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INTRODUCTION/AIMS: Terminal Schwann cells (tSCs) are nonmyelinating Schwann cells present at the neuromuscular junction (NMJ) with multiple integral roles throughout their lifespan. There is no known gene differentiating tSCs from myelinating Schwann cells, making their isolation and investigation challenging. In this work we investigated genes expressed within tSCs. METHODS: A novel dissection technique was utilized to isolate the tSC-containing NMJ band from the sternomastoid muscles of S100-GFP mice. RNA was isolated from samples containing: (a) NMJ bands (tSCs with nerve and muscle), (b) nerve, and (c) muscle, and microarray genetic expression analysis was conducted. Data were validated by quantitative real-time polymerase chain reaction (qRT-PCR) and immunofluorescent staining. To identify genes specific to tSCs compared with other NMJ components, analysis of variance and rank-order analysis were performed using the Partek Genomic Suite. RESULTS: Microarray analysis of the tSC-enriched NMJ band revealed upregulation (by 4- to 12-fold) of several genes unique to the NMJ compared with muscle or nerve parts alone (P < .05). Among these genes, Tbx21 (or T-bet) was identified, which showed a 12-fold higher expression at the NMJ compared with sciatic nerve (P < .002). qRT-PCR analysis showed Tbx21 mRNA expression was over ninefold higher (P < .05) in the NMJ relative to muscle and nerve. Tbx21 protein colocalized with tSCs and was not noted in myelinating SCs from sciatic nerve. DISCUSSION: We found TBX21 to be expressed in tSCs. Additional studies will be performed to determine the functional significance of TBX21 in tSCs. These studies may enhance the investigative tools available to modulate tSCs to improve motor recovery after nerve injury.

With the rapid development of the automotive industry and the wide application of 5G network technology, there are more and more Telematics Box (T-Box) equipped with intelligent operating systems in vehicles and they are becoming more and more complex. Because it is connected to the on-board CAN bus internally and interconnects with mobile phone /PC through the cloud platform externally, the security of T-Box must be fully guaranteed, to make the automotive more secure. T-Box can realize remote control function, so the T-Box information security problem has been paid more and more attention. In this paper, the T-Box were tested from multiple dimensions by using various methods, and the results were statistically analyzed, and the corresponding protection strategies were proposed for the corresponding security risks.

Complex RNA–RNA interactions are increasingly known to play key roles in numerous biological processes from gene expression control to ribonucleoprotein granule formation. By contrast, the nature of these interactions and characteristics of their interfaces, especially those that involve partially or wholly structured RNAs, remain elusive. Herein, we discuss different modalities of RNA–RNA interactions with an emphasis on those that depend on secondary, tertiary, or quaternary structure. We dissect recently structurally elucidated RNA–RNA complexes including RNA triplexes, riboswitches, ribozymes, and reverse transcription complexes. These analyses highlight a reciprocal relationship that intimately links RNA structure formation with RNA–RNA interactions. The interactions not only shape and sculpt RNA structures but also are enabled and modulated by the structures they create. Understanding this two-way relationship between RNA structure and interactions provides mechanistic insights into the expanding repertoire of noncoding RNA functions, and may inform the design of novel therapeutics that target RNA structures or interactions.

Organ laterality refers to the left-right asymmetry in disposition and conformation of internal organs and is established during embryogenesis. The heart is the first organ to display visible left-right asymmetries through its left-sided positioning and rightward looping. Here, we present a new zebrafish loss-of-function allele for tbx5a, which displays defective rightward cardiac looping morphogenesis. By mapping individual cardiomyocyte behavior during cardiac looping, we establish that ventricular and atrial cardiomyocytes rearrange in distinct directions. As a consequence, the cardiac chambers twist around the atrioventricular canal resulting in torsion of the heart tube, which is compromised in tbx5a mutants. Pharmacological treatment and ex vivo culture establishes that the cardiac twisting depends on intrinsic mechanisms and is independent from cardiac growth. Furthermore, genetic experiments indicate that looping requires proper tissue patterning. We conclude that cardiac looping involves twisting of the chambers around the atrioventricular canal, which requires correct tissue patterning by Tbx5a.

Bacterial T-boxes are regulatory mRNA regions that control the transcription or translation of factors involved in amino acid supply. T-boxes act by directly binding to non-aminoacylated tRNA in response to amino acid starvation. Three studies now capture three-dimensional structures of tRNA–T-box complexes and reveal a set of RNA features that are required for the recognition of specific tRNAs and modulation of gene expression.

Cardiovascular development is a complex process that starts with the formation of symmetrically located precardiac mesodermal precursors soon after gastrulation and is completed with the formation of a four-chambered heart with distinct inlet and outlet connections. Multiple transcriptional inputs are required to provide adequate regional identity to the forming atrial and ventricular chambers as well as their flanking regions; i.e., inflow tract, atrioventricular canal, and outflow tract. In this context, regional chamber identity is widely governed by regional activation of distinct T-box family members. Over the last decade, novel layers of gene regulatory mechanisms have been discovered with the identification of non-coding RNAs. microRNAs represent the most well-studied subcategory among short non-coding RNAs. In this study, we sought to investigate the functional role of distinct microRNAs that are predicted to target T-box family members. Our data demonstrated a highly dynamic expression of distinct microRNAs and T-box family members during cardiogenesis, revealing a relatively large subset of complementary and similar microRNA–mRNA expression profiles. Over-expression analyses demonstrated that a given microRNA can distinctly regulate the same T-box family member in distinct cardiac regions and within distinct temporal frameworks, supporting the notion of indirect regulatory mechanisms, and dual luciferase assays on Tbx2, Tbx3 and Tbx5 3′ UTR further supported this notion. Overall, our data demonstrated a highly dynamic microRNA and T-box family members expression during cardiogenesis and supported the notion that such microRNAs indirectly regulate the T-box family members in a tissue- and time-dependent manner.

T-box 5 (TBX5) protein belongs to the T-box family whose members play a crucial role in cell-type specification, morphogenesis and organogenesis. TBX5 is a transcription factor important for cardiac development and upper limbs formation and its haploinsufficiency causes Holt-Oram syndrome (HOS). An increase in TBX5 dosage also leads to HOS, suggesting that TBX5 is a dose-sensitive transcription factor that needs to be tightly regulated but the molecular mechanisms involved remain unclear. In this work we report the cloning and functional analysis of human TBX5 promoter region 1 (upstream of exon 1) and promoter region 2 (upstream of exon 2), that probably regulate the transcription of the different transcript variants. In silico analysis showed several binding sites for cardiac and skeletal related transcription factors (TFs) and their functionality was assessed using promoter-luciferase constructions and TF-expressing vectors. MEF2A (Myocyte enhancer factor 2 A) was shown to positively regulate both TBX5 promoters, while EGR1 (early growth response 1) repressed both promoters. SOX9 (SRY (sex determining region Y)-box 9) repressed only the activity of promoter region 2. Interestingly, YY1 (Yin and yang 1) repressed promoter region 1 (that regulates the expression of variant 1 and 3), but activated promoter region 2 (that regulates the expression of variant 4). In conclusion, this work provides novel insights toward the better understanding of TBX5 transcriptional regulation by cardiac- and skeletal-related TFs. Portuguese Foundation for Science and Technology (FCT)Portuguese Foundation for Science and Technology [UIDB/04326/2020]; FCTPortuguese Foundation for Science and TechnologyEuropean Commission [SFRH/BD/141918/2018] info:eu-repo/semantics/publishedVersion

