active process, which represses or enables the access of transcription machinery to genes in response to external stimuli, including hypoxia. However, in hypoxia, the specific requirement, as well as the molecular mechanism by which the chromatin-remodeling complexes regulate gene expression, remains unclear. In this study, we report that the Brahma (BRM) and Brahma-related gene 1 (BRG1) ATPase-containing SWI/SNF chromatin-remodeling complexes promote the expression of the hypoxia-inducible transcription factor 1α (HIF1α) and HIF2α genes and also promote hypoxic induction of a subset of HIF1 and HIF2 target genes. We show that BRG1 or BRM knockdown in Hep3B and RCC4T cells reduces hypoxic induction of HIF target genes, while reexpression of BRG1 or BRM in BRG1/BRM-deficient SW13 cells increases HIF target gene activation. Mechanistically, HIF1 and HIF2 increase the hypoxic induction of HIF target genes by recruiting BRG1 complexes to HIF target gene promoters, which promotes nucleosome remodeling of HIF target gene promoters in a BRG1 ATPase-dependent manner. Importantly, we found that the function of BRG1 complexes in hypoxic SW13 and RCC4T cells is dictated by the HIF-mediated hypoxia response and could be opposite from their function in normoxic SW13 and RCC4T cells.
Hypoxia (Hx) is a common characteristic of many solid tumors. The Hx intratumoral microenvironment stabilizes hypoxia-inducible transcription factor 1α (HIF1α) and HIF2α, which are normally degraded under normoxia (Nx). The stabilized HIF1α and HIF2α proteins translocate to the nucleus, where they dimerize with the constitutive nuclear protein ARNT (the aryl hydrocarbon receptor nuclear translocator, also called HIF1β) to form HIF1α/ARNT (HIF1) and HIF2α/ARNT (HIF2) heterodimers. HIF1 and HIF2 bind to HIF binding sites (HBS) on HIF target gene promoters and/or enhancers and transactivate genes involved in neovascularization, glycolysis, cellular proliferation, and metastasis. Thus, the HIF-mediated Hx transcriptional response is critical for tumor progression by allowing cancer cells to adapt to a low-oxygen environment (1–4). However, recent reports indicate that the HIF2- and particularly the HIF1-mediated Hx response can activate tumor-suppressive genes, such as Scgb3a, Bnip3, Bnip3L, Nix, MYC inhibitor Mxi, p21, and p27, in a cell-type-specific manner (5–10).
It is well established that multiple transcription factors (TFs) are required to achieve maximal activation of target genes in response to a specific stimulus such as Hx. This multifactorial transcription complex has been termed the “enhanceosome.” Individual TFs in the enhanceosome complex may promote transcription by recruiting RNA polymerase II/general TFs and/or by recruiting chromatin-modifying enzymes such as histone acetylase, CBP and p300, and the chromatin-remodeling SWI/SNF complex. In the context of the enhanceosome associated with the Hx response, two additional TFs, STAT3 and USF2, are required for maximal Hx response (11, 12). STAT3 and USF2 function in the Hx response by recruiting CBP and p300 to the HIF1 and HIF2 target genes, respectively (11, 12). Despite several reports indicating that HIFs activate their target genes by recruiting CBP and p300 (13–15), our recent studies, using chromatin immunoprecipitation, did not find a significant role for HIF in recruiting CBP and p300 to HIF target genes (11, 12). Therefore, our recent findings lead to an important question in the field of Hx: how does HIF activate target gene transcription in Hx cells?
SWI/SNF complexes regulate transcription through nucleosome disruption and reconstruction in an ATP-dependent manner. SWI/SNF complexes can be divided into the BAF complexes, containing the Brahma-related gene 1 (BRG1) or Brahma (BRM) ATPase, and the PBAF complexes, containing the BRG1 ATPase (16–19). BAF and PBAF complexes share nine protein subunits (beta-actin, BAF53, BAF60, BAF57, BAF155, BAF170, SNF5, BAF45, and BRG1) but can be distinguished by the unique protein subunit BAF250a or BAF250b present in the BAF complex, whereas PBAF contains BAF200, BAF180, and BRD7. While other subunits are important for the integrity of the SWI/SNF complex and its targeting to DNA, the BRG1 or BRM ATPase is thought to be absolutely required for the function of SWI/SNF; thus, a mutation or loss of expression of the BRG1 or BRM ATPase inactivates the whole complex (16–19).
We hypothesized that HIF recruits SWI/SNF complexes to HIF target genes, increasing Hx induction of HIF targets. In this study, we have probed the interplay between HIF and BRM/BRG1 SWI/SNF complexes. Our studies showed that there are SWI/SNF-dependent and -independent Hx-inducible genes. Functional studies indicated that the BRG1 complex amplifies HIF-mediated hypoxia responses. The role of the BRG1 complex in Hx cells is determined by the function of the HIF-mediated Hx response. These findings further our understanding of the role of BRG1 complexes in cancer cells under Hx.
Hep3B and Hep3B/ARNT short hairpin RNA (shRNA) cells were cultured in minimal essential medium with Earle's balanced salt solution (MEM/EBSS; HyClone) containing 10% fetal bovine serum (FBS), 2 mM l-glutamine, 1 mM sodium pyruvate, 100,000 U/liter penicillin-streptomycin, 1.5 g/liter sodium bicarbonate, and 1× nonessential amino acids (NEAA). SW13, RCC4, RCC4T, U2OS, and 293T cells were grown in high-glucose Dulbecco's modified Eagle medium (DMEM; HyClone) with 10% FBS, 2 mM l-glutamine, 100,000 U/liter penicillin-streptomycin, and 1× NEAA. Prior to Hx treatment, 25 mM HEPES was added to the growth medium, and cells were incubated under Nx (21% O2) or Hx (1.5% O2) for 12 to 16 h.
