TBX3 Represses TBX2 Under the Control of the PRC2 Complex In

Oh et al. Oncogenesis (2019) 8:27 https://doi.org/10.1038/s41389-019-0137-z Oncogenesis

ARTICLE Open Access TBX3 represses TBX2 under the control of the PRC2 complex in skeletal muscle and rhabdomyosarcoma Teak-Jung Oh1, Abhinav Adhikari 2, Trefa Mohamad3, Aiysha Althobaiti2 and Judith Davie2

Abstract TBX2 and TBX3 function as repressors and are frequently implicated in oncogenesis. We have shown that TBX2 represses p21, p14/19, and PTEN in rhabdomyosarcoma (RMS) and skeletal muscle but the function and regulation of TBX3 were unclear. We show that TBX3 directly represses TBX2 in RMS and skeletal muscle. TBX3 overexpression impairs cell growth and migration and we show that TBX3 is directly repressed by the polycomb repressive complex 2 (PRC2), which methylates histone H3 lysine 27 (H3K27me). We found that TBX3 promotes differentiation only in the presence of early growth response factor 1 (EGR1), which is differentially expressed in RMS and is also a target of the PRC2 complex. The potent regulation axis revealed in this work provides novel insight into the effects of the PRC2 complex in normal cells and RMS and further supports the therapeutic value of targeting of PRC2 in RMS.

Introduction phenotypes and the presence of myogenic markers such 4

1234567890():,; 1234567890():,; 1234567890():,; 1234567890():,; Rhabdomyosarcoma (RMS) is the most common soft as myogenic regulatory factors (MRFs) , yet these factors tissue pediatric sarcoma, which is thought to largely arise appear to be inactive in RMS5. from the skeletal muscle lineage1. The more common The T-box family of transcription factors are highly form of the disease is the embryonal subtype (ERMS), conserved and related throughout all metazoan lineages. characterized by loss of heterozygosity at the 11p15 locus, They share a common DNA-binding domain known as a region which harbors insulin-like growth factor 2. the T-box motif and participate in diverse types of orga- Alveolar RMS (ARMS) is the more aggressive form of nogenesis and developmental regulation6. The T-box RMS that is characterized by t(2;13)(q35;q14) or t(1;13) motif binds to the core sequence GGTGTGA known as (q36;q14) translocations. The translocations result in the T-element7. Distinct from most members of the T- chimeric transcripts that fuse the 5′ portion of the paired box family, TBX2 is known as a potent transcriptional box proteins 3 or 7 (PAX3 or PAX7), including an intact repressor that functions in both embryonic development, DNA-binding domain, to the transactivation domain of a and if deregulated, tumorigenesis8. The oncogenic forkhead transcription factor (FKHR), creating novel potential of TBX2 was first identified by its ability to − − PAX3-FKHR (t(2;13)(q35;q14)) or PAX7-FKHR (t(1;13) bypass cellular senescence in a Bmi / mouse embryo (q36;q14)) fusion proteins2,3. RMS is diagnosed by fibroblast model9. TBX2 has been shown to promote cell observation of unique skeletal muscle cell morphology proliferation and block cellular senescence by repressing CDKN1A (p21), CDKN2A (p14/19ARF)10, and PTEN11. Another member of the T-box family, TBX3, is also Correspondence: Judith Davie ([email protected]) 1School of Molecular and Cellular Biology, University of Illinois at Urbana- characterized as a transcriptional repressor and develop- Champaign, Urbana, IL 61801, USA mental regulator12. TBX2 and TBX3 are highly related 2 Department of Biochemistry and Molecular Biology and Simmons Cancer and thought to have arisen from a gene duplication Institute, Southern Illinois University School of Medicine, Carbondale, IL 62901, 13 USA event . In the case of deregulation, TBX3 also acquires Full list of author information is available at the end of the article.

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the potential to become an oncogene in melanoma cells14. (RH30 and RH28) with an expression plasmid for TBX311. TBX3 has been found to be overexpressed in melanoma15 As anticipated, we observed that TBX3 was upregulated and several sarcomas16. TBX3 has been shown to promote (Fig. 1a). Upon the upregulation of TBX3, we found that tumor metastasis and the migration of breast cancer TBX2 was downregulated (Fig. 1b) in RH30, RH28, and cells17. In MCF-12A breast epithelial cells and B16 mouse RD cells. The degree of TBX3 overexpression in RMS melanoma cells, TBX3 has been shown to directly repress cells corresponded to the degree of TBX2 repression in TBX2 under the control of TGF-β118. Our lab has shown each cell line tested (Fig. 1a, b). The repression of TBX2 that TBX3 is expressed throughout skeletal muscle dif- by TBX3 was confirmed at the protein level in RD, RH28, ferentiation, whereas the expression of TBX3 in RMS cell and RH30 cell lines (Fig. 1c). For the RD2 cell line, RNA lines is almost completely abrogated11. results were inconsistent but the protein analysis con- The polycomb repressive complexes 1 and 2 (PRC1/2) firmed that TBX3 repression of TBX2 could be observed are critical in the precise and accurate regulation of in these cells as well (Fig. 1d). development in many physiological systems including To understand if TBX2 and TBX3 reciprocally repres- skeletal muscle19. PRC1 and PRC2 work synergistically sed each other’s expression, we asked if TBX2 could also and indispensably together for transcriptional repression repress TBX3. RMS cell lines were transiently transfected of target gene by ubiquitylation of histone H2A lysine 119 with a shRNA construct against TBX227 and assayed for residue and methylation of histone 3 lysine 27 (H3K27) expression of TBX2 and TBX3. As anticipated, we residues, respectively20. The catalytic subunit of PRC2 is observed that TBX2 was depleted (Fig. 1e), but saw no enhancer of zeste 2 (Ezh2)21. upregulation of TBX3 (Fig. 1f). Thus, depletion of TBX2 Many studies have revealed that EZH2 is overexpressed does not upregulate TBX3. In fact, TBX3 was even more in a large number of cancers22. In RMS, pharmacological strongly downregulated in the absence of TBX2, indicat- inhibition of EZH2 in ERMS resulted in reduced pro- ing that TBX2 may play a role in activating TBX3 in RMS. liferation and pharmacological inhibition or depletion of Our data showed that TBX3 represses TBX2 in RMS cell EZH2-induced myogenic differentiation in these cells23. lines so we asked if this expression correlation could also EZH2 depletion in ARMS has also been shown to inhibit be observed in additional sarcomas using available patient proliferation and initiate apoptosis24. JARID2, a founding data. Sarcoma expression data for TBX3 and TBX2 were member of Jumonji family of proteins, is known to be a downloaded from The Cancer Genome Atlas (TCGA) substoichiometric component of PRC2 that appears to (https://cancergenome.nih.gov/) database using GDAC function in targeting PRC2 activity25. JARID2 is highly Firehose. Two cohorts of the top one-third TBX3 and expressed in ARMS and contributes to the inhibition of TBX2 expressing samples were combined and duplicates differentiation in ARMS cells26. samples were removed. A correlation analysis was per- In this work, we show that TBX3 represses TBX2 under formed and a scatter plot was generated for TBX3 and the control of the PRC2 complex in RMS and skeletal TBX2 expression (Fig. 1g). TBX2 and TBX3 were found to muscle. RMS cells and proliferating skeletal muscle cells be inversely correlated (Spearman correlation coefficient contain high levels of EZH2 that represses TBX3 and (rs) = −0.5173, p < 0.0001, N = 113), which was also evi- allows TBX2 expression. Depletion of EZH2 upregulates dent from two distinct clusters on the heatmap with TBX3 which then down regulates TBX2. We also show opposite expression (N = 263) (Fig. 1h). that the early growth response gene EGR1 is correlated To characterize the cellular effects of TBX3 in RMS, we with the induction of differentiation and repressed by generated stable RH30 cell lines expressing TBX3 and PRC2 in ARMS. Discovery of this novel PRC2-TBX3- three independent clonal isolates were used for further TBX2 genetic axis has important implications for under- analysis. Expression of exogenous TBX3 was confirmed at standing the mechanisms that drive proliferation and both the mRNA level (Fig. 2a) and the protein level (Fig. differentiation in RMS and skeletal muscle. 2c). We found that stable overexpression of TBX3 resulted in high levels of TBX3 mRNA expression (Fig. 2a) and Results repressed TBX2 mRNA expression (Fig. 2b). The repres- TBX3 represses TBX2 sion of TBX2 by TBX3 could also be observed at the We have previously shown that TBX2 is highly protein level (Fig. 2c). TBX3 was detected with antibodies expressed in RMS while TBX3 is not11,27. In skeletal against TBX3 and the V5 epitope tag only present on muscle, TBX2 is expressed in proliferating myoblasts, but exogenous TBX3. In each case, the degree of over- sharply downregulated upon differentiation while TBX3 is expressed TBX3 correlated to the degree of TBX2 expressed throughout myogenesis and highly expressed repression, confirming that TBX3 represses TBX2. during differentiation11,27. To understand the potential To determine if the repression of TBX2 is directly role of TBX3 in RMS, we transiently transfected RMS cell mediated by TBX3, chromatin immunoprecipitation lines representing both ERMS (RD and RD2) and ARMS (ChIP) assays were performed using antibodies against

