Thyroid Hormone Receptor β Suppression of RUNX2 Is Mediated by Brahma-Related Gene 1–Dependent Chromatin Remodeling

Noelle E Gillis; Thomas H Taber; Eric L Bolf; Caitlin M Beaudet; Jennifer A Tomczak; Jeffrey H White; Janet L Stein; Gary S Stein; Jane B Lian; Seth Frietze; Frances E Carr

doi:10.1210/en.2018-00128

. 2018 May 9;159(6):2484–2494. doi: 10.1210/en.2018-00128

Thyroid Hormone Receptor β Suppression of RUNX2 Is Mediated by Brahma-Related Gene 1–Dependent Chromatin Remodeling

Noelle E Gillis ^1,², Thomas H Taber ¹, Eric L Bolf ^1,², Caitlin M Beaudet ¹, Jennifer A Tomczak ¹, Jeffrey H White ¹, Janet L Stein ^2,³, Gary S Stein ^2,³, Jane B Lian ^2,³, Seth Frietze ^2,⁴, Frances E Carr ^1,^2,^✉

PMCID: PMC6692870 PMID: 29750276

Abstract

Thyroid hormone receptor β (TRβ) suppresses tumor growth through regulation of gene expression, yet the associated TRβ-mediated changes in chromatin assembly are not known. The chromatin ATPase brahma-related gene 1 (BRG1; SMARCA4), a key component of chromatin-remodeling complexes, is altered in many cancers, but its role in thyroid tumorigenesis and TRβ-mediated gene expression is unknown. We previously identified the oncogene runt-related transcription factor 2 (RUNX2) as a repressive target of TRβ. Here, we report differential expression of BRG1 in nonmalignant and malignant thyroid cells concordant with TRβ. BRG1 and TRβ have similar nuclear distribution patterns and significant colocalization. BRG1 interacts with TRβ, and together, they are part of the regulatory complex at the RUNX2 promoter. Loss of BRG1 increases RUNX2 levels, whereas reintroduction of TRβ and BRG1 synergistically decreases RUNX2 expression. RUNX2 promoter accessibility corresponded to RUNX2 expression levels. Inhibition of BRG1 activity increased accessibility of the RUNX2 promoter and corresponding expression. Our results reveal a mechanism of TRβ repression of oncogenic gene expression: TRβ recruitment of BRG1 induces chromatin compaction and diminishes RUNX2 expression. Therefore, BRG1-mediated chromatin remodeling may be obligatory for TRβ transcriptional repression and tumor suppressor function in thyroid tumorigenesis.

A functional interaction between TRβ and BRG is identified as critical for regulation of the oncogene RUNX2 and may reveal novel tumor suppression action in thyroid cancer.

Altered gene-expression programming in cancer cells is often a consequence of a loss of function of cell type–specific transcriptional control mechanisms (1). Dysregulation of the transcription factor thyroid hormone receptor β (TRβ), a member of the thyroid hormone receptor family, through genomic modifications and epigenetic silencing, is characteristic of thyroid, breast, and other solid tumors (2, 3). Despite a recognized role as a tumor suppressor and a positive prognostic indicator, the mechanisms by which TRβ regulates tumor growth are not clear (4–7).

TRβ is a ligand-dependent transcription factor that mediates the effects of the thyroid hormone T₃ in a number of biological processes, including development, growth, and differentiation. TRβ regulatory functions involve both genomic and nongenomic actions. Recent studies indicate that TRβ tumor-suppressive effects are mediated through intracellular signaling pathways, including phosphatidylinositol 3-kinase/Akt (8), Ras/MAPK (4), and Janus kinase–signal transducers and activators of transcription (6), as well as the induction of the mesenchymal-to-epithelial transition (9). At the genomic level, TRβ mediates the effects of T₃ via the regulation of gene expression through the recruitment of coregulators and chromatin remodeling complexes. TRβ has been shown to induce reorganization of chromatin and local nucleosome structures via interaction with genomic regulatory elements to alter target gene transcription (10, 11). Previous studies revealed that loss of nuclear corepressor 1, a cofactor integral to TRβ genomic action, is correlated with tumor growth and metastasis (12). The mode by which TRβ acts to upregulate gene expression has been studied extensively; however, the ligand-dependent and -independent, TRβ-mediated gene-repression mechanisms remain poorly understood. Disruption of TRβ in cancer is therefore expected to alter the assembly of coregulator complexes needed for the regulation of gene-expression programs.

The mammalian homologs of switch/sucrose nonfermentable (SWI/SNF) chromatin ATPases, brahma-related gene 1 (BRG1; SMARCA4), and brahma (SMARCA2), as well as BRG1-associated factors (BAFs), modulate cell-cycle functions and chromatin structure in cancer, as well as developmental programming (10, 13–17). These mutually exclusive ATPases, in combination with BAFs, mediate changes in the nuclear microenvironment, characteristic of cancer. In particular, BRG1 is known to disrupt the chromatin architecture of target promoters and has been implicated in both the activation and repression of nuclear hormone receptor (NR)–mediated gene expression (14, 18). BRG1 has a documented association with various NRs, notably the estrogen, progesterone, and androgen receptors, and has been identified as critical for maintaining transcriptional control over gene-expression programs (14). The loss of NR function or imbalance of NR-BRG1 interaction may lead to altered BRG1 activity and dysregulation of tumorigenic programming.

The involvement of BRG1 in cellular transformation and subsequent tumor development is complex and highly context dependent. The loss of function of BRG1 and related complexes is associated with cell-cycle defects and tumor formation, leading to the prospect that BRG1 may function as a tumor suppressor in many cancers (19, 20). BRG1 mutations occur frequently in human cancers and are a putative therapeutic target (21, 22). However, delineation of the role of BRG1 in tumor growth is incomplete, and the impact of interactions with tumor suppressors, such as TRβ, is unknown.

We previously defined the gene encoding the oncogenic transcription factor runt-related transcription factor 2 (RUNX2) as a repressive target of TRβ in thyroid cells (23). RUNX2 is a prometastatic transcription factor that is crucial for proper osteogenesis and is an integral factor in skeletal maintenance (24). However, it has been identified as a contributor to invasion and metastasis in a variety of cancers, including thyroid cancer (25–27). Notably, loss of TRβ expression results in increased RUNX2 levels, likely contributing to thyroid cancer progression (23). In the current study, we demonstrate a functional interaction between BRG1 and TRβ in thyroid cancer cells and show that BRG1 enhances TRβ-mediated repression of RUNX2 gene expression. We conclude that BRG1-mediated chromatin remodeling is a critical factor in TRβ-mediated suppression of RUNX2 gene expression.