Control mRNA (1027281; Qiagen) or small interfering RNAs (siRNAs) specific for human BRM (L-017253-00; Dharmacon) or BRG1 (L-010431-00; Dharmacon) mRNA were transfected into Hep3B cells at 50% confluence by using HiPerFect transfection reagent (Qiagen) according to the manufacturer's protocol. Thirty-two hours posttransfection, cells were cultured at 21% or 1.5% O2 for 12 to 16 h and were then collected for analysis of mRNA or protein. To stably knock down BRM or BRG1 mRNA, Hep3B or RCC4T cells were transduced with pLKO.1 lentiviruses expressing shRNAs targeting mRNA of either BRM (TRCN0000020329 or TRCN0000020332; Open Biosystems), BRG1 (TRCN0000015549 or TRCN0000015550; Open Biosystems), or both, and transduced cells were selected by puromycin treatment. To stably knock down HIF1α or HIF2α in SW13 or RCC4T cells, the cells were transduced with pLKO.1 lentiviruses expressing shRNAs targeting mRNA of HIF1α (TRCN000003810; Open Biosystems) or HIF2α (TRCN000003806; Open Biosystems), and transduced cells were selected by puromycin treatment. The specificity of the HIF1α or HIF2α shRNA was tested previously (11, 12).
The pBJ5 human BRG1 (hBRG1) (Addgene plasmid 17873, deposited by Jerry Crabtree) (20) and pBABE hBRM (Addgene plasmid 1961, deposited by Robert Kingston) (21) constructs were purchased from Addgene. The pBJ5 hBRG1 plasmid was used as the template for the generation of an ATPase-dead BRG1 construct by PCR-mediated mutation of amino acid lysine 785 to arginine (K785R) and amino acid threonine 786 to serine (T786S). These constructs were used for transient-transfection experiments in SW13 cells using Lipofectamine reagent (18324-012; Invitrogen) and Plus reagent (11514-015; Invitrogen). Typically, 2 × 105 cells per well in 6-well plates were transfected with 1 μg of either pBABE hBRM or pBJ5 hBRG1 (wild type [WT] or ATPase dead) or with 500 ng each of pBABE hBRM and pBJ5 hBRG1. Thirty-two hours after transfection, cells were placed under Nx or Hx for 16 h, and protein and mRNA were collected for analyses. The expression plasmids of mouse HIF1α (mHIF1α) or mouse HIF2α under the control of the elongation factor 1 (EF1) promoter have been described elsewhere (22). Hep3B/mHIF1α or Hep3B/mHIF2α cells were generated by transfection of EF1/mHIF1α or EF1/mHIF2α DNA and hygromycin selection.
Whole-cell lysates were prepared, and protein concentrations were determined. Western blot analysis was performed using standard protocols with the following primary antibodies: an anti-HIF1α monoclonal antibody (MAb) (610959, detecting human HIF1α protein; BD Bioscience), an anti-HIF1α polyclonal antibody (pAb) (NB 100-134, detecting both human and mouse HIF1α protein; Novus Biologicals), an anti-HIF2α MAb (D9E3, detecting both human and mouse HIF2α protein; Cell Signaling), an anti-ARNT MAb (NB 100-124; Novus Biologicals), an anti-BRM pAb (SC-6450; Santa Cruz), an anti-BRG1 pAb (SC-10768; Santa Cruz), an anti-Flag MAb (F3165; Sigma), and an antiactin pAb (SC-1616; Santa Cruz). Densitometric analysis was performed using ImageJ software (freely available at http://rsb.info.nih.gov/ij/).
RNA was isolated from cells using the RNeasy Plus minikit (Qiagen) and was then reverse transcribed using the iScript Advanced cDNA synthesis kit (Bio-Rad). mRNA levels were quantified by reverse transcription-quantitative PCR (RT-qPCR) using iQ Sybr green supermix (Bio-Rad) in triplicate on the CFX384 real-time system (Bio-Rad). All primer sets designed to measure mRNA levels or used in chromatin immunoprecipitation (ChIP) were validated for their specificity and amplification efficiency (85% to 110%) using melt curve analysis, RT-qPCR product sequencing, and standard dilution analysis. RT-qPCR results were normalized using the ΔΔCT method; 18S rRNA and β-actin were used as reference genes, and untreated Nx samples were used as controls. At least three independent experiments were performed to generate the results presented in the figures.
ChIP assays were performed as described previously (12). An anti-ARNT antibody (NB 100-110; Novus Biologicals) and an anti-BRG1 pAb (SC-10768; Santa Cruz) were used for protein-DNA complex precipitation; rabbit preimmune serum served as a control. DNA from input or immunoprecipitated samples was assayed using Sybr green-based qPCR methods with specific primers designed to amplify the CA9, EPO, HIF1α, or HIF2α promoters or with primers for the exons/introns as nonspecific controls as described previously (12).
The BRG1 cDNA in pBJ5 hBRG1 was PCR amplified and inserted into the pcDNA3.1 Flag vector, generating a BRG1 expression vector with a Flag tag at the C terminus of the BRG1 protein. HEK293T cells were cotransfected either with BRG1-Flag, mouse HIF1α, or mouse HIF2α constructs alone or with BRG1 plus mHIF1 or BRG1 plus mHIF2α and were placed under Hx for 16 h for analysis of BRG1-HIFα interactions. Cleared cell lysates were incubated with M2–anti-Flag–agarose beads (Sigma) to pull down Flag-tagged BRG1. The lysate samples and eluents from the beads were then assayed for protein expression and precipitation or coprecipitation.
Hep3B cells or SW13 cells transfected with an empty vector or a vector expressing WT BRG1 or ATPase-dead BRG1 in 10-cm dishes (~60% confluence) were cultured under Nx or Hx for 16 h. Nuclei were isolated using the EZ Nucleosomal DNA Prep kit (catalog no. D5220; Zymo Research) and were treated with 0.5 U of micrococcal nuclease (MNase) for 10 min at room temperature. Following MNase incubation, digestion was stopped using 5× MN Stop buffer, and nucleosomal DNA was purified and precipitated according to the manufacturer's instructions. Following purification, nucleosomal DNA was used for Sybr green-based qPCR to determine nucleosome positioning on the CA9 and LDHA promoters. Overlapping qPCR primer sets were designed from positions +407 to −836 relative to the transcription start site for the CA9 promoter and +36 to −1070 relative to the transcription start site for the LDHA promoter to generate amplicons of 150 bp, the size of DNA associated with one nucleosome. All primer sets designed to measure nucleosome positioning were validated for specificity and amplification efficiency (80% to 120%) using melt curve analysis and standard dilution analysis. qPCR results were normalized using the ΔCT method. For CA9 in Hep3B cells, a region spanning positions +273 to +407 was used as an internal control, since this region did not differ between Nx and Hx cells. For CA9 in SW13 cells, a region spanning positions −214 to −87 was used as an internal control, since this region did not differ between Nx and Hx cells or between cells transfected with BRG1-Flag and those transfected with mBRG1-Flag. For LDHA, a region spanning positions +20 to +117 was used as an internal control. Three independent tests were performed in triplicate for each experiment, and results for representative samples are presented.