Extra-embryonic mesoderm (ExM)-composed of the earliest cells that traverse the primitive streak-gives rise to the endothelium as well as haematopoietic progenitors in the developing yolk sac. How a specific subset of ExM becomes committed to a haematopoietic fate remains unclear. Here we demonstrate using an embryonic stem cell model that transient expression of the T-box transcription factor Eomesodermin (Eomes) governs haemogenic competency of ExM. Eomes regulates the accessibility of enhancers that the transcription factor stem cell leukaemia (SCL) normally utilizes to specify primitive erythrocytes and is essential for the normal development of Runx1+ haemogenic endothelium. Single-cell RNA sequencing suggests that Eomes loss of function profoundly blocks the formation of blood progenitors but not specification of Flk-1+ haematoendothelial progenitors. Our findings place Eomes at the top of the transcriptional hierarchy regulating early blood formation and suggest that haemogenic competence is endowed earlier during embryonic development than was previously appreciated.

T-box riboswitches constitute a large family of tRNA-binding leader sequences that play a central role in gene regulation in many gram-positive bacteria. Accurate inference of the tRNA binding to T-box riboswitches is critical to predict their cis-regulatory activity. However, there is no central repository of information on the tRNA binding specificities of T-box riboswitches, and de novo prediction of binding specificities requires advanced knowledge of computational tools to annotate riboswitch secondary structure features. Here, we present the T-box Riboswitch Annotation Database (TBDB, https://tbdb.io), an open-access database with a collection of 23,535 T-box riboswitch sequences, spanning the major phyla of 3,632 bacterial species. Among structural predictions, the TBDB also identifies specifier sequences, cognate tRNA binding partners, and downstream regulatory targets. To our knowledge, the TBDB presents the largest collection of feature, sequence, and structural annotations carried out on this important family of regulatory RNA.

Organ laterality refers to the left-right asymmetry in disposition and conformation of internal organs and is established during embryogenesis. The heart is the first organ to display visible left-right asymmetries through its left-sided positioning and rightward looping. Here, we present a new zebrafish loss-of-function allele for tbx5a, which displays defective rightward cardiac looping morphogenesis. By mapping individual cardiomyocyte behavior during cardiac looping, we establish that ventricular and atrial cardiomyocytes rearrange in distinct directions. As a consequence, the cardiac chambers twist around the atrioventricular canal resulting in torsion of the heart tube, which is compromised in tbx5a mutants. Pharmacological treatment and ex vivo culture establishes that the cardiac twisting depends on intrinsic mechanisms and is independent from cardiac growth. Furthermore, genetic experiments indicate that looping requires proper tissue patterning. We conclude that cardiac looping involves twisting of the chambers around the atrioventricular canal, which requires correct tissue patterning by Tbx5a.

The mouse T-box transcription factors T and Tbx6 are co-expressed in the primitive streak and have unique domains of expression; T is expressed in the notochord, while Tbx6 is expressed in the presomitic mesoderm. T-box factors are related through a shared DNA binding domain, the T-domain, and can therefore bind to similar DNA sequences at least in vitro. We investigated the functional similarities and differences of T and Tbx6 DNA binding and transcriptional activity in vitro and their interaction genetically in vivo. We show that at one target, Dll1, the T-domains of T and Tbx6 have different affinities for the binding sites present in the mesoderm enhancer. We further show using in vitro assays that T and Tbx6 differentially affect transcription with Tbx6 activating expression tenfold higher than T, that T and Tbx6 can compete at target gene enhancers, and that this competition requires a functional DNA binding domain. Next, we addressed whether T and Tbx6 can compete in vivo. First, we generated embryos that express Tbx6 at greater than wild-type levels embryos and show that these embryos have short tails, resembling the T heterozygous phenotype. Next, using the dominant-negative TWis allele, we show that Tbx6+/− TWis/+ embryos share similarities with embryos homozygous for the Tbx6 hypomorphic allele rib-vertebrae, specifically fusions of several ribs and malformation of some vertebrae. Finally, we tested whether Tbx6 can functionally replace T using a knockin approach, which resulted in severe T null-like phenotypes in chimeric embryos generated with ES cells heterozygous for a Tbx6 knockin at the T locus. Altogether, our results of differences in affinity for DNA binding sites and transcriptional activity for T and Tbx6 provide a potential mechanism for the failure of Tbx6 to functionally replace T and possible competition phenotypes in vivo., Summary: Mouse Tbx6 fails to compensate for heterozygous loss of T; instead ectopic Tbx6 in the T expression-domain in knockin embryos generates T null-like phenotypes suggestive of competition.

RATIONALE: The heartbeat is organized by the cardiac conduction system (CCS), a specialized network of cardiomyocytes. Patterning of the CCS into atrial node versus ventricular conduction system (VCS) components with distinct physiology is essential for the normal heartbeat. Distinct node versus VCS physiology has been recognized for more than a century, but the molecular basis of this regional patterning is not well understood. OBJECTIVE: To study the genetic and genomic mechanisms underlying node versus VCS distinction and investigate rhythm consequences of failed VCS patterning. METHODS AND RESULTS: Using mouse genetics, we found that the balance between T-box transcriptional activator, Tbx5, and T-box transcriptional repressor, Tbx3, determined the molecular and functional output of VCS myocytes. Adult VCS-specific removal of Tbx5 or overexpression of Tbx3 re-patterned the fast VCS into slow, nodal-like cells based on molecular and functional criteria. In these cases, gene expression profiling showed diminished expression of genes required for VCS-specific fast conduction but maintenance of expression of genes required for nodal slow conduction physiology. Action potentials (APs) of Tbx5-deficient VCS myocytes adopted nodal-specific characteristics, including increased AP duration and cellular automaticity. Removal of Tbx5 in-vivo precipitated inappropriate depolarizations in the atrioventricular (His)-bundle associated with lethal ventricular arrhythmias. TBX5 bound and directly activated cis-regulatory elements at fast conduction channel genes required for fast physiological characteristics of the VCS action potential, defining the identity of the adult VCS. CONCLUSIONS: The CCS is patterned entirely as a slow, nodal ground state, with a T-box dependent, physiologically dominant, fast conduction network driven specifically in the VCS. Disruption of the fast VCS gene regulatory network (GRN) allowed nodal physiology to emerge, providing a plausible molecular mechanism for some lethal ventricular arrhythmias.