Materials and Methods

Culture of thyroid cell lines

Immortalized thyroid cell lines, derived from nonmalignant thyroid epithelial cells (Nthy-ORI), and papillary (BHP2-7), follicular (WRO), and anaplastic (SW 1736) carcinomas were cultured in RPMI 1640 growth media with l-glutamine (300 mg/L), sodium pyruvate, and nonessential amino acids (1%; Cellgro/Mediatech), supplemented with 10% fetal bovine serum (Life Technologies) and penicillin-streptomycin (200 IU/L; Cellgro/Mediatech). Cells were maintained at 37°C, 5% CO₂, and 100% humidity. Thyroid tumor cell lines were generously provided by Dr. John Copland III (Mayo Clinic). The Nthy-ORI cell line was obtained from the European Collection of Cell Cultures. All cell lines were authenticated (22 January 2018) using short tandem repeat profiles and the Promega GenePrint10 System in the Advanced Genome Technologies Core at the University of Vermont.

Immunoblot analysis

Proteins were isolated from whole cells in lysis buffer (20 mM Tris-HCl, pH 8, 137 mM NaCl, 10% glycerol, 1% Triton X-100, and 2 mM EDTA) containing Protease Inhibitor Cocktail 78410 (Thermo Fisher Scientific), as previously described (23). Nuclear proteins were prepared with NE-PER Nuclear and Cytoplasmic Extraction Reagents 78833 (Thermo Fisher Scientific) with the addition of Protease Inhibitor Cocktail 781410 (Thermo Fisher Scientific) per the manufacturer’s protocol. Proteins were resolved by polyacrylamide gel electrophoresis on 10% SDS gels EC60752 (Life Technologies) and immobilized onto nitrocellulose membranes (GE Healthcare Dharmacon) by electroblot (Bio-Rad Laboratories). Specific proteins were detected by immunoblotting using primary antibodies against TRβ (1:500; RRID: AB_628038; Santa Cruz Biotechnology), BAF60 (1:1000; RRID: AB_2192137; Santa Cruz Biotechnology), BRG1 (1:2000; RRID: AB_10861578; Abcam), and BAF57 (1:1000; RRID: AB_11156559; Abcam). Immunoreactive proteins were detected by enhanced chemiluminescence (Thermo Fisher Scientific) using horseradish peroxidase-conjugated mouse anti-rabbit (1:10,000; RRID: AB_2339149; Jackson ImmunoResearch) or goat anti-mouse (1:10,000, RRID: AB_437779; Calbiochem). Specific details of antibodies and their use are summarized in Table 1. Proteins were visualized by VersaDoc MP3000 (Bio-Rad Laboratories) and intensities quantified by Image Laboratory (Bio-Rad Laboratories).

Table 1.

Antibody Table

Antigen	Use	Manufacturer; Catalog Number	RRID	Dilution or Amount
BRG1	WB	Abcam; ab110641	AB_10861578	1:2000
	IP, ChIP	Cell Signaling Technology; CS493605	AB_2193944	4 μg/200 μg protein
	IP, ChIP	Cell Signaling Technology; CS493605	AB_2193944	2 μg/50 μg chromatin
	IF	Sigma-Aldrich; 4200195	AB_10634191	1:500
TRβ	WB	Santa Cruz Biotechnology; sc738	AB_628038	1:500
	IP	Thermo Fisher Scientific; MA1-216	AB_2287303	4 μg/200 μg protein
	IF	Abcam; ab15545	AB_301955	1:500
	ChIP	Thermo Fisher Scientific; PA5-29684	AB_2493188	2 μg/50 μg chromatin
BAF60	WB	Santa Cruz Biotechnology; sc514400	AB_2192137	1:1000
BAF57	WB	Abcam; ab131328	AB_11156559	1:1000
H3K27ac	ChIP	Abcam; ab4729	AB_2118291	2 μg/50 μg chromatin
H1.2	ChIP	Abcam; ab4086	AB_2117983	2 μg/50 μg chromatin
β-Actin	WB	Rockland Immunochemicals; 600-401-886	AB_2223521	1:10,000
Mouse anti-rabbit HRP	WB	Jackson ImmunoResearch; 211-035-109	AB_2339149	1:10,000
Goat anti-mouse HRP	WB	Calbiochem; 401253	AB_437779	1:10,000
Rabbit IgG	ChIP	Cell Signaling Technology; CS2729S	AB_1031062	2 μg/50 μg chromatin

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Abbreviations: ChIP, chromatin immunoprecipitation; H3K27ac, histone 3 lysine 27 acetylation; HRP, horseradish peroxidase; IF, immunofluorescence; IP, immunoprecipitation; WB, Western blot.

Analysis of thyroid cancer patient sample data

Publically available microarray expression data, deposited in the National Center for Biotechnology Information Gene Expression Omnibus Database (GSE76039, GSE3467), were analyzed using GEOR2 (www.ncbi.nlm.nih.gov/gds) to reveal differential expression of BRG1 across the spectrum of thyroid cancers.

RNA extraction and real-time quantitative RT-PCR

Total RNA was extracted using the RNeasy Plus Kit (Qiagen), according to the manufacturer’s protocol. cDNA was then generated using 5× RT MasterMix (Applied Biological Materials). Gene expression was quantified by quantitative RT-PCR (qRT-PCR) using 2× BrightGreen MasterMix (Applied Biological Materials) on a QuantStudio 3 Real-Time PCR System (Applied Biosystems). qRT-PCR primer sequences are summarized in Supplemental Table 1. Fold change in gene expression compared with endogenous controls was calculated using the ΔΔCt method.

Immunofluorescence

Proteins were detected in whole cells by immunofluorescence. Cells, seeded in six-well dishes on coverslips coated with 0.5% gelatin (Sigma-Aldrich), were grown overnight in phenol red-free RPMI 1640 (Cellgro) at 37°C, 100% humidity, and 5% CO₂. The cells, packed on ice, were washed with PBS (Cellgro) and fixed with 3.7% formalin in PBS for 10 minutes. After two washes in PBS, cells were permeabilized in 0.025% Triton X-100 (Sigma-Aldrich) for 20 minutes and then washed in PBS with 3% BSA (PBSA; Sigma-Aldrich). Cells were blocked with 10% normal goat serum (Jackson ImmunoResearch) in PBSA with 0.3% Triton X-100 (PBSAT) and then washed in PBSAT. Primary antibodies, diluted in PBSAT with 10% normal goat serum, were added, and cells were incubated overnight at 4°C at 100% humidity. Cells were washed with PBSAT and incubated with secondary antibodies diluted in PBSAT at room temperature for 1 hour. After washes in PBSA, cells were incubated at room temperature for 15 minutes in 4′,6-diamidino-2-phenylindole nucleic acid stain (Life Technologies) in PBSA. After washes in PBSA, cells were mounted on slides using Prolong Gold (Life Technologies). Specific proteins were detected by immunofluorescence with antibodies targeting TRβ (1:500; RRID: AB_301955; Abcam) and BRG1 (1:500; RRID: AB_10634191; Sigma-Aldrich). Images were captured using a Zeiss LSM 510 confocal microscope. Quantitation of colocalization was determined using Volocity 3D Rendering Software (Perkin Elmer).

