The T-box transcription factor TBX3 drives proliferation by direct repression of the p21WAF1 cyclin-dependent kinase inhibitor
© Willmer et al. 2016
Received: 20 January 2016
Accepted: 12 April 2016
Published: 22 April 2016
TBX3, a member of the T-box family of transcription factors, is essential in development and has emerged as an important player in the oncogenic process. TBX3 is overexpressed in several cancers and has been shown to contribute directly to tumour formation, migration and invasion. However, little is known about the molecular basis for its role in development and oncogenesis because there is a paucity of information regarding its target genes. The cyclin-dependent kinase inhibitor p21WAF1 plays a pivotal role in a myriad of processes including cell cycle arrest, senescence and apoptosis and here we provide a detailed mechanism to show that it is a direct and biologically relevant target of TBX3.
Using a combination of luciferase reporter gene assays and in vitro and in vivo binding assays we show that TBX3 directly represses the p21WAF1 promoter by binding a T-element close to its initiator. Furthermore, we show that the TBX3 DNA binding domain is required for the transcriptional repression of p21WAF1 and that pseudo-phosphorylation of a serine proline motif (S190) located within this domain may play an important role in regulating this ability. Importantly, we demonstrate using knockdown and overexpression experiments that p21WAF1 repression by TBX3 is biologically significant and required for TBX3-induced cell proliferation of chondrosarcoma cells.
Results from this study provide a detailed mechanism of how TBX3 transcriptionally represses p21WAF1 which adds to our understanding of how it may contribute to oncogenesis.
Members of the T-box transcription factor family play crucial roles in embryonic development [1–4] and there is overwhelming evidence to show that they impact on several cancers by functioning as tumour suppressors and/or tumour promoters [5–13]. For example, TBX3 is critical for the formation of, amongst other structures, the heart, limbs and mammary glands and haploinsufficiency of the human TBX3 gene results in ulnar-mammary syndrome which is characterized by hypoplasia of these structures [14–16]. On the other hand, the overexpression of TBX3 is a feature of a wide range of cancers and it is a key driver of several oncogenic processes including proliferation, migration and invasion [9, 11, 12, 16–20]. There are also a few studies that have described a tumour suppressor role for TBX3 in cervical, uterine and bladder tumours, glioblastomas and fibrosarcomas [13, 16]. Considering the plethora of biological processes that it contributes to, TBX3 must clearly be regulating a number of genes. However, to date very few TBX3 target genes have been identified.
An intact p19ARF-Mdm2-p53-p21WAF pathway is important for protecting cells from becoming cancerous because it negatively regulates the cell cycle and promotes senescence and apoptosis . The cyclin-dependent kinase inhibitor p14ARF is responsible for the inhibition of MDM2-mediated ubiquitination and subsequent degradation of p53 [22, 23]. TBX3 is able to bypass senescence and/or evade apoptosis by transcriptionally repressing p14ARF/p19ARF via a variant T-element [8, 24–26]. This has led to the speculation that TBX3 may bind this degenerate T-element with low affinity and that it may impact on the above processes by targeting the p19ARF-Mdm2-p53-p21WAF pathway at multiple points for example by repressing p19ARF, p53 and p21WAF (hereafter referred to as p21) and/or activating Mdm2 . Indeed, there is evidence to suggest that TBX3 may also repress p21 transcriptionally  and we and others have shown an inverse correlation between the ability of TBX3 to promote proliferation of chondrosarcoma, mammary epithelial and hepatic progenitor cells and p21 levels [13, 29, 30]. However, the mechanism by which TBX3 regulates p21 has yet to be elucidated and whether this regulation is biological relevant still needs to be confirmed.
Here we show, using in vitro and in vivo assays, that p21 is a direct target of TBX3. Importantly, we provide a full characterisation of the mechanism by which TBX3 represses p21 and we demonstrate that this is required for TBX3′s pro-proliferative role in chondrosarcomas.
TBX3 is a direct transcriptional repressor of p21 gene expression
TBX3 binds the p21 promoter in vitro and in vivo
In order to validate this result in vivo, chromatin immunoprecipitation assays were performed. TBX3-bound DNA was immunoprecipitated from SW1353 control and SW1353 shTBX3 cells and the DNA was subjected to qRT-PCR with primers spanning a region of the p21 promoter containing the T-121 bp. The results show that in the control cells, there was an approximately 3.2 fold enrichment of TBX3 occupancy on the p21 promoter but not on the GAPDH control (Fig. 3d). As expected, this occupancy was significantly reduced in shTBX3 cells. These data suggest that, in vivo, TBX3 binds a region of the p21 promoter containing the T-121 bp.