Typically, HEK293T cells were grown to ~50% confluence in 6-well plates and were transfected with 400 ng of a luciferase reporter driven by either the HIF1α or the HIF2α promoter, 200 ng β-galactosidase (β-Gal), 300 ng triple-mutation HIF1α (HIF1αTM), 300 ng of STAT3, and 300 ng of BRG1 (pBJ5 hBRG1) or no BRG1 by using the Fugene transfection reagent. Thirty-six hours after transfection, cells were collected into 400 μl 1× reporter lysis buffer (Promega) and were assayed for β-Gal activity and luciferase activity using a luminometer. Promoter activation by HIF1, STAT3, and BRG1 was corrected for β-Gal transfection efficiency and is presented as fold induction relative to promoter activities from an empty-vector control. Results are averages for three experiments.
For proliferation assays, 20,000 to 40,000 cells/well were plated in growth medium in 24-well plates. The following day, the cell medium was replaced with complete medium containing 25 mM HEPES and was placed under Nx or Hx. The medium was changed every 2 days, and every 24 h, 3 wells of cells per treatment were collected by trypsinization and were counted by hemacytometer to yield an average number of cells/day. For scratch assays, cells were plated at 80% confluence in normal growth medium in duplicate in 6-well plates. The next day, when the cells formed a monolayer, the monolayers were scratched with a pipette tip. Then the cells were washed with phosphate-buffered saline (PBS) twice to remove detached cells and serum. The cells were then incubated under Nx or Hx for the remainder of the experiment in serum-free medium containing HEPES (25 mM). Photographs of the scratches at the same location in the well were taken daily, and the width of the scratch was measured using ImageJ software. Duplicate measurements were averaged, and the average measurements were graphed as the percentage of closure of the scratch compared to the initial scratch (day zero). For clonogenic survival assays, 1,000 cells/well were plated in duplicate in 6-well plates in normal growth medium. The medium was changed every 2 days. Then, 6 days after plating, cells were washed, and colonies were stained with crystal violet dye and were counted.
One-way analysis of variance was performed unless otherwise stated. Error bars in figures indicate ±1 standard deviation. Asterisks indicate statistical significance as follows: *, P < 0.05; **, P < 0.01. Controls for statistical analysis are specified in each figure. All experiments were performed at least three separate times.
Due to their high levels of HIF1α and HIF2α protein and high levels of induction of HIF target genes, Hep3B cells are often used in Hx studies (23–25). To determine the role of SWI/SNF chromatin-remodeling complexes in the Hx response, Hep3B cells were transfected either with a nontargeting control siRNA or with siRNAs against BRM, BRG1, or both BRM and BRG1. BRM and BRG1 siRNAs significantly reduced the mRNA (Fig. 1A) and protein (Fig. 1B) levels of BRM and BRG1, respectively, in Nx and Hx Hep3B cells. Interestingly, knockdown of BRG1 consistently reduced the Hx induction of HIF1 target genes CA9 and ANGPTL4 (Fig. 1C) and HIF2 target genes EPO and PTPRB (Fig. 1D), while knockdown of BRM reduced the level of EPO induction but not that of CA9, ANGPTL4, or PTPRB (Fig. 1C and andD).D). Simultaneous knockdown of BRG1 and BRM did not further reduce the level of HIF target gene induction. Importantly, the mRNA and protein levels of HIF1α, HIF2α, and ARNT were not affected by transient knockdown of BRM, BRG1, or both BRM and BRG1 (Fig. 1A and andB).B). These data suggested that both the BRG1 and the BRM complex are involved in the Hx response in Hep3B cells. However, the BRG1 complex appears to have a broader and more important role than the BRM complex in the Hx response in Hep3B cells.
Transient BRG1 or BRM knockdown using siRNA may be insufficient to remove premade BRG1/BRM proteins in SWI/SNF complexes that are already associated with their target gene promoters. Thus, we stably reduced the levels of BRG1 or BRM mRNA (Fig. 2A) and protein (Fig. 2B) in Hep3B cells by using shRNAs. Compared to that for Hep3B cells with shRNA against green fluorescent protein (Hep3B/GFP shRNA cells), Hep3B/BRG1 shRNA cells exhibited greatly reduced Hx induction of all HIF target genes analyzed, including CA9, ANGPTL4, GLUT1, PGK1, EPO, PAI1, PTPRB, ADM, and VEGF (Fig. 2C and andD;D; also data not shown), while Hep3B/BRM shRNA cells exhibited slightly reduced induction of CA9 and EPO but not of ANGPTL4 and PTPRB (Fig. 2C and andD).D). The reduced Hx induction of HIF target genes in Hep3B/BRG1 shRNA cells was likely due to reduced mRNA and protein levels of HIF1α and HIF2α (Fig. 2A and andB),B), suggesting an important role of the BRG1 complex in maintaining HIF1α and HIF2α gene expression in Hep3B cells.