Immunoprecipitation

Nuclear protein extract was precleared with Protein G Dynabeads. Protein (200 μg) was incubated with either an isotype-specific IgG control antibody (mouse or rabbit) or an anti-TRβ (4 μg; RRID: AB_2287303; Thermo Fisher Scientific) or anti-BRG1 (4 μg; RRID: AB_2193944; Cell Signaling Technology) antibody for 1 hour at 4°C. Protein G Dynabeads (50 μL) were added to the reaction and incubated for 1 hour at 4°C. The beads were then washed three times with DNA binding buffer [20 mM HEPES, pH 7.6, 30 mM KCl, 1 mM EDTA, 10 mM (NH₄)₂SO₄, 1 mM dithiothreitol (DTT), and 0.2% (v/v) Tween-20]. Samples were eluted with Laemmli sample buffer and DTT and analyzed via immunoblot.

Small interfering RNA transfection

Loss of function was assayed by small interfering RNA (siRNA) using On Target Plus siRNAs, targeting human SMARCA4 (L-010431-00-0005; GE Healthcare Dharmacon) with nontargeting siRNA (D-001810-10-20; GE Healthcare Dharmacon) as control. Cells were plated at a density of 2.5 × 10⁵ per well and transfected with 50 to 200 nM siRNA with Oligofectamine per the manufacturer’s protocol (Invitrogen/Life Technologies). The effect of knockdown of BRG1 on endogenous RUNX2 and metastatic markers was determined after 24 hours.

DNA pulldown assay

The proteins that bind to the native sequence of the “A” oligo of the RUNX2 P1 proximal promoter were characterized by DNA pulldown. The reverse oligo was biotinylated at the 3′-OH terminus (Enzo Life Sciences); the complimentary forward strand was added and annealed after the reaction. Biotinylated oligonucleotide (0.75 fmol) was then added to 50 µL streptavidin-coupled T1 Dynabeads (Invitrogen) with 200 µL DNA binding buffer [20 mM HEPES, pH 7.6, 30 mM KCl, 10 mM (NH₄)₂SO₄, 1 mM EDTA, 1 mM DTT, 0.2% (v/v) Tween-20] in a total reaction volume of 250 µL. The reactions proceeded at 4°C for 1 hour with constant agitation. Beads were then washed twice with 200 µL DNA binding buffer. Next, 350 µg nuclear lysate was precleared for 15 minutes with 120 µL prewashed Streptavidin-coupled T1 Dynabeads (Invitrogen) with 2 µg sheared herring sperm DNA (Promega). KCl was added to each reaction to achieve a final concentration of 300 mM, and 20 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1 mM EDTA, and 0.5% (v/v) Nonidet P-40 was added to bring the final reaction volume to 750 µL. Reactions proceeded for 1.5 hours at 4°C with constant agitation. Beads were then washed with 350 µL 20 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1 mM EDTA, and 0.5% (v/v) Nonidet P-40 and washed 1× with H₂O. Samples were analyzed via immunoblot.

Chromatin immunoprecipitation

Binding to the RUNX2 promoter region was determined by chromatin immunoprecipitation (ChIP)–qRT-PCR. Cultured human thyroid cells were crosslinked with 1% formaldehyde for 10 minutes, neutralized with 1.25 M glycine, rinsed twice with PBS, pelleted, and frozen. Cells were lysed in the presence of protease inhibitors (Roche), and chromatin was extracted and sonicated to 200 to 500 bp using a Covaris S220 Focused Ultrasonicator. Sonicated cell lysate was incubated with 2 μg indicated antibody and rotated overnight at 4°C. Protein G Dynabeads (20 µL; Invitrogen) were blocked in 0.5% BSA, resuspended in lysis buffer, added to the lysate/antibody mix, and rotated at 4°C for 3 hours. Complexes were washed extensively, eluted, and incubated at 65°C overnight to reverse crosslinks. Samples were treated with ribonuclease A, incubated with Proteinase K, phenol/chloroform extracted, and ethanol precipitated. Pellets were resuspended in 70 μL 10 mM Tris-HCl, pH 8. DNA was amplified with Q5 High-Fidelity DNA Polymerase (New England Biolabs), according to the manufacturer’s recommendations. Analysis of P1 and P2 transcripts of the RUNX2 gene used promoter-specific primers (Supplemental Table 1). Chromatin-binding proteins were detected using antibodies targeting TRβ (2 μg; RRID: AB_2493188; Thermo Fisher Scientific), histone 3 lysine 27 acetylation (H3K27ac; 2 μg; RRID: AB_2118291; Abcam), H1.2 (2 μg; RRID: AB_2117983; Abcam), BRG1 (2 μg; RRID: AB_2193944; Cell Signaling Technology), and rabbit IgG (2 μg; RRID: AB_1031062; Cell Signaling Technology).

DNase hypersensitivity assay

Promoter accessibility was determined via the DNase hypersensitivity assay. Chromatin samples were obtained, as described previously. Chromatin (2 μg) was treated with 0.5 unit DNase I (Qiagen) for 15 minutes at room temperature in 1× DNase Incubation Buffer (10 mM Tris-HCl, pH 7.6, 2.5 mM MgCl₂, 0.5 mM CaCl₂). Reactions were terminated by the addition of 50 mM EDTA and vortexing. Crosslinking was reversed by incubating the samples at 95°C for 20 minutes. DNA fragments were purified using a PureLink PCR Clean-Up Column (Invitrogen). DNA was used as a template for qRT-PCR using primers specific to the RUNX2 P1 promoter (Supplemental Table 2).

Lentiviral constructs and stable cell line generation

The lentiviral backbone vector pCDH-MSCV-EF1-GFP-Puro (System Biosciences) was digested with EcoRI and NotI. The coding sequence for TRβ was generated by PCR from pDest-515 Flag-TRβ (23) and ligated into the lentiviral expression vector. Viral particles containing pCDH-MSCV-EF1-GFP-Puro (pCDH-Empty) and pCDH-MSCV-EF1-GFP-Puro-TRβ (pCDH-TRβ) were generated in human embryo kidney 293 cells. The resulting viral particles were used to transduce SW 1736. The resulting cells were cultured, as described previously, with the addition of 1 µg/ml puromycin (Gold Biotechnology).

