T-box Domain Research Articles

The Glu86 Residue in TBX4 Proves Critical for Human Lung Development.

T-box transcription factors are a group of evolutionarily conserved T-box-containing regulators of mesoderm specification and development. Heterozygous single nucleotide variants (SNVs) or copy-number variant (CNV) deletions involving dosage-sensitive TBX4 have been associated with pulmonary arterial hypertension (PAH), ischiocoxopodopatellar syndrome with or without PAH, and lethal lung developmental disorders (LLDDs), including acinar dysplasia (AcDys), congenital alveolar dysplasia (CAD), and other unspecified primary pulmonary hypoplasias. Loss- and gain-of-function variants have been proposed to cause pediatric PAH and LLDDs, and adult forms of PAH, respectively. Of more than 50 missense SNVs scattered across the entire TBX4, only three have been reported in patients with LLDDs, all mapping to the T-box domain. Here, we report a recurrence of a pathogenic substitution Glu86Lys identified in an unrelated patient with AcDys. In silico predictions of the conformational changes of TBX4 resulting from this and another substitution, Glu86Gln, suggest the loss of most intermolecular hydrogen bonds involving residue 86, including those with Tyr230 that directly interact with DNA. Functional assays on the TBX4 variants in fetal lung fibroblasts confirmed their deleterious character. We propose that Glu86 is critically involved in maintaining TBX4 structure and function essential for airway branching during early stages of human lung development. Substitutions of this residue may act in a dominant negative manner, leading to AcDys and CAD.

An inherited TBX3 alteration in a prenatal case of ulnar-mammary syndrome: Clinical assessment and functional characterization in Drosophila melanogaster.

Ulnar mammary syndrome (UMS) results from heterozygous variants in the TBX3 gene and impacts limb, tooth, hair, apocrine gland, and genitalia development. The expressivity of UMS is highly variable with no established genotype-phenotype correlations. TBX3 belongs to the Tbx gene family, which encodes transcription factors characterized by the presence of a T-box DNA-binding domain. We describe a fetus exhibiting severe upper limb defects and harboring the novel TBX3:c.400 C > T (p.P134S) variant inherited from the mother who remained clinically misdiagnosed until prenatal diagnosis. Literature revision was conducted to uncover the TBX3 clinical and mutational spectrum. Moreover, we generated a Drosophila humanized model for TBX3 to study the developmental consequences of the p.P134S as well as of other variants targeting different regions of the protein. Phenotypic analysis in flies, coupled with in silico modeling on the TBX3 variants, suggested that the c.400 C > T is UMS-causing and impacts TBX3 localization. Comparative analyses of the fly phenotypes caused by the expression of all variants, demonstrated that missense changes in the T-box domain affect more significantly TBX3 activity than variants outside this domain. To improve the clinicians' recognition of UMS, we estimated the frequency of the main clinical features of the disease. Core features often present pre-pubertally include defects of the ulna and/or of ulnar ray, hypoplastic nipples and/or areolas and, less frequently, genitalia anomalies in young males. These results enhance our understanding of the molecular basis and the clinical spectrum of UMS, shedding light on the functional consequences of TBX3 variants in a developmental context.

Disruption of T-box transcription factor eomesa results in abnormal development of median fins in Oujiang color common carp Cyprinus carpio.

Median fins are thought to be ancestors of paired fins which in turn give rise to limbs in tetrapods. However, the developmental mechanisms of median fins remain largely unknown. Nonsense mutation of the T-box transcription factor eomesa in zebrafish results in a phenotype without dorsal fin. Compared to zebrafish, the common carp undergo an additional round of whole genome duplication, acquiring an extra copy of protein-coding genes. To verify the function of eomesa genes in common carp, we established a biallelic gene editing technology in this tetraploidy fish through simultaneous disruption of two homologous genes, eomesa1 and eomesa2. We targeted four sites located upstream or within the sequences encoding the T-box domain. Sanger sequencing data indicated the average knockout efficiency was around 40% at T1-T3 sites and 10% at T4 site in embryos at 24 hours post fertilization. The individual editing efficiency was high to about 80% at T1-T3 sites and low to 13.3% at T4 site in larvae at 7 days post fertilization. Among 145 mosaic F0 examined at four months old, three individuals (Mutant 1-3) showed varying degrees of maldevelopment in the dorsal fin and loss of anal fin. Genotyping showed the genomes of all three mutants were disrupted at T3 sites. The null mutation rates on the eomesa1 and eomesa2 loci were 0% and 60% in Mutant 1, 66.7% and 100% in Mutant 2, and 90% and 77.8% in Mutant 3, respectively. In conclusion, we demonstrated a role of eomesa in the formation and development of median fins in Oujiang color common carp and established an method that simultaneously disrupt two homologous genes with one gRNA, which would be useful in genome editing in other polyploidy fishes.