Pseudo-phosphorylation of TBX3 at Serine-Proline 190 (SP190) regulates its ability to repress p21
Down-regulation of p21 by TBX3 mediates its pro-proliferative effects in chondrosarcoma cells
Pseudo-phosphorylation of TBX3 at SP190 reduces its ability to promote cell proliferation
Despite a growing list of reports implicating TBX3 in development and oncogenesis, little is known about the molecular basis for its role in these processes because there is a paucity of information regarding its target genes. Of relevance to this study is the evidence suggesting that during embryonic development and cancer, TBX3 impacts on cell proliferation by repressing key cell cycle regulators [26, 28, 29, 32–34]. For example, an intact p19ARF-Mdm2-p53-p21 pathway is important for protecting cells from immortalisation and results from a previous study revealed that depleting TBX3 in chondrosarcoma cells led to an increase in p19ARF, p53 and p21 protein levels which was accompanied by a decrease in cell proliferation . This raised the possibility that TBX3 may promote cell proliferation by disabling this pathway at multiple points for example by repressing p19ARF, p53 and p21 and/or activating Mdm2. Indeed, TBX3 has been reported to directly repress p14ARF/p19ARF and our observation that p21 levels increase when TBX3 is depleted in the mouse chondrosarcoma ATDC5 cells, which do not express p53 , implies that TBX3 must also repress p21 independently of p19ARF-p53. While this is consistent with other reports suggesting that p21 may be a direct TBX3 target gene [14, 35], the mechanism by which TBX3 regulates p21 and the biological context in which this regulation occurs has yet to be elucidated. The current study shows that p21 mRNA and protein levels increased when TBX3 was knocked down in chondrosarcoma cells and provides compelling in vitro and in vivo data to show that TBX3 directly represses p21 which is important for TBX3-induced cell proliferation.
This study provides a full characterisation of the repression of p21 by TBX3 in chondrosarcoma cells. Consistent with p21 being a direct TBX3 target gene, the TBX3 DNA binding domain (T-box) was shown to be required for this repression. Interestingly, Pflugfelder and Fischer also recently reported that two ulnar mammary syndrome associated TBX3 DNA binding domain mutations (H187Y and R130S/G129A) could both abrogate its ability to repress a p21 promoter driving a luciferase reporter . Furthermore, we show that a highly conserved T-element at -121 bp mediates, in part, the repression of p21 by TBX3. This site was previously shown to be required for the repression of p21 by TBX2 but we have been unable to detect TBX2 protein in ATDC5 or SW1353 cells and therefore it is unlikely to be regulating p21 in these cells . It would therefore be interesting to determine whether the highly homologous TBX2 and TBX3 have redundant functions in regulating the p21 promoter at this site in cancers where they are both expressed or whether the availability of cofactors may determine which one of the two regulates p21. The repression domain (RD1) in the C-terminus of the TBX3 protein was also shown to be required for the repression of p21. This is consistent with reports that have indicated that of the two repression domains in the TBX3 protein, RD1 is the dominant one . Indeed, RD1 was also recently reported to be required for the repression of TBX2 by TBX3 and it has previously been shown to be essential for the ability of TBX3 to inhibit apoptosis and immortalise primary embryonic fibroblasts [31, 35, 37]. Furthermore, the majority of ulnar mammary syndrome-associated TBX3 C-terminal mutants that exhibit loss of function have been reported to lack the dominant repression domain .
