TBX2 controls a proproliferative gene expression program in melanoma.

Senescence shapes embryonic development, plays a key role in aging, and is a critical barrier to cancer initiation, yet how senescence is regulated remains incompletely understood. TBX2 is an antisenescence T-box family transcription repressor implicated in embryonic development and cancer. However, the repertoire of TBX2 target genes, its cooperating partners, and how TBX2 promotes proliferation and senescence bypass are poorly understood. Here, using melanoma as a model, we show that TBX2 lies downstream from PI3K signaling and that TBX2 binds and is required for expression of E2F1, a key antisenescence cell cycle regulator. Remarkably, TBX2 binding in vivo is associated with CACGTG E-boxes, present in genes down-regulated by TBX2 depletion, more frequently than the consensus T-element DNA binding motif that is restricted to Tbx2 repressed genes. TBX2 is revealed to interact with a wide range of transcription factors and cofactors, including key components of the BCOR/PRC1.1 complex that are recruited by TBX2 to the E2F1 locus. Our results provide key insights into how PI3K signaling modulates TBX2 function in cancer to drive proliferation.

In situ regeneration of inner hair cells in the damaged cochlea by temporally regulated co-expression of Atoh1 and Tbx2.

Cochlear inner hair cells (IHCs) are primary sound receptors, and are therefore a target for developing treatments for hearing impairment. IHC regeneration in vivo has been widely attempted, although not yet in the IHC-damaged cochlea. Moreover, the extent to which new IHCs resemble wild-type IHCs remains unclear, as is the ability of new IHCs to improve hearing. Here, we have developed an in vivo mouse model wherein wild-type IHCs were pre-damaged and nonsensory supporting cells were transformed into IHCs by ectopically expressing Atoh1 transiently and Tbx2 permanently. Notably, the new IHCs expressed the functional marker vGlut3 and presented similar transcriptomic and electrophysiological properties to wild-type IHCs. Furthermore, the formation efficiency and maturity of new IHCs were higher than those previously reported, although marked hearing improvement was not achieved, at least partly due to defective mechanoelectrical transduction (MET) in new IHCs. Thus, we have successfully regenerated new IHCs resembling wild-type IHCs in many respects in the damaged cochlea. Our findings suggest that the defective MET is a critical barrier that prevents the restoration of hearing capacity and should thus facilitate future IHC regeneration studies.

Revisiting the Potency of Tbx2 Expression in Transforming Outer Hair Cells into Inner Hair Cells at Multiple Ages In Vivo.

The mouse auditory organ cochlea contains two types of sound receptors: inner hair cells (IHCs) and outer hair cells (OHCs). Tbx2 is expressed in IHCs but repressed in OHCs, and neonatal OHCs that misexpress Tbx2 transdifferentiate into IHC-like cells. However, the extent of this switch from OHCs to IHC-like cells and the underlying molecular mechanism remain poorly understood. Furthermore, whether Tbx2 can transform fully mature adult OHCs into IHC-like cells is unknown. Here, our single-cell transcriptomic analysis revealed that in neonatal OHCs misexpressing Tbx2, 85.6% of IHC genes, including Slc17a8, are upregulated, but only 38.6% of OHC genes, including Ikzf2 and Slc26a5, are downregulated. This suggests that Tbx2 cannot fully reprogram neonatal OHCs into IHCs. Moreover, Tbx2 also failed to completely reprogram cochlear progenitors into IHCs. Lastly, restoring Ikzf2 expression alleviated the abnormalities detected in Tbx2+ OHCs, which supports the notion that Ikzf2 repression by Tbx2 contributes to the transdifferentiation of OHCs into IHC-like cells. Our study evaluates the effects of ectopic Tbx2 expression on OHC lineage development at distinct stages of either male or female mice and provides molecular insights into how Tbx2 disrupts the gene expression profile of OHCs. This research also lays the groundwork for future studies on OHC regeneration.

Tbx2 and Tbx3 regulate cell fate progression of the otic vesicle for inner ear development.