Functional Characterization of Cooperating MGA Deletions in RUNX1::RUNX1T1 Acute Myeloid Leukemia

Max-gene associated (MGA) is a dual-specificity transcription factor that is recurrently mutated in 2-5% of all cancers and commonly seen in lung adenocarcinoma, endometrial carcinoma, and colorectal cancer. Our group has previously described heterozygous loss-of-function mutations in 9% of RUNX1::RUNX1T1 pediatric AMLs. Similar loss-of-function mutations are seen in other hematopoietic malignancies, including chronic lymphocytic leukemia (CLL), natural killer/T-cell lymphoma, B-cell acute lymphoblastic leukemia (B-ALL), and T-cell acute lymphoblastic leukemia (T-ALL). MGA is a component of the non-canonical polycomb repressive complex (ncPRC1.6) containing an amino-terminal T-box domain and a carboxy-terminal Myc-like basic helix-loop-helix (bHLH) domain which are used to regulate MAX-network and T-box family targets. In hematological malignancies such as RUNX1::RUNX1T1 AMLs, MGA mutations disrupt or remove the Myc-like bHLH domain. The role of MGA in normal hematopoietic regulation and leukemic development are understudied. This is likely due to the lack of in vivo MGA deficiency models which are embryonic lethal. We hypothesize that the loss of MGA leads to disruption of the ncPRC1.6 complex, causing a loss in global transcriptional repression of many pathways, including Myc and E2F target genes, leading to enhanced hematopoietic cell growth and self-renewal, as well as shortening the latency of AML driven by RUNX1::RUNXT1. To test this hypothesis, we designed a multidisciplinary approach to characterize the hematopoietic function of MGA. IP-Westerns of overexpressed flag-tagged MGA and its subsequent variants in K562 cells revealed that MGA truncations disrupt interactions with components of the ncPRC1.6 complex. Because these truncations lead to the loss of MGA's molecular function, we next generated CRISPR/Cas9 to knockout MGA in Molm13 cells. Using RNA-Seq and CUT&RUN to assess the genomic localization of MGA, we determined that MGA binds to an average of 85,119 peaks and 17.7% of those sites are at transcription start sites, including cell cycle genes such as CCND2. Deletion of MGA led to the removal of repressive H3K27me3 marks and an increase in MYC binding as well as active histone marks such as H3K4me3 and H3K27Ac at the transcription start site of CCND2. This led to an increase in CCND2 expression (logFC 1.56, FDR 1.56E-12) which is supported by in vitro EdU assays showing a 10.43% increase in cells cycling into S-phase. To further define the hematopoietic function of MGA, we designed a conditional knockout Mga mouse model with LoxP sites flanking exon 3, leading to deletion of Mga in hematopoietic stem and progenitor cells (HSPCs) when crossed with Vav1-cre. Transcriptome profiling analysis of HSPCs isolated from heterozygous (Mga+/) and homozygous (Mga-/-) mice reveals that Mga-/- mice, but not Mga+/-, leads to up-regulation of cell cycle (FDR 4.2E-06), Myc (FDR 6.3E-10), and E2f (FDR 2.9E-17) signaling pathways. Ex vivo serial CFU replating and cell proliferation assays support this, showing that Mga-/- leads to increased stem cell self-renewal and cell growth. However, bone marrow and spleen analysis of both Mga+/- and Mga-/- mice reveal no changes in immunophenotype when compared to WT controls. Expression of RUNX1::RUNX1T1 isoform 9a (AML-ETO9a) in Mga+/- or Mga-/- HSPCs reveals a significant increase (69% and 74% respectively, p<0.01) in leukemic stem cell self-renewal, when compared to WT controls expressing AML-ETO9a at week 4. When transplanted in mice, Mga+/- and Mga-/- HSPCs expressing AML-ETO9a led to a rapid expansion of blasts (GFP+, lin-, cKit+) found in the peripheral blood as well as a shortened latency compared to wild-type controls expressing AML-ETO9a (Figure 1). These studies demonstrated MGA as a regulator of hematopoietic cell proliferation (Figure 2), and how MGA deficiency cooperates with oncogenes to create more aggressive leukemia. Because MGA mutations occur in many tumor types, these data also provide functional insights into ncPRC1.6 activity in leukemia and other tumor types with recurrent mutations in MGA or other components of this complex. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

TBX5 variant with the novel phenotype of mixed‑type total anomalous pulmonary venous return in Holt‑Oram Syndrome and variable intrafamilial heart defects.