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Fig. 1 TBX3 represses TBX2 in RMS. a, b The expression construct pEF-TBX3 (TBX3) or pEF empty vector (EV) was transiently transfected into RD, RH28, and RH30 cell lines and assayed by qRT-PCR using primers against TBX3 (a) and TBX2 (b). Error bars, standard errors (S.E.) and ***p ≤ 0.001 versus EV. c Protein extracts from the transiently transfected isolates in (a) were used for western blot analysis with antibodies against the indicated proteins. d pEF-TBX3 (TBX3) and pEF (EV) were transiently transfected into RD2 cells and examined by western bot analysis against the indicated proteins. GAPDH was used as the loading control. e, f A short-hairpin RNA construct against TBX2 (shTBX2) or a scrambled control (scr) construct was transiently transfected into RD, RH28, and RH30 cell lines and assayed by qRT-PCR using primers against TBX2 (e) and TBX3 (f). Error bars, S.E. and ***p ≤ 0.001 versus EV. TBX3 and TBX2 are inversely correlated in sarcoma. (g) Scatter plot representation of mRNA expression of TBX3 and TBX2 in

sarcoma patients from The Cancer Genome Atlas (TCGA) (rs = −0.5173, p < 0.0001, N = 113). (h) mRNA expression data for TBX3 and TBX2 in sarcoma patients were clustered and downloaded using cBioPortal (N = 263). Red represents higher expression while blue represents decreased expression

TBX3 and primers against the TBX2 promoter and RH30 that the degree of enrichment was greatly enhanced in the cells either overexpressing TBX3 or vector only. We TBX3 overexpression cell line. found that TBX3 was enriched on the TBX2 promoter, conﬁrming that TBX3 directly represses the TBX2 pro- TBX3 overexpression impairs proliferation, migration, and moter (Fig. 2d). Intriguingly, this enrichment could be anchorage-independent growth of RH30 cells but does not observed in both RH30 vectors, only cells that express low promote differentiation endogenous levels of TBX3 and in RH30 cells over- Previous work in the lab has shown that TBX2 plays a expressing TBX3 (Fig. 2d). Given the low level of endo- critical role in maintaining oncogenic properties such as genous TBX3 expression in RMS cells, it is not surprising proliferation, migration, and anchorage-independent

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Fig. 2 TBX3 directly represses TBX2 in RH30 cells. a pEF-TBX3 (TBX3) or pEF-empty vector (EV) was stably transfected into RH30 cells and the expression of TBX3 in three independent isolates was assayed by qRT-PCR analysis. Error bars, S.E. and ***p ≤ 0.001 versus EV. b Expression of TBX2 in the cells described in (a) was measured by qRT-PCR analysis. Error bars, S.E. and **p ≤ 0.01, ***p ≤ 0.001 versus EV. c Protein extracts from the stably transfected isolates in (a) were used for western blot analysis with antibodies against the indicated proteins. GAPDH was used as a loading control. d TBX3 binds to the TBX2 promoter. ChIP assays were performed on RH30 EV and RH30 TBX3-1 cells with antibodies against TBX3 or IgG and primers to the TBX2 promoter. Error bars, S.E. and **p ≤ 0.01, ***p ≤ 0.001 versus IgG growth in RMS11,27. TBX3 has been implicated in onco- additional unknown mechanisms. The migration ability of genesis in breast cancer and melanoma14,17. To determine RH30 cells stably overexpressing TBX3 was assayed by a if TBX3 inhibited oncogenesis by repressing TBX2 or scratch wound assay and we found that TBX3 also instead had a TBX2 independent role in promoting inhibited cell migration as assayed by wound recovery oncogenesis in RMS cells, we assayed for the growth (Fig. 3d). The anchorage-independent growth of RH30 properties of RH30 cells expressing exogenous TBX3. We cells stably overexpressing TBX3 was examined by a soft found that proliferation was strongly inhibited by TBX3 agar assay and all cell lines showed an inhibition of and closely correlated with the degree of TBX3 expression anchorage-independent growth (Fig. 3e). Together, these in each clonal isolate (Fig. 3a). As TBX3 represses TBX2, results strongly suggest that TBX3 does not appear to which we showed to be required for proliferation27,we confer oncogenic properties in RMS cells and appears asked if restoration of TBX2 could reverse the effect of instead to function as a tumor suppressor by repressing TBX3 overexpression. We found that proliferation was TBX2. partially restored when TBX2 expression was rescued To investigate if overexpression of TBX3 in RH30 cells using TBX2 overexpression construct in TBX3 over- could promote differentiation as we observed with the expressing RH30 cells (Fig. 3b). Intriguingly, these cells depletion of TBX227, we subjected the TBX3 over- contained even higher levels of TBX2 than RH30 cells expressing cells to differentiation conditions. The cell (Fig. 3c), yet proliferation was not fully rescued. These lines were then assayed for expression of myosin heavy results strongly suggest that TBX3 does not indepen- chain (MyHC) by immunoﬂuorescence. RH30 cells dently promote proliferation in RMS cells and rather, expressing exogenous TBX3 did not show the presence of functions to inhibit proliferation by repressing TBX2 and MyHC positive cells (Fig. 3f). We also assayed for

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Fig. 3 (See legend on next page.)