BACKGROUND T-box transcription factor protein 21 (TBX21) is expressed in immune cells and some tumor cells. Defects in TBX21 gene can cause Th1/Th2 imbalance, which is closely related to tumorigenesis. The expression and clinical value of TBX21 in skin cutaneous melanoma (SKCM) are not clear. MATERIAL AND METHODS RNA-Seq expression and clinical information were downloaded from The Cancer Genome Atlas (TCGA) and Genotype-Tissue Expression (GTEx) databases. Wilcoxon signed-rank test and logistic regression were used to explore the relationship between TBX21 expression and clinical parameters such as gender, stage, etc. The correlation between clinicopathological characteristics and overall survival of SKCM patients was estimated by Cox regression and the Kaplan-Meier method. Gene set enrichment analysis (GSEA) and protein-protein interaction (PPI) were conducted to analyze the potential mechanism of TBX21 in the progression of SKCM. RESULTS Compared with normal samples, TBX21 was significantly upregulated in SKCM tissues. SKCM patients with lower TBX21 expression might have a worse prognosis than those with higher TBX21 expression according to Kaplan-Meier survival analysis. Cox analysis also reached the same conclusion: TBX21 was an independent prognostic indicator. GSEA showed that the highly expressed phenotypes in TBX21 were enriched to varying degrees with various signaling pathways. PPI network showed the top 10 proteins that were closely related to TBX21. CONCLUSIONS TBX21 expression was significantly correlated with the prognosis of SKCM patients and was found to be involved in a great many immunological pathways that affect the occurrence and development of tumors.

T-box riboswitches constitute a large family of tRNA-binding leader sequences that play a central role in gene regulation in many gram-positive bacteria. Accurate inference of the tRNA binding to T-boxes is critical to predict their cis-regulatory activity. However, there is no central repository of information on the tRNA binding specificities of T-box riboswitches and de novo prediction of binding specificities requires advance knowledge of computational tools to annotate riboswitch secondary structure features. Here we present T-box annotation Database (TBDB,https://tbdb.io), an open-access database with a collection of 23,497 T-box sequences, spanning the major phyla of 3,621 bacterial species. Among structural predictions, the TBDB also identifies specifier sequences, cognate tRNA binding partners, and downstream regulatory target. To our knowledge, the TBDB presents the largest collection of feature, sequence, and structural annotations carried out on this important family of regulatory RNA.

BackgroundTBX5 is a transcription factor that has an important role in development of heart. TBX5 variants in the region encoding the T-box domain have been shown to cause cardiac defects, such as atrial septal defect or ventricular septal defect, while TBX5 variants have also been identified in a few cardiomyopathy patients and considered causative. We identified a TBX5 variant (c.791G>A, p.Arg264Lys), that is over-represented in cardiomyopathy patients. This variant is located outside of the T-box domain, and its pathogenicity has not been confirmed by functional analyses.ObjectiveTo investigate whether the TBX5 R264K is deleterious and could contribute to the pathogenesis of cardiomyopathy.Methods and resultsWe developed mice expressing Tbx5 R264K. Mice homozygous for this variant displayed compensated dilated cardiomyopathy; mild decreased fractional shortening, dilatation of the left ventricle, left ventricular wall thinning and increased heart weight without major heart structural disorders. There was no difference in activation of the ANF promotor, a transcriptional target of Tbx5, compared to wild-type. However, analysis of RNA isolated from left ventricular samples showed significant increases in the expression of Acta1 in left ventricle with concomitant increases in the protein level of ACTA1.ConclusionsMice homozygous for Tbx5 R264K showed compensated dilated cardiomyopathy. Thus, TBX5 R264K may have a significant pathogenic role in some cardiomyopathy patients independently of T-box domain pathway.

Pulmonary arterial hypertension (PAH) is a lethal disease [1]. Although mutations in BMPR2 and other genes have been reported, the genetic causes in large numbers of patients, especially with sporadic PAH, remain unknown. In 2013, Kerstjens-Frederikse et al. first reported TBX4 mutations in patients with PAH [2]. TBX4 is an essential transcription factor for the development of the hindlimbs and lungs [3]. In European countries, the frequency of TBX4 mutation was reported as 2.4–4.1% in adult-onset PAH [2, 4] and as 7.5–30% in child-onset PAH [2, 5] (Fig. 27.1). However, its frequency in Asian patients with PAH has yet to be studied.

Somites are a pair of epithelial spheres beside a neural tube and are formed with an accurate periodicity during embryogenesis in vertebrates. It has been known that Hes7 is one of the core clock genes for somitogenesis, and its expression domain is restricted in the presomitic mesoderm (PSM). However, the molecular mechanism of how Hes7 transcription is regulated is not clear. Here, using transgenic mice and luciferase-based reporter assays and in vitro binding assays, we unravel the mechanism by which Hes7 is expressed exclusively in the PSM. We identified a Hes7 essential region residing −1.5 to −1.1 kb from the transcription start site of mouse Hes7, and this region was indispensable for PSM-specific Hes7 expression. We also present detailed analyses of cis-regulatory elements within the Hes7 essential region that directs Hes7 expression in the PSM. Hes7 expression in the PSM was up-regulated through the E-box, T-box, and RBPj-binding element in the Hes7 essential region, presumably through synergistic signaling involving mesogenin1, T-box6 (Tbx6), and Notch. Furthermore, we demonstrate that Tbx18, Ripply2, and Hes7 repress the activation of the Hes7 essential region by the aforementioned transcription factors. Our findings reveal that a unified transcriptional regulatory network involving a Hes7 essential region confers robust PSM-specific Hes7 gene expression.

Vertebrate eye development and function critically depend on the regulation of proliferation of retinal pigment epithelium (RPE) cells. Hence, a thorough analysis of the molecular parameters controlling RPE cell proliferation is crucial for our understanding of the physiology of this cell type both in health and in disease. The T-box transcription factor TBX2 is an important cell cycle regulator in development and oncogenesis, but its specific role in RPE cell proliferation is far from clear. The purpose of the present study is to investigate whether TBX2 plays any role in regulating RPE cell proliferation.The expression of TBX2 in RPE cells was analyzed in wildtype mice and ARPE-19 cells by co-staining for RPE-specific markers and cell proliferation. In vitro, the role of TBX2 was studied by manipulating its levels using RNAi and analyzing the effects on DNA synthesis and cell growth and on gene expression at the RNA and protein levels.Here, we find that TBX2 is expressed in RPE cells both in vivo and in vitro. Specific knockdown of TBX2 in the human RPE cell line ARPE-19 leads to an accumulation of cells at G1. This cell cycle arrest is accompanied by changes in the levels of known cell cycle regulators and, in particular, by an increase in the levels of the tumor-suppressor gene CCAAT/enhancer-binding protein delta (CEBPD). In fact, simultaneous knockdown of both TBX2 and CEBPD interferes with the reduction in cell proliferation brought about by TBX2 reduction alone.Our results provide novel insights into the regulatory mechanisms of cell proliferation in the RPE and may contribute to our understanding of normal RPE maintenance and its pathology in degenerative and proliferative disorders of the eye.

Recently, it was discovered that exposure to mainstream antibiotics activate numerous bacterial riboregulators that control antibiotic resistance genes including metabolite-binding riboswitches and other transcription attenuators. However, the effects of commonly used antibiotics, many of which exhibit RNA-binding properties, on the widespread T-box riboswitches, remain unknown. In Staphylococcus aureus, a species-specific glyS T-box controls the supply of glycine for both ribosomal translation and cell wall synthesis, making it a promising target for next-generation antimicrobials. Here, we report that specific protein synthesis inhibitors could either significantly increase T-box-mediated transcription antitermination, while other compounds could suppress it, both in vitro and in vivo. In-line probing of the full-length T-box combined with molecular modelling and docking analyses suggest that the antibiotics that promote transcription antitermination stabilize the T-box:tRNA complex through binding specific positions on stem I and the Staphylococcal-specific stem Sa. By contrast, the antibiotics that attenuate T-box transcription bind to other positions on stem I and do not interact with stem Sa. Taken together, our results reveal that the transcription of essential genes controlled by T-box riboswitches can be directly modulated by commonly used protein synthesis inhibitors. These findings accentuate the regulatory complexities of bacterial response to antimicrobials that involve multiple riboregulators.