Variants in T-box transcription factor 5 (TBX5) can result in a wide phenotypic spectrum, specifically in the heart and the limbs. TBX5 has been implicated in causing non-syndromic cardiac defects and Holt-Oram syndrome (HOS). The present study investigated the underlying molecular etiology of a family with heterogeneous heart defects. The proband had mixed-type total anomalous pulmonary venous return (mixed-type TAPVR), whereas her mother had an atrial septal defect. Genetic testing through trio-based whole-exome sequencing was used to reveal the molecular etiology. A nonsense variant was identified in TBX5 (c.577G>T; p.Gly193*) initially showing co-segregation with a presumably non-syndromic presentation of congenital heart disease. Subsequent genetic investigations and more complete phenotyping led to the correct diagnosis of HOS, documenting the novel association of mixed-type TAPVR with HOS. Finally, protein modeling of the mutant TBX5 protein that harbored this pathogenic nonsense variant (p.Gly193*) revealed a substantial drop in the quantity of non-covalent bonds. The decrease in the number of non-covalent bonds suggested that the resultant mutant dimer was less stable compared with the wild-type protein, consequently affecting the protein's ability to bind DNA. The present findings extended the phenotypic cardiac defects associated with HOS; to the best of our knowledge, this is the first association of mixed-type TAPVR with TBX5. Prior to the current analysis, the molecular association of TAPVR with HOS had never been documented; hence, this is the first genetic investigation to report the association between TAPVR and HOS. Furthermore, it was demonstrated that the null-variants reported in the T-box domain of TBX5 were associated with a wide range of cardiac and/or skeletal anomalies on both the inter-and intrafamilial levels. In conclusion, genetic testing was highlighted as a potentially powerful approach in the prognostication of the proper diagnosis.

Abstract P331: Upregulated TBX1 by genetic variants are associated with human congenital heart disease

Congenital heart disease (CHD) is the most common human birth defect worldwide. The cause of CHD is so far not well understood. Uncovering genetic factors leading to CHD is still a pressing task to be solved. TBX1 is one of the transcription factors early expressed in embryonic cardiac progenitors. In animal models, imbalanced TBX1 activity leads to cardiac defects. Given the dosage effect of TBX1, it is possible that genetic variant altering TBX1 function or expression level would affect heart development and contribute to CHD. In order to study the association of genetic variants of TBX1 and CHD susceptibility, we performed genetic screening in 409 CHD patients and 213 healthy controls. Bioinformatic and in vitro functional studies were performed to evaluate the impact of genetic variants. One single nucleotide polymorphism (SNP), rs41260844, in TBX1 promoter region was identified to be associated with CHD. The minor allele of rs41260844 is associated with higher CHD risk and shows increased TBX1 promoter activity (Fig A). Further study showed the minor allele attenuates TBX1 promoter binding affinity with nuclear protein(s) (Fig B). In addition, a novel case-specific missense rare mutation, p.P164L, in T-box domain was identified and predicted as a deleterious mutation. Functional analysis showed a trend of increased TBX1 function with the rare mutation. In summary, we concluded that a higher TBX1 expression level or activity is associated with CHD susceptibility, which could affect TBX1 downstream targets and thus disrupt the balance of the complex regulation network during cardiogenesis.

TBX5 R264K acts as a modifier to develop dilated cardiomyopathy in mice independently of T-box pathway.

TBX5 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. To investigate whether the TBX5 R264K is deleterious and could contribute to the pathogenesis of cardiomyopathy. We 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. Mice 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.