Since the BRG1 complex is more important than the BRM complex in HIFα (Fig. 2) and HIF target gene (Fig. 1) regulation in Hep3B cells, we focused specifically on BRG1. To determine the function of BRG1 in HIF target gene induction, we established Hep3B cells that have higher levels of HIFα protein, but lower levels of BRG1 protein, than wild-type Hep3B cells. First, we stably transfected Hep3B cells with wild-type mouse HIF1α or HIF2α cDNAs under the control of the human elongation factor 1 (EF1) promoter. We used mouse HIF1α or HIF2α cDNA, not human HIF cDNA, because mouse and human HIFα proteins are functionally interchangeable and there are antibodies that specifically recognize the human HIF1α proteins. Hep3B/mHIF1α and Hep3B/mHIF2α cells expressed 3- to 6-fold more (endogenous and transfected) HIF1α or HIF2α mRNA than parental Hep3B cells under Nx but 10-fold more under Hx (Fig. 3A). The increased levels of mouse HIF1α or HIF2α mRNA under Hx were due to increased expression of the mHIFα gene, which is enhanced by the PGK1 promoter (an Hx-induced promoter), located upstream of the EF1 promoter in the vector (Fig. 3A). Relative to the parental Hep3B cells, Hep3B/mHIF1α cells exhibited 2- to 3-fold higher induction of HIF1 target genes but 20 to 30% reduced induction of HIF2 target genes, while Hep3B/mHIF2α cells exhibited 2- to 3-fold higher induction of HIF2 target genes but 20 to 30% reduced induction of HIF1 target genes (Fig. 3B and andCC).
Then BRG1 was stably knocked down in Hep3B/mHIF1α or Hep3B/mHIF2α cells (Fig. 4A and andB).B). In agreement with the results presented in Fig. 2A and andB,B, stable knockdown of BRG1 significantly reduced endogenous HIF1α and HIF2α mRNA levels compared to those in Hep3B/mHIF1α or Hep3B/mHIF2α cells, as detected by qPCR primers located in the 3′ untranslated region (3′ UTR) of human HIFα mRNA (Fig. 3D). Interestingly, Hep3B/mHIF1α/BRG1 shRNA or Hep3B/mHIF2α/BRG1 shRNA cells exhibited levels of mouse HIF1α or mouse HIF2α mRNA similar to those in Hep3B/mHIF1α or Hep3B/mHIF2α cells (Fig. 3D), as detected by qPCR primers located in the 3′ UTR (from the vector) of the mouse HIF mRNAs. In agreement with the mRNA analysis, endogenous human HIF1α protein levels in Hx Hep3B/mHIF1α/BRG1 shRNA and Hep3B/HIF2α/BRG1 shRNA cells were significantly reduced, as detected by an antibody specifically recognizing endogenous human HIF1α protein (Fig. 4B, anti-HIF1α human). However, the Hx Hep3B/mHIF1α/BRG1 shRNA or Hep3B/HIF2α/BRG1 shRNA cells expressed higher levels of total HIF1α or HIF2α protein, respectively, as detected by an antibody that recognized total HIFα protein (Fig. 4B, anti-HIF1α human/mouse or anti-HIF2α human/mouse). Since Hep3B/GFP shRNA cells express levels of HIF1α and HIF2α proteins lower than those of Hep3B/mHIF1α/BRG1 shRNA and Hep3B/mHIF2α/BRG1 shRNA cells, respectively, the Hep3B/GFP shRNA cells, not the Hep3B/mHIF1α or Hep3B/mHIF2α cells, were used as controls in the subsequent HIF target gene studies. Interestingly, Hx induction of HIF1 target genes CA9 and ENO2 in Hep3B/mHIF1α/BRG1 shRNA cells (Fig. 4C) and of HIF2 target genes EPO and PTPRB in Hep3B/mHIF2α/BRG1 shRNA cells (Fig. 4D) were still significantly reduced, demonstrating the functional importance of the BRG1 complex in HIF target gene activation in Hep3B cells.
To further investigate the role of the BRG1 and BRM complexes in the Hx response, we transiently transfected plasmids expressing BRM, BRG1, or both into SW13 human adrenal adenocarcinoma cells, a cell line that is deficient in BRM and BRG1 (26), as confirmed by our Western blot analysis (Fig. 5A). Reexpression of BRM and particularly BRG1 significantly increased HIF2α mRNA levels (Fig. 5C) in Nx and Hx SW13 cells and HIF2α protein levels in Hx SW13 cells (Fig. 5B). However, BRG1 and BRM complexes did not regulate HIF1α and ARNT expression in SW13 cells (Fig. 5B and andCC).
Although no HIFα protein was detected in Nx SW13/BRM or SW13/BRG1 cells, these cells exhibited increased expression of HIF1 target genes CA9 and ANGPTL4 (Fig. 5D) and the HIF2 target gene PAI1 under Nx (Fig. 5E), demonstrating a significant function of BRG1 and BRM complexes in regulating the basal expression of HIF target genes. Furthermore, Hx SW13/BRG1 or SW13/BRM cells also exhibited significantly increased induction of HIF1 targets CA9 and ANGPTL4 (Fig. 5D) despite normal mRNA and protein levels of HIF1α and ARNT (Fig. 5B and andC),C), supporting a role for BRG1 and BRM complexes in activating HIF1 target genes. Due to increased expression of HIF2α protein in SW13/BRG1 or SW13/BRM cells, we were not able to conclude whether increased expression of the HIF2 target gene PAI1 under Hx was a function of increased HIF2α protein levels or a function of the restored BRG1/BRM complex (Fig. 5E). To determine whether the ATPase activity of BRG1 is required for the expression of HIF target genes, we transfected SW13 cells with an ATPase-dead version of BRG1 (mBRG1) (Fig. 6). Although similar protein levels of WT BRG1 and mBRG1 were detected (Fig. 6A), WT BRG1 but not ATPase-dead BRG1 was able to increase the expression of HIF2α (Fig. 6C) and HIF target genes (Fig. 6D and andE).E). In summary, we determined that while the BRG1/BRM complex is not absolutely required for the HIF-mediated Hx response, BRG1 and BRM complexes promote HIF target gene transcription in Nx and Hx SW13 cells. In agreement with data from Hep3B cells, BRG1 appears to be more potent than BRM in enhancing the Hx response of SW13 cells.