Summary T-box transcription factors T/Brachyury homolog A (Ta) and Tbx16 are essential for correct mesoderm development in zebrafish. The downstream transcriptional networks guiding their functional activities are poorly understood. Additionally, important contributions elsewhere are likely masked due to redundancy. Here, we exploit functional genomic strategies to identify Ta and Tbx16 targets in early embryogenesis. Surprisingly, we discovered they not only activate mesodermal gene expression but also redundantly regulate key endodermal determinants, leading to substantial loss of endoderm in double mutants. To further explore the gene regulatory networks (GRNs) governing endoderm formation, we identified targets of Ta/Tbx16-regulated homeodomain transcription factor Mixl1, which is absolutely required in zebrafish for endoderm formation. Interestingly, we find many endodermal determinants coordinately regulated through common genomic occupancy by Mixl1, Eomesa, Smad2, Nanog, Mxtx2, and Pou5f3. Collectively, these findings augment the endoderm GRN and reveal a panel of target genes underlying the Ta, Tbx16, and Mixl1 mutant phenotypes., Graphical Abstract, Highlights • Ta and Tbx16 redundantly regulate genes through common cis-regulatory modules • Ta and Tbx16 control expression of mixl1 and other key endodermal regulators • Mixl1 occupies endodermal CRMs with Smad2, Eomesa, Nanog, Mxtx2, and Pou5f3 • Ta and tbx16 double mutants fail to correctly form liver, pancreas, and gut tube, Endoderm contributes to the respiratory and gastrointestinal tracts and all associated organs. How transcription factors control endodermal progenitor specification and expansion is not completely understood. Here, Nelson et al. identify a key redundant requirement for T-box factors Ta and Tbx16 in zebrafish endoderm formation and explore the downstream transcriptional programs.

T-box factors comprise an archaic family of evolutionary conserved transcription factors that regulate patterns of gene expression essential for embryonic development. The T-box transcription factor 3 (TBX3), a member of this family, is expressed in several tissues and plays critical roles in, among other structures, the heart, mammary gland and limbs and haploinsufficiency of the human TBX3 gene is the genetic basis for the autosomal dominant disorder, ulnar-mammary syndrome. Overexpression of TBX3 on the other hand has been linked to several cancers including melanoma, breast, pancreatic, liver, lung, head and neck, ovarian, bladder carcinomas and a number of sarcoma subtypes. Furthermore, there is strong evidence that TBX3 promotes oncogenesis by impacting proliferation, tumour formation, metastasis as well as cell survival and drug resistance. More recently, TBX3 was however shown to also have tumour suppressor activity in fibrosarcomas and thus its functions in oncogenesis appear to be context dependent. Identification of the upstream regulators of TBX3 and the molecular mechanism(s) underpinning its oncogenic roles will make valuable contributions to cancer biology.

HPV16 is the most carcinogenic human papillomavirus and causes >50% of cervical cancers, the majority of anal cancers and 30% of oropharyngeal squamous cell carcinomas. HPV carcinogenesis relies on the continuous expression of the two main viral oncoproteins E6 and E7 that target >150 cellular proteins. Among them, epigenetic modifiers, including DNA Methyl Transferases (DNMT), are dysregulated, promoting an aberrant methylation pattern in HPV-positive cancer cells. It has been previously reported that the treatment of HPV-positive cervical cancer cells with DNMT inhibitor 5-aza-2'-deoxycytidine (5azadC) caused the downregulation of E6 expression due to mRNA destabilization that was mediated by miR-375. Recently, the T-box transcription factor 2 (TBX2) has been demonstrated to repress HPV LCR activity. In the current study, the role of TBX2 in E6 repression was investigated in HPV16 cervical cancer cell lines following 5azadC treatment. A decrease of E6 expression was accompanied by p53 and p21 restoration. While TBX2 mRNA was upregulated in 5azadC-treated SiHa and Ca Ski cells, TBX2 protein was not detectable. Furthermore, the overexpression of TBX2 protein in cervical cancer cells did not allow the repression of E6 expression. The TBX2 transcription factor is therefore unlikely to be associated with the repression of E6 following 5azadC treatment of SiHa and Ca Ski cells. © 2020 Spandidos Publications. All rights reserved.

Background The heterozygous microdeletion of chromosome 22q11.2 results in a spectrum of disorders, including DiGeorge syndrome (DGS) and velocardiofacial syndrome (VCFS), with phenotypic features that can include the classic triad of congenital heart disease (CHD), thymic aplasia and hypoparathyroidism. Such microdeletions are usually detectable by fluorescence in situ hybridization (FISH). Case presentation We report a case of a twenty-three year-old female who presented with clinical features of chromosome 22q11.2 deletion syndrome including cardiac anomalies, hypoparathyroidism and dysmorphic facial features. FISH did not reveal a 22q11.2 microdeletion. Further genetic analysis showed T box-1 (TBX1) heterozygous mutation. Conclusions The TBX1 gene plays a significant role in the development of fourth pharyngeal arch structures. Mutations of TBX1, which is found at chromosome 22q11.21 can be responsible for the development of syndromes classically associated with chromosome 22q11.2 deletions. This case emphasizes that the TBX1 gene, among other genes, can be responsible for the developmental anomalies seen in these syndromes.

T-box riboregulators are a class of cis-regulatory RNAs that govern the bacterial response to amino acid starvation by binding, decoding and reading the aminoacylation status of specific transfer RNAs. Here we provide a high-resolution crystal structure of a full-length T-box from Mycobacterium tuberculosis that explains tRNA decoding and aminoacylation sensing by this riboregulator. Overall, the T-box consists of decoding and aminoacylation sensing modules bridged by a rigid pseudoknot structure formed by the mid-region domains. Stem-I and the Stem-II S-turn assemble a claw-like decoding module, while the antiterminator, Stem-III, and the adjacent linker form a tightly interwoven aminoacylation sensing module. The uncharged tRNA is selectively recognized by an unexpected set of favorable contacts from the linker region in the aminoacylation sensing module. A complex structure with a charged tRNA mimic shows that the extra moiety dislodges the linker, which is indicative of the possible chain of events that lead to alternative base-pairing and altered expression output.

International audience; Transcription factors that coordinate migration, differentiation or proliferation of enteric nervous system (ENS) precursors are not well defined. To identify novel transcriptional regulators of ENS development, we performed microarray analysis at embryonic day (E) 17.5 and identified many genes that were enriched in the ENS compared to other bowel cells. We decided to investigate the T-box transcription factor Tbx3, which is prominently expressed in developing and mature ENS. Haploinsufficiency for TBX3 causes ulnar-mammary syndrome (UMS) in humans, a multi-organ system disorder. TBX3 also regulates several genes known to be important for ENS development. To test the hypothesis that Tbx3 is important for ENS development or function, we inactivated Tbx3 in all neural crest derivatives, including ENS progenitors using Wnt1-Cre and a floxed Tbx3 allele. Tbx3 fl/fl; Wnt1-Cre conditional mutant mice die shortly after birth with cleft palate and difficulty feeding. The ENS of mutants was well-organized with a normal density of enteric neurons and nerve fiber bundles, but small bowel glial cell density was reduced. Despite this, bowel motility appeared normal. Furthermore, although Tbx3 is expressed in cardiac neural crest, Tbx3 fl/fl; Wnt1-Cre mice had structurally normal hearts. Thus, loss of Tbx3 within neural crest has selective effects on Tbx3-expressing neural crest derivatives.

