Mutations In TBX5 Research Articles

Loss of Tbx1 induces bone phenotypes similar to cleidocranial dysplasia.

T-box transcription factor, TBX1, is the major candidate gene for 22q11.2 deletion syndrome (DiGeorge/ Velo-cardio-facial syndrome) characterized by facial defects, thymus hypoplasia, cardiovascular anomalies and cleft palates. Here, we report that the loss of Tbx1 in mouse (Tbx1(-/-)) results in skeletal abnormalities similar to those of cleidocranial dysplasia (CCD) in humans, which is an autosomal-dominant skeletal disease caused by mutations in RUNX2. Tbx1(-/-) mice display short stature, absence of hyoid bone, failed closure of fontanelle, bifid xiphoid process and hypoplasia of clavicle and zygomatic arch. A cell-type-specific deletion of Tbx1 in osteochondro-progenitor (Tbx1(OPKO)) or mesodermal (Tbx1(MKO)) lineage partially recapitulates the Tbx1(-/-) bone phenotypes. Although Tbx1 expression has not been previously reported in neural crest, inactivation of Tbx1 in the neural crest lineage (Tbx1(NCKO)) leads to an absence of the body of hyoid bone and postnatal lethality, indicating an unanticipated role of Tbx1 in neural crest development. Indeed, Tbx1 is expressed in the neural crest-derived hyoid bone primordium, in addition to mesoderm-derived osteochondral progenitors. Ablation of Tbx1 affected Runx2 expression in calvarial bones and overexpression of Tbx1 induced Runx2 expression in vitro. Taken together, our current studies reveal that Tbx1 is required for mesoderm- and neural crest-derived osteoblast differentiation and normal skeletal development. TBX1 mutation could lead to CCD-like bone phenotypes in human.

A mutation in the Kozak sequence of GATA4 hampers translation in a family with atrial septal defects.

Atrial septal defect (ASD) is the most common congenital heart defect clinically characterized by an opening in the atrial septum. Mutations in GATA4, TBX5, and NKX2-5 underlie this phenotype. Here, we report on the identification of a novel -6 G>C mutation in the highly conserved Kozak sequence in the 5'UTR of GATA4 in a small family presenting with two different forms of ASD. This is the first time a mutation in the Kozak sequence has been linked to heart disease. Functional assays demonstrate reduced GATA4 translation, though the GATA4 transcript levels remain normal. This leads to a reduction of GATA4 protein level, consequently diminishing the ability of GATA4 to transactivate target genes, as demonstrated by using the GATA4-driven Nppa (ANF) promoter. In conclusion, we identified a mutation in the GATA4 Kozak sequence that likely contributes to the pathogenesis of ASD. In general, it points to the importance of accurate protein level regulation during heart development and emphasizes the need to analyze the entire transcribed region when screening for mutations.

Novel TBX1 loss-of-function mutation causes isolated conotruncal heart defects in Chinese patients without 22q11.2 deletion.

BackgroundTBX1 and CRKL haploinsufficiency is thought to cause the cardiac phenotype of the 22q11.2 deletion syndrome. However, few unequivocal mutations of TBX1 and CRKL have been discovered in isolated conotrucal heart defects (CTDs) patients. The aim of the study was to screen the mutation of TBX1 and CRKL in isolated CTDs Chinese patients without 22q11.2 deletion and identify the pathomechanism of the missense mutations.MethodsWe enrolled 199 non-22q11.2 deletion patients with CTDs and 139 unrelated healthy controls. Gene sequencing were performed for all of them. The functional data of mutations were obtained by in vitro transfection and luciferase experiments and computer modelling.ResultsScreening of the TBX1 coding sequence identified a de novo missense mutation (c.385G → A; p.E129K) and a known polymorphism (c.928G → A; p.G310S). In vitro experiments demonstrate that the TBX1E129K variant almost lost transactivation activity. The TBX1G310S variant seems to affect the interaction of TBX1 with other factors. Computer molecular dynamics simulations showed the de novo missense mutation is likely to affect TBX1-DNA interaction. No mutation of CRKL gene was found.ConclusionsThese observations suggest that the TBX1 loss-of-function mutation may be involved in the pathogenesis of isolated CTDs. This is the first human missense mutation showing that TBX1 is a candidate causing isolated CTDs in Chinese patients without 22q11.2 deletion.