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(see figure on previous page) Fig. 3 TBX3 inhibits oncogenic properties but does not promote differentiation of RH30 cells. a TBX3 inhibits proliferation. Identical number of cells were seeded and cells were counted every 2 days. Error bars, S.E. and *p ≤ 0.05 and ***p ≤ 0.001 versus EV. b TBX2 partially rescues the proliferation defect. The RH30 TBX3-1 cell line was transiently transfected with pEF TBX2 (TBX2) or pEF empty vector (EV) and assayed for proliferation as in (a) except cell counts were performed every 24 h. c mRNA expression of TBX3 and TBX2 in the cells shown in (b) was assayed by qRT-PCR. Error bars, S.E. and ***p ≤ 0.001 versus EV. d TBX3 inhibits migration. Scratch assays were performed on RH30 cells overexpressing TBX3 (TBX3) or pEF (EV). Images were taken at ×100 magnification. Migration rate is quantitated in the right panel. Error bars, S.E. and **p ≤ 0.01, ***p ≤ 0.001 versus EV. e Soft agar assays on RH30 TBX3-1 and EV cell lines. Images were taken at ×100 magnification and the number of colonies was quantified by counting in five random fields. Error bars, S.E. and ***p ≤ 0.001 versus EV. f RH30 overexpressing TBX3 cell lines were assayed by immunofluorescence with myosin heavy chain antibodies (MyHC) following 4 days of differentiation (D4). Images were taken at ×100 and scale bars represent 100 μm. DAPI was used to stain nuclei. g mRNA expression of differentiation specific genes is not upregulated in RH30 cells overexpressing TBX3 in the presence (D4) or absence (UD) of differentiation conditions as assayed by qRT-PCR. Error bars, S.E.

additional markers of differentiation such as TROPONIN2 EZH2 is upregulated in RMS and targets TBX3 (TNNI2) and MYOGENIN (MYOG) in cells under normal As has been previously shown23,24,26, the catalytic sub- growth conditions and under differentiation conditions. unit of the PRC2 complex, EZH2, is upregulated in RMS We found no upregulation of these markers under either cell lines when compared to C2C12 cells (Fig. 5a). We condition (Fig. 3g). These data show that TBX3 expres- asked if depletion of EZH2 in RH30 cells altered the sion in RH30 cells is not sufficient to induce myogenic expression of TBX2 or TBX3. EZH2 mRNA was depleted differentiation. with a shRNA construct (shEZH2) in RH30 cells and stable cell lines expressing shEZH2 were selected. Two Overexpression of TBX3 impairs proliferation and clones were further characterized and we noted that in migration and promotes differentiation of RD cells both cell lines, EZH2 was modestly depleted at the level of Our results suggested that TBX3 functions as a tumor mRNA (Fig. 5b) and protein (Fig. 5e). These results are in suppressor in ARMS by repression of TBX2. To extend contrast to results obtained with a transient approach these finding to the ERMS subtype, we established stable with siRNA against EZH2 in RH30 cells, which resulted in cell lines overexpressing TBX3 in RD cells and char- a severe downregulation of EZH2 and apoptosis24. Con- acterized three independent clonal isolates. We found sistent with these results, we found that the cell lines that exogenous TBX3 mRNA was highly expressed in recovered in our approach were only modestly depleted each cell line (Fig. 4a) and TBX2 mRNA was repressed for EZH2. We found that TBX3 mRNA was upregulated (Fig. 4b). Cell proliferation assays showed an impaired upon EZH2 depletion in RH30 cells (Fig. 5c). We then proliferation capability of these cells which correlated examined TBX2 mRNA expression and found that TBX2 with the degree of TBX3 overexpression and TBX2 mRNA was accordingly downregulated (Fig. 5d). These repression (Fig. 4c). Scratch wound assays also showed results were confirmed at the protein level as well (Fig. reduced migration in RD cells expressing exogenous 5e). TBX3 (Fig. 4d). Thus, TBX3 appears to function as an To examine the effects of EZH2 depletion in RH30 cells, inhibitor of cell growth and migration in both ERMS and we examined cell growth properties of the cells. Cell ARMS cells. To determine if TBX3 could promote dif- proliferation assays showed that depletion of EZH2 ferentiation in RD cells, cells overexpressing TBX3 were inhibited RH30 cell growth (Fig. 5f). The migration and subjected to differentiation conditions. Cells were assayed motility of RH30 cells were examined by a scratch assay for the expression of MyHC by immunofluorescence and and both of the EZH2 depletion lines showed slower we found that MyHC cells were observable when TBX3 scratch recovery than the control (Fig. 5g). Anchorage- was expressed (Fig. 4e). Not only was the presence of independent growth was assayed by soft agar assays and MyHC observed but multinucleate myotubes could also showed that EZH2 depletion inhibited anchorage- be observed upon TBX3 overexpression. When expres- independent growth (Fig. 5h). Thus, depletion of EZH2 sion of differentiation specific genes including TNNI2 inhibits proliferation, migration, and anchorage- (Fig. 4f) and MYOG (Fig. 4g) were examined, upregula- independent growth of RH30 cells. tion of these genes was observed. MYOG was also We next asked if depletion of EZH2 could promote assayed at the protein level and the degree of MYOG differentiation in RH30 cells. No overt signs of differ- upregulation closely correlated with the level of upregu- entiation were observed in the earlier siRNA approach lated TBX3 (Fig. 4h). Together, these results show that which led to apoptosis24. In this prior study, cells were TBX3 can induce differentiation in RD cells, in contrast grown in growth medium. Here, RH30 cells modestly to what was seen in RH30 cells. depleted for EZH2 were subject to differentiation

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Fig. 4 (See legend on next page.)