To determine whether BRG1 binds to the promoters of HIF1α, HIF2α, and HIF target genes, chromatin immunoprecipitation (ChIP) was performed using antibodies against BRG1 or ARNT (positive control) in Nx or Hx Hep3B cells. As expected, the level of ARNT binding on the promoters of HIF target genes CA9 and EPO was significantly increased in Hx Hep3B cells (Fig. 7A and andB),B), while little ARNT binding was detected on the HIF1α gene in either Nx or Hx Hep3B cells (Fig. 7C). Surprisingly, we detected significant ARNT binding on the HIF2α promoter in Hx Hep3B cells (Fig. 7D), although HIF2α is not a HIF target gene in this cell type (Fig. 1A). Interestingly, we detected BRG1 binding on the promoters of HIF target genes CA9 (Fig. 7A) and EPO (Fig. 7B) and on the promoters of HIF2α and HIF1α (much less) in Nx Hep3B cells (Fig. 7C and andD),D), consistent with the fact that BRG1 activity is required for the expression of these genes in Nx Hep3B cells (Fig. 2). Interestingly, BRG1 binding on the promoters of CA9, EPO, and HIF2α (not HIF1α) was increased by Hx (Fig. 7A to toDD).
To assess whether increased BRG1 binding in Hx cells is HIF/ARNT dependent, similar ChIP assays were conducted in Nx and Hx Hep3B/ARNT shRNA cells. As expected, ARNT knockdown significantly reduced ARNT association on all four promoters in Hx Hep3B cells. Interestingly, BRG1 binding on the promoters of HIF target genes CA9 and EPO was significantly decreased in Hx Hep3B/ARNT shRNA cells (Fig. 7A and andB).B). However, ARNT knockdown did not change BRG1 binding on the HIF1α or HIF2α promoter (Fig. 7C and andD).D). To provide further evidence that HIF1 and HIF2 proteins recruit the BRG1 complex to HIF target gene promoters, we performed coimmunoprecipitation (co-IP) experiments in Hx HEK293T cells cotransfected with Flag-tagged BRG1 and wild-type mouse HIF1α or HIF2α expression plasmids. Pulldown of BRG1-Flag protein coprecipitated endogenous as well as transfected HIF1α protein (Fig. 7E, center). BRG1-Flag also coprecipitated transfected HIF2α protein (Fig. 7E, bottom). However, no endogenous HIF2α co-IP was observed, likely due to low levels of endogenous HIF2α expression in 293T cells. To further test the role of the BRG1 complex in activating HIF1α and HIF2α gene transcription, we performed HIF1α and HIF2α promoter reporter gene assays. Transfection of BRG1 alone did not change HIF1α or HIF2α promoter activity (data not shown). However, coexpression of BRG1 enhanced the ability of HIF1α and STAT3 to activate the HIF1α and HIF2α promoters (Fig. 7F). These data suggest that while there is basal binding of BRG1 on HIF target genes, Hx increases the binding of BRG1 to HIF target genes in an ARNT/HIF-dependent manner. Additionally, BRG1 also binds to and activates HIF1α and HIF2α promoters in an ARNT-independent manner.
Previous studies have suggested that the induction of primary response genes in lipopolysaccharide (LPS)-stimulated macrophages is BRG1 independent (27, 28). These primary response genes typically have CpG-rich promoters (27, 28). Thus, we selected a subset of HIF target genes with CpG island promoters to see if their induction requires BRG1/BRM activity. Since all these genes (GLUT1, LDHA, PDK1, and PGK1) are HIF1-specific targets (23, 25), we addressed this question in Hep3B/mHIF1α/BRG1 shRNA cells, a cell line that has high levels of HIF1α protein despite stable BRG1 knockdown (Fig. 4B). Although Hx induction of CA9 and ENO2 was markedly reduced in Hep3B/mHIF1/BRG1 shRNA cells (Fig. 4C), GLUT1, LDHA, PDK1, and PGK1 exhibited normal or higher than normal levels of induction (Fig. 8A). Additionally, Hx induction of these HIF1 target genes was not significantly enhanced by reexpression of BRM, BRG1, or both BRM and BRG1 in SW13 cells (Fig. 8B). Also, reduction of BRG1 activity in Hep3B cells and reconstitution of BRG1 or BRM in SW13 cells did not change the levels of these genes under Nx (Fig. 8A and andB).B). In agreement with the expression data, the protein levels of BRG1 on the PGK1 promoter (0.3% and 0.5% of input in Nx and Hx Hep3B cells, respectively) were much lower than those on the promoters of the BRG1-dependent genes CA9 and EPO (Fig. 7A and andB).B). Taken together, our data support the novel concept that HIF target genes can be divided into BRG1/BRM-dependent or -independent genes.
To better understand how BRG1 promotes HIF target gene transcription in Hx Hep3B cells and whether there is a nucleosomal binding difference between BRG1-dependent and BRG1-independent HIF targets, we performed a qPCR-based nucleosome scanning assay (NUSA) on the promoters of the BRG1-dependent HIF1 target gene CA9 (positions −836 to +345) and the BRG1-independent HIF1 target gene LDHA (positions −1070 to +36) in Nx and Hx Hep3B cells. We found that in Nx Hep3B cells, there was significant nucleosome association on the CA9 promoter in the area from −337 to +63, as judged by lower sensitivity to micrococcal nuclease (MNase) digestion (reflected by increased detection by qPCR), than in the area from −836 to −337, which is more sensitive to MNase (as reflected by decreased detection by qPCR) (Fig. 9A). However, Hx reduced nucleosome association on the CA9 promoter in the area from −337 to +63, an area important for RNA polymerase II and HIF/ARNT binding (the HBS is at −16) (29), as judged by increased sensitivity to MNase digestion (reflected by reduced detection by qPCR) (Fig. 9A). To test whether the BRG1 complex is responsible for the increased sensitivity of the CA9 promoter to MNase in Hx Hep3B cells, similar experiments were performed in Hep3B/mHIF1α/BRG1 shRNA cells. Interestingly, no MNase sensitivity difference was observed among different regions of the CA9 promoter in Nx cells (Fig. 9B) or between Nx and Hx Hep3B/mHIF1α/BRG1 shRNA cells (Fig. 9B), suggesting that BRG1 is responsible for the Hx-induced nucleosome remodeling observed on the CA9 promoter. Although Hx increased the MNase sensitivity in the region of the LDHA promoter from −31 to −214 (Fig. 9C), this change was BRG1 independent, since Hx-induced nucleosome remodeling of the LDHA promoter was also observed in Hep3B/mHIF1α/BRG1 shRNA cells (Fig. 9D). These results provide direct evidence that BRG1 regulates the chromatin remodeling of a BRG1-dependent HIF target gene, CA9, but has no role in the chromatin remodeling of a BRG1-independent HIF target gene, LDHA.
