- Open Access
The chromatin remodelling factor BRG1 is a novel binding partner of the tumor suppressor p16INK4a
© Becker et al; licensee BioMed Central Ltd. 2009
- Received: 17 October 2008
- Accepted: 16 January 2009
- Published: 16 January 2009
CDKN2A/p16 INK4a is frequently altered in human cancers and it is the most important melanoma susceptibility gene identified to date. p16INK4a inhibits pRb phosphorylation and induces cell cycle arrest, which is considered its main tumour suppressor function. Nevertheless, additional activities may contribute to the tumour suppressor role of p16INK4a and could help explain its specific association with melanoma predisposition. To identify such functions we conducted a yeast-two-hybrid screen for novel p16INK4a binding partners.
We now report that p16INK4a interacts with the chromatin remodelling factor BRG1. We investigated the cooperative roles of p16INK4a and BRG1 using a panel of cell lines and a melanoma cell model with inducible p16INK4a expression and BRG1 silencing. We found evidence that BRG1 is not required for p16INK4a-induced cell cycle inhibition and propose that the p16INK4a-BRG1 complex regulates BRG1 chromatin remodelling activity. Importantly, we found frequent loss of BRG1 expression in primary and metastatic melanomas, implicating this novel p16INK4a binding partner as an important tumour suppressor in melanoma.
This data adds to the increasing evidence implicating the SWI/SNF chromatin remodelling complex in tumour development and the association of p16INK4a with chromatin remodelling highlights potentially new functions that may be important in melanoma predisposition and chemoresistance.
- Chromatin Remodelling
- Primary Melanoma
- Induce Cell Cycle Arrest
- Chromatin Remodelling Complex
The cyclin dependent kinase inhibitor p16INK4a is frequently inactivated in human cancers and is a highly penetrant melanoma susceptibility gene; more than 50 germline mutations have been identified in high-risk melanoma-prone families . The principal function of p16INK4a is to inhibit cell cycle progression by preventing the cyclin dependent kinases CDK4 and CDK6 from phosphorylating the retinoblastoma protein, pRb. In the presence of p16INK4a, pRb remains hypophosphorylated and forms active pRb-E2F transcriptional repressor complexes that silence genes required for S-phase entry. Consequently, ectopic expression of p16INK4a promotes pRb-dependent G1 cell cycle arrest and senescence. Moreover, functional p16INK4a is commonly maintained in pRb-deficient tumors (reviewed by Sherr & Roberts ), and this underscores the dependency of p16INK4a on the pRb pathway.
Hypophosphorylated pRb can repress gene transcription at least partly by remodelling chromatin structure through its interactions with proteins such as HDAC1, BRM and BRG1 [3–5]. As the catalytic core of the SWI/SNF chromatin remodelling complex, the interaction between BRG1 and pRb was proposed to recruit the complex to E2F responsive promoters and enhance pRb transcriptional repressor activity.  There is also evidence that BRG1 acts upstream of pRb by repressing cyclin D1 expression  and upregulating the expression of the CDK inhibitors p21Waf1, p15INK4b and p16INK4a [7–9] to maintain pRb in an active, hypophosphorylated state. Not surprisingly, BRG1 may function as a tumor suppressor; BRG1 hemizygous mice are susceptible to tumors , while complete loss of BRG1 potentiates lung cancer development  and BRG1 is silenced or mutated in human tumor cell lines derived from breast, ovarian, lung, brain and colon cancers [4, 12]. BRG1 is also lost in established neuroendocrine carcinomas and adenocarcinomas of the cervix , and the loss of BRG1 expression in lung cancer is associated with a poor prognosis [14, 15].
In this study, it is identified for the first time that BRG1 specifically associates with p16INK4a in vivo, and that both proteins are frequently lost in human melanomas. Although both BRG1 and p16INK4a regulate pRb activity we found no evidence that p16INK4a and BRG1 co-operate in cell cycle regulation. Targeted silencing of BRG1 did not diminish pRb-dependent p16INK4a activities; p16INK4a retained potent cell cycle inhibitory activity and induced senescence in the presence and absence of BRG1. Contrary to previous reports, that BRG1-deficient cells are relatively resistant to p16INK4a-induced cell cycle arrest , we show that pRb activity is BRG1-independent and thus, BRG1 does not influence p16INK4a-mediated cell cycle inhibition. Together with the frequent loss in primary melanomas the novel BRG1 interaction with the melanoma associated tumor suppressor p16INK4a implies an important role for BRG1 in melanoma.