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(see figure on previous page) Fig. 4 TBX3 inhibits proliferation and promotes differentiation in RD cells. a, b RD cells were transfected with pEF-TBX3 (TBX3) or pEF (EV) and three independent clones were characterized. mRNA expression of TBX3 (a) and TBX2 (b) was assayed by qRT-PCR. Error bars, S.E. and **p ≤ 0.01, ***p ≤ 0.001 versus EV. c TBX3 inhibits proliferation. RD cells overexpressing TBX3 shown in (a) were assayed for proliferation by a cell counting assay. Error bars, S.E. and **p ≤ 0.01, ***p ≤ 0.001 versus EV. d Scratch assays were performed on RH30 cells overexpressing TBX3 (TBX3) or pEF (EV). Images were taken at ×100 magnification. Migration rate is quantitated in the right panel. Error bars, S.E. and *p ≤ 0.05, ***p ≤ 0.001 versus EV. e RD cells overexpressing TBX3 were assayed by immunofluorescence with antibodies against MyHC after 4 days differentiation (D4) DAPI was used to stain nuclei. Images were taken at ×100 and scale bars represent 100 μm. Nuclei per myofiber are quantitated in the right panel. f, g mRNA expression of TNNI2 (f) and MYOG (g) were examined in RD cells overexpressing TBX3 by qRT-PCR after 4 days of differentiation (D4). Error bars, S.E. and *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001 versus EV. h Protein extracts from RD cells as in (f, g) were used for western blot assays with antibodies against MYOG. GAPDH was used as a loading control

conditions and examined for MyHC by immuno- the results obtained here, we examined EGR1 mRNA fluorescence. We found that RH30 cells depleted for expression in RH30 cells depleted for EZH2 and found EZH2 showed MyHC expression, indicating that deple- that EGR1 mRNA was upregulated upon EZH2 depletion tion of EZH2 could promote differentiation (Fig. 6a). We (Fig. 7a). A target gene of EGR1, NDRG1, was accordingly also found that mRNA expression of differentiation spe- upregulated as well (Fig. 7b). EGR1 mRNA expression was cific genes including TNNI2 (Fig. 6b), MYOD1 (Fig. 6c), also examined in RH30 cells expressing TBX3 and we saw and MYOG (Fig. 6d) were upregulated. The upregulation no upregulation of EGR1 mRNA (Fig. 7c), strongly sug- of MYOG and MyHC was confirmed at the protein level gesting that EGR1 is required to promote differentiation. as well (Fig. 6e). Taken together, the data clearly showed We also found that inhibition of EZH2 activity with the that modest depletion of EZH2 inhibits proliferation and chemical inhibitor GSK126 upregulated EGR1 mRNA in promotes differentiation. RH30 cells (Fig. 7d) and this upregulation could be EZH2 has been shown to promote differentiation in detected at the protein level as well (Fig. 7e). To confirm ERMS23 and we sought to understand if EZH2 also that EGR1 is a direct target of EZH2 in RH30 cells, we regulated TBX3 in these cells as well. shEZH2 constructs performed ChIP analysis and found that EZH2 (Fig. 7f) were transiently transfected into RD cells and we found and H3K27me (Fig. 7g) were found on the EGR1 promoter that depletion of EZH2 upregulated TBX3 mRNA and in these cells. We also asked if transient depletion of inhibited TBX2 mRNA (Fig. 6f). As our EZH2 depletion EGR1 could inhibit differentiation in RD cells stably results in RH30 differed from previously published work, expressing ectopic TBX3. We found that transient we sought to understand if our shRNA approach could depletion of EGR1 reduced the number and size of recapitulate what has been shown with an siRNA myofibers observed (Fig. 7h). While the depletion of EGR1 approach in RD cells23. RD cells were grown to con- achieved by this approach was modest, quantification of fluence, transiently transfected with shEZH2, and sub- the MyHC protein level showed that the downregulation jected to differentiation conditions for 2 days prior to of MyHC closely correlated with the depletion of EGR1 immunofluorescence with MyHC antibodies. We found (Fig. 7i). that MyHC was observed by immunofluorescence (Fig. 6g), consistent with what was observed with a siRNA The PRC2-TBX3-TBX2 genetic axis exists in normal skeletal approach23. Parallel samples were analyzed for EZH2 muscle depletion by mRNA expression (Fig. 6h). Stable cell lines To understand the relevance of these results in normal expressing shEZH2 could not be recovered, suggesting skeletal muscle, we used C2C12 cells depleted for JARID2 that EZH2 is critical for cell survival in ERMS cells. and EZH2 with shRNA constructs32 and assayed for the Our results showed that depletion of EZH2 promotes effect on Tbx3 and Tbx2 mRNA expression. We found differentiation in both ARMS and ERMS cells. However, that Tbx3 mRNA was upregulated when EZH2 (Fig. 8a) or overexpression of TBX3 only promoted differentiation in JARID2 (Fig. 8b) were depleted. Tbx2 mRNA was corre- ERMS cells. Recently, we have shown that the early spondingly downregulated upon the depletion of EZH2 response growth factor, EGR1, is highly expressed in (Fig. 8a) or JARID2 (Fig. 8b). ChIP assays were used to ERMS and weakly expressed in ARMS. Ectopic expression assay for the presence of histone H3 lysine 27 trimethy- of EGR1 in RH30 cells promoted differentiation28. EGR1 lation (H3K27me3) on the Tbx3 promoter and we found has been shown to be repressed by PRC2 under the that H3K27me3 was enriched on this promoter (Fig. 8c). control of the SS18-SSX fusion protein in synovial sar- Depletion of JARID2 resulted in a loss of this enrichment, comas29. EGR1 is also a target of the PRC2 complex in indicating that H3K27me3 on the Tbx3 promoter is acute myeloid leukemia30 and in chondrogenesis31.To dependent on JARID2 and the PRC2 complex (Fig. 8c). To determine if the expression of EGR1 was correlated with determine if TBX3 directly repressed TBX2 as we had

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A B C D E EZH2 TBX3 TBX2 RH30 RMS C2C12 1.2 25 1.2 1 20 *** 1 0.8 0.8 RH30 RH28 RD RD2 UD D6 * 15 * ** shEZH2-1 shEZH2-2 scr 0.6 0.6 old Expression old Expression EZH2 F 10