The morphogenesis of the otic vesicle (OV) to form inner ear organs serves as an excellent model system to understand cell fate acquisition on a single cell level. Tbx2 and Tbx3 (Tbx2/3) encode closely related T-box transcription factors that are expressed widely in the mammalian OV. Inactivation of both genes in the OV (Tbx2/3cKO) results in failed morphogenesis into inner ear organs. To understand the basis of these defects, single cell RNA-sequencing (scRNA-seq) was performed on the OV lineage, in controls versus Tbx2/3cKO embryos. We identified a multipotent population termed otic progenitors in controls that are marked by expression of the known otic placode markers Eya1, Sox2, and Sox3 as well as new markers Fgf18, Cxcl12, and Pou3f3. The otic progenitor population was increased three-fold in Tbx2/3cKO embryos, concomitant with dysregulation of genes in these cells as well as reduced progression to more differentiated states of prosensory and nonsensory cells. An ectopic neural population of cells was detected in the posterior OV of Tbx2/3cKO embryos but had reduced maturation to delaminated neural cells. As all three cell fates were affected in Tbx2/3cKO embryos, we suggest that Tbx2/3 promotes progression of multipotent otic progenitors to more differentiated cell types in the OV.

miR-486-5p Attenuates Steroid-Induced Adipogenesis and Osteonecrosis of the Femoral Head Via TBX2/P21 Axis.

Enhanced adipogenic differentiation of mesenchymal stem cells (MSCs) is considered as a major risk factor for steroid-induced osteonecrosis of the femoral head (SOFNH). The role of microRNAs during this process has sparked interest. miR-486-5p expression was down-regulated significantly in femoral head bone tissues of both SONFH patients and rat models. The purpose of this study was to reveal the role of miR-486-5p on MSCs adipogenesis and SONFH progression. The present study showed that miR-486-5p could significantly inhibit adipogenesis of 3T3-L1 cells by suppressing mitotic clonal expansion (MCE). And upregulated expression of P21, which was caused by miR-486-5p mediated TBX2 decrease, was responsible for inhibited MCE. Further, miR-486-5p was demonstrated to effectively inhibit steroid-induced fat formation in the femoral head and prevented SONFH progression in a rat model. Considering the potent effects of miR-486-5p on attenuating adipogenesis, it seems to be a promising target for the treatment of SONFH.

Cardiac Transcription Factors and Regulatory Networks.

Cardiac development is a fine-tuned process governed by complex transcriptional networks, in which transcription factors (TFs) interact with other regulatory layers. In this chapter, we introduce the core cardiac TFs including Gata, Hand, Nkx2, Mef2, Srf, and Tbx. These factors regulate each other's expression and can also act in a combinatorial manner on their downstream targets. Their disruption leads to various cardiac phenotypes in mice, and mutations in humans have been associated with congenital heart defects. In the second part of the chapter, we discuss different levels of regulation including cis-regulatory elements, chromatin structure, and microRNAs, which can interact with transcription factors, modulate their function, or are downstream targets. Finally, examples of disturbances of the cardiac regulatory network leading to congenital heart diseases in human are provided.

Transcription of biological aging markers (ANRIL, P16INK4a, TBX2, and TERRA) and their correlations with severity of sulfur mustard exposure in veterans.

Sulfur mustard (SM) exposure has delayed harmful effects, including premature biological aging. This study aimed to evaluate the expression of aging markers (i.e., ANRIL, P16INK4a, TBX2, and TERRA) and assess their correlation with the severity of SM exposure in the long term. The study was conducted on two volunteer groups. 1) SM-exposed group, exposed to SM once in 1987 during the war; divided into three subgroups based on the injury severity, asymptomatic (without any clinical signs), mild, and severe; 2) Non-exposed group. In the SM-exposed group, ANRIL transcript was decreased, especially in subgroups of mild and severe. TBX2 transcript was also decreased in the total SM-exposed group. This decrease was more significant in the mild and severe subgroups than in asymptomatic ones. P16INK4a transcript was increased in the SM-exposed group, especially in the asymptomatic subgroup. The increase in TERRA transcript was also significant in all subgroups. There was a positive correlation between the TERRA transcript and the severity of injury, while this correlation was negative for the ANRIL. It is concluded that the delayed toxicity of SM may be associated with dysregulation of aging markers leading to premature cellular aging. These markers' alterations differed according to the severity of SM injury.