The T-box genes are essential transcription factors during limb development. In Drosophila, Dorsocross (Doc) and optomotor-blind (omb), members of the Tbx2 and Tbx6 families, are best studied in the Drosophila wing development. Despite prominently expressed in leg discs, the specific function of these genes in leg growth is still not revealed. Here we demonstrated that Doc and omb regulated the morphogenesis of leg intermediate regions in a functionally redundant manner. Loss of Doc or omb individually did not result in any developmental defects of the legs, but loss of both genes induced significant defects in femur and proximal tibia of the adult legs. These genes located in the dorsal domain, where the Doc region expanded and cross-overlapped with the omb region corresponding to the presumptive leg intermediate region. We detected that the normal epithelial folds in the leg discs were disrupted along with dorsal repression of cell proliferation and activation of cell apoptosis when Doc and omb were both reduced. Furthermore, the dorsal expression of dachshund (dac), a canonical leg developmental gene specifying the leg intermediate region, was maintained by Doc and omb. Meanwhile, the Notch pathway was compromised in the dorsal domain when these genes were reduced, which might contribute to the joint defect of the adult leg intermediate regions. Our study provides cytological and genetic evidence for understanding the redundant function of Doc and omb in leg morphogenesis.

The T-box (TBX) genes are part of an evolutionarily conserved family of transcription factors involved in organ development. They serve key roles in a number of molecular mechanisms, including proliferation, cell fate and organ identity. In addition, previous studies suggest that TBX genes have essential functions in the tumorigenesis and progression of various types of cancer. For example, TBX proteins served significant roles in carcinogenesis, proliferation and differentiation, senescence and apoptosis, invasion and migration, mesenchymal-epithelial and epithelial-mesenchymal transition, oncogenic signaling pathways and drug sensitivity. However, the exact mechanisms by which TBX genes carry out these functions have not yet been fully elucidated. The present review focuses on the role of TBX genes in cancer, with the aim of further clarifying their function. As altered levels of TBX proteins have detrimental consequences in numerous types of cancer, there is a need for further research into TBX genes, which this review may aid through providing a comprehensive insight into the topic.

T-box transcription factors play important roles in vertebrate mesoderm formation. Eomesodermin is involved in the initial step of the prospective mesodermal cells recruited near the primitive streak. Then T or Brachyury gene is responsible for general and axial mesodermal development. Tbx6, on the other hand, promotes paraxial mesodermal development while suppressing neural differentiation. Here, we studied differentiative properties of mouse ES cells (mESCs) with its Tbx6 expression regulated under the Tet-off system. mESCs were treated with noggin to promote neural differentiation. When Tbx6 was simultaneously turned on, later neural differentiation of these cells hardly occurred. Next, mESCs were subjected to formation of the embryoid bodies (EBs). When Tbx6 was turned on during EB formation, the rate of later cardiac troponin T (cTnT)-positive cells increased. If the cells were further treated with a wnt inhibitor KY02111 after EB formation, a synergistic increase of cTnT-positive cells occurred. Tbx6 expression in mESCs influenced the constituent ratio of the cardiac myosin light chain types, such that atrial species markedly increased over ventricular ones. These results are coincident with the function of Tbx6 in normal development, in that Tbx6 strongly suppressed neural differentiation while promoting cardiac development in a cooperative manner with wnt inhibition., Highlights • Tbx6 expression in mouse ES cells (mESCs) inhibited neural differentiation. • Tbx6 expression in mESCs increased cardiac muscle synergistically with wnt inhibitor. • Tbx6 expression increased atrial myosin light chains over ventricular chains.

Embryonic development relies on activating and repressing regulatory influences that are faithfully integrated at the core promoter of individual genes. In vertebrates, the basal machinery recognizing the core promoter includes TATA-binding protein (TBP) and two TBP-related factors. In Xenopus embryos, the three TBP family factors are all essential for development and are required for expression of distinct subsets of genes. Here, we report on a non-canonical TBP family-insensitive (TFI) mechanism of transcription initiation that involves mesoderm and organizer gene expression. Using TBP family single- and triple-knockdown experiments, α-amanitin treatment, transcriptome profiling and chromatin immunoprecipitation, we found that TFI gene expression cannot be explained by functional redundancy, is supported by active transcription and shows normal recruitment of the initiating form of RNA polymerase II to the promoter. Strikingly, recruitment of Gcn5 (also known as Kat2a), a co-activator that has been implicated in transcription initiation, to TFI gene promoters is increased upon depletion of TBP family factors. TFI genes are part of a densely connected TBP family-insensitive T-box-Otx2-Gsc interaction network. The results indicate that this network of genes bound by Vegt, Eomes, Otx2 and Gsc utilizes a novel, flexible and non-canonical mechanism of transcription that does not require TBP or TBP-related factors., Highlighted article: A network of embryonic genes, many of which are expressed in the mesoderm and the organiser, can initiate transcription through a non-canonical mechanism.

Summary Combinatorial action of transcription factors (TFs) with partially overlapping expression is a widespread strategy to generate novel gene-expression patterns and, thus, cellular diversity. Known mechanisms underlying combinatorial activity require co-expression of TFs within the same cell. Here, we describe the mechanism by which two TFs that are never co-expressed generate a new, intersectional expression pattern in C. elegans embryos: lineage-specific priming of a gene by a transiently expressed TF generates a unique intersection with a second TF acting on the same gene four cell divisions later; the second TF is expressed in multiple cells but only activates transcription in those where priming occurred. Early induction of active transcription is necessary and sufficient to establish a competent state, maintained by broadly expressed regulators in the absence of the initial trigger. We uncover additional cells diversified through this mechanism. Our findings define a mechanism for combinatorial TF activity with important implications for generation of cell-type diversity., Graphical Abstract, Highlights • Lineage-specific priming enables asymmetric gene expression in L/R neuron pairs • Transient, lineage-specific TFs prime a locus for later activation by a bilateral TF • An early active transcriptional state is necessary and sufficient for priming • Maintenance of asymmetric primed state occurs in a symmetric regulatory environment, Studying neuronal specification in C. elegans, Charest et al. reveal that lineage-specific priming by a transiently expressed transcription factor allows activation of the primed gene by another transcription factor acting four cell divisions later. The findings suggest that the intersection of temporally segregated transcription factors contributes to cell-specific gene expression and cell diversification.