BRG1 binds p16INK4a
pRb pathway in human cell lines
pRb pathway proteins in cell lines
p16INK4a requires pRB to induce cell cycle arrest
p16INK4a does not require BRG1 to promote cell cycle arrest or induce cell senescence
Silencing of BRG1 had no significant impact on the proliferation rate or cell cycle distribution of the WMM1175_p16INK4a cell line. In the absence of BRG1, p16INK4a retained the ability to inhibit the proliferation of the WMM1175 cells (Figure 5C), and this was associated with arrest in the G1-phase of the cell cycle with a concomitant S-phase inhibition (Figure 5D) that was maintained over the five-day induction period (data not shown). Moreover, the silencing of BRG1 had no impact on the ability of p16INK4a to totally prevent outgrowth of colonies upon low seeding density (Figure 5E).
BRG1 is lost in melanoma
BRG1 is frequently lost in melanomas
The p16INK4a tumor suppressor has a critical influence on melanoma tumorigenesis. We have now shown that the chromatin remodelling factor BRG1 is a novel binding partner of p16INK4aand confirm this interaction in vivo. More importantly, we show that loss of BRG1 occurs frequently in primary and metastatic melanomas and propose that BRG1 may play an important role as a tumor suppressor in this cancer.
We have also shown that p16INK4a requires pRb, but not BRG1 to promote cell cycle arrest. This differs from several previous findings in the literature but agrees with others: It has been suggested that the pRb-BRG1 interaction is required for the pRb repression of E2F-target genes such as cyclin E and cyclin A, and thereby cell cycle arrest. According to this hypothesis, cells lacking BRG1 would harbor only inactive pRb, thus conferring resistance to p16INK4a induced growth arrest [5, 16]. These findings differ from those of Bultman et al.  who did not observe a functional interaction between pRB and BRG1 in their murine models and Kang et al. , who showed that the BRG1-pRB interaction was not required for BRG1 induced cell cycle arrest in SW-13 cells. In contrast to our work, Kang et al.  used long-term BRG1 expression, which caused growth arrest in SW-13 cells, and showed that BRG1 bound the p21Waf1 promoter and upregulated its expression 3–7 days after BRG1 expression. This was sufficient to induce cell cycle arrest and senescence independent of the BRG1 ability to complex with pRb. In this study we have clearly demonstrated that p16INK4a requires pRb, but not BRG1, to promote cell cycle arrest. Our data is mainly based on the thorough analysis of a well-defined melanoma cell model, with inducible physiological relevant expression levels of p16INK4a and the use of highly specific BRG1-silencing molecules. In this model, p16INK4a induction promotes rapid G1-cell cycle arrest followed by cellular senescence, and these functions were not affected by silencing of BRG1.
Chromatin changes, which involve chromatin remodelling, are an important step in p16INK4/pRb dependent senescence . It was recently shown that the BRG1 homologue, BRM, forms an initiating component of heterochromatin complexes during the senescence of melanocytes . BRG1 has also been implicated in senescence of melanocytes, as it has been identified in the SWI/SNF complex facilitating transcription in response to IGFBP7, the latter itself being an important player in oncogenic BRAF-induced senescence . However, our data show that p16INK4a is able to promote senescence in WMM1175 melanoma cells in the absence of BRG1 indicating that the p16INK4a/pRb senescence pathway does not require BRG1.
As the catalytic component of the SWI/SNF chromatin remodelling complex, BRG1 facilitates unwinding of DNA helices bound to and wrapped around histone structures. The SWI/SNF chromatin remodelling complex can be recruited by specific DNA binding molecules such as transcriptional activators or repressors and directed to specific DNA targets. For instance, BRG1 promotes p53 dependent transcription by interacting with this tumor suppressor [27, 28], while it functions as a co-repressor of E2F dependent transcription by associating with the E2F transcriptional repressor pRb . Furthermore, BRG1 has recently been reported to promote transcriptional activity of the melanocyte specific transcription factor MITF-M . MITF-M plays an important role in melanocyte proliferation and survival (reviewed by Goding)  and activates the expression of p16INK4a . It is possible that the p16INK4a interaction with BRG1 modulates any one or more of these functions. For example it is tempting to speculate that p16INK4a influences MITF-M transcriptional activity via its association with BRG1. This would create an important feedback loop between MITF-M and p16INK4a. We are currently investigating the impact of p16INK4a on these BRG1 specific chromatin remodelling functions.