T-box transcription factor 2 mediates antitumor immune response in cutaneous squamous cell carcinoma by regulating the expression of programmed death ligand 1.

BACKGROUND: Cutaneous squamous cell carcinoma (CSCC) is the second largest nonmelanoma skin cancer in humans; effective treatment options for metastatic CSCC are still in short. In this study, we aimed to explore the function of T-box transcription factor 2 (TBX2) in CSCC. METHODS: The expression level of TBX2 was determined in CSCC samples and cell lines. Programmed death ligand 1 (PD-L1) expression was also analyzed in human CSCC samples. Furthermore, SCC13 cells were transfected with TBX2-DN (loss of function) or normal TBX2 to check its role in regulating PD-L1. RESULTS: The expression level of TBX2 was positively correlated with the stage of CSCC. CSCC tumor cell lines have significantly higher expression levels of TBX2 than normal skin cell lines, and SCC13 cells showed the highest expression. PD-L1 expressions were upregulated during the progression of CSCC, and positively correlated with TBX2. Furthermore, PD-L1 expression increased in SCC13 cells overexpressing TBX2. However, TBX2 did not regulate the activation of IFNγ signal, but mediated the expression of interferon regulatory factor 1 (IRF1) and PD-L1 in both SCC13 and PDV cells. CONCLUSION: TBX2 could mediate antitumor immune response in CSCC by regulating the expression of PD-L1 through IRF1. It might be a prognostic marker in CSCC and synergistic target for PD-1 immunotherapy.

Complex functional redundancy of Tbx2 and Tbx3 in mouse limb development.

The limb phenotypes of Tbx2 and Tbx3 mutants are distinct: loss of Tbx2 results in isolated duplication of digit 4 in the hindlimb while loss of Tbx3 results in anterior polydactyly and posterior oligodactly in the forelimb. In the face of such disparate phenotypes, we sought to determine whether Tbx2 and Tbx3 have functional redundancy during development of the mouse limb. We found that sequential loss of alleles generates defects that are not simply additive of those observed in single mutants and that multiple structures in both the forelimb and hindlimb display compound sensitivity to decreased gene dosage.

TBX2/3 is required for regeneration of dorsal-ventral and medial-lateral polarity in planarians.

The molecular mechanisms responsible for axis establishment during non-embryonic processes remain elusive. The planarian flatworm is an ideal model organism to study body axis polarization and patterning in vivo. Here, we identified a homolog of the TBX2/3 in the planarian Dugesia japonica. RNA interference (RNAi) knockdown of TBX2/3 results in the ectopic formation of protrusions in the midline of the dorsal surface which shows an abnormal expression of midline and ventral cell markers. Additionally, the TBX2/3 RNAi animals also show the duplication of expression of the boundary marker at the lateral edge. Furthermore, TBX2/3 is expressed in muscle cells and co-expressed with bmp4. Inhibition of bone morphogenetic protein (BMP) signaling reduces the expression of TBX2/3 at the midline. These results suggest that TBX2/3 RNAi results in phenotypic characters caused by inhibition of the BMP signal, indicating that TBX2/3 is required for DV and ML patterning, and might be a downstream gene of BMP signaling.

A role of Hey2 transcription factor for right ventricle development through regulation of Tbx2-Mycn pathway during cardiac morphogenesis.