To test if the ATPase activity of BRG1 is required for nucleosome remodeling of the CA9 promoter, we performed NUSA experiments for the CA9 promoter in Nx and Hx SW13 cells transfected with an empty vector (control) or with a vector carrying WT or ATPase-dead BRG1. Interestingly, significant nucleosome remodeling of the CA9 promoter was observed in Hx SW13 cells expressing WT BRG1 (Fig. 10A) but not ATPase-dead BRG1 (Fig. 10B) in comparison with control Hx SW13 cells. These data are consistent with expression data showing that Hx induction of CA9 is increased by reexpression of WT but not ATPase-dead BRG1 (Fig. 6).
To determine the role of BRG1 in Nx and Hx cancer cells, SW13 cells were stably transduced with HIF1α, HIF2α, or scrambled (Scrm) shRNAs, followed by transient transfection with an empty vector or a WT BRG1 expression plasmid. HIF1α protein was similarly expressed in Hx SW13 cells transfected with an empty vector or a BRG1 expression vector, and its expression was significantly reduced by HIF1α shRNA (Fig. 11A). HIF2α protein was detected only in Hx SW13 cells transfected with a WT BRG1 expression vector, and its expression was reduced in Hx SW13/HIF2α shRNA cells carrying a BRG1 expression vector (SW13/HIF2α shRNA+BRG1 cells) (Fig. 11A). In agreement with the data shown in Fig. 5C, HIF2α mRNA levels were increased by the reintroduction of BRG1, whereas HIF1α and ARNT mRNA levels were not changed in these cells (Fig. 11B). As expected, Hx induction of CA9 and ANGPTL4 was significantly reduced in SW13/HIF1α shRNA cells, but not in SW13/HIF2α shRNA cells (Fig. 11C), since these cells express little HIF2α mRNA (Fig. 11B). Relative to SW13/Scrm+Ctrl cells, Nx and Hx SW13/Scrm shRNA+BRG1 cells exhibited significantly increased CA9 and ANGPTL4 expression. However, relative to SW13/Scrm shRNA+BRG1 cells, SW13/HIF1α shRNA+BRG1 cells exhibited reduced CA9 and ANGPTL4 expression under Hx but not under Nx. The levels of CA9 and ANGPTL4 in Nx and Hx SW13/HIF2α shRNA+BRG1 cells were also similar to those in SW13/Scrm shRNA+BRG1 cells, indicating that these genes are activated primarily by HIF1 and not by HIF2.
We then performed a proliferation assay for these cells (Fig. 11D). Hx completely blocked the proliferation of SW13/Scrm shRNA+Ctrl cells, in contrast to Nx SW13/Scrm shRNA+Ctrl cells, which grew steadily from day 1 to day 3. These data suggested that Hx not only stops SW13 cell proliferation but also slightly reduces the number of SW13 cells. Stable knockdown of HIF1α significantly enhanced cell proliferation at day 1 under Hx; however, proliferation plateaued at day 1, and there was no further growth during days 2 to 3. Like SW13/Scrm shRNA+Ctrl cells, Hx SW13/HIF2α shRNA+Ctrl cells exhibited slightly reduced numbers, which is expected, since these cells do not express HIF2α mRNA. Consistent with a tumor-suppressive function of BRG1 (26, 30, 31), Nx SW13/Scrm shRNA+BRG1 cells exhibited slower growth than Nx SW13/Scrm+Ctrl cells. However, these cells in which BRG1 was reexpressed continued to grow under Nx. Interestingly, under Hx, SW13/Scrm shRNA+BRG1 cells exhibited increased cell number reduction at day 2 and then recovered a little at day 3, providing strong evidence for increased cell number reduction due to BRG1 reexpression. SW13/HIF1α shRNA+BRG1 cells proliferated similarly to SW13/HIF1 shRNA+Ctrl cells under Hx at day 1, likely because of delayed manifestation of BRG1 activity during day 1, consistent with the observation that reexpression of BRG1 did not reduce Nx cell numbers on day 1. Although the numbers of SW13/HIF1α shRNA+BRG1 cells began to decrease on days 2 to 3 relative to the numbers of SW13/HIF1α shRNA+Ctrl cells, proliferation was still higher than in SW13/Scrm shRNA+BRG1 cells, suggesting that HIF1α knockdown could partially compensate for the proliferation-inhibitory and cell number reduction functions of BRG1 under Hx. This implies that the inhibitory function of BRG1 under Hx likely occurs through regulation of the HIF1-mediated Hx response. In addition, SW13/HIF2α shRNA+BRG1 cells displayed more proliferation than SW13/Scrm+BRG1 cells on days 1 and 2, suggesting that HIF2α also plays a proliferation-inhibitory role in Hx SW13 cells. In summary, these data support a proliferation-suppressive role of BRG1 in Nx SW13 cells and also indicate that under Hx, BRG1 reexpression aggravates the reduction in cell numbers by enhancing the HIF-mediated Hx response.
To further explore the role of BRG1 in HIF-independent and -dependent tumorigenesis, we stably knocked down BRG1, HIF1α, HIF2α, BRG1 plus HIF1α, or BRG1 plus HIF2α in RCC4T cells, a renal cell carcinoma cell line, using shRNAs (Fig. 12A). In contrast to the results for Hep3B cells, BRG1 knockdown did not reduce HIF1α or HIF2α protein levels (Fig. 12A). As expected, CA9 levels were greatly reduced in Hx RCC4T/HIF1α shRNA cells, but increased in Hx RCC4T/HIF2α shRNA cells, consistent with previous findings suggesting that CA9 is a HIF1 target gene in RCC4T cells (12, 32). ANGPTL4 levels were decreased in both Hx RCC4T/HIF1α shRNA cells and Hx RCC4T/HIF2α shRNA cells, suggesting that ANGPTL4 is a common target gene of HIF1 and HIF2 in RCC4T cells (Fig. 12B). BRG1 shRNA reduced Hx induction of both genes (Fig. 12B), demonstrating, in another cell line, that BRG1 is important for HIF target gene expression. Hx induction of CA9 was further decreased in RCC4T/BRG1+HIF1α shRNA cells relative to Hx RCC4T/BRG1 shRNA cells or RCC4T/HIF1α shRNA cells. CA9 induction in RCC4T/BRG1+HIF2α shRNA cells was intermediate between those in RCC4T/HIF2α shRNA cells and RCC4T/BRG1 shRNA cells (Fig. 12B). However, Hx induction of ANGPTL4, a HIF1/HIF2 target gene, in RCC4T/BRG1+HIF1α shRNA cells and RCC4T/BRG1+HIF2α shRNA cells was similar to that in RCC4T/HIF1α shRNA cells and RCC4T/HIF2α shRNA cells, respectively (Fig. 12B).