Regardless of the function of the BRG1-p16INK4a complex, it is evident that BRG1 expression can be lost relatively early in melanoma development, with a significant proportion (> 70%) of primary melanomas showing no detectable BRG1 expression, while BRM expression was usually maintained in these tumors (< 20% loss). Overall, the rate of BRG1 loss was high in melanomas and comparable to that of p16INK4a , which suggests that selection against BRG1 expression arises relatively early in melanoma genesis. The fact that, additionally to the frequent loss of either tumor suppressor, a high proportion of melanomas show loss of both proteins correlates with our data showing BRG1-independence of the p16INK4a cell cycle regulatory functions and this suggests BRG1 independent and dependent functions of p16INK4a. BRG1 is proposed to be an important modulator of chromatin in melanocytic cells. In particular, BRG1 promotes transcriptional activity of the melanocyte specific transcription factor MITF-M , reduction of BRG1 expression in zebrafish embryos leads to a reduction in neural crest derived cells including melanocytes  and thirdly we found BRG1 expression in normal, primary human melanocytes. Therefore we propose that BRG1 is a vital melanoma associated tumour suppressor, which is frequently lost in the initial stages of the disease.
The identification of BRG1 as a potential tumor suppressor in melanoma adds to the increasing evidence implicating the SWI/SNF chromatin remodelling complex in tumor development. BRG1 mutations have been identified in small cell lung carcinomas  and loss of BRG1 expression or mislocalisation of BRG1 to the cytoplasm has been associated with poor prognosis in this malignancy [14, 15]. Another study showed that 71% of neuroendocrine carcinomas of the cervix had lost BRG1 expression  and BRG1 has been implicated in breast cancer through its role in estrogen receptor dependent transcription , its interaction with the breast cancer susceptibility gene BRCA1  and because BRG1 haploinsufficient mice are prone to mammary tumors . Furthermore, BRG1 is often lost or mutated in various tumor cell lines including cells derived from pancreatic, ovarian, lung, brain and colon cancer . In primary melanoma, the chromosomal region of BRG1 (19p13.2) is not deleted at high frequency , nevertheless, translocations in this chromosomal region have been associated with the disease in three cases .
We have identified BRG1 as a novel binding partner of the tumor suppressor p16INK4a and confirmed this interaction in normal cells. Together with our immunohistologic data confirming frequent BRG1 loss in primary melanomas, this implicates BRG1 as an important tumor suppressor in melanoma.
Yeast two-hybrid screen
The Matchmaker2 Gal4 yeast two-hybrid system (Clontech, Mountain View, CA, USA) was used to screen for p16INK4a binding partners in the Y190 yeast strain with p16INK4a cloned into the pAS2 vector in frame to the Gal4 binding domain and a human brain cDNA library cloned into the pACT2 vector in frame with the Gal4 transactivation domain (Clontech, Mountain View, CA, USA) according to the manufacturers instructions.
U2OS, SAOS-2 (osteosarcoma), HCT116 (colon cancer), NCI-H1299 (lung cancer, are referred to as H1299 throughout this manuscript), C33A (cervical cancer), SW-13 (adrenocarcinoma), WS-1 (normal human fibroblasts) and WMM1175_wtp16 (melanoma) cells were grown in DMEM media with 10% foetal bovine serum and in case of WMM1175_wtp16 cells this was supplemented with 250 μg/ml Hygromycin and 500 μg/ml geneticin (Invitrogen, Carlsbad, CA, USA). Transfections were performed with FuGene (Roche, Mannheim, Germany).
Stable BRG1 silenced p16INK4a inducible WMM1175 clones
× 105 WMM1175_wtp16 cells were transfected with 4 μg of a BRG1 targeting siRNA (5'gatccGCATGCACCAGATGCACAAgttcaagagaCTTGTGCATCTGGTGCATGttttttggaaa3') cloned into the pSilencerPuro vector (Ambion, Austin, Texas, USA) or a control siRNA, targeting the luciferase gene, in the same vector supplied by Ambion. After selection with puromycin (2 μg/ml media) clones appeared after 20 days and were expanded, maintained with DMEM media including hygromycin, geneticin and puromycin and tested for BRG1 silencing and p16INK4a inducibility.