International audience; The arterial and venous poles of the mammalian heart are hotspots of congenital heart defects (CHD) such as those observed in 22q11.2 deletion (or DiGeorge) and Holt-Oram syndromes. These regions of the heart are derived from late differentiating cardiac progenitor cells of the Second Heart Field (SHF) located in pharyngeal mesoderm contiguous with the elongating heart tube. The T-box transcription factor Tbx1, encoded by the major 22q11.2 deletion syndrome gene, regulates SHF addition to both cardiac poles from a common progenitor population. Despite the significance of this cellular addition the mechanisms regulating the deployment of common progenitor cells to alternate cardiac poles remain poorly understood. Here we demonstrate that Tbx5, mutated in Holt-Oram syndrome and essential for venous pole development, is activated in Tbx1 expressing cells in the posterior region of the SHF at early stages of heart tube elongation. A subset of the SHF transcriptional program, including Tbx1 expression, is subsequently downregulated in Tbx5 expressing cells, generating a transcriptional boundary between Tbx1-positive arterial pole and Tbx5-positive venous pole progenitor cell populations. We show that normal downregulation of the definitive arterial pole progenitor cell program in the posterior SHF is dependent on both Tbx1 and Tbx5. Furthermore, retinoic acid (RA) signaling is required for Tbx5 activation in Tbx1-positive cells and blocking RA signaling at the time of Tbx5 activation results in atrioventricular septal defects at fetal stages. Our results reveal sequential steps of cardiac progenitor cell patterning and provide mechanistic insights into the origin of common forms of CHD.

T-box (TBX) transcription factors are evolutionary conserved genes and master transcriptional regulators. In mammals, TBX2 subfamily (TBX2, TBX3, TBX4, and TBX5) genes are expressed in the developing lung bud and tracheae. Our group previously showed that the expression of TBX2 subfamily was significantly high in human normal lungs, but markedly suppressed in lung adenocarcinoma (LUAD). To further elucidate their role in LUAD pathogenesis, we first confirmed abundant expression of protein products of the four members by immunostaining in adult human normal lung tissues. We also found overall suppressed expression of these genes and their corresponding proteins in a panel of human LUAD cell lines. Transient over-expression of each of the genes in human (NCI-H1299), and mouse (MDA-F471) derived lung cancer cells was found to significantly inhibit growth and proliferation as well as induce apoptosis. Genome-wide transcriptomic analyses on NCI-H1299 cells, overexpressing TBX2 gene subfamily, unraveled novel regulatory pathways. These included, among others, inhibition of cell cycle progression but more importantly activation of the histone demethylase pathway. When using a pattern-matching algorithm, we showed that TBX's overexpression mimic molecular signatures from azacitidine treated NCI-H1299 cells which in turn are inversely correlated to expression profiles of both human and murine lung tumors relative to matched normal lung. In conclusion, we showed that the TBX2 subfamily genes play a critical tumor suppressor role in lung cancer pathogenesis through regulating its methylating pattern, making them putative candidates for epigenetic therapy in LUAD.

International audience; A small network of spontaneously active Tbx3+ cardiomyocytes forms the cardiac conduction system (CCS) in adults. Understanding the origin and mechanism of development of the CCS network are important steps towards disease modeling and the development of biological pacemakers to treat arrhythmias. We found that Tbx3 expression in the embryonic mouse heart is associated with automaticity. Genetic inducible fate mapping revealed that Tbx3+ cells in the early heart tube are fated to form the definitive CCS components, except the Purkinje fiber network. At mid-fetal stages, contribution of Tbx3+ cells was restricted to the definitive CCS. We identified a Tbx3+ population in the outflow tract of the early heart tube that formed the atrioventricular bundle. Whereas Tbx3+ cardiomyocytes also contributed to the adjacent Gja5+ atrial and ventricular chamber myocardium, embryonic Gja5+ chamber cardiomyocytes did not contribute to the Tbx3+ sinus node or to atrioventricular ring bundles. In conclusion, the CCS is established by progressive fate restriction of a Tbx3+ cell population in the early developing heart, which implicates Tbx3 as a useful tool for developing strategies to study and treat CCS diseases.

T-box riboswitches are cis-regulatory RNA elements that regulate the expression of proteins involved in amino acid biosynthesis and transport by binding to specific tRNAs and sensing their aminoacylation state. While the T-box modular structural elements that recognize different parts of a tRNA have been identified, the kinetic trajectory describing how these interactions are established temporally remains unclear. Using smFRET, we demonstrate that tRNA binds to the riboswitch in two steps, first anticodon recognition followed by the sensing of the 3’ NCCA end, with the second step accompanied by a T-box riboswitch conformational change. Studies on site-specific mutants highlight that specific T-box structural elements drive the two-step binding process in a modular fashion. Our results set up a kinetic framework describing tRNA binding by T-box riboswitches, and suggest such binding mechanism is kinetically beneficial for efficient, co-transcriptional recognition of the cognate tRNA ligand., eLife digest Living organisms depend upon a group of chemicals called amino acids to survive. Amino acids are the building blocks of proteins, and proteins have many important roles within and around cells. Bacteria regulate certain genes to ensure they have the right balance of different amino acids to survive. By controlling the availability of certain proteins that help them to make or collect certain amino acids, bacteria can control their overall amino acid balance. Before a protein is made, a molecular machine called RNA polymerase must first copy the information in a gene to make a molecule called a messenger RNA (mRNA). The mRNA is then translated to make the protein from individual amino acids. In this process, each amino acid needs to be first attached to another molecule called a transfer RNA (tRNA). In many bacteria species, the mRNAs involved in making or transporting amino acids contain structures called T-boxes. These structures guide the RNA polymerase to make more of the mRNAs when the levels of the amino acid become too low. A T-box, however, does not sense the level of the amino acid directly. Instead it senses the number of tRNA molecules that do not carry an amino acid. Zhang, Chetnani et al. examined a particular T-box interacting with tRNA using pairs of fluorescent dyes to detect distances between molecules. The T-box first recognizes a part of the tRNA called the anticodon to make sure it binds the correct type of tRNA. It then changes its shape to detect whether the tRNA is attached to an amino acid. This two-step process is driven by multiple structural elements within the T-box, and the flexibility of the T-box plays a critical role. A cell’s survival depends on it keeping amino acid levels under control. Understanding how bacteria do this could lead to new antibiotic drugs that target the T-box to kill cells. This study also provides insights into the workings of mRNA components like T-boxes – a type of riboswitch – which is an unusual means of controlling gene activity.

T-box (TBX) transcription factors are evolutionary conserved genes and master transcriptional regulators. In mammals, TBX2 subfamily (TBX2, TBX3, TBX4, and TBX5) genes are expressed in the developing lung bud and tracheae. Our group previously showed that the expression of TBX2 subfamily was significantly high in human normal lungs, but markedly suppressed in lung adenocarcinoma (LUAD). To further elucidate their role in LUAD pathogenesis, we first confirmed abundant expression of protein products of the four members by immunostaining in adult human normal lung tissues. We also found overall suppressed expression of these genes and their corresponding proteins in a panel of human LUAD cell lines. Transient over-expression of each of the genes in human (NCI-H1299), and mouse (MDA-F471) derived lung cancer cells was found to significantly inhibit growth and proliferation as well as induce apoptosis. Genome-wide transcriptomic analyses on NCI-H1299 cells, overexpressing TBX2 gene subfamily, unraveled novel regulatory pathways. These included, among others, inhibition of cell cycle progression but more importantly activation of the histone demethylase pathway. When using a pattern-matching algorithm, we showed that TBX's overexpression mimic molecular signatures from azacitidine treated NCI-H1299 cells which in turn are inversely correlated to expression profiles of both human and murine lung tumors relative to matched normal lung. In conclusion, we showed that the TBX2 subfamily genes play a critical tumor suppressor role in lung cancer pathogenesis through regulating its methylating pattern, making them putative candidates for epigenetic therapy in LUAD.