After confirming that BRG1 regulates HIF target genes in RCC4T cells, we performed several functional assays for each of the types of cells mentioned above cultured under Nx or Hx (Fig. 12C to toE).E). First, we performed clonogenic assays (Fig. 12C) and found that Hx did not affect the clonogenic survival of control RCC4T/Scrm cells, likely due to the opposing effects of HIF1 and HIF2: HIF1 inhibits, and HIF2 promotes, the clonogenic survival of RCC4T cells under Hx (Fig. 12C). BRG1 knockdown significantly reduced cell survival under Nx, indicating that BRG1 promotes the clonogenic survival of Nx RCC4T cells (Fig. 12C). However, there was no survival difference between Nx and Hx RCC4T/BRG1 shRNA cells, consistent with the observation that hypoxia did not change cell survival. Knockdown of HIF1α or HIF2α on top of BRG1 knockdown also did not change the survival of these cells, likely because BRG1 knockdown itself had already reduced the activity of HIF1 and HIF2 to sufficiently low levels, masking the effects of HIF1α or HIF2α knockdown.
We also performed proliferation assays and found that Hx increased the proliferation of RCC4T/Scrm cells (Fig. 12D). In addition, knockdown of HIF1α or HIF2α increased or decreased cell proliferation, respectively, suggesting a proliferation-inhibitory role for HIF1 and a proliferation-promoting role for HIF2 (Fig. 12D). Interestingly, BRG1 knockdown increased cell proliferation, suggesting that BRG1 inhibits cell proliferation in Nx RCC4T cells. However, under Hx, BRG1 knockdown reduced cell proliferation relative to that in Nx RCC4T/BRG1 shRNA cells and prevented the Hx-induced increase in proliferation seen in RCC4T/Scrm cells, suggesting that the BRG1 complex promotes RCC4T cell proliferation during Hx. Knockdown of HIF1α or HIF2α on top of BRG1 knockdown did not change cell proliferation from that in BRG1 knockdown cells (Fig. 12D), likely because BRG1 knockdown reduced the activity of HIF1 and HIF2 to sufficiently low levels, concealing the effects of further HIF1α or HIF2α knockdown.
Finally, we performed a wound-healing cell migration assay (Fig. 12E). We found that Hx slightly reduced the migration of RCC4T/Scrm shRNA cells at 48 and 72 h but not at 24 h. Interestingly, migration in RCC4T/HIF1α shRNA cells was increased over that in control cells under Hx, indicating that the HIF1-mediated Hx response inhibits migration in RCC4T cells. In contrast, migration was not significantly changed by HIF2α knockdown, although there was a trend toward reduced cell migration with HIF2α knockdown. Nx RCC4T/BRG1 shRNA cells exhibited reduced migration at all three time points, suggesting that the BRG1 complex promotes RCC4T cell migration under Nx. However, under Hx, BRG1 knockdown increased cell migration over that for Hx RCC4T/Scrm shRNA cells at all three time points, suggesting a migration-suppressive function of BRG1 under Hx. The migration-suppressive role of the BRG1 complex in Hx RCC4T cells is consistent with the migration-suppressive role of the hypoxia response in this cell type. Again, knockdown of HIF1α on top of BRG1 knockdown did not significantly change cell migration from that in BRG1 knockdown cells (Fig. 12E). However, knockdown of HIF2α in addition to BRG1 knockdown reduced cell migration at 24 and 48 h but not at 72 h. In conclusion, the BRG1 complex can have opposing functions in Nx and Hx RCC4T cells, and the function of the BRG1 complex in Hx RCC4T cells is in agreement with the function of the HIF-mediated Hx response for RCC4T cell survival, proliferation, and migration in vitro.
While Hx-mediated HIFα protein stability and the major players in the Hx signaling pathway are well established (1–4), how HIF activates its target genes is much less understood. Since HIF is not the main recruiter of p300 and CBP (11, 12), we investigated the role of HIF in recruiting the BRG1/BRM complexes in the Hx response.
To date, the conclusions from three published papers concerning the role of BRG1/BRM complexes in the Hx response (33–35) do not agree with each other and are inaccurate. Kenneth et al. showed that knockdown of BRG1 (but not BRM) or other components of the BRG1 complex, such as BAF57, BAF155, and BAF170, in U2OS cells significantly decreased the levels of HIF1α (but not HIF2α) mRNA and protein (33). Since they did not detect BRG1 protein binding on the promoter(s) of a HIF target gene(s) (33), they concluded that BRG1 has no coactivator role in the HIF-mediated Hx response. In contrast, Wang et al. showed that knockdown of BRM alone or BRM plus BRG1 but not BRG1 alone in Hep3B cells reduced Hx induction of EPO (34). In their second paper, they showed that knockdown of both BRG1 and BRM in Hep3B cells reduced Hx induction of LDHA and PGK1 (35). Thus, Wang et al. suggested that the BRM complex served as a coactivator for the HIF-mediated Hx response, including the activation of genes such as LDHA and PGK1, which are BRG1/BRM-independent HIF target genes, as we reported here. We think we can now reconcile the discrepancy between our data and those of Kenneth et al. and Wang et al. We believe that the data of Wang and colleagues could be explained by the ability of BRG1/BRM to regulate HIF1α gene expression; thus, BRG1/BRM knockdown in Hep3B cells reduced HIF1α levels and reduced the Hx induction of all HIF target genes, including LDHA and PGK1, which are BRG1 independent. Kenneth et al. did not detect the binding of BRG1/BRM protein on the promoter(s) of a HIF target gene(s), although they did not specify the genes they assessed (33). We speculate that they might have selected BRG1-independent HIF target genes, such as GLUT1, for their BRG1 binding assay, since GLUT1 was one of the two genes they used for functional readout in response to BRG1 knockdown (33). Thus, our data clearly addressed the inconsistencies between these published papers and provide strong evidence for the roles of BRG1 and BRM complexes in regulating HIF1α expression and in the HIF-mediated Hx response.