A basic helix-loop-helix transcription factor Hey2 is expressed in the ventricular myocardium and endocardium of mouse embryos, and Hey2 null mice die perinatally showing ventricular septal defect, dysplastic tricuspid valve and hypoplastic right ventricle. In order to understand region-specific roles of Hey2 during cardiac morphogenesis, we generated Hey2 conditional knockout (cKO) mice using Mef2c-AHF-Cre, which was active in the anterior part of the second heart field and the right ventricle and outflow tract of the heart. Hey2 cKO neonates reproduced three anomalies commonly observed in Hey2 null mice. An earliest morphological defect was the lack of right ventricular extension along the apico-basal axis at midgestational stages. Underdevelopment of the right ventricle was present in all cKO neonates including those without apparent atresia of right-sided atrioventricular connection. RNA sequencing analysis of cKO embryos identified that the gene expression of a non-chamber T-box factor Tbx2 was ectopically induced in the chamber myocardium of the right ventricle. Consistently, mRNA expression of the Mycn transcription factor, which was a cell cycle regulator transcriptionally repressed by Tbx2, was down regulated, and the number of S-phase cells was significantly decreased in the right ventricle of cKO heart. These results suggest that Hey2 plays an important role in right ventricle development during cardiac morphogenesis, at least in part, through mitigating Tbx2-dependent inhibition of Mycn expression.

Combined genomic and proteomic approaches reveal DNA binding sites and interaction partners of TBX2 in the developing lung.

BACKGROUND: Tbx2 encodes a transcriptional repressor implicated in the development of numerous organs in mouse. During lung development TBX2 maintains the proliferation of mesenchymal progenitors, and hence, epithelial proliferation and branching morphogenesis. The pro-proliferative function was traced to direct repression of the cell-cycle inhibitor genes Cdkn1a and Cdkn1b, as well as of genes encoding WNT antagonists, Frzb and Shisa3, to increase pro-proliferative WNT signaling. Despite these important molecular insights, we still lack knowledge of the DNA occupancy of TBX2 in the genome, and of the protein interaction partners involved in transcriptional repression of target genes. METHODS: We used chromatin immunoprecipitation (ChIP)-sequencing and expression analyses to identify genomic DNA-binding sites and transcription units directly regulated by TBX2 in the developing lung. Moreover, we purified TBX2 containing protein complexes from embryonic lung tissue and identified potential interaction partners by subsequent liquid chromatography/mass spectrometry. The interaction with candidate proteins was validated by immunofluorescence, proximity ligation and individual co-immunoprecipitation analyses. RESULTS: We identified Il33 and Ccn4 as additional direct target genes of TBX2 in the pulmonary mesenchyme. Analyzing TBX2 occupancy data unveiled the enrichment of five consensus sequences, three of which match T-box binding elements. The remaining two correspond to a high mobility group (HMG)-box and a homeobox consensus sequence motif. We found and validated binding of TBX2 to the HMG-box transcription factor HMGB2 and the homeobox transcription factor PBX1, to the heterochromatin protein CBX3, and to various members of the nucleosome remodeling and deacetylase (NuRD) chromatin remodeling complex including HDAC1, HDAC2 and CHD4. CONCLUSION: Our data suggest that TBX2 interacts with homeobox and HMG-box transcription factors as well as with the NuRD chromatin remodeling complex to repress transcription of anti-proliferative genes in the pulmonary mesenchyme.

TBX2, a Novel Regulator of Labour.