We characterized the Andrias davidianus T-box 1 (Tbx1) gene. Tbx1 expression was high in testis and low in other examined tissues. Immunohistochemistry detected tbx 1 expression in somatic and germ cells 62 days post-hatching (dph), prior to gonad differentiation. At 210 dph, after gonad differentiation, tbx1 was expressed in spermatogonia and testis somatic cells and in granulosa cells in ovary. Tbx1 expression was up-regulated in ovary after high temperature treatment. In the neomale, tbx 1 expression showed a similar profile to normal males, and vice-versa for genetic male. Over-expression of tbx1 in females after injection of TBX1 protein down-regulated the female-biased genes cyp19a and foxl2 and up-regulated the male-biased amh gene. When tbx 1 was knocked down by tbx 1/siRNA, cyp19a and foxl2 expression was up-regulated, and expression of amh , cyp26a , dmrt1 , and wt1 was down-regulated. Results suggest that tbx1 influenced sex-related gene expression and participates in regulation of A. davidianus testis development.

Owing to a multitude of functions, there is barely a tissue or a cellular process that is not being regulated by Notch signaling. To allow the Notch signal to be deployed in numerous contexts, many different mechanisms have evolved to regulate the level, duration and spatial distribution of Notch activity. To identify novel effectors of Notch signaling in Drosophila melanogaster, we analyzed the whole transcriptome of the wing and eye imaginal discs in which an activated form of Notch was overexpressed. Selected candidate genes from the transcriptome analysis were subjected to genetic interaction experiments with Notch pathway components. Among the candidate genes, T-box encoding gene, Dorsocross (Doc) showed strong genetic interaction with Notch ligand, Delta. Genetic interaction between them resulted in reduction of eye size, loss of cone cells, and cell death, which represent prominent Notch loss of function phenotypes. Immunocytochemical analysis in Df(3L)DocA/Dl 5f trans-heterozygous eye discs showed accumulation of Notch at the membrane. This accumulation led to decreased Notch signaling activity as we found downregulation of Atonal, a Notch target and reduction in the rate of Notch-mediated cell proliferation. Doc mutant clones generated by FLP-FRT system showed depletion in the expression of Delta and subsequent reduction in the Notch signaling activity. Similarly, Doc overexpression in the eye discs led to modification of Delta expression, loss of Atonal expression and absence of eye structure in pharate adults. Taken together, our results suggest that Doc regulates the expression of Delta and influence the outcome of Notch signaling in the eye discs.

The Drosophila Tbx20 homologs midline and H15 act as selector genes for ventral fate in Drosophila legs. midline and H15 expression defines the ventral domain of the leg and the two genes are necessary and sufficient for the development of ventral fate. Ventral-specific expression of midline and H15 is activated by Wingless (Wg) and repressed by Decapentaplegic (Dpp). Here we identify VLE, a 5 kb enhancer that drives ventral specific expression in the leg disc that is very similar to midline expression. Subdivision of VLE identifies two regions that mediate both activation and repression and third region that only mediates repression. Loss- and gain-of-function genetic mosaic analysis shows that the activating and repressing regions respond to Wg and Dpp signaling respectively. All three repression regions depend on the activity of Mothers-against-decapentaplegic, a Drosophila r-Smad that mediates Dpp signaling, and respond to ectopic expression of the Dpp target genes optomoter-blind and Dorsocross 3. However, only one repression region is responsive to loss of schnurri, a co-repressor required for direct repression by Dpp-signaling. Thus, Dpp signaling restricts midline expression through both direct repression and through the activation of downstream repressors. We also find that midline and H15 expression are both subject to cross-repression and feedback inhibition. Finally, a lineage analysis indicates that ventral midline-expressing cells and dorsal omb-expressing cells do not mix during development. Together this data indicates that the ventral-specific expression of midline results from both transcriptional regulation and from a lack of cell-mixing between dorsal and ventral cells., Summary: Leg expression of the selector gene midline in Drosophila is controlled both by transcriptional regulation and by the lack of mixing between dorsal and ventral cells in the distal leg.

T-Box transcription factors are expressed throughout the gestational period and coordinate a variety of embryonic events that enable proper development, from the first differentiation of embryonic and extraembryonic tissues until final organogenesis. Although the T-Box gene family comprises essential roles in early cellular differentiation, in adult tissues it has also been associated with cancer development. In spite of their common T-Box regulatory binding domain, T-Box family members utilize different cofactors and different spatiotemporal expression patterns to confer their specificity in diverse developmental processes. The earliest expression note of T-Box factors can be observed even before fertilization in primordial germ cells and just after zygotic gene activation (around the eight-cell blastomere stage). Thus, particularly the early stages of development are highly influenced by these key regulators in line with the notion that T-Box mutations lead to developmental disorders and even lethality. In this review, we summarize recently acquired findings on T-Box factors to provide a comprehensive overview on their role during early embryogenesis.

The transcription factor brachyury (T, BRA) is one of the first markers of gastrulation and lineage specification in vertebrates. Despite its wide use and importance in stem cell and developmental biology, its functional genomic targets in human cells are largely unknown. Here, we use differentiating human embryonic stem cells to study the role of BRA in activin A-induced endoderm and BMP4-induced mesoderm progenitors. We show that BRA has distinct genome-wide binding landscapes in these two cell populations, and that BRA interacts and collaborates with SMAD1 or SMAD2/3 signalling to regulate the expression of its target genes in a cell-specific manner. Importantly, by manipulating the levels of BRA in cells exposed to different signalling environments, we demonstrate that BRA is essential for mesoderm but not for endoderm formation. Together, our data illuminate the function of BRA in the context of human embryonic development and show that the regulatory role of BRA is context dependent. Our study reinforces the importance of analysing the functions of a transcription factor in different cellular and signalling environments., Summary: In differentiating hESCs, BRACHYURY has cell type-specific targets and functions: it acts with SMAD1 to promote mesoderm specification, while in the endoderm it interacts with SMAD2/3.