Using rescue assays in TBX3 knockdown and overexpression chondrosarcoma cell culture models, the repression of p21 by TBX3 was shown to be physiologically relevant because it was required for TBX3-induced cell proliferation. Importantly, a highly conserved serine-proline motif at amino acid 190 in the TBX3 DNA-binding domain (SP190) was shown to regulate its ability to bind and transcriptionally repress p21 and consequently, its ability to promote proliferation. Indeed, when the serine residue at this site was mutated to glutamic acid (E) to mimic phosphorylation, the ability of TBX3 to repress the p21 promoter was abrogated and this mutant could also not promote proliferation. SP motifs are consensus sites for many kinases including the MAP kinases  and CDKs  and this data suggest that post-translational mechanisms such as phosphorylation may be important to regulate TBX3 function. Indeed, there is evidence that phosphorylation of TBX3 regulates its ability to transcriptionally repress its target genes. For example, phosphorylation of TBX3 at SP692 and SP720 by the p38 mitogen-activated protein (MAP) kinase and AKT3 respectively can enhance its ability to transcriptionally repress its well-known target, E-cadherin [17, 40]. The kinase responsible for phosphorylating TBX3 at SP190 was not identified in this study however, we are currently investigating the possibility that it may be ERK which, like TBX3, can act as either an oncoprotein or a tumour suppressor . Furthermore, ERK has been shown to negatively regulate cell proliferation in human hepatoma cells [41, 42]. It is therefore possible that phosphorylation of TBX3 by ERK may provide a mechanism by which ERK can inhibit proliferation and future experiments should investigate this possibility because it has implications for our understanding of TBX3′s oncogenic role.
It is important to note that p21 expression and activity are tightly regulated both transcriptionally and post-transcriptionally and we therefore cannot exclude the possibility that, in addition to TBX3, p21 may be subject to regulation by other factors in our study. Indeed, in response to stimuli such as DNA damage, oxidative stress, cytokines and mitogens, p21 can be transcriptionally regulated by p53, Sp1, Sp3, p63, AP4, Klf, and c-Myc and post-translationally by E3 ubiquitin ligase complexes and phosphorylation by AKT1 [43, 44]. Furthermore, in chondrosarcomas as well as in other cancers, TBX3 has been implicated in several oncogenic processes including promoting cell proliferation, tumour formation, migration and invasion which suggest that it must be regulating several other target genes. Indeed, it is possible that in addition to p21, TBX3 may promote cell proliferation by repressing other negative regulators of the cell cycle such as PTEN, p14ARF/p19ARF and p53.
TBX3 is essential in development and has emerged as an important player in the genesis of several cancers where it directly contributes to proliferation, tumour formation, migration and invasion. However, little is known about the molecular basis for its roles in these processes because its full repertoire of target genes has not been identified. Here we show that TBX3 promotes proliferation by directly repressing the CDKI p21. This repression is shown to involve the TBX3 DNA binding domain and is mediated by a T-element at -121 bp of the p21 promoter. The significance of our findings may have implications for the design of anti-cancer drugs to treat TBX3-driven cancers.
The wild-type p21 luciferase reporter construct (WTp21) contains a 2.4 kb fragment of the human p21 promoter inserted upstream of a 2.6 kb luciferase (LUC) reporter gene . This construct was kindly provided by Professor Chris Marshall of the Institute of Cancer Research, UK. The mutant p21 luciferase reporter construct (Mutp21) contains a disrupted T-element in the initiator of the p21 promoter generated by site-directed mutagenesis as previously described . The p21-pRc/CMV plasmid, which was prepared in the laboratory of Prof William Kaelin, was obtained from Addgene (Addgene plasmid 20814) [18, 46]. The human pCMV-TBX3-HA expression construct was kindly provided by Dr Christine Campbell of the Cleveland Clinic Foundation, USA. The TBX3 DNA-binding mutant (DBM) was generated by site-directed mutagenesis using the following primer 5′ CATTACCAAGTCGGGAgGGgGAATGTTTCCTCCATTTAAAG 3′. Using the pCMV-TBX3-HA expression construct, the TBX3 C-Terminal region was removed by cutting with BglII (at position 1121 bp) and SpeI (50 bp after stop translation site) thus generating the TBX3 N-terminal construct. The TBX3 SP190A and E mutant constructs were generated by site-directed mutagenesis using the following primers: SP190A: 5′CATTCACCCGGACgcgCCCGCTACTGGGGAACAGTGGATG3′, SP190E: 5′CATTCACCCGGACgagCCGGCTACTGGGGAACAGTGGATG3′.