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Fig. 5 EZH2 is upregulated in RMS and targets TBX3. a Expression of EZH2 was examined by western blot assays for RMS cell lines and proliferating C2C12 cells (UD) and after 6 days of differentiation (D6). Replicate blots are quantitated in the lower panel. GAPDH was used as a loading control. b–d RH30 cells were transfected with shEZH2 and scrambled control (scr) and two independent clones were characterized following selection for stable isolates. Expression of EZH2 (b), TBX3 (c), and TBX2 (d) were assayed by qRT-PCR. Error bars, S.E. and *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001 versus scr. e Protein extracts from the cells in (b) were used for western blot assays with antibodies against the indicated proteins. GAPDH was used as a loading control. f Depletion of EZH2 impairs proliferation. RH30 cells as in (b) were seeded with the same number of cells and harvested for cell counts every 2 days. Error bars, S.E. and ***p ≤ 0.001 versus scr. g Scratch assays were performed for the RH30 cells as in (b). Images were taken at ×100 magnification. Migration rate was quantitated in the lower panel. Error bars, S.E. and *p ≤ 0.05, ***p ≤ 0.001 versus scr. h Soft agar assays were performed on the cells as in (b). Images were taken at ×100 magnification and the number of colonies quantified by counting in five random fields. Error bars, S.E. and ***p ≤ 0.001 versus scr

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A B C MyHCDAPI Merge 3X TNNI2 MYOD1 n 12 4 o

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Fig. 6 EZH2 represses TBX3 in RD cells and promotes differentiation of ARMS and ERMS cells. a RH30 shEZH2 cell lines were assayed by immunofluorescence with antibodies against MyHC after 4 days of differentiation. DAPI was used to stain the nuclei. Images were taken at ×100 and scale bars represent 100 μm. b–d The mRNA expression of TNNI2 (b), MYOD1 (c), and MYOG (d) were assayed in RH30 shEZH2 cell lines after 4 days of differentiation (D4) by qRT-PCR. Error bars, S.E. and **p ≤ 0.01, ***p ≤ 0.001 versus scr. e Protein extracts from RH30 cells as in (b) were used for western blot analysis with antibodies against the indicated proteins. GAPDH was used as the loading control. f EZH2 represses TBX3 in RD cells. RD cells were transiently transfected with shEZH2 and scr control and assayed by mRNA expression of the indicated genes by qRT-PCR. Error bars, S.E. and ***p ≤ 0.001 versus scr. g RD cells were transiently transfected with shEZH2 and scr control and assayed by immunofluorescence with antibodies against MyHC after 2 days of differentiation (D2). DAPI was used to stain nuclei. Images were taken at ×100 and scale bars represent 100 μm. Nuclei per myofiber are quantitated in the right panel. Error bars, S.E., and n.s. represent not statistically significant versus scr. h The mRNA expression of EZH2 in the cells shown in (g) was assayed by qRT-PCR and shown in the right panel. Error bars, S.E. and ***p ≤ 0.001 versus scr

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A B C EGR1 NDRG1 EGR1 6 *** 4 *** 1.2 5 3.5 ***

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Fig. 7 EGR1 is correlated with differentiation and repressed by EZH2 in RH30 cells. a, b mRNA expression of EGR1 (a) and NDRG1 (b) was assayed by qRT-PCR in RH30 shEZH2 and scr cell lines. Error bars, S.E. and ***p ≤ 0.001 versus scr. c TBX3 does not induce EGR1. mRNA expression of EGR1 was assayed by qRT-PCR. Error bars, S.E. and *p ≤ 0.05, **p ≤ 0.01 versus scr. n.s. represents not statistically significant. d, e Inhibition of EZH2 induces EGR1. RH30 cells were treated with the indicated concentration of GSK126 or DMSO (vehicle control) for 72 h and harvested for RNA (d) and protein (e). Error bars, S.E. and *p ≤ 0.05, ***p ≤ 0.001 versus DMSO. f EZH2 binds the EGR1 promoter. ChIP assays were performed on RH30 cells using antibodies against EZH2 and primers against the EGR1 promoter. Error bars, S.E. and ***p ≤ 0.001 versus IgG. g The EGR1 promoter contains H3K27me3. ChIP assays were performed on RH30 cells using antibodies against H3K27me3 and primers against the EGR1 promoter. Error bars, S.E. and ***p ≤ 0.001 versus IgG. h Stable RD cell lines overexpressing TBX3 were transiently transfected with shEGR1 or scr when confluent and assayed by immunofluorescence for MyHC after 2 days of differentiation conditions (D2). DAPI was used to stain nuclei. Images were taken at ×100 and scale bars represent 100 μm. Nuclei per myofiber are quantitated in the right panel. Error bars, S.E. and **p ≤ 0.01 versus scr. i Cells as in (h) were analyzed for protein expression with the indicated antibodies and replicate blots are quantitated in the right panel. TUBULIN was used as the loading control. Error bars, S.E. and **p ≤ 0.01 versus scr. n.s. represents not statistically significant

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A TBX3 TBX2 B TBX3 TBX2 14 1.2 1.2 e 10

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C Tbx3 promoter D E F 0.06 Tbx2 promoter 0.009 Tbx2 *** *** * C2C12 3.5 0.05 scr 0.008 scr *** 3 shJarid2 0.007 ssion shJarid2 e 0.04 ** EV Tbx3 KO 0.006 2.5 0.03 ut 0.005 2 TBX3

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I J

Tbx2 DIFFERENTIATION 1.4 ns 1.2 TBX3 1 p21 / p14 0.8 /PTEN 0.6 MYOBLAST 0.4 PRC-2 0.2 TBX2 Relative Gene expression 0 DMSO GSK126 RMS CELLS C2C12 Tbx3 KO PROLIFERATION MYOTUBE

Fig. 8 JARID2 and EZH2 directly repress TBX3 in skeletal muscle. a, b C2C12 cells were transfected with shEzh2 (a) or shJarid2 (b) and scr control. Stable isolates were assayed by qRT-PCR for mRNA expression of Tbx2 and Tbx3. Error bars, S.E. and, ***p ≤ 0.001 versus scr. c Methylation of the Tbx3 promoter is dependent on JARID2. ChIP assays were performed on C2C12 cell lines with scr or shJarid2 using antibodies against H3K27me3 and primers to the Tbx3 promoter. Error bars S.E. and ***p ≤ 0.001 as indicated. d TBX3 binds the Tbx2 promoter. ChIP assays were performed on C2C12 cell lines with scr or shJarid2 using antibodies against TBX3 and primers to the Tbx2 promoter. Error bars S.E. and *p ≤ 0.05, **p ≤ 0.01 as indicated. e Tbx3 KO cells analyzed by the western blot assay. Cells transfected with EV without Tbx3 guide RNA are labeled EV. f Tbx2 is upregulated upon Tbx3 deletion as assayed by qRT-PCR. Error bars S.E. and ***p ≤ 0.001. g Cells transfected with EV without Tbx3 guide RNA (EV) were treated with 1.25 μM GSK126 24 h post seeding for 48 h before assaying gene expression by qRT-PCR. Error bars S.E. and ***p ≤ 0.001 as indicated. h Inhibition of EZH2 does not lead to repression of Tbx2 in Tbx3 depleted cells. Tbx3 KO cells and control (EV) were treated and assayed for gene expression as in (g). Error bars S.E. and ***p ≤ 0.001 versus EV. n.s. represents not statistically signiﬁcant. i Inhibition of EZH2 does not alter Tbx2 expression in Tbx3 depleted cells. Tbx3 KO cells were treated and assayed as in (g). n.s. represents not statistically signiﬁcant. j Model of the PRC2-TBX3-TBX2 genetic axis in rhabdomyosarcoma and skeletal muscle. The PRC2 complex is highly expressed during proliferation in myoblasts and RMS (in orange) which represses TBX3 and allows TBX2 expression, which represses p21/p14/PTEN that are required for differentiation (in green). A decline in PRC2 expression derepresses TBX3 which represses TBX2 and promotes differentiation and myotube formation in skeletal muscle