Statistics

All statistical analyses were performed using GraphPad Prism software (GraphPad Software). A Fisher t test (P ≤ 0.05) was used for paired comparisons. A two-way ANOVA, followed by a multiple comparisons test (P ≤ 0.05), was used to compare groups. Data are represented as means ± SD.

Results

BRG1 is differentially expressed in thyroid cells

Loss of function of the SWI/SNF chromatin remodeling complex has been linked to tumorigenesis in a variety of cancers. The enzymatic activity of BRG1, a mammalian homolog of SWI/SNF chromatin ATPases, is necessary for NR-mediated transcriptional regulation to control tumorigenic expression programs (14). Therefore, we established an expression profile of BRG1 and two of its associated factors (Baf57 and Baf60) in a panel of representative thyroid cell lines. Immunoblot analyses revealed reduced expression of BRG1 in papillary (BHP2-7)- and follicular (WRO)-differentiated thyroid cancer cells compared with nonmalignant thyroid cells (Nthy-ORI). BRG1 protein expression was reduced fourfold in de-differentiated anaplastic (SW 1736) thyroid cancer cells (Fig. 1A and 1B). A similar expression pattern was observed at the mRNA level (Fig. 1C). Baf57 expression showed no significant difference between cell lines at the protein level, and Baf60 was significantly reduced only in the BHP2-7 cell line. There was no significant difference in expression of Baf57 or Baf60 mRNA in these thyroid cell lines. To determine the clinical relevance of our observations, BRG1 mRNA levels were assessed from microarray expression data obtained from patient samples (28, 29). These results indicate that BRG1 expression is significantly reduced in poorly differentiated and anaplastic thyroid tumors compared with matched normal tissue (Fig. 1D). We previously determined a similar expression profile of TRβ in both tissue microarrays and our thyroid cancer cell lines: reduced TRβ expression in poorly differentiated and aggressive tumors (23). These observations prompted us to explore the potential for an interaction between the two nuclear factors as a mechanism for tumor suppression.

Figure 1. — BRG1 expression is reduced in thyroid cancer cell lines and thyroid cancer tissue samples. (A) Representative immunoblot illustrates BRG1, Baf57, and Baf60 protein expression in thyroid cell lines derived from a spectrum of thyroid cancer subtypes. (B) The histogram illustrates quantitation of BRG1, Baf57, and Baf60 protein levels, standardized to β-actin, averaged from three independent experiments performed in triplicate. Error bars are SD; significance compared with nonmalignant cells (Nthy-ORI) is indicated. (C) The graph illustrates BRG1, Baf57, and Baf60 mRNA levels, standardized to glyceraldehyde 3-phosphate dehydrogenase (GAPDH), averaged from three independent experiments performed in triplicate. Error bars are SD; significance compared with nonmalignant cells (Nthy-ORI) is indicated. (D) Publically available microarray expression data (GSE76039, GSE3467), analyzed using GEOR2, reveals differential expression of BRG1 in thyroid cancer patient tumor samples. Error bars are SD; significance compared with normal thyroid tissue is indicated. *P < 0.05; **P < 0.01; ****P < 0.0001. ADU, arbitrary densitometric units; ATC, anaplastic thyroid cancer; PDTC, poorly differentiated thyroid carcinoma; PTC, papillary thyroid cancer.

BRG1 associates with TRβ in thyroid cells

Immunofluorescence microscopy revealed that BRG1 and TRβ have similar nuclear distribution patterns and substantial colocalization. BRG1 and TRβ are both localized to the nucleus and show substantial overlap in nonmalignant thyroid cells, as well as in papillary, follicular, and anaplastic thyroid cancer cells (Fig. 2A). Quantitation of the overlay signal of BRG1 and TRβ in nonmalignant, differentiated, and de-differentiated malignant thyroid cells reveals a high concordance in all thyroid cell images (Fig. 2B). Thus, TRβ and BRG1 have similar nuclear distribution patterns and substantial colocalization, implicating potential interaction.

Figure 2. — BRG1 and TRβ are colocalized and associate with the *RUNX2* promoter. (A) Immunofluorescence illustrates detection of TRβ and BRG1 proteins in whole-thyroid cells. (B) Quantitation of the colocalization of TRβ and BRG1 in each thyroid cell line demonstrates a high concordance in localization. (C) TRβ is detected by immunoblot after immunoprecipitation from nonmalignant thyroid nuclear lysate using an anti-BRG1 antibody. (D) BRG1 is detected by immunoblot after immunoprecipitation from nonmalignant thyroid nuclear lysate using an anti-TRβ antibody. (E) BRG1 and TRβ are both detected by immunoblot after DNA pulldown using oligos containing the native sequence of the RUNX2 promoter. Binding is lost when an oligo containing a mutant thyroid hormone response element (TRE) is used, and when a 10× unlabeled competitor probe is used. (F) ChIP–qRT-PCR shows enrichment of BRG1 and TRβ at the RUNX2 promoter in nonmalignant cells (Nthy-ORI) but not in anaplastic thyroid cancer cells (SW 1736). Values were calculated as fold enrichment compared with the matched IgG antibody control. Error bars are SD; significance compared with IgG is indicated. *P < 0.05; ***P < 0.001. DAPI, 4′,6-diamidino-2-phenylindole; IB, immunoblot; IP, immunoprecipitation.

We next determined whether the colocalization reflects TRβ and BRG1 interaction within the context of nuclear regulatory complexes. Reciprocal coimmunoprecipitation demonstrates that BRG1 and TRβ form a complex in nuclear extracts isolated from nonmalignant thyroid cells (Fig. 2C and 2D). Coimmunoprecipitation of BRG1 and TRβ is not dependent on the presence of T₃ (Supplemental Fig. 1). We previously identified putative thyroid hormone receptor response elements (TREs) in the RUNX2 P1 promoter (Supplemental Fig. 2) (23). Given that TRβ suppresses RUNX2 tumor promoter activity, we further investigated the association of BRG1 with the TRβ-RUNX2 TRE complex by DNA pulldown assays. After incubation of nuclear protein lysate with an oligonucleotide containing the native sequence of the RUNX2 promoter at site A (−105 to −49), we isolated the resulting protein complexes and detected both TRβ and BRG1 by immunoblot (Fig. 2E). Importantly, BRG1 binding was lost when an oligonucleotide containing a mutated TRE was used in this assay. This suggests that TRβ must be present for BRG1 recruitment to this site. ChIP demonstrated that both BRG1 and TRβ are enriched at the RUNX2 P1 promoter region in nonmalignant thyroid cells compared with the isotype IgG controls in nonmalignant thyroid cells. However, in anaplastic thyroid cancer cells, where both TRβ and BRG1 expression is reduced, there was no significant enrichment of either factor (Fig. 2F). Loss of function of the TRβ-BRG1 regulatory complex may therefore contribute to the increased RUNX2 expression observed in anaplastic thyroid cancer.