T-box genes often exhibit dynamic expression patterns, and their expression levels can be crucial for normal function. Despite the importance of these genes, there is little known about T-box gene regulation. We have focused on the Caenorhabditis elegans gene tbx-2 to understand how T-box gene expression is regulated, and here we demonstrate TBX-2 itself directly represses its own expression in a negative autoregulatory loop. tbx-2 is essential for normal pharyngeal muscle development, and a tbx-2 promoter gfp fusion (Ptbx-2::gfp) is transiently expressed in the pharynx during embryogenesis and in a small number of head neurons in larvae and adults. Reduced tbx-2 function resulted in ectopic Ptbx-2::gfp expression in the seam cells and gut in larvae and adults. Mutation of potential T-box binding sites within the tbx-2 promoter resulted in a similar pattern of ectopic Ptbx-2::gfp expression, and chromatin immunoprecipitation analyses show TBX-2 binds these sites in vivo. This pattern of ectopic Ptbx-2::gfp expression in tbx-2 mutants was very similar to that observed in mutants affecting the NF-Y complex, and our results comparing tbx-2 and nfyb-1 single- and double mutants suggest TBX-2 and NF-Y function in a single pathway to repress the tbx-2 promoter. The tbx-2 promoter is the first direct target identified for TBX-2, and we used it to ask whether SUMOylation is essential for TBX-2 repression. RNAi knockdown of SUMOylation pathway components led to ectopic Ptbx-2::gfp expression in the seam cells and gut. Ectopic Ptbx-2::gfp also was observed in the syncytial hypodermis, suggesting either the tbx-2 promoter is repressed by other SUMOylation dependent mechanisms, or that decreased SUMOylation leads to stable changes in seam cell nuclei as they fuse with the syncytial hypodermis. We suggest negative autoregulation is an important mechanism that allows precise control of tbx-2 expression levels and may allow rapid changes in gene expression during development.

In addition to their cell-autonomous roles in mesoderm development, the zebrafish T-box transcription factors no tail a (ntla) and spadetail (spt/tbx16) are required for medial floor plate (MFP) formation. Posterior MFP cells are completely absent in zebrafish embryos lacking both Ntla and Spt function, and genetic mosaic analyses have shown that the two T-box genes promote MFP development in a non-cell-autonomous manner. On the basis of these observations, it has been proposed that Ntla/Spt-dependent mesoderm-derived signals are required for the induction of posterior but not anterior MFP cells. To investigate the mechanisms by which Ntla and Spt regulate MFP development, we have used photoactivatable caged morpholinos (cMOs) to silence these T-box genes with spatiotemporal control. We find that posterior MFP formation requires Ntla or Spt activity during early gastrulation, specifically in lateral margin-derived cells that converge toward the midline during epiboly and somitogenesis. Nodal signaling-dependent MFP specification is maintained in the absence of Ntla and Spt function; however, midline cells in ntla;spt morphants exhibit aberrant morphogenetic movements, resulting in their anterior mislocalization. Our findings indicate that Ntla and Spt do not differentially regulate MFP induction along the anterior-posterior axis; rather, the T-box genes act redundantly within margin-derived cells to promote the posterior extension of MFP progenitors.

Drosophila Midline (Mid) is an ortholog of vertebrate Tbx20, which plays roles in the developing heart, migrating cranial motor neurons, and endothelial cells. Mid functions in cell-fate specification and differentiation of tissues that include the ectoderm, cardioblasts, neuroblasts, and egg chambers; however, a role in the somatic musculature has not been described. We identified mid in genetic and molecular screens for factors contributing to somatic muscle morphogenesis. Mid is expressed in founder cells (FCs) for several muscle fibers, and functions cooperatively with the T-box protein H15 in lateral oblique muscle 1 and the segment border muscle. Mid is particularly important for the specification and development of the lateral transverse (LT) muscles LT3 and LT4, which arise by asymmetric division of a single muscle progenitor. Mid is expressed in this progenitor and its two sibling FCs, but is maintained only in the LT4 FC. Both muscles were frequently missing in mid mutant embryos, and LT4-associated expression of the transcription factor Krüppel (Kr) was lost. When present, LT4 adopted an LT3-like morphology. Coordinately, mid misexpression caused LT3 to adopt an LT4-like morphology and was associated with ectopic Kr expression. From these data, we concluded that mid functions first in the progenitor to direct development of LT3 and LT4, and later in the FCs to influence whichever of these differentiation profiles is selected. Mid is the first T-box factor shown to influence LT3 and LT4 muscle identity and, along with the T-box protein Optomotor-blind-related-gene 1 (Org-1), is representative of a new class of transcription factors in muscle specification.

In tetrapods, Tbx4, Tbx5 and Hox cluster genes are crucial for forelimb and hindlimb development and mutations in these genes are responsible for congenital limb defects. The molecular basis of their integrated mechanisms of action in the context of limb development remains poorly understood. We studied Tbx4 and Hoxc10 due to their overlapping loss of function phenotypes and co-localized expression in hindlimb buds. We report an extensive overlap between Tbx4 and Hoxc10 genome occupancy and their putative target genes. Tbx4 and Hoxc10 interact directly with each other, have the ability to bind to a previously unrecognised Tbox-Hox composite DNA motif and show synergistic activity when acting on reporter genes. Pitx1, the master gene for hindlimb specification, also shows extensive genomic colocalization with Tbx4 and Hoxc10. Genome occupancy by Tbx4 in hindlimb buds is similar to Tbx5 occupancy in forelimbs. In contrast, another Hox factor, Hoxd13, also interacts with Tbx4/Tbx5 but Hoxd13 antagonises Tbx4/Tbx5-dependent transcriptional activity. Collectively, the modulation of Tbx-dependent activity by Hox factors acting on common DNA targets may integrate different developmental processes for balanced formation of proportionate limbs.

Tbx6 is a T-box transcription factor with multiple roles in embryonic development as evidenced by dramatic effects on mesoderm cell fate determination, left/right axis determination, and somite segmentation in mutant mice. The expression of Tbx6 is restricted to the primitive streak and presomitic mesoderm, but some of the phenotypic features of mutants are not easily explained by this expression pattern. We have used genetically-inducible fate mapping to trace the fate of Tbx6-expressing cells in wild-type and mutant embryos to explain some of the puzzling features of the mutant phenotype. We created an inducible Tbx6-creERT2 transgenic mouse in which cre expression closely recapitulates endogenous Tbx6 expression both temporally and spatially. Using a lacZ-based Cre reporter and timed tamoxifen injections, we followed temporally overlapping cohorts of cells that had expressed Tbx6 and found contributions to virtually all mesodermally-derived embryonic structures as well as the extraembryonic allantois. Contribution to the endothelium of major blood vessels may account for the embryonic death of homozygous mutant embryos. In mutant embryos, Tbx6-creERT2-traced cells contributed to the abnormally segmented anterior somites and formed the characteristic ectopic neural tubes. Retention of cells in the mutant tail bud indicates a deficiency in migratory behavior of the mutant cells and the presence of Tbx6-creERT2-traced cells in the notochord, a node derivative provides a possible explanation for the heterotaxia seen in mutant embryos., Summary: Embryonic cells that transiently express the transcription factor, Tbx6, during the process of gastrulation have been tracked in later development in wild-type and Tbx6 homozygous mutant embryos, where they give rise to the ectopic neural tubes characteristic of the mutant phenotype.

The T-box transcription factors T-bet and Eomesodermin (Eomes) instruct discrete stages in NK cell development. However, their role in the immune response of mature NK cells against pathogens remains unexplored. We used an inducible deletion system to elucidate the cell-intrinsic role of T-bet and Eomes in mature NK cells during the course of mouse CMV infection. We show both T-bet and Eomes to be necessary for the expansion of virus-specific NK cells, with T-bet upregulation induced by IL-12 signaling and STAT4 binding to a conserved enhancer region upstream of the Tbx21 loci. Interestingly, our data suggest maintenance of virus-specific memory NK cell numbers and phenotype was dependent on T-bet, but not Eomes. These findings uncover a nonredundant and stage-specific influence of T-box transcription factors in the antiviral NK cell response.