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seen in RH30 cells, we performed ChIP assays with anti- differentiation19, this maintains repression of the antibodies against TBX3 and primers against the Tbx2 proproliferative TBX3 and expression of the proproliferative moter. We found that TBX3 binds the Tbx2 promoter and TBX2 when PRC2 is expressed. In RMS, deregulated that this binding is enriched upon depletion of JARID2, PRC2 activity leads to constitutive expression of TBX2 which induces TBX3 expression (Fig. 8d). To confirm the which functions to drive proliferation by direct repression PRC2-TBX3-TBX2 axis in skeletal muscle, we ablated of CDKN1A, CDKN2A, and PTEN while inhibiting the Tbx3 in C2C12 cells using a CRISPR/Cas9 approach. activity of MYOD1 and MYOG27. Synthetic guides directed to the first and second exons of The repression of TBX3 by the PRC2 complex has been Tbx3 were used to generate individual stable cell lines that previously observed34. Genome wide profiling of a PRC2 were screened by genomic DNA analysis and protein component, SUZ12, and histone H3 trimethylated at expression by sequencing (data not shown) and western lysine 27 (H3K27me3), identified a group of developmental blot (Fig. 8e), respectively. The Cas9 containing plasmid regulator genes that are repressed by PRC2 to maintain without a guide sequence was used as the control (EV). pluripotency and thus poised for activation during ES cell Tbx2 mRNA was assayed in the Tbx3 KO cell line and we differentiation and TBX3 was included in this group of found that Tbx2 was upregulated, confirming that TBX3 developmental regulators34. These results are consistent represses TBX2 (Fig. 8f). To confirm that EZH2 represses with what we observe in this study. Tbx3, C2C12 EV cells were treated with the EZH2 inhi- TBX3 had previously been shown to represses TBX2 bitor, GSK126. We found that Tbx3 was upregulated expression by directly associating with the TBX2 pro- when EZH2 was inhibited (Fig. 8g). As a positive control moter in a breast cancer cell line, MCF-12A18. However, for GSK126, we also examined the expression of Myog, unlike what has been observed in those previous studies which has been shown to be upregulated immediately which showed that TBX3 has oncogenic potential, we upon the depletion of EZH233 and we found that Myog found no evidence that TBX3 promotes oncogenesis in was upregulated (Fig. 8g). To confirm the requirement of RMS. Instead, we found that TBX3 acts as a tumor sup- TBX3 to mediate the EZH2 activation of TBX2, we pressor in RMS, largely due to its repression of TBX2. treated EV and Tbx3 KO cells with GSK126. As antici- Upregulation of the PRC2 complex has been shown be pated, Tbx3 mRNA was not activated in the Tbx3 KO correlated with many cancer types35. JARID2, a non- cells upon EZH2 inhibition (Fig. 8h). TBX2 levels catalytic, substoichiometric component of the PRC2 remained high in the Tbx3 KO cells despite EZH2 inhi- complex, has been shown to be upregulated in RMS, bition, confirming that the loss of EZH2 is not sufficient where it promotes cell viability and inhibits differentia- to repress TBX2 in the absence of TBX3. The expression tion26. The catalytic subunit of EZH2 has been shown to of Myog was also examined here and we saw no upregu- be highly expressed in ERMS and siRNA-mediated lation of Myog in the Tbx3 KO cells (Fig. 8h). This result is depletion or pharmacological inhibition of EZH2 inhi- further support for the high levels of TBX2 in the Tbx3 bits proliferation and induce differentiation of ERMS23. KO cells as Myog is directly regulated by MYOD1 and we EZH2 was also shown to be required for viability of have shown that TBX2 profoundly inhibits MRF activ- ARMS by repressing FBX03224. Treatment of RH30 or RD ity27. We also compared Tbx2 expression in Tbx3 KO cells with siEZH2 leads to robust transient depletion of cells treated with vehicle control or GSK126 and found no EZH2 after 24 h and induced apoptosis but did not induce change in Tbx2 mRNA expression (Fig. 8i), again con- MYOGENIN or MYOD1. Transient inhibition of EZH2 firming that TBX3 is required for the loss of EZH2 to lead has been shown to induce MYOGENIN in skeletal muscle – to the repression of Tbx2. cells33,36 38. Our lab has recently shown that transient depletion of EZH2 or JARID2 does result in transient Discussion upregulation of MYOGENIN and MYOD1 as others have The role of TBX3 in regulating the tumorigenesis of seen, but sustained depletion of either factor leads to a RMS was unknown before our work. We show that TBX3 block in Wnt signaling, which inhibits MYOD1 and blocks acts to repress TBX2 in both RMS and normal muscle. differentiation32. RMS cells are deregulated in Wnt sig- TBX3 does not appear to promote oncogenesis of RMS naling39, so a similar mechanism is not anticipated in cells, unlike what has been observed in melanoma and RMS. Here, we found that RH30 cells with a modest breast cancer cells14,17. Our results reveal the novel sustained depletion of EZH2 generated by stable selection genetic axis of the PRC2 complex, TBX3, and TBX2 (Fig. of a shRNA construct against EZH2 led to a decrease in 8j). JARID2 recruits the PRC2 complex to repress TBX3, proliferation and an induction of differentiation, including which then relieves the TBX3 repression of TBX2, the upregulation of MYOD1 and MYOG. allowing TBX2 expression. In normal skeletal muscle, We show here that EZH2 represses TBX3, which allows where the PRC2 complex is highly expressed in muscle the expression of the potent oncogene TBX2. This genetic precursor cells and sharply downregulated upon axis also acts in skeletal muscle and provides a molecular