Mouse anti-β-actin (AC-74, Sigma, Castle Hill, NSW, Australia), mouse anti-Flag (M2, Sigma, Castle Hill, NSW, Australia), rabbit anti p16INK4a antibody (Western and immunohistochemistry, N-20, SantaCruz, Santa Cruz, CA, USA), mouse anti-p16INK4a antibody (immunoprecipitation, 2B4D11, Zymed Laboratories, San Francisco, CA, USA), mouse anti-BRG1 antibody (Western, G7, SantaCruz, Santa Cruz, CA, USA), rabbit anti-BRG1 antibody (immunohistochemistry, H-88, Santa Cruz, Santa Cruz, CA, USA), rabbit anti-MYC (A14, SantaCruz, Santa Cruz, CA, USA), Ki67 (MIB-1, Dako, Glostrup, Denmark), goat anti-BRM (Western, N-19, Santa Cruz, Santa Cruz, CA, USA), rabbit anti-BRM (immunohistochemistry, ), mouse anti-CDK4 (C8218, Sigma, Castle Hill, NSW, Australia), mouse anti-CDK6 (MS-451-P0, Neomarker, Union City, CA, USA), rabbit anti-phosphorylated pRb (Ser807/811, Cell Signalling, Boston, MA, USA), mouse anti-pRb (G3-245, BD Pharmingen, Franklin Lakes, NJ, USA), mouse anti-topoisomerase II (Ab1, Oncogene, San Diego, CA, USA),
hours post seeding U2OS cells (2 × 106), they were transfected with 7 μg pCMV-Myc5b-p16 and either 10.5 μg pcDNA3-BRG1-Flag  or 10.5 μg pCMV-Flag5b vector (Promega, Madison, Wisconsin, USA). Cells were harvested 24 hours post transfection, lysed in IP-buffer (50 mM Tris pH7.4, 150 mM NaCl, 1 mM EDTA, 1% NP-40, 0.25% sodium deoxycholate, protease inhibitors (Complete tablets, Roche, Mannheim, Germany)) and immunoprecipitation was performed with mouse-anti-Flag antibody or a matched mouse IgG coupled to tosyl-activated Dynal beads (Dynal Biotech, Oslo, Norway) following the manufacturers instructions. Proteins were separated on a 5–15% gradient SDS-PAGE gel, transferred to PVDF membranes (Millipore, Billerica, MA, USA) and probed for FLAG-BRG1 and MYC-p16INK4a with the mouse-anti-FLAG antibody or a rabbit anti-p16INK4a antibody.
For immunoprecipitations of endogenous BRG1 WMM1175_wtp16 cells were induced to express p16INK4a with 4 mM IPTG or mock treated for 72 hours; alternatively passage 20 WS-1 human dermal fibroblasts were used. Nuclear pellets were produced using low salt buffer (10 mM HEPES pH 7.9, 10 mM KCl, 0.1 mM EDTA, 0.1 mM EGTA, 1 mM DTT) and lyzed in IP-buffer with protease inhibitors. 5 mg of nuclear lysate was used for immunoprecipitation using a mouse anti-p16INK4a antibody or a matched mouse IgG. Protein antibody complexes were purified using protein-A-agarose (Santa Cruz, Santa Cruz, CA, USA). Immunoblotting was performed as described above, endogenous BRG1 was detected with a mouse anti-BRG1 antibody.
SW-13 cells were seeded at 105 cells on cover slips into 6-well plates and transfected 24 hours post seeding with 1 μg pCMV-MYC5b-p16 and 1.5 μg pcDNA3-BRG1-FLAG. Cells were fixed with methanol:acetone (1:1) for one minute, washed with PBS and probed with mouse anti-FLAG and rabbit anti-MYC antibodies and secondary Alexa Fluor 594 nm goat-anti-mouse and Alexa Fluor 488 nm goat anti-anti-rabbit antibodies (Invitrogen, Carlsbad, CA, USA). Images were taken with a BX-51 microscope and a SPOT camera and a FV1000 confocal microscope (Olympus, Center Valley, PA, USA).
WMM1175_p16INK4a cells silenced for BRG1 or expressing a control silencing molecule were seeded after 1, 3, 5 days induction with 4 mM IPTG at 4 × 104 cells on cover slips and fixed 8 hours later with 2% formaldehyde, 0.2% glutaraldehyde, 7 mM Na2HPO4, 1.5 mM KH2PO4, 140 mM NaCl, and 2.6 mM KCl and stained for SA-β-galactosidase, DAPI, Ki67, p16INK4a and BRG1. Images were taken with a BX-51 microscope and a SPOT camera (Olympus, Center Valley, PA, USA).
μg total cell lysate or nuclear lysate was separated on 15% SDS-PAGE gels or 5–15% gradient SDS-page gels, transferred to PVDF membranes (Millipore, Billerica, MA, USA) and probed for β-actin p16INK4a, BRG1, BRM, pRb, CDK6, phoshorylated pRb and p16INK4a.