Background and Objectives: Therapeutic interventions targeting molecular factors involved in the transition from uterine quiescence to overt labour are not substantially reducing the rate of spontaneous preterm labour. The identification of novel rational therapeutic targets are essential to prevent the most common cause of neonatal mortality. Based on our previous work showing that Tbx2 (T-Box transcription factor 2) is a putative upstream regulator preceding progesterone withdrawal in mouse myometrium, we now investigate the role of TBX2 in human myometrium. Materials and Methods: RNA microarray analysis of (A) preterm human myometrium samples and (B) myometrial cells overexpressing TBX2 in vitro, combined with subsequent analysis of the two publicly available datasets of (C) Chan et al. and (D) Sharp et al. The effect of TBX2 overexpression on cytokines/chemokines secreted to the myometrium cell culture medium were determined by Luminex assay. Results: Analysis shows that overexpression of TBX2 in myometrial cells results in downregulation of TNFα- and interferon signalling. This downregulation is consistent with the decreased expression of cytokines and chemokines of which a subset has been previously associated with the inflammatory pathways relevant for human labour. In contrast, CXCL5 (C-X-C motif chemokine ligand 5), CCL21 and IL-6 (Interleukin 6), previously reported in relation to parturition, do not seem to be under TBX2 control. The combined bioinformatical analysis of the four mRNA datasets identifies a subset of upstream regulators common to both preterm and term labour under control of TBX2. Surprisingly, TBX2 mRNA levels are increased in preterm contractile myometrium. Conclusions: We identified a subset of upstream regulators common to both preterm and term labour that are activated in labour and repressed by TBX2. The increased TBX2 mRNA expression in myometrium collected during a preterm caesarean section while in spontaneous preterm labour compared to tissue harvested during iatrogenic preterm delivery does not fit the bioinformatical model. We can only explain this by speculating that the in vivo activity of TBX2 in human myometrium depends not only on the TBX2 expression levels but also on levels of the accessory proteins necessary for TBX2 activity.

Transcriptional repression of the ectodomain sheddase ADAM10 by TBX2 and potential implication for Alzheimer's disease.

BACKGROUND: The ADAM10-mediated cleavage of transmembrane proteins regulates cellular processes such as proliferation or migration. Substrate cleavage by ADAM10 has also been implicated in pathological situations such as cancer or Morbus Alzheimer. Therefore, identifying endogenous molecules, which modulate the amount and consequently the activity of ADAM10, might contribute to a deeper understanding of the enzyme's role in both, physiology and pathology. METHOD: To elucidate the underlying cellular mechanism of the TBX2-mediated repression of ADAM10 gene expression, we performed overexpression, RNAi-mediated knockdown and pharmacological inhibition studies in the human neuroblastoma cell line SH-SY5Y. Expression analysis was conducted by e.g. real-time RT-PCR or western blot techniques. To identify the binding region of TBX2 within the ADAM10 promoter, we used luciferase reporter assay on deletion constructs and EMSA/WEMSA experiments. In addition, we analyzed a TBX2 loss-of-function Drosophila model regarding the expression of ADAM10 orthologs by qPCR. Furthermore, we quantified the mRNA level of TBX2 in post-mortem brain tissue of AD patients. RESULTS: Here, we report TBX2 as a transcriptional repressor of ADAM10 gene expression: both, the DNA-binding domain and the repression domain of TBX2 were necessary to effect transcriptional repression of ADAM10 in neuronal SH-SY5Y cells. This regulatory mechanism required HDAC1 as a co-factor of TBX2. Transcriptional repression was mediated by two functional TBX2 binding sites within the core promoter sequence (- 315 to - 286 bp). Analysis of a TBX2 loss-of-function Drosophila model revealed that kuzbanian and kuzbanian-like, orthologs of ADAM10, were derepressed compared to wild type. Vice versa, analysis of cortical brain samples of AD-patients, which showed reduced ADAM10 mRNA levels, revealed a 2.5-fold elevation of TBX2, while TBX3 and TBX21 levels were not affected. CONCLUSION: Our results characterize TBX2 as a repressor of ADAM10 gene expression and suggest that this regulatory interaction is conserved across tissues and species.

Prognostic significance of high metabolic activity in breast cancer: PET signature in breast cancer.