On the Spectrum with T‐box Brain Protein 1: A Transcription Factor Protein Critical for Normal Neural Development

The Independent School MSOE Center for BioMolecular Modeling SMART Team used 3‐D modeling and printing technology to examine structure‐function relationships and applications of T‐box brain protein 1 (TBR1). The overall function of TBR1 is to act as a transcription factor protein. Located in the nucleus, this protein aids in vertebrate embryonic brain and neural development, specifically acting as an essential component in cerebral cortex development. TBR1 has the same general structure as other transcription factor protein homologs of the T‐box family, which all share a common DNA‐binding motif (the T‐box) and can form homodimers. Structurally, TBR1 has been studied to show how specific residues in its active site could lead to autism spectrum disorders (among other neurological diseases) if it does not align properly with DNA during transcription. Within the T‐box domain at Position 271, a tryptophan sidechain is located in the center of the motif that stabilizes the domain through contact with surrounding hydrophobic amino acids and ensures that DNA‐binding residues are correctly positioned. At Position 374, an asparagine residue also stabilizes the domain and ensures that DNA‐binding sites are positioned correctly by forming hydrogen bonds with its surrounding residues. Studies suggest that positively‐charged lysine amino acids that are close to the negatively‐charged phosphate backbone of DNA, such as those at Position 288 and Position 389, are critical for the binding of the protein to the minor groove of DNA. Additionally, TBR1 has been shown to act as either a repressor or activator in gene regulation of several important neurological cascades. Therefore, while not all the mechanisms are understood and many of its applications are still theoretical at this time, TBR1 has been shown to be involved with attention and memory. When this transcription factor protein malfunctions, its deregulation has been linked to Parkinson's disease and Alzheimer's disease. It has also been linked to several autism‐related disorder genes and a small percentage of unrelated sporadic autism disorders. Finally, TBR1 may be linked to language and speech disorders and synesthesia, though more research must be conducted to understand its exact function in these areas.Support or Funding InformationSupported and Funded by The Independent School; Wichita, KS 67207This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

DNA‐binding specificity of TBX5 and its coding variants associated with Holt‐Oram Syndrome

Transcription factors (TFs) are important sequence‐specific DNA binding proteins that regulate gene expression and control cellular state. Evaluation of the DNA‐binding properties of transcription factors is fundamental for understanding their possible roles in controlling gene regulatory networks. TBX5 is a transcription factor that has been identified as an important regulator in organ development. TBX5 mutations have been associated to Holt‐Oram Syndrome, a genetic disorder that affects upper limb formation and often the heart. We aim to determine the specificity of DNA‐binding of TBX5 and its T‐Box DNA‐binding domain and how it changes when the protein mutates. We have cloned TBX5 and its T‐Box domain that contains a His‐ and Strep‐Tag affinity tags that we plan to use for purifying after we overexpress it in E. coli cells. We will then analyze the DNA‐binding specificity of TBX5 and the changes it conveys when it mutates using Cognate Site Identification by High‐Throughput Systematic Evolution of Ligands by Exponential enrichment (HT‐SELEX), as this will allow us to better understand the impact of TBX5 mutations on its function.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Mga Modulates Bmpr1a Activity by Antagonizing Bs69 in Zebrafish.

MAX giant associated protein (MGA) is a dual transcriptional factor containing both T-box and bHLHzip DNA binding domains. In vitro studies have shown that MGA functions as a transcriptional repressor or activator to regulate transcription of promotors containing either E-box or T-box binding sites. BS69 (ZMYND11), a multidomain-containing (i.e., PHD, BROMO, PWWP, and MYND) protein, has been shown to selectively recognizes histone variant H3.3 lysine 36 trimethylation (H3.3K36me3), modulates RNA Polymerase II elongation, and functions as RNA splicing regulator. Mutations in MGA or BS69 have been linked to multiple cancers or neural developmental disorders. Here, by TALEN and CRISPR/Cas9-mediated loss of gene function assays, we show that zebrafish Mga and Bs69 are required to maintain proper Bmp signaling during early embryogenesis. We found that Mga protein localized in the cytoplasm modulates Bmpr1a activity by physical association with Zmynd11/Bs69. The Mynd domain of Bs69 specifically binds the kinase domain of Bmpr1a and interferes with its phosphorylation and activation of Smad1/5/8. Mga acts to antagonize Bs69 and facilitate the Bmp signaling pathway by disrupting the Bs69–Bmpr1a association. Functionally, Bmp signaling under control of Mga and Bs69 is required for properly specifying the ventral tailfin cell fate.

Egr2 and 3 Inhibit T-bet-Mediated IFN-γ Production in T Cells.