TRβ and BRG1 cooperatively suppress RUNX2 expression in thyroid cells

We next examined the functional relationship between TRβ-BRG1 complex formation and RUNX2 gene expression. Upon knockdown of BRG1 by siRNA transfection in nonmalignant thyroid cells (Fig. 3A), RUNX2 mRNA levels increase (Fig. 3B). This indicated that BRG1 may be directly participating in the regulation of this gene. Additionally, BRG1 knockdown led to an increase in expression of three putative prometastatic RUNX2 target genes: AVEGFA, cyclin D1, and MMP9, further emphasizing the importance of maintenance of transcriptional control over RUNX2 for tumor suppression (Fig. 3C). To determine whether reintroduction of BRG1 could rescue RUNX2 repression, we used SW 1736 cells that have been modified to express TRβ stably (Fig. 4A). Overexpression of BRG1 by transient transfection in anaplastic thyroid cancer cells (Fig. 4B) did not reduce RUNX2 expression, suggesting that the presence of TRβ is necessary for repressive activity of RUNX2 (Fig. 4C). Indeed, expression of TRβ alone reduced RUNX2 expression in these cells, whereas reintroduction of both BRG1 and TRβ further repressed RUNX2 expression. These results indicate a cooperative mechanism of action of TRβ and BRG1 for suppression of RUNX2 expression.

Figure 3. — BRG1 silencing leads to increased RUNX2 and RUNX2 target gene expression. (A) Representative immunoblot illustrates that transfection of nonmalignant thyroid cells (Nthy-ORI) with siBRG1 results in a concentration-dependent decrease in BRG1 protein. (B) Knockdown of BRG1 also results in corresponding decreases in BRG1 mRNA. Loss of BRG1 results in increased RUNX2 mRNA levels. Data are the averaged fold change in transfected cells normalized to mock-transfected cells. Graph summarizes quantitation of three separate experiments performed in triplicate; values are standardized to GAPDH. Error bars are SD; significance compared with nontargeting control is indicated. (C) Knockdown of BRG1 increases Runx2 target gene expression: MMP9, cyclin D1, and VEGF. Data are the averaged fold change in transfected cells normalized to mock-transfected cells. Graph summarizes quantitation of three separate experiments performed in triplicate; values are standardized to GAPDH. Error bars are SD; significance compared with nontargeting control is indicated. *P < 0.05; ***P < 0.001; ****P < 0.0001.

Figure 4. — BRG1 mediates RUNX2 suppression. (A) Representative immunoblot shows increased TRβ expression after lentiviral transduction of SW 1736 (SW) cells compared with cells transduced with an empty vector (EV). (B) qRT-PCR results show that transient transfection with a BRG1 expression vector increases BRG1 mRNA levels in both the SW-EV transduced cell line and the SW-TRβ transduced cell line. Error bars are SD; significance compared with empty vector control is indicated. (C) qRT-PCR results show changes in RUNX2 mRNA levels after stably transduced cells were transiently transfected with a BRG1 expression vector. Overexpression of BRG1 in the absence of TRβ does not repress RUNX2. BRG1 transfection in the presence of TRβ represses RUNX2 further than TRβ alone. Error bars are SD; significance compared with empty vector control is indicated. *P < 0.05; ***P < 0.001. WT, wild-type.

BRG1 mediates RUNX2 promoter accessibility

BRG1, as the catalytic subunit of a major chromatin remodeling complex, functions to facilitate chromatin remodeling directed by its binding partners. With the use of ChIP, followed by targeted qRT-PCR, we detected changes in enrichment of H3K27ac, a histone modification indicative of active transcription. In nonmalignant cells, where RUNX2 expression is low, there was no enrichment of H3K27ac at the RUNX2 promoter. However, in anaplastic cells, where RUNX2 expression is high, we observed 10-fold enrichment of this active histone mark (Fig. 5A). We assessed the RUNX2 P1 promoter accessibility in nonmalignant and anaplastic thyroid cells using DNase I hypersensitivity assays, followed by targeted qRT-PCR. Sensitivity to DNase I digestion was significantly increased in anaplastic thyroid cancer cells when compared with nonmalignant cells, which indicates a more accessible chromatin state (Fig. 5B). These findings are consistent with the previously observed expression pattern of RUNX2 in our thyroid cancer cells (23). Addition of the BRG1 inhibitor PF1-3 (30), which blocks catalytic function, resulted in increased sensitivity to DNase digestion in nonmalignant thyroid cells, with no change in anaplastic thyroid cancer cells (Fig. 5C). BRG1 has been shown to mediate chromatin compaction at promoters that are repressed by the progesterone receptor (PR), specifically via deposition of the histone variant H1.2 into proximal promoter regions (31). We hypothesized that BRG1 may facilitate chromatin remodeling for negative regulation directed by TRβ in a similar manner. In agreement with this report, we observed a robust decrease in H1.2 enrichment upon pharmacological inhibition of BRG1 (Fig. 5D). Therefore, BRG1 mediates heterochromatinization of the RUNX2 promoter, which facilitates TRβ-mediated suppression of RUNX2 expression.

Figure 5. — BRG1 mediates RUNX2 promoter accessibility. (A) The active chromatin modification, H3K27ac, is enriched at the RUNX2 promoter in anaplastic thyroid cancer cells (SW 1736) but not in nonmalignant thyroid cells (Nthy-ORI). Values were calculated as fold enrichment compared with the matched IgG antibody control. Error bars are SD; significance compared with IgG is indicated. (B) The RUNX2 promoter is more accessible in anaplastic thyroid cancer cells (SW 1736) than nonmalignant (Nthy-ORI) by the DNase I hypersensitivity assay. Values were calculated as relative sensitivity to DNase digestion compared with a known heterochromatinized site. Error bars are SD; significance compared with nonmalignant cells is indicated. (C) Treatment with a BRG1 inhibitor (PF1-3, 30 µM) for 6 hours results in an increase in sensitivity to DNase I digestion of the RUNX2 promoter in nonmalignant cells (Nthy-ORI). Error bars are SD; significance compared with vehicle control is indicated. (D) Treatment with PFI-3 results in a loss of H1.2 enrichment at the RUNX2 promoter detected by ChIP. Values were calculated as fold enrichment compared with the matched IgG antibody control. Error bars are SD; significance compared with vehicle control is indicated. *P < 0.05; **P < 0.01; ***P < 0.001.