High metabolic activity, reflected in increased glucose uptake, is one of the hallmarks of many cancers including breast cancer. However, not all cancers avidly take up glucose, suggesting heterogeneity in their metabolic demand. Thus, we aim to generate a genomic signature of glucose hypermetabolism in breast cancer and examine its clinical relevance. To identify genes significantly associated with glucose uptake, gene expression data were analyzed together with the standardized uptake values (SUVmax) of 18F-fluorodeoxy-glucose on positron emission tomography (PET) for 11 breast cancers. The resulting PET signature was evaluated for prognostic significance in four large independent patient cohorts (n = 5417). Potential upstream regulators accountable for the high glucose uptake were identified by gene network analysis. A PET signature of 242 genes was significantly correlated with SUVmax in breast cancer. In all four cohorts, high PET signature was significantly associated with poorer prognosis. The prognostic value of this PET signature was further supported by Cox regression analyses (hazard ratio 1.7, confidential interval 1.48-2.02; P < 0.001). The PET signature was also strongly correlated with previously established prognostic genomic signatures such as PAM50, Oncotype DX, and NKI. Gene network analyses suggested that MYC and TBX2 were the most significant upstream transcription factors in the breast cancers with high glucose uptake. A PET signature reflecting high glucose uptake is a novel independent prognostic factor in breast cancer. MYC and TBX2 are potential regulators of glucose uptake.

A transcription factor network controls cell migration and fate decisions in the developing zebrafish pineal complex.

The zebrafish pineal complex consists of four cell types (rod and cone photoreceptors, projection neurons and parapineal neurons) that are derived from a single pineal complex anlage. After specification, parapineal neurons migrate unilaterally away from the rest of the pineal complex whereas rods, cones and projection neurons are non-migratory. The transcription factor Tbx2b is important for both the correct number and migration of parapineal neurons. We find that two additional transcription factors, Flh and Nr2e3, negatively regulate parapineal formation. Flh induces non-migratory neuron fates and limits the extent of parapineal specification, in part by activation of Nr2e3 expression. Tbx2b is positively regulated by Flh, but opposes Flh action during specification of parapineal neurons. Loss of parapineal neuron specification in Tbx2b-deficient embryos can be partially rescued by loss of Nr2e3 or Flh function; however, parapineal migration absolutely requires Tbx2b activity. We conclude that cell specification and migration in the pineal complex are regulated by a network of at least three transcription factors.

Neonatal Lung Disease Associated with TBX4 Mutations.

Variable lung disease was documented in 2 infants with heterozygous TBX4 mutations; their clinical presentations, pathology, and outcomes were distinct. These findings demonstrate that TBX4 gene mutations are associated with neonatal respiratory failure and highlight the wide spectrum of clinicopathological outcomes that have implications for patient diagnosis and management.

TBX2-positive cells represent a multi-potent mesenchymal progenitor pool in the developing lung.

BACKGROUND: In the embryonic mammalian lung, mesenchymal cells act both as a signaling center for epithelial proliferation, differentiation and morphogenesis as well as a source for a multitude of differentiated cell types that support the structure of the developing and mature organ. Whether the embryonic pulmonary mesenchyme is a homogenous precursor pool and how it diversifies into different cell lineages is poorly understood. We have previously shown that the T-box transcription factor gene Tbx2 is expressed in the pulmonary mesenchyme of the developing murine lung and is required therein to maintain branching morphogenesis. METHODS: We determined Tbx2/TBX2 expression in the developing murine lung by in situ hybridization and immunofluorescence analyses. We used a genetic lineage tracing approach with a Cre line under the control of endogenous Tbx2 control elements (Tbx2cre), and the R26mTmG reporter line to trace TBX2-positive cells in the murine lung. We determined the fate of the TBX2 lineage by co-immunofluorescence analysis of the GFP reporter and differentiation markers in normal murine lungs and in lungs lacking or overexpressing TBX2 in the pulmonary mesenchyme. RESULTS: We show that TBX2 is strongly expressed in mesenchymal progenitors in the developing murine lung. In differentiated smooth muscle cells and in fibroblasts, expression of TBX2 is still widespread but strongly reduced. In mesothelial and endothelial cells expression is more variable and scattered. All fetal smooth muscle cells, endothelial cells and fibroblasts derive from TBX2+ progenitors, whereas half of the mesothelial cells have a different descent. The fate of TBX2-expressing cells is not changed in Tbx2-deficient and in TBX2-constitutively overexpressing mice but the distribution and abundance of endothelial and smooth muscle cells is changed in the overexpression condition. CONCLUSION: The fate of pulmonary mesenchymal progenitors is largely independent of TBX2. Nevertheless, a successive and precisely timed downregulation of TBX2 is necessary to allow proper differentiation and functionality of bronchial smooth muscle cells and to limit endothelial differentiation. Our work suggests expression of TBX2 in an early pulmonary mesenchymal progenitor and supports a role of TBX2 in maintaining the precursor state of these cells.