T-bet is important for differentiation of cytotoxic CD8 and Th1 CD4 T cells. We have discovered that Egr2 and 3 are potent inhibitors of T-bet function in CD4 and CD8 effector T cells. Egr2 and 3 were essential to suppress Th1 differentiation in Th2 and Th17 conditions in vitro and also to control IFN-γ–producing CD4 and CD8 T cells in response to virus infection. Together with Egr2 and 3, T-bet is induced in naive T cells by Ag stimulation, but Egr2 and 3 expression was inhibited by Th1–inducing cytokines. We found that Egr2 and 3 physically interact with the T-box domain of T-bet, blocking T-bet DNA binding and inhibiting T-bet–mediated production of IFN-γ. Thus, Egr2 and 3 are antagonists of T-bet function in effector T cells and are important for the control of inflammatory responses of T cells.

TBX20 loss-of-function mutation responsible for familial tetralogy of Fallot or sporadic persistent truncus arteriosus.

Congenital heart disease (CHD), the most common form of developmental abnormality in humans, remains a leading cause of morbidity and mortality in neonates. Genetic defects have been recognized as the predominant causes of CHD. Nevertheless, CHD is of substantial genetic heterogeneity and the genetic defects underlying CHD in most cases remain unclear. In the current study, the coding regions and splicing junction sites of the TBX20 gene, which encodes a T-box transcription factor key to cardiovascular morphogenesis, were sequenced in 175 unrelated patients with CHD, and a novel heterozygous TBX20 mutation, p.K274X, was identified in an index patient with tetralogy of Fallot (TOF). Genetic analysis of the proband's available family members showed that his father, elder brother and son had also TOF. In addition, his father and elder brother had also atrial septal defect, and his niece had persistent truncus arteriosus and ventricular septal defect. Analysis of the pedigree revealed that the mutation co-segregated with CHD transmitted in an autosomal dominant fashion, with complete penetrance. The nonsense mutation, which was absent in the 800 control chromosomes, was predicted to produce a truncated protein with only the amino terminus and partial T-box domain left. Functional analyses by using a dual-luciferase reporter assay system showed that the mutant TBX20 lost the ability to transactivate the target gene ANF. Furthermore, the mutation reduced the synergistic activation between TBX20 and NKX2.5 as well as GATA4, two other transcriptional factors previously associated with various CHD, encompassing TOF. This study firstly links TBX20 loss-of-function mutation to familial TOF or sporadic persistent truncus arteriosus, providing novel insight into the molecular pathogenesis of CHD.

Tbx20 drives cardiac progenitor formation and cardiomyocyte proliferation in zebrafish

Tbx20 is a T-box transcription factor that plays essential roles in the development and maintenance of the heart. Although it is expressed by cardiac progenitors in all species examined, an involvement of Tbx20 in cardiac progenitor formation in vertebrates has not been previously described. Here we report the identification of a zebrafish tbx20 mutation that results in an inactive, truncated protein lacking any functional domains. The cardiac progenitor population is strongly diminished in this mutant, leading to the formation of a small, stretched-out heart. We found that overexpression of Tbx20 results in an enlarged heart with significantly more cardiomyocytes. Interestingly, this increase in cell number is caused by both enhanced cardiac progenitor cell formation and the proliferation of differentiated cardiomyocytes, and is dependent upon the activity of Tbx20's T-box and transcription activation domains. Together, our findings highlight a previously unappreciated role for Tbx20 in promoting cardiac progenitor formation in vertebrates and reveal a novel function for its activation domain in cardiac cell proliferation during embryogenesis.

The Roles of T-Box Genes in Vertebrate Limb Development.

Members of the T-box gene family have diverse roles during embryogenesis and many play critical roles in the developing limb. This is exemplified by the fact that, in humans, mutations in T-box genes are associated with several congenital syndromes that include limb defects as part of their characteristic spectrum of abnormalities. T-box genes encode for evolutionary conserved transcription factors that include both transcriptional activators and repressors. The hallmark of T-box gene members is the presence of the eponymous DNA-binding T-box domain. There are 17 mammalian T-box genes, which based on the sequence homology of the T-box domain, are grouped into five subfamilies, namely, T, Tbx1, Tbx2, Tbx6, and Tbr1. At least nine T-box genes are expressed during limb development with distinct and dynamic expression patterns. All four members of Tbx2 subfamily (Tbx2, Tbx3, Tbx4, Tbx5) and three members of Tbx1 (Tbx1, Tbx15, Tbx18), Brachyury (T) and Eomes (Tbr2) are expressed in the developing limb.