Cell lines and culture condition
SW1353 chondrosarcoma cells (American Type Culture Collection) were cultured in Dulbecco’s modified Eagle’s medium (DMEM) (Highveld Biological), supplemented with 10 % fetal bovine serum, 100 U/ml penicillin and 100 µg/ml streptomycin. ATDC5 chondrosarcoma cells were maintained in Dulbecco’s modified Eagle medium: nutrient mixture F-12 (DMEM/F12) (1:1) supplemented with 5 % FBS, 100 U/ml penicillin, 100 µg/ml streptomycin, 10 μg/ml human transferrin (T Sigma Alderich, USA), and 3 × 10−8 M sodium selenite (Sigma Alderich, USA). All cells were maintained in a 37 °C incubator (95.0 % air/5 % CO2, 65 % humidity).
Generation of stable TBX3 knock down cell lines
Stable knockdown of TBX3 in SW1353 and ATDC5 cell lines was achieved with the following siRNA sequence cloned into the pSuper.neo/GFP (Oligoengine) shRNA expression vector as previously described [11, 13]. Cells were transfected with the pSuper.neo/GFP expression vector containing sequences targeted to TBX3, or the nonspecific control using FugeneHD (Roche Diagnostics, Basel, Switzerland). Stable transfectants were selected by the addition of 400 μg/ml (ATDC5 cells) or 800 μg/ml (SW1353 cells) of G418 (Invitrogen) to the growth medium.
Generation of stable TBX3 overexpression cell lines
Tbx3 overexpressing cell lines were generated by stably transfecting SW1353 cells with a FLAG-tagged pCMV-empty vector, pCMV-Tbx3 (kindly provided by Professor Colin Goding at the Ludwig Institute of Cancer Research, UK) using FuGENE®HD (Roche Molecular Biochemicals, Mannheim, Germany) according to the manufacturer’s instructions. Stable transfectants were selected for with 800 μg/ml G418 antibiotic (Promega, USA). Effective overexpression of Tbx3 was assessed by western blot analysis.
Transfections and luciferase assays
SW1353 or ATDC5 cells were plated at 7 × 104 cells per well in a 12 well plate. One day later cells were transiently transfected using XtremeGENE HP DNA transfection reagent (Roche), according to the manufacturer’s instructions. 200 ng of the relevant p21 reporter plasmid and varying amounts of the TBX3 expression construct or empty vector were transfected. 10 ng of the RL-CMV vector (Promega), which contains the cytomegalovirus promoter driving the expression of a renilla reporter, was used as an internal control for transfection efficiency. The Dual-Luciferase Reported Assay System (Promega, USA) and a Luminoskan Ascent luminometer (Thermo Labsystems) were used to determine relative luciferase and renilla activity.
Small interferring RNA (siRNA) sequences and transfections
Transient knockdown of p21 expression was achieved using ON-TARGETplus SMARTpool Human CDKN1A siRNA (sip21#1) (Thermo Scientific) and sip21#2 (SI00299810) from Qiagen. shTBX3, shCtrl, FLAG-TBX3 and FLAG-Empty cells were transfected siRNAs to p21 or control ((non-silencing) siRNA (1027310; Qiagen) using HiPerFect (Qiagen) according to the manufacturer’s instructions.
Chromatin immunoprecipitation assay (ChIP)
90 % confluent SW1353 cells were fixed with formaldehyde, washed in PBS and collected by centrifugation. Following this the chromatin was extracted and subjected to sonication to obtain DNA fragments of between 300 and 500 bp. The protein-bound DNA was then immunoprecipitated with A/G PLUS Agarose beads (Santa Cruz Biotechnology Inc) and an antibody against TBX3 (8ug, sc-17871, Santa Cruz Biotechnology Inc.) or IgG (8ug, negative control, Santa Cruz Biotechnology) was added overnight. The beads were then washed and the cross-links reversed by heating at 65 °C. The DNA to which TBX3 bound was purified using the PCR purification kit (Qiagen, USA) according to the manufacturer’s instructions. qRT-PCR analysis was performed using primers spanning the T-element in the initiator of the p21 promoter (Forward-5′-GGGGCGGTTGTATATCAGGG-3′, Reverse-5′-TCTCACCTCCTCTGAGTGCC-3′). Crossing values (Ct) of TBX3 and IgG DNA were adjusted by normalising against the Ct value of 1 % of input DNA and the ΔΔCt method was used to determine fold enrichment. The equation was used as follows: 2−(∆Ct1−∆Ct2) (∆Ct1 = TBX3, ∆Ct2 = IgG).