Identification of TBX2 and TBX3 variants in patients with conotruncal heart defects by target sequencing.

BACKGROUND: Conotruncal heart defects (CTDs) are heterogeneous congenital heart malformations that result from outflow tract dysplasia; however, the genetic determinants underlying CTDs remain unclear. Increasing evidence demonstrates that dysfunctional TBX2 and TBX3 result in outflow tract malformations, implying that both of them are involved in CTD pathogenesis. We screened for TBX2 and TBX3 variants in a large cohort of CTD patients (n = 588) and population-matched healthy controls (n = 300) by target sequencing and genetically analyzed the expression and function of these variants. RESULTS: The probably damaging variants p.R608W, p.T249I, and p.R616Q of TBX2 and p.A192T, p.M65L, and p.A562V of TBX3 were identified in CTD patients, but none in controls. All altered amino acids were highly conserved evolutionarily. Moreover, our data suggested that mRNA and protein expressions of TBX2 and TBX3 variants were altered compared with those of the wild-type. We screened PEA3 and MEF2C as novel downstream genes of TBX2 and TBX3, respectively. Functional analysis revealed that TBX2R608W and TBX2R616Q variant proteins further activated HAS2 promoter but failed to activate PEA3 promoter and that TBX3A192T and TBX3A562V variant proteins showed a reduced transcriptional activity over MEF2C promoter. CONCLUSIONS: Our results indicate that the R608W and R616Q variants of TBX2 as well as the A192T and A562V variants of TBX3 contribute to CTD etiology; this was the first association of variants of TBX2 and TBX3 to CTDs based on a large population.

Epigenetic Suppression of the T-box Subfamily 2 (TBX2) in Human Non-Small Cell Lung Cancer.

(1) The TBX2 subfamily of transcription factors (TBXs 2, 3, 4 and 5) are markedly down-regulated in human non-small cell lung cancer (NSCLC) and exert tumor suppressor effects in lung malignancy. Yet, mechanisms underlying suppressed expression of the TBX2 subfamily in NSCLC are elusive. Here, we interrogated probable epigenetic mechanisms in suppressed expression of the TBX2 subfamily in human NSCLC. (2) TBX2 subfamily gene expression and methylation levels in NSCLC and normal lung tissues were surveyed using publicly available RNA-sequence and genome-wide methylation datasets. Methylation ß-values of the four genes were statistically compared between NSCLCs and normal lung tissues, correlated with gene expression levels, and interrogated with clinicopathological variables. Expression and methylation levels of TBXs were quantified in NSCLC cells using real-time PCR and methylation-specific PCR assays, respectively. Effects of the DNA methyltransferase inhibitor 5-azacytidine (Aza) on TBX2 subfamily expression were assessed in NSCLC cells. Impact of TBX2 subfamily expression on Aza-treated cells was evaluated by RNA interference. (3) All four TBXs were significantly hypermethylated in NSCLCs relative to normal lung tissues (p < 0.05). Methylation ß-values of the genes, with exception of TBX2, were significantly inversely correlated with corresponding mRNA expression levels (p < 0.05). We found no statistically significant differences in hypermethylation levels of the TBX2 subfamily by clinicopathological features including stage and tobacco history. Expression levels of the TBX genes were overall suppressed in NSCLC cells relative to normal alveolar cells. Members of the subfamily were significantly hypermethylated in all tested NSCLC cell lines relative to normal alveolar cells. Treatment with Aza induced the expression of the TBX2 subfamily concomitant with NSCLC cell growth inhibition. Further, simultaneous knockdown of the four TBX genes markedly reduced anti-growth effects of Aza in NSCLC cells. (4) Our study sheds light on new epigenetic profiles in the molecular pathogenesis of human NSCLC.