Discussion

Chromatin remodeling is an essential element of genetic regulation, wherein the availability of binding sites and upstream enhancer regions is dynamically controlled. To interact with such regulatory elements and to regulate gene expression, TRβ induces reorganization of local nucleosome structure, resulting in changes in chromatin accessibility (32). Most of the seminal studies that have characterized TRβ-mediated chromatin remodeling have done so in the context of gene activation (11, 32, 33). The mechanisms underlying gene repression by TRβ are much less well understood. There have been corepressors (e.g., nuclear corepressor, SMRT) identified as essential for gene repression by TRβ (34–36); however, there is a limited understanding of the signaling events that lead to TRβ-directed chromatin compaction or how alternative TRβ-mediated complexes dynamically regulate gene-expression programs. In this study, we present evidence that TRβ associates with the catalytic subunit of the human SWI/SNF complex, BRG1, at a negatively regulated promoter to facilitate chromatin compaction and gene repression.

BRG1 activity has been described as critical for NR-mediated transcriptional regulation, yet interaction between BRG1 and TRβ has not been previously reported. Our data show that TRβ and BRG1 cooperate to regulate the expression of the RUNX2 oncogene. Furthermore, our functional studies indicate that the presence of TRβ is necessary for BRG1-mediated chromatin remodeling to occur at this promoter. Loss of this cooperative transcriptional regulation of RUNX2 in our anaplastic thyroid cancer cells likely contributes to their tumorigenic phenotype. Increased RUNX2 expression has been implicated in a variety of cancers, including thyroid cancer, as a contributor to metastasis and a poor prognostic marker (23, 26). Although our analysis has focused on suppression of RUNX2, we hypothesize that TRβ-BRG1 complexes exhibit transcriptional control over other sites throughout the genome. This cooperative regulation may be integral to maintaining cellular homeostasis and a normal, noncancerous phenotype. As TRβ tumor suppressor function is disrupted across the spectrum of thyroid cancer, altered BRG1 may contribute to tumorigenic gene-expression programming.

Mutations of the gene encoding BRG1 are thought to occur in ∼20% of all human cancers (19). The loss of function of BRG1 is associated with cell-cycle defects and tumor formation in some cancer types and reduced tumor growth in others. Overexpression of BRG1 is associated with tumorigenesis and may correlate with poor outcomes in triple-negative breast cancer (37) and invasive ductal carcinomas (38), providing support for its use as a potential therapeutic target. The fact that BRG1 has been associated with both tumor growth and suppression denotes a tissue and cell type-dependent function. This could also reflect the loss of NR function, or imbalance of NR-BRG1 interaction. For example, the liganded PR, in association with BRG1, induces heterochromatinization, gene silencing, and reduced proliferation in PR-positive breast cancer cells, emphasizing the importance of the BRG1-NR context (31). Further delineation of how BRG1 integrates complex signaling pathways may be important for determining the therapeutic benefit of BRG1 attenuation through pharmacological intervention. Although BRG1 inhibitors alone are unable to slow cell growth, they may enhance susceptibility of aggressive cancers to standard chemotherapies (14). Deeper understanding of BRG1 interaction with transcription factors with tumor suppressor functions, such as TRβ, can inform further development of pharmaceuticals targeting BRG1 and their use in a clinical setting as a precision medicine.

In summary, our findings demonstrate a mechanism for TRβ-mediated gene repression through BRG1-dependent chromatin remodeling. As a putative tumor suppressor, the loss of TRβ function is known to contribute to thyroid cancer growth. A TRβ mutant, PV, which exhibits resistance to thyroid hormone, is known to facilitate the development of follicular thyroid cancer in mouse models (39). However, it is not known whether the PV mutation disrupts the interaction with BRG1 or regulation of RUNX2 expression. Our studies have uncovered a concordant loss of BRG1 and TRβ and a functional link in suppression of an oncogenic target, RUNX2. Our analysis suggests that BRG1 expression varies between subtypes of thyroid cancer and may be dependent on the driver mutation. Elucidation of the TRβ-BRG1 regulatory network in thyroid and other cancers is critical to understanding the mechanisms of TRβ-mediated tumor suppression. Additional characterization of factors that impact TRβ-BRG1 complex formation would give further insight into the functional consequences of deregulation of the nuclear microenvironment on thyroid cancer progression.

Supplementary Material

Supplemental Datas

Click here for additional data file.^{(129.2KB, pdf)}

Acknowledgments

The BRG1 expression vector was generously provided by Drs. Kevin Trotter and Trevor Archer (National Institutes of Health). Lentiviral constructs were made with the assistance of Dr. Jon Ramsey, University of Vermont (UVM) Cancer Translational Research Laboratory, UVM. The automated DNA sequencing, authentication of cell lines, and other molecular analyses were performed in the Vermont Integrative Genomics Resource, supported by the UVM Cancer Center, Lake Champlain Cancer Research Organization, and UVM Larner College of Medicine. Microscopy images and analyses were conducted within the Microscopy Imaging Center, UVM.

Financial Support: This work was supported by grants from the National Institutes of Health (3P01CA082834-15S1 to G.S.S., J.L.S., and F.E.C.; R37 DE012528 to J.B.L.; R21CA209229 and 1R21AI121528 to S.F.); American Cancer Society Institutional Research Grant at UVM to S.F.; and Lake Champlain Cancer Research Organization (LCCRO-032338 to F.E.C.). We also acknowledge research support provided by a National Science Foundation Major Research Instrumentation grant, MRI-1429003: Acquisition of a High Performance Computing Cluster for Multidisciplinary Research and Education at the University of Northern Colorado (to S.F.).

Disclosure Summary: The authors have nothing to disclose.

Glossary

Abbreviations:

BAF: brahma-related gene 1–associated factor
BRG1: brahma-related gene 1
ChIP: chromatin immunoprecipitation
DTT: dithiothreitol
GAPDH: glyceraldehyde 3-phosphate dehydrogenase
H3K27ac: histone 3 lysine 27 acetylation
NR: nuclear hormone receptor
PBSA: PBS with 3% BSA
PBSAT: PBS with 3% BSA with 0.3% Triton X-100
PR: progesterone receptor
qRT-PCR: quantitative RT-PCR
RUNX2: runt-related transcription factor 2
siRNA: small interfering RNA
SWI/SNF: switch/sucrose nonfermentable
TRE: thyroid hormone receptor response element
TRβ: thyroid hormone receptor β
UVM: University of Vermont

References

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Supplemental Datas

Click here for additional data file.^{(129.2KB, pdf)}

[B1] 1. Bradner JE, Hnisz D, Young RA. Transcriptional addiction in cancer. Cell. 2017;168(4):629–643. [DOI] [PMC free article] [PubMed] [Google Scholar]

[B2] 2. Kim WG, Cheng SY. Thyroid hormone receptors and cancer. Biochim Biophys Acta. 2013:1830(7):3928–3936. [DOI] [PMC free article] [PubMed] [Google Scholar]