Genetic mutation analysis in Japanese patients with non-syndromic congenital heart disease.

Congenital heart disease (CHD) is the most common birth defect occurring in humans and some transcriptional factors have been identified as causative. However, additional mutation analysis of these genes is necessary to develop effective diagnostic and medical treatment methods. We conducted sequence analysis of the coding regions of NKX2.5, GATA4, TBX1, TBX5, TBX20, CFC1 and ZIC3 in 111 Japanese patients with non-syndromic CHD and 9 of their relatives. All patient samples were also analyzed by multiplex ligation-dependent probe amplification using probes involved in chromosome deletion related to CHD. Five novel variations of TBX5, GATA4 and TBX20 were detected in 6 of the patients, whereas none were found in 200 controls. The TBX5 variation p.Pro108Thr, located in the T-box domain, was identified in a patient with tricuspid atresia, an exon-intron boundary variation of GATA4 (IVS4+5G>A) was detected in a Tetralogy of Fallot patient and an 8p23 microdeletion was detected in one patient with atrioventricular septal defect and psychomotor delay. A total of seven non-synonymous polymorphisms were found in the patients and controls. Accumulation of novel variations of genes involving the cardiac development may be required for better understanding of CHD.

Novel ENU-Induced Mutation in Tbx6 Causes Dominant Spondylocostal Dysostosis-Like Vertebral Malformations in the Rat.

Congenital vertebral malformations caused by embryonic segmentation defects are relatively common in humans and domestic animals. Although reverse genetics approaches in mice have provided information on the molecular mechanisms of embryonic somite segmentation, hypothesis-driven approaches cannot adequately reflect human dysmorphology within the population. In a N-ethyl-N-nitrosourea (ENU) mutagenesis project in Kyoto, the Oune mutant rat strain was isolated due to a short and kinked caudal vertebra phenotype. Skeletal staining of heterozygous rats showed partial loss of the cervical vertebrae as well as hemivertebrae and fused vertebral blocks in lumbar and sacral vertebrae. In homozygous embryos, severe displacement of the whole vertebrae was observed. The Oune locus was genetically mapped to rat chromosome 1 using 202 backcross animals and 50 genome-wide microsatellite markers. Subsequently, a miss-sense mutation in the Tbx6 gene was identified in the critical region. Although the mutation is located within the T-box domain near a predicted dimmer-interface, in vitro experiments revealed that the Tbx6 variant retains normal DNA binding ability and translational efficiency. However, the variant has decreased transcriptional activation potential in response to Notch-mediated signaling. Recently, it was reported that a dominant type of familial spondylocostal dysostosis is caused by a stoploss mutation in TBX6. Thus, we propose that partial dysfunction of Tbx6 leads to similar congenital vertebral malformations in both humans and rats. The Oune strain could be a unique animal model for dominant spondylocostal dysostosis and is useful for molecular dissection of the pathology of congenital vertebral malformations in humans.

Growth, Structure, and Stability of the High-Index TbOx(112) Surface on Cu(111)

Low-energy electron microscopy and microillumination low-energy electron diffraction (μLEED) were used to investigate the structural properties of TbOx films grown on Cu(111) in ultrahigh vacuum. Our results reveal that the morphology and structure of the terbia films depend sensitively on the surface temperature during growth. Deposition at room temperature produces terbia films that uniformly cover the surface but lack long-range order that is detectable with LEED. Annealing in O2 induces the formation of three-dimensional TbOx islands that grow as dendrites and cause the disordered terbia to dewet from the Cu(111) substrate. LEED measurements show that the terbia dendrites are crystalline with a hexagonal (1.4 × 1.4) structure with respect to the Cu(111) surface, which is consistent with cubic fluorite-like TbOx(111). In contrast to room temperature growth, deposition at 475 °C produces mainly rectangular TbOx islands that coexist with smaller quantities of irregularly shaped islands. Utilizing μLEED, we identify the rectangular domains as the cubic fluorite-like TbOx(112) structure while the irregularly shaped islands are TbOx(111). The TbOx(112) structure has a rectangular unit cell of dimensions (3.58 Å × 8.86 Å) that grows in registry with the Cu(111) surface by aligning the short side of the rectangular cell parallel to a close-packed direction of Cu(111). Analysis shows that the longer lattice vector of TbOx(112) coincides with every fourth row of the Cu(111) surface, while the shorter lattice vector has a 5:7 coincidence relation with Cu(111). Lastly, we find that the TbOx(112) phase is less stable than the (111) phase and incorporates into the TbOx(111) domains above 530 °C.