Cell proliferation assay
WMM1175_p16INK4a cells silenced for BRG1 or expressing a control silencing molecule were seeded at 103 cells per well in 96 well plates. For each day one plate was assayed for MTS levels using a CellTitre 96 Aqueous One Solution Proliferation assay (Promega, Madison, Wisconsin, USA) according to the manufacturer's protocol using a Victor2 1420 Multilable counter (Perkin Elmer).
Cell cycle distribution
105 cells were seeded per well into 6-well plates and 24 hours later transfected with 1 μg pCMV-MYC5b-p16 and/or 1.5 μg pcDNA3-BRG1-FLAG or 2.5 μg pCMV-MYC5b vector plus 250 ng pEGFPspectrin. Total transfected DNA was adjusted to 2.75 μg with pCMV-MYC5b vector. Cells were harvested 48 hours post transfection and fixed in 4°C 70% ethanol for at least 1 hour and stained with 50 μg/ml propidium iodide and 50 μg/ml RNasesA in PBS and analyzed using a FACScalibur and ModFit software (Becton Dickinson, Franklin Lakes, NJ, USA). Percent S-phase change was calculated (percent S-phase vector control – percent S-phase sample) × 100/percent S-phase vector control.
WMM1175_wtp16 cells expressing a siRNA targeting BRG1 or a control siRNA molecule targeting luciferase were induced for 1, 3 or 5 days with 4 mM IPTG or mock treated. For each time point the cell cycle distribution was determined as described above.
Paraffin-embedded formaldehyde fixed primary (Breslow depth of invasion > 2 mm) or metastatic melanomas were cut at 4 mm onto Superfrost Plus slides and dried at 60°C for 1 hour. Sections were rehydrated through histolene and ethanol, heated in antigen retrieval buffer (Dako, Glostrup, Denmark) overnight at 70°C. Slides were placed in 3% hydrogen peroxide for 10 min then blocked for 1 hour with 50% normal goat serum (Serum Australis, Tamworth, NSW, Australia) diluted in 1% Tween 20/tris buffered saline (TTBS). Samples were incubated with primary antibodies for 1 hour at dilutions indicated. For p16INK4a and BRG1 slides were incubated for 30 minutes with biotinylated goat anti-rabbit (Dako, Glostrup, Denmark) diluted 1:400 in TBT (1%BSA in TTBS) and finally for 30 minutes with biotinylated-HRP/streptavidin (Invitrogen, Carlsbad, CA, USA) diluted in TBT. Antibodies were detected using 3,3'-diaminobenzidine tetrachloride (DAB; Invitrogen, Carlsbad, CA, USA), counter stained with Mayers Haemotoxylin (Sigma, Castle Hill, NSW, Australia), dehydrated and mounted using Normount (Fronine, Riverstone, NSW, Australia). For BRM, slides were incubated for 1 hour with Alexa Fluor goat anti-rabbit 594 nm (Invitrogen, Carlsbad, CA, USA) diluted 1:1000 with DAPI (Sigma, Castle Hill, NSW, Australia) diluted 1:2000 in TBT. Slides were washed then mounted using 3% n-propylgallate/50% glycerol. Primary antibodies used were mouse anti-p16 (1:200), rabbit anti-BRG1 (1:100) and rabbit anti-BRM1 (1:400). Sections were scored for staining intensity as 0 (equal to control), 1 (very weak positive)), 2 (positive) and 3 (strong positive) and the proportion of tumor tissue with positive staining as 0 (none), 1 (< 10%), 2 (< 50%) and 3 (> 50%). Tumors were considered to have detectable positive staining when the (intensity score) × (proportion staining score) was > 1. Only tumor samples with enough tissue for staining of at least two of the proteins were included in the study. Appropriate negative and positive controls were used with each batch of immunostaining. This study is covered by the Sydney South West Area Health Service Ethics Review Committee (RPAH Zone) Protocol No. X08-0155 & HREC Ref. 08/RPAH/262 – "Histological and Immunohistological Analysis of Melanocytic Tumours".
We thank Dr Mei Huang for providing the BRG1 expression plasmid. This study was supported by the University of Sydney Cancer Research Fund, the Cancer Council NSW, the Cancer Institute NSW and the National Health and Medical Research Council of Australia, NHMRC. HR is an NHMRC RD Wright Fellow, SH is supported by a scholarship from the German Academic Exchange Service, DAAD, and the Cancer Institute NSW, RS is a Cancer Institute NSW Clinical Research Fellow and LS is a Cameron Melanoma Research Fellow of the Melanoma and Skin Cancer Research Institute, University of Sydney.
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