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explanation of previous results from our lab. We have negative prior to the study and following the study. C2C12 shown that TBX2 is expressed in proliferating myoblasts myoblasts (ATCC, Manassas, VA, USA) were grown in and is sharply downregulated upon differentiation, while DMEM supplemented with 10% FBS. To induce differ- TBX3 is expressed throughout myogenesis and upregu- entiation, cells were grown to 90% confluence and the lated during differentiation11. Here, we establish that the media switched to DMEM supplemented with 2% horse down regulation of EZH2 upon skeletal muscle differ- serum (Hyclone, Fisher Scientific). Cells were grown in entiation leads to the upregulation of TBX3 and the differentiation medium for the number of days indicated in subsequent downregulation of TBX2. each experiment. Our results also showed that TBX3 is not sufficient to induce myogenic differentiation in ARMS, while depletion Drugs of EZH2 is sufficient to promote differentiation. These GSK126 (Cayman Chemicals) was dissolved in DMSO results were correlated with the expression of EGR1, at a concentration of 28.5 mM and 0.1% DMSO was used which we have shown acts as a tumor suppressor in RMS as the vehicle control. Blasticidin (Corning, Fisher Sci- that can induce differentiation in ARMS upon ectopic entific) was dissolved in ddH2O at 10 mg/ml and used at expression28. We found that EGR1 is a target of EZH2 10 μg/ml in RH30 cells and 2 μg/ml in RD cells. Pur- repression. EGR1 has also been shown to be a target of the omyocin (Corning, Fisher Scientific) was dissolved in 40 PAX-FOXO1 fusion that characterizes ARMS . Thus, ddH2O at 10 mg/ml and used at 2 μg/ml in all cell lines. EGR1 is both repressed by EZH2 and destabilized by Drugs were diluted in DMEM for use at the indicated PAX3-FOXO1 in ARMS. It remains to be determined if concentrations. ERG1 is a target of PRC2 in ERMS cells, but it may be that the lack of PAX3-FOXO1 in these cells is sufficient to Plasmids allow EGR1 expression. Consistent with this hypothesis, TBX3 was cloned into the pEF TOPO (Invitrogen, we observed that the rescue of EGR1 expression by EZH2 Thermo Fisher) vector as previously described11. pEF was by inhibition or depletion was relatively modest when used as the vector control and contains the gene for compared to the level of EGR1 present in ERMS cells. blasticidin resistance. shRNA against TBX2 and scr con- The PRC2-TBX3-TBX2 axis discovered in this work has trol were previously described27. shRNA constructs are in significant implications for both RMS and skeletal muscle. the pLKO.1 vector, which contains the gene for pur- The PRC2 complex serves as a central mediator between omycin resistance. proliferation and differentiation and understanding the functions of its many downstream targets offers new Cell transfection insight into how skeletal muscle grows and repairs. Cells were transfected with the indicated plasmids using TBX2 serves as a potent oncogene in RMS and many calcium phosphate according to standard protocols or other cancers when overexpressed and thus, under- Turbofect transfection reagent (Fisher Scientific) accord- standing the deregulation of TBX2 through the PRC2- ing to manufacturer’s protocol. Transient transfections TBX3-TBX2 axis offers insight in new approaches to were harvested at 48 h post transfection or as indicated. inhibit tumor growth of RMS and additional cancers. This Stable cell lines were made by transfecting cells with work also provides a mechanistic understanding of the linearized plasmids and selecting for drug resistant colo- effect of PRC2 in RMS and offers additional support to nies. Glass rings were used to isolate individual clones. the therapeutic value of PRC2 inhibition in RMS. Cells were recovered by trypsinization and transferred to single wells for propagation. Materials and methods Cell culture Chromatin immunoprecipitation (ChIP) RD and RH30 cells (ATCC) were grown in Dulbecco’s ChIP assays were performed and quantified as described modified Eagle medium (DMEM) supplemented with 10% previously41 with the following modifications: A total of fetal bovine serum (FBS) (Hyclone, Fisher Scientific, Wal- 1×107 cells were used for each immunoprecipitation tham, MA, USA) according to standard protocols. RD2 and and protein A agarose beads (Life Technologies, RH28 were obtained from Denis Guttridge, Medical Uni- Carlsbad, CA, USA) were used to immunoprecipitate the versity of South Carolina, and grown as described above. All antibody–antigen complexes. Primers are described in cell lines were authenticated by Bio-Synthesis (Lewisville, Supplemental Table 1 and the antibodies used are listed in TX, USA) using STR analysis on September 14, 2011. Cell Supplemental Table 2. The real time PCR was performed in lines are routinely screened for mycoplasma infection using triplicate. The results are represented as percentage of IP the LookOut Mycoplasma quantitative polymerase chain over input signal (% Input). All ChIP assays shown are reaction (qPCR) Detection Kit (Sigma, St. Louis, MO, USA). representative of four independent experiments. Standard All cell lines in this study were found to be mycoplasma error from the mean was calculated and plotted as error bar.

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Immunofluorescence indicated time. Scratch area was measured using ImageJ Cells were grown on cover slips, fixed with paraf- software (NIH). All scratch assays shown are representa- ormaldehyde, blocked with 10% goat serum, 1.0 % NP-40 tive of four independent experiments. in phosphate buffered saline (PBS) for 1 h and washed with PBS. Primary antibodies against MyHC(MF20, Soft agar assay Developmental Studies Hybridoma Bank, Iowa City, IA, Soft agar assays were carried out in 60 mm culture USA) were incubated overnight at 4 °C, washed with PBS, dishes in which 2 ml of 0.7% Noble agar (Fisher Scientific) and detected by Alexa Fluor-488 goat anti-mouse anti- in 1× DMEM with 10% FBS was overlaid with 2 ml of body (Life Technologies). Cell nuclei were stained by 0.35% agar in 1× DMEM with 10% FBS containing the incubating with 1 μM DAPI (Life Technologies) for 5 min. cells. Cells of each clone (3 × 105) were plated in triplicate. One milliliter of culture medium was added to the top of Proliferation assay each plate every 5 days and cells were grown at 37 °C in a 4 A total of 2 × 10 cells per well were seeded in six-well CO2 incubator for 30 days. The plates were stained with plates and on the indicated days, cells were counted under a 1 ml of 0.05% Crystal Violet (Fisher Scientific) for >1 h light microscope using a hemocytometer. Cell viability was and colonies were counted using a dissecting microscope. determined by trypan blue staining. Cell counting was All assays shown are representative of four independent performed in duplicate for four blinded biological replicates. experiments.