[B3] 3. Aranda A, Martínez-Iglesias O, Ruiz-Llorente L, García-Carpizo V, Zambrano A. Thyroid receptor: roles in cancer. Trends Endocrinol Metab. 2009;20(7):318–324. [DOI] [PubMed] [Google Scholar]

[B4] 4. Martínez-Iglesias O, Garcia-Silva S, Tenbaum SP, Regadera J, Larcher F, Paramio JM, Vennström B, Aranda A. Thyroid hormone receptor beta1 acts as a potent suppressor of tumor invasiveness and metastasis. Cancer Res. 2009;69(2):501–509. [DOI] [PubMed] [Google Scholar]

[B5] 5. Kim WG, Zhu X, Kim DW, Zhang L, Kebebew E, Cheng SY. Reactivation of the silenced thyroid hormone receptor β gene expression delays thyroid tumor progression. Endocrinology. 2013;154(1):25–35. [DOI] [PMC free article] [PubMed] [Google Scholar]

[B6] 6. Park JW, Zhao L, Cheng SY. Inhibition of estrogen-dependent tumorigenesis by the thyroid hormone receptor β in xenograft models. Am J Cancer Res. 2013;3(3):302–311. [PMC free article] [PubMed] [Google Scholar]

[B7] 7. Kim WG, Zhao L, Kim DW, Willingham MC, Cheng SY. Inhibition of tumorigenesis by the thyroid hormone receptor β in xenograft models. Thyroid. 2014;24(2):260–269. [DOI] [PMC free article] [PubMed] [Google Scholar]

[B8] 8. Furuya F, Hanover JA, Cheng SY. Activation of phosphatidylinositol 3-kinase signaling by a mutant thyroid hormone beta receptor. Proc Natl Acad Sci USA. 2006;103(6):1780–1785. [DOI] [PMC free article] [PubMed] [Google Scholar]

[B9] 9. García-Silva S, Aranda A. The thyroid hormone receptor is a suppressor of ras-mediated transcription, proliferation, and transformation. Mol Cell Biol. 2004;24(17):7514–7523. [DOI] [PMC free article] [PubMed] [Google Scholar]

[B10] 10. Shi YB, Matsuura K, Fujimoto K, Wen L, Fu L. Thyroid hormone receptor actions on transcription in amphibia: the roles of histone modification and chromatin disruption. Cell Biosci. 2012;2(1):42. [DOI] [PMC free article] [PubMed] [Google Scholar]

[B11] 11. Ramadoss P, Abraham BJ, Tsai L, Zhou Y, Costa-e-Sousa RH, Ye F, Bilban M, Zhao K, Hollenberg AN. Novel mechanism of positive versus negative regulation by thyroid hormone receptor β1 (TRβ1) identified by genome-wide profiling of binding sites in mouse liver. J Biol Chem. 2014;289(3):1313–1328. [DOI] [PMC free article] [PubMed] [Google Scholar]

[B12] 12. Martínez-Iglesias O, Alonso-Merino E, Aranda A. Tumor suppressive actions of the nuclear receptor corepressor 1. Pharmacol Res. 2016;108:75–79. [DOI] [PubMed] [Google Scholar]

[B13] 13. Muchardt C, Yaniv M. When the SWI/SNF complex remodels...the cell cycle. Oncogene. 2001;20(24):3067–3075. [DOI] [PubMed] [Google Scholar]

[B14] 14. Trotter KW, Archer TK. The BRG1 transcriptional coregulator. Nucl Recept Signal. 2008;6:e004. [DOI] [PMC free article] [PubMed] [Google Scholar]

[B15] 15. Romero OA, Sanchez-Cespedes M. The SWI/SNF genetic blockade: effects in cell differentiation, cancer and developmental diseases. Oncogene. 2014;33(21):2681–2689. [DOI] [PubMed] [Google Scholar]

[B16] 16. Wu JI. Diverse functions of ATP-dependent chromatin remodeling complexes in development and cancer. Acta Biochim Biophys Sin (Shanghai). 2012;44(1):54–69. [DOI] [PubMed] [Google Scholar]

[B17] 17. Ho L, Crabtree GR. Chromatin remodelling during development. Nature. 2010;463(7280):474–484. [DOI] [PMC free article] [PubMed] [Google Scholar]

[B18] 18. Euskirchen GM, Auerbach RK, Davidov E, Gianoulis TA, Zhong G, Rozowsky J, Bhardwaj N, Gerstein MB, Snyder M. Diverse roles and interactions of the SWI/SNF chromatin remodeling complex revealed using global approaches. PLoS Genet. 2011;7(3):e1002008. [DOI] [PMC free article] [PubMed] [Google Scholar]

[B19] 19. Wilson BG, Roberts CW. SWI/SNF nucleosome remodellers and cancer. Nat Rev Cancer. 2011;11(7):481–492. [DOI] [PubMed] [Google Scholar]

[B20] 20. Kadoch C, Crabtree GR. Mammalian SWI/SNF chromatin remodeling complexes and cancer: Mechanistic insights gained from human genomics. Sci Adv. 2015;1(5):e1500447. [DOI] [PMC free article] [PubMed] [Google Scholar]

[B21] 21. Shain AH, Pollack JR. The spectrum of SWI/SNF mutations, ubiquitous in human cancers. PLoS One. 2013;8(1):e55119. [DOI] [PMC free article] [PubMed] [Google Scholar]

[B22] 22. Kadoch C, Hargreaves DC, Hodges C, Elias L, Ho L, Ranish J, Crabtree GR. Proteomic and bioinformatic analysis of mammalian SWI/SNF complexes identifies extensive roles in human malignancy. Nat Genet. 2013;45(6):592–601. [DOI] [PMC free article] [PubMed] [Google Scholar]

[B23] 23. Carr FE, Tai PW, Barnum MS, Gillis NE, Evans KG, Taber TH, White JH, Tomczak JA, Jaworski DM, Zaidi SK, Lian JB, Stein JL, Stein GS. Thyroid hormone receptor-β (TRβ) mediates runt-related transcription factor 2 (RUNX2) expression in thyroid cancer cells: A novel signaling pathway in thyroid cancer. Endocrinology. 2016;157(8):3278–3292. [DOI] [PMC free article] [PubMed] [Google Scholar]

[B24] 24. Liu JC, Lengner CJ, Gaur T, Lou Y, Hussain S, Jones MD, Borodic B, Colby JL, Steinman HA, van Wijnen AJ, Stein JL, Jones SN, Stein GS, Lian JB. Runx2 protein expression utilizes the Runx2 P1 promoter to establish osteoprogenitor cell number for normal bone formation. J Biol Chem. 2011;286(34):30057–30070. [DOI] [PMC free article] [PubMed] [Google Scholar]