Western blot analyses
Cells were harvested and protein prepared as described previously . Primary antibodies used were as follows: rabbit polyclonal anti-TBX3 (Zymed 42-4800 or Abcam 99302) and polyclonal rabbit anti-p38 (Sigma-Aldrich, M0800), monoclonal mouse anti-HA (Sigma-Aldrich, H9658), and polyclonal rabbit anti-p21 (Santa Cruz Biotechnology Inc., C-19). Signal was detected using peroxidase-conjugated goat anti-mouse or anti-rabbit antibodies (1:5000) and visualised by enhanced chemiluminescence (ECL) (Pierce, USA).
Quantitative real-time polymerase chain reaction (qRT-PCR)
RNA was extracted from cells using the high pure RNA isolation kit (Roche, Germany) according to the manufacturer’s instructions and reverse transcription performed using 1ug of RNA and the InProm-II™ reverse transcription system (Promega A3800, USA) according to the manufacturer’s instructions. The reactions were carried out using 2 μl of cDNA, Power SYBR® Green PCR Master Mix (Applied Biosystems, UK) and primers to amplify the human TBX3 and p21 genes (Qiagen, USA). qRT-PCR was performed using the StepOnePlus™ PCR system (Applied Biosystems) using the following parameters: denaturation for 15 min at 95 °C and combined annealing and extension for 35 cycles at 60 °C for 1 min. Samples were prepared in triplicate and non-template controls were run to detect contamination or non-specific amplification. The 2−ΔΔCt method employed to analyse results and relative mRNA expression levels of TBX3 and p21 were normalised to mRNA levels of glucuronidase-beta (GUSB).
DNA affinity immunoblot (DAI)
Oligonucleotide probes p21WT (5′-CTCGAGGCCAGCTGAGGTGTGAGCAGCTGCCG-3′) and p21MUT (5′-CTCGAGGCCAGCTGAGGCTCGAGCAGCTGCCG-3′) were annealed with their complementary strands. SW1353 were harvested and nuclear extracts isolated as described previously . Alternatively cells were transfected with pCMV empty vector, WT TBX3, TBX3 SP190A or TBX3 SP190E constructs and whole cell lysate harvested. 20 µg of this protein was analysed by western blotting to confirm that the constructs were expressed at equal levels in the cell before continuing. For the DAI, biotinylated p21 probe was immobilized on Dynabeads Streptavidin (Dynal Invitrogen) according to the manufacturer’s instructions. For each reaction, 40 μg protein extract was incubated with 1 μg DNA probe in binding buffer (20 mM Tris–HCL;pH7.6, 50 mM MaCl, 1 mM MgCl2, 0.2 mM EDTA, 5 % glycerol, 0.5 mM DTT and 2 µg poly dI/dC) in a final volume of 200 µl. The protein and probe bound beads were pulled down with a magnet, extensively washed with binding buffer and then boiled in 25 μl of 2 × SDS-PAGE sample buffer before SDS-polyacrylamide gel electrophoresis followed by immunoblotting with an antibody to HA.
Growth curve and MTT assays
Short-term growth of cells was compared to that of controls over a 3-day period, as described previously . 3 × 104 cells were seeded in triplicate in 24-well plates on day 0 and treated with siRNA for 2–3 days. Alternatively cell proliferation was assessed using the MTT cell proliferation assay kit. At the appropriate times, 10 μl MTT reagent was added to each well to a final concentration of 0.5 mg/ml and cells were incubated at 37 °C in 9 % CO2 for 4 h. To each well, 100 μl of solubilization solution was added and the cells incubated overnight, after which the absorbance measured at 595 nm. Cell growth graphs were constructed of the cell viability as absorbance measured for each transfection treatment.
Statistical significance was determined using the student T test (Excel, Windows XP). Significance was accepted at p < 0.05.
SP designed this study and edited and finalised the manuscript. SH, TW and JP carried out the experiments and drafted the manuscript. SH and TW contributed equally to the work published in this manuscript. All authors read and approved the final manuscript.
This work was supported by grants from the SA Medical Research Council, the National Research Foundation (NRF), Cancer Association of South Africa (CANSA), Cancer Research Initiative of South Africa (CARISA) and the University of Cape Town. The content is solely the responsibility of the authors and does not necessarily represent the official views of the funding agencies.
The authors declare that they have no competing interests.
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