We reported here, for the first time, that the BRG1 complex also regulates HIF2α gene expression in Hep3B (Fig. 2) and SW13 (Fig. 5) cells and that the BRM complex also activates HIF2α gene expression in SW13 cells (Fig. 5). In addition, since we used multiple cell lines, we showed for the first time that the interplay between the BRG1/BRM complex and HIFα genes is cell type specific. For example, BRG1, but not the BRM complex, activates both HIF1α and HIF2α in Hep3B cells (Fig. 2), while both the BRG1 and BRM complexes activate HIF2α but not HIF1α in SW13 cells (Fig. 5). However, the BRG1/BRM complex does not regulate HIF1α and HIF2α gene expression in RCC4T cells (Fig. 12). Currently, we are not able to determine the reasons for these phenomena. For example, we found that the negligible role of BRM in regulating HIFα gene expression in Hep3B cells is not due to low BRM expression, since BRM is highly expressed in Hep3B cells (data not shown), and overexpression of BRM in Hep3B cells also does not induce HIFα gene expression (data not shown). Additionally, the BRG1/BRM-independent expression of the HIF1α gene in SW13 cells is not due to high HIF1α expression, since HIF1α levels are similar in SW13 and Hep3B cells (data not shown).
We also established, for the first time, that the BRG1 complex acts as a coactivator of HIF target genes by showing that BRG1 binds to the promoters of the HIF target genes CA9 and EPO (Fig. 7). Using a nucleosome scanning assay, we showed that the BRG1 complex is involved in remodeling the CA9 promoter in Hx cells (Fig. 9), providing direct evidence for a coactivator role of the BRG1 complex in HIF target gene activation. Moreover, we showed that Hx-induced nucleosome remodeling (Fig. 10) and transcription activation (Fig. 6) of CA9 are dependent on the ATPase activity of BRG1. While Hx-mediated increases in BRG1 recruitment to the EPO promoter have been reported previously (34), we furthered these studies by showing that HIF/ARNT is responsible for increased BRG1 recruitment to HIF target genes during Hx (Fig. 7). Also, we showed that BRG1 interacts with HIF1α and HIF2α proteins, as assessed by co-IP. Thus, our results provide a novel mechanism, suggesting that HIF/ARNT activates its target genes by recruiting BRG1 complexes to HIF target gene promoters. BRG1 recruitment increases nucleosome remodeling and enhances transcription in an ATPase-dependent manner.
BRG1, BRM, and particularly the non-ATPase components of the SWI/SNF complexes are frequently inactivated in multiple cancers (26, 30, 31, 36, 37). Thus, the SWI/SNF complex has long been regarded as a tumor suppressor. For example, reintroduction of BRG1 into cells lacking BRG1 expression was sufficient to reverse their transformed phenotype and to induce growth arrest and a flattened morphology (26). This is consistent with our data for SW13 cells showing that reintroduction of BRG1 into Nx SW13 cells reduced cell proliferation (Fig. 11D). For the first time, we analyzed the role of the BRG1 complex in the proliferation of Hx cells. In agreement with a proliferation-suppressive role of the HIF1-mediated Hx response in SW13 cells, we found that BRG1 reexpression amplified the proliferation-suppressive activity of the HIF-mediated hypoxia response, aggravating the reduction in cell numbers, which is reversed by HIF1α knockdown (Fig. 11D). Unexpectedly, we found that the effects of BRG1 on the proliferation of SW13 cells and the reduction in their numbers differ between days 0 to 1 and days 2 to 3. This difference is likely due to a lag period in BRG1 function; therefore, the proliferation effects observed during days 0 to 1 are BRG1 independent but HIF dependent, since HIF levels are stably reduced prior to BRG1 transfection. Unfortunately, we were not able to perform clonogenic survival or wound-healing migration assays in SW13 cells because stable transfection of BRG1 promotes cell senescence, as reported previously (38).
The role of the BRG1 complex in RCC4T cells is particularly interesting. Under Hx, HIF1- and HIF2-mediated Hx responses play opposite roles in cell survival, proliferation, and migration, as reported previously (39). Despite the opposing effects of HIF1 and HIF2, and despite the fact that BRG1 knockdown reduced both HIF1- and HIF2-mediated Hx responses, we found that the function of BRG1 in Hx RCC4T cells is closely related to the net effect of the Hx response on cell survival, proliferation, and migration. Another interesting observation is that the BRG1 complex has opposite cell proliferation and migration functions in Nx versus Hx RCC4T cells. For example, the BRG1 complex inhibits cell proliferation under Nx but increases cell proliferation under Hx. In addition, the BRG1 complex promotes cell migration under Nx but decreases cell migration under Hx. These findings are particularly important, providing evidence, for the first time, that the Hx tumor microenvironment could be a determinant for the function of the BRG1 complex in Hx cells.
In summary, this study confirms that the BRG1 complex regulates the expression of the HIF1α gene and establishes a novel role of BRG1 and BRM complexes in HIF2α gene transcription. In addition, we separate the function of BRG1/BRM in regulating HIFα genes from their role in HIF target gene activation. Furthermore, we report, for the first time, that activation of a subset of HIF target genes is BRG1/BRM independent. Importantly, we found that increased BRG1 recruitment to HIF target genes is an important way for HIF/ARNT to activate the expression of its target genes. In addition, our functional studies with SW13 and RCC4T cells indicated that the role of the BRG1 complex in cell proliferation, migration, and survival in hypoxic cells is dictated by the role of HIF-mediated hypoxia responses.
This work was supported by grants from the National Cancer Institute (RO1CA134687 to Cheng-Jun Hu). Johnny A. Sena was supported by Research Supplemental to Promote Diversity (NCI) from 1 June 2010 to 30 May 2012.
We thank Trevor Williams for thoughtful discussions and reading of the manuscript.
Published ahead of print 29 July 2013