Exome Sequencing Identifies a c.148-1G>C Mutation of TBX5 in a Holt-Oram Family with Unusual Genotype-Phenotype Correlations

Background/Aims: Congenital heart defects (CHD) can occur with upper limbs deformities. Holt-Oram syndrome is the main type of heart-hand syndromes, characterized by upper limb radial ray malformations, CHD and/or conduction abnormalities. Mutations of the TBX5 gene, most of which are found within the T-box domain, are one cause of the disease. We aimed to find the cause of the disease in a family with two children exhibiting symptoms of Holt-Oram syndrome while the parents tend to be normal. Methods: Chromosomal microarray analysis and exome sequencing were applied in the proband segments bearing the specific mutation and single nucleotide variants (SNVs) suspected of being involved in the disease were analyzed by polymerase chain reaction and direct sequencing. Results: A splice acceptor site mutation c.148-1G>C of TBX5 was detected in both the father and the proband. The mutation may result in an aberrant transcript which will most probably undergo nonsense-mediated decay (NMD) system resulting in haploinsufficiency of TBX5 protein. In the meantime, 3 candidated SNVs were detected. Conclusions: c.148-1G>C of TBX5 should be the pathogenic cause of the disease in this family. Works have been done to find a possible explanation of the unusual genotype-phenotype correlations in this family and further studies are still needed.

Novel mutations in the transcriptional activator domain of the human TBX20 in patients with atrial septal defect.

Background. The relevance of TBX20 gene in heart development has been demonstrated in many animal models, but there are few works that try to elucidate the effect of TBX20 mutations in human congenital heart diseases. In these studies, all missense mutations associated with atrial septal defect (ASD) were found in the DNA-binding T-box domain, none in the transcriptional activator domain. Methods. We search for TBX20 mutations in a group of patients with ASD or ventricular septal defect (VSD) using the High Resolution Melting (HRM) method and DNA sequencing. Results. We report three missense mutations (Y309D, T370O, and M395R) within the transcriptional activator domain of human TBX20 that were associated with ASD. Conclusions. This is the first association of TBX20 transcriptional activator domain missense mutations with ASD. These findings could have implications for diagnosis, genetic screening, and patient follow-up.

Lysine 313 of T-box Is Crucial for Modulation of Protein Stability, DNA Binding, and Threonine Phosphorylation of T-bet

A T-box-containing protein expressed in T cells (T-bet) is a key transcription factor involved in the regulation of Th cell differentiation. Although T-bet-deficient CD4(+) T cells fail to produce IFN-γ and typically differentiate into Th2 cells in vitro, ectopic overexpression of T-bet elevates IFN-γ and suppresses production of IL-2 and Th2 cytokines through different mechanisms. Despite the importance of the T-bet protein level, the regulatory mechanisms that control T-bet protein stability are largely unknown. In this study, we found that T-bet underwent proteasomal degradation via ubiquitination at Lys-313. Despite its robust accumulation following lysine mutation, T-bet(K313R) failed to increase IFN-γ production because of diminished DNA binding activity, as demonstrated in the crystal structure of T-bet-DNA complex. Strikingly, T-bet(K313R) entirely lost the ability to suppress IL-2 production and Th2 cell development; this was due to loss of its interaction with NFAT1. We further identified that the T-bet(K313R) reduced the phosphorylation of T-bet at Thr-302, and that threonine phosphorylation was essential for T-bet interaction with NFAT1 and suppression of NFAT1 activity. Retroviral transduction of T-bet(T302A) into T-bet-deficient cells restored IFN-γ levels compared with those induced by wild-type T-bet, but this mutant failed to inhibit IL-2 and Th2 cytokine production. Collectively, these data show that Lys-313 in the T-box domain is essential for controlling T-bet protein stability via ubiquitin-dependent degradation, T-bet binding to the IFN-γ promoter, and for the interaction with and suppression of NFAT1. Thus, multiple posttranslational modifications of T-bet are involved in fine-tuning cytokine production during Th cell development.