Quantitative real time PCR (qRT-PCR) Western blot Trizol (Life Technologies) was used for RNA extraction Cell extracts were made by lysing PBS washed cell from cells. Two micrograms of total RNA was treated pellets in the radioimmunoprecipitation assay buffer with DNase (Promega, Madison, WI, USA) and reverse supplemented with protease inhibitors (Complete pro- transcribed with MultiScribe MuLV reverse transcriptase tease inhibitor, Roche Diagnostics, Indianapolis, IN, USA) (Life Technologies). Forty nanograms cDNA was used for and clear lysates obtained by centrifugation. Protein qPCR amplification (Life Technologies) with SYBR green concentration was determined by the Bradford’s assay PCR master mix (Life Technologies). Negative controls (Bio-Rad, Hercules, CA, USA) and 50 µg of protein was included no RT samples where no reverse transcriptase loaded for each well of sodium dodecyl sulfate poly- was added for each RNA sample. All quantitative RT-PCR acrylamide gel electrophoresis. Resolved proteins were (qRT-PCR) was performed in triplicate and three inde- then transferred onto a PVDF membrane using a tank pendent RNA samples were assayed for each time point. blotter (Bio-Rad). Membranes were blocked with 5% milk qRT-PCR data were calculated using the comparative Ct in 1× Tris-buffered saline plus Tween 20 (TBST) and method (Life Technologies). Standard deviations from the followed by incubation with primary antibody overnight mean of the [Δ] Ct values were calculated from three at 4 °C. After washing membranes with 1× TBST, mem- independent RNA samples. Primers used are listed in branes were incubated with the corresponding secondary Supplemental Table 1. Where possible, intron spanning antibody, washed with 1× TBST and incubated with primers were used. qRT-PCR gene expression data are chemiluminescent substrate according to the manu- shown using the format of relative gene expression facture’s protocol (Pierce SuperSignal, Fisher Scientific) (Relative Fold Change). A fold change was calculated for and visualized by autoradiography and/or an iBright each sample pair and then normalized to the fold change FL1000 Imager (Invitrogen, Thermo Fisher Scientific, observed at HPRT and/or 18s rRNA. Waltham, MA, USA). Blots were quantitated using ImageJ software (NIH) or iBright Analysis software. Four biolo- Scratch assay gical replicates were performed for each western blot Cell mobility was assayed by scraping a straight line with assay. a 20 µl pipet tip on a monolayer of cells grown to 100% confluency. Cell debris was removed by washing. To obtain Genome editing of Tbx3 gene the same field during the image acquisition, marks were Guide sequences for the Tbx3 gene were chosen using created near the scratch line. The plate was placed in a an online CRISPR Design Tool (http://tools.genome- CO2 incubator at 37 °C for the indicated hours. Percentage engineering.org). Guide containing DNA oligonucleo- migration rates were calculated using following formula: tides was designed and cloned into pSpCas9(BB)-2A-Puro (PX459) V2.0 as described42. pSpCas9(BB)-2A-Puro % ¼ ½À = Ã Migration rate A0 At A0 100 (PX459) V2.0 was a gift from Feng Zhang (Addgene plasmid #62987; http://n2t.net/addgene:62987; RRID: where “A0” is the area of the scratch immediately after the Addgene_62987). Following selection of individual pur- scratch was made, and “At” is the area of the scratch after omycin resistant clones, genomic DNA was isolated using

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Trizol (Invitrogen, Thermo Fisher Scientific, Waltham, Supplementary Information accompanies this paper at (https://doi.org/ MA, USA) and PCR amplified targeted regions were 10.1038/s41389-019-0137-z). sequenced. Received: 28 November 2018 Revised: 12 February 2019 Accepted: 20 March 2019 The Cancer Genome Atlas (TCGA) sarcoma expression data analysis Using cBioPortal (www.cbioportal.org), mRNA expression data for TBX3 and TBX2 in sarcoma patients References (TCGA, Provisional, 265) were visualized on a heatmap 1. Dagher, R. & Helman, L. Rhabdomyosarcoma: an overview. Oncologist 4,34–44 (1999). and clustered. The mRNA expression Z-score (RNAseq 2. Barr, F. G. et al. Rearrangement of the PAX3 paired box gene in the V2 RSEM) was set to default (±2). For further analysis, paediatric solid tumour alveolar rhabdomyosarcoma. Nat. Genet. 3, mRNA expression (RNA Seq V2 RSEM) data of TBX3 113–117 (1993). 3. Galili, N. et al. 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Cancer 12, 117 (2013). We thank Ms. Yae Ji Kim for technical assistance with many of the described 15. Rodriguez,M.,Aladowicz,E.,Lanfrancone,L.&Goding,C.R.Tbx3repressesE- experiments. This study was initiated under grant 159609 from the American cadherin expression and enhances melanoma invasiveness. Cancer Res. 68, Cancer Society, Illinois Division to J.D. T.M. was supported by the Higher 7872–7881 (2008). Committee for Education Development Agency (HCED) in Iraq, which also 16. Willmer, T., Cooper, A., Sims, D., Govender, D. & Prince, S. The T-box supplied reagent costs to support this work. J.D. was supported by the transcription factor 3 is a promising biomarker and a key regulator of the National Institute of Arthritis and Musculoskeletal and Skin Diseases (NIAMS) of oncogenic phenotype of a diverse range of sarcoma subtypes. Onco- the National Institutes of Health under Award Number RAR068622. The genesis 5, e199 (2016). content is solely the responsibility of the authors and does not necessarily 17. Peres, J. et al. The highly homologous T-box transcription factors, TBX2 and represent the official views of the National Institutes of Health. TBX3, have distinct roles in the oncogenic process. Genes Cancer 1, 272–282 (2010). 18. Li, J. et al. The anti-proliferative function of the TGF-beta1 signaling pathway Author details involves the repression of the oncogenic TBX2 by its homologue TBX3. J. Biol. 1School of Molecular and Cellular Biology, University of Illinois at Urbana- Chem. 289,35633–35643 (2014). Champaign, Urbana, IL 61801, USA. 2Department of Biochemistry and 19. Juan, A. H. et al. Polycomb EZH2 controls self-renewal and safeguards the Molecular Biology and Simmons Cancer Institute, Southern Illinois University transcriptional identity of skeletal muscle stem cells. Genes Dev. 25, School of Medicine, Carbondale, IL 62901, USA. 3Biology Department, College 789–794 (2011). of Science, Salahaddin University, Erbil, Kurdistan 44002, Iraq 20. Spivakov, M. & Fisher, A. G. Epigenetic signatures of stem-cell identity. Nat. Rev. Genet. 8, 263 (2007). 21. Kuzmichev, A., Nishioka, K., Erdjument-Bromage, H., Tempst, P. & Reinberg, D. Conflict of interest Histone methyltransferase activity associated with a human multiprotein The authors declare that they have no conflict of interest. complex containing the enhancer of zeste protein. Genes Dev. 16,2893–2905 (2002). 22. Kim,K.H.&Roberts,C.W.TargetingEZH2incancer.Nat. Med. 22,128–134 Publisher’s note (2016). Springer Nature remains neutral with regard to jurisdictional claims in 23. Ciarapica, R. et al. Pharmacological inhibition of EZH2 as a promising differ- published maps and institutional affiliations. entiation therapy in embryonal RMS. BMC Cancer 14, 139 (2014).

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