[B25] 25. Niu DF, Kondo T, Nakazawa T, Oishi N, Kawasaki T, Mochizuki K, Yamane T, Katoh R. Transcription factor Runx2 is a regulator of epithelial-mesenchymal transition and invasion in thyroid carcinomas. Lab Invest. 2012;92(8):1181–1190. [DOI] [PubMed] [Google Scholar]

[B26] 26. Sancisi V, Borettini G, Maramotti S, Ragazzi M, Tamagnini I, Nicoli D, Piana S, Ciarrocchi A. Runx2 isoform I controls a panel of proinvasive genes driving aggressiveness of papillary thyroid carcinomas. J Clin Endocrinol Metab. 2012;97(10):E2006–E2015. [DOI] [PMC free article] [PubMed] [Google Scholar]

[B27] 27. Sancisi V, Gandolfi G, Ambrosetti DC, Ciarrocchi A. Histone deacetylase inhibitors repress tumoral expression of the proinvasive factor RUNX2. Cancer Res. 2015;75(9):1868–1882. [DOI] [PubMed] [Google Scholar]

[B28] 28. He H, Jazdzewski K, Li W, Liyanarachchi S, Nagy R, Volinia S, Calin GA, Liu CG, Franssila K, Suster S, Kloos RT, Croce CM, de la Chapelle A. The role of microRNA genes in papillary thyroid carcinoma. Proc Natl Acad Sci USA. 2005;102(52):19075–19080. [DOI] [PMC free article] [PubMed] [Google Scholar]

[B29] 29. Landa I, Ibrahimpasic T, Boucai L, Sinha R, Knauf JA, Shah RH, Dogan S, Ricarte-Filho JC, Krishnamoorthy GP, Xu B, Schultz N, Berger MF, Sander C, Taylor BS, Ghossein R, Ganly I, Fagin JA. Genomic and transcriptomic hallmarks of poorly differentiated and anaplastic thyroid cancers. J Clin Invest. 2016;126(3):1052–1066. [DOI] [PMC free article] [PubMed] [Google Scholar]

[B30] 30. Vangamudi B, Paul TA, Shah PK, Kost-Alimova M, Nottebaum L, Shi X, Zhan Y, Leo E, Mahadeshwar HS, Protopopov A, Futreal A, Tieu TN, Peoples M, Heffernan TP, Marszalek JR, Toniatti C, Petrocchi A, Verhelle D, Owen DR, Draetta G, Jones P, Palmer WS, Sharma S, Andersen JN. The SMARCA2/4 ATPase domain surpasses the bromodomain as a drug target in SWI/SNF-mutant cancers: Insights from cDNA rescue and PFI-3 inhibitor studies. Cancer Res. 2015;75(18):3865–3878. [DOI] [PMC free article] [PubMed] [Google Scholar]

[B31] 31. Nacht AS, Pohl A, Zaurin R, Soronellas D, Quilez J, Sharma P, Wright RH, Beato M, Vicent GP. Hormone-induced repression of genes requires BRG1-mediated H1.2 deposition at target promoters. EMBO J. 2016;35(16):1822–1843. [DOI] [PMC free article] [PubMed] [Google Scholar]

[B32] 32. Grøntved L, Waterfall JJ, Kim DW, Baek S, Sung MH, Zhao L, Park JW, Nielsen R, Walker RL, Zhu YJ, Meltzer PS, Hager GL, Cheng SY. Transcriptional activation by the thyroid hormone receptor through ligand-dependent receptor recruitment and chromatin remodelling. Nat Commun. 2015;6(1):7048. [DOI] [PMC free article] [PubMed] [Google Scholar]

[B33] 33. Shi YB. Unliganded thyroid hormone receptor regulates metamorphic timing via the recruitment of histone deacetylase complexes. Curr Top Dev Biol. 2013;105:275–297. [DOI] [PMC free article] [PubMed] [Google Scholar]

[B34] 34. Astapova I, Dordek MF, Hollenberg AN. The thyroid hormone receptor recruits NCoR via widely spaced receptor-interacting domains. Mol Cell Endocrinol. 2009;307(1-2):83–88. [DOI] [PMC free article] [PubMed] [Google Scholar]

[B35] 35. Shimizu H, Astapova I, Ye F, Bilban M, Cohen RN, Hollenberg AN. NCoR1 and SMRT play unique roles in thyroid hormone action in vivo. Mol Cell Biol. 2015;35(3):555–565. [DOI] [PMC free article] [PubMed] [Google Scholar]

[B36] 36. Wang D, Xia X, Liu Y, Oetting A, Walker RL, Zhu Y, Meltzer P, Cole PA, Shi YB, Yen PM. Negative regulation of TSHalpha target gene by thyroid hormone involves histone acetylation and corepressor complex dissociation. Mol Endocrinol. 2009;23(5):600–609. [DOI] [PMC free article] [PubMed] [Google Scholar]

[B37] 37. Wu Q, Madany P, Akech J, Dobson JR, Douthwright S, Browne G, Colby JL, Winter GE, Bradner JE, Pratap J, Sluder G, Bhargava R, Chiosea SI, van Wijnen AJ, Stein JL, Stein GS, Lian JB, Nickerson JA, Imbalzano AN. The SWI/SNF ATPases are required for triple negative breast cancer cell proliferation. J Cell Physiol. 2015;230(11):2683–2694. [DOI] [PMC free article] [PubMed] [Google Scholar]

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PERMALINK

Thyroid Hormone Receptor β Suppression of RUNX2 Is Mediated by Brahma-Related Gene 1–Dependent Chromatin Remodeling

Noelle E Gillis

Thomas H Taber

Eric L Bolf

Caitlin M Beaudet

Jennifer A Tomczak

Jeffrey H White

Janet L Stein

Gary S Stein

Jane B Lian

Seth Frietze

Frances E Carr

Abstract

Materials and Methods

Culture of thyroid cell lines

Immunoblot analysis

Table 1.

Analysis of thyroid cancer patient sample data

RNA extraction and real-time quantitative RT-PCR

Immunofluorescence

Immunoprecipitation

Small interfering RNA transfection

DNA pulldown assay

Chromatin immunoprecipitation

DNase hypersensitivity assay

Lentiviral constructs and stable cell line generation

Statistics

Results

BRG1 is differentially expressed in thyroid cells

Figure 1.

BRG1 associates with TRβ in thyroid cells

Figure 2.

TRβ and BRG1 cooperatively suppress RUNX2 expression in thyroid cells

Figure 3.

Figure 4.

BRG1 mediates RUNX2 promoter accessibility

Figure 5.

Discussion

Supplementary Material

Acknowledgments

Glossary

Abbreviations:

References

Associated Data

Supplementary Materials

ACTIONS

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RESOURCES

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