543miR-19a replacement rescues cardiac and fin defects in zebrafish model of holt-oram syndrome

Purpose: MicroRNAs (miRNAs) are short, single-stranded RNAs that anneal with complementary sequences in mRNAs thereby suppressing protein expression and often decreasing mRNA stability. Due to the high number of genes that are potentially targeted by one miRNA and to the relatively low number of different miRNAs coded in the genome, miRNAs are the best candidates to orchestrate gene expression in embryonic development and in response to inducing signals, by integrating distinct transcription factors pathways. Indeed, the manipulation of miRNA expression or function can have a profound impact on cellular phenotype. miRNA are critically involved in almost all the biological processes in health and disease including several cardiovascular disorders. Tbx5 is a transcription factor crucial for heart development and implicated in cardiac function. In human, TBX5 mutations are associated with Holt-Oram Syndrome (HOS), which is characterized by upper limb and congenital heart defects. In mouse model of HOS, expression profiling revealed that Tbx5 regulates hundreds of genes through complex transcriptional networks, acting both directly on gene expression and indirectly as “regulating of regulators”. Our data indicate that miRNAs can be important effectors of Tbx5. The goal of our project is to better characterize the miRNAs/Tbx5 regulatory network with the aim: a) to understand the molecular roots of the complex HOS phenotype; b) to verify if it might be possible, by miRNA modulation, to restore the aberrant pathways generated by Tbx5 alteration. Methods: Using zebrafish as model systems of HOS, we characterized the miRNA profiles of wild type (WT) and Tbx5-depleted embryos by NGS. The impact of differentially expressed miRNAs on cardiac development of WT embryos and Tbx5 morphants was investigated by gain and loss of function experiments. Results: Among the identified miRNAs responsive to Tbx5 dosage we focused on miR-19a, whose expression decreases in HOS zebrafish embryos. We showed that: a) miR-19a dysregulation affects heart development; b) miR-19a modulates the expression of genes important for epicardial development and retinoic acid metabolism; c) miR-19a replacement was able to rescue cardiac and pectoral fin defects and increase the viability of Tbx5 morphants. Conclusions-Our data demonstrate that in zebrafish the modulation of a single miRNA is sufficient to reduce the defects generated by a complex syndrome like HOS, supporting enthusiasm for the continued exploration of miRNAs as a new class of drug target.

Disruption of myocardial Gata4 and Tbx5 results in defects in cardiomyocyte proliferation and atrioventricular septation

Mutations in GATA4 and TBX5 are associated with congenital heart defects in humans. Interaction between GATA4 and TBX5 is important for normal cardiac septation, but the underlying molecular mechanisms are not well understood. Here, we show that Gata4 and Tbx5 are co-expressed in the embryonic atria and ventricle, but after E15.5, ventricular expression of Tbx5 decreases. Co-localization and co-immunoprecipitation studies demonstrate an interaction of Gata4 and Tbx5 in the developing atria and ventricles, but the ventricular interaction declines after E14.5. Gata4(+/-);Tbx5(+/-) mouse embryos display decreased atrial and ventricular myocardial thickness at E11.5, prior to cardiac septation. To determine the cell lineage in which the interaction was functionally significant in vivo, mice heterozygous for Gata4 in the myocardium or endocardium and heterozygous for Tbx5 (Gata4(MyoDel/wt);Tbx5(+/-) and Gata4(EndoDel/wt);Tbx5(+/-), respectively) were generated. Gata4(MyoDel/wt);Tbx5(+/-) mice displayed embryonic lethality, thin myocardium with reduced cell proliferation, and atrioventricular septation defects similar to Gata4;Tbx5 compound heterozygotes while Gata4(EndoDel/wt);Tbx5(+/-) embryos were normal. Cdk4 and Cdk2, cyclin-dependent kinases required for myocardial development and septation were reduced in Gata4(+/-);Tbx5(+/-) hearts. Cdk4 is a known direct target of Gata4 and the regulation of Cdk2 in the developing heart has not been studied. Chromatin immunoprecipitation and transactivation studies demonstrate that Gata4 and Tbx5 directly regulate Cdk4 while only Tbx5 activates Cdk2 expression. These findings highlight the mechanisms by which disruption of the Gata4 and Tbx5 interaction in the myocardium contributes to cardiac septation defects in humans.

Mammalian TBX1 preferentially binds and regulates downstream targets via a tandem T-site repeat.

Haploinsufficiency or mutation of TBX1 is largely responsible for the etiology of physical malformations in individuals with velo-cardio-facial/DiGeorge syndrome (VCFS/DGS/22q11.2 deletion syndrome). TBX1 encodes a transcription factor protein that contains an evolutionarily conserved DNA binding domain termed the T-box that is shared with other family members. All T-box proteins, examined so far, bind to similar but not identical consensus DNA sequences, indicating that they have specific binding preferences. To identify the TBX1 specific consensus sequence, Systematic Evolution of Ligands by Exponential Enrichment (SELEX) was performed. In contrast to other TBX family members recognizing palindrome sequences, we found that TBX1 preferentially binds to a tandem repeat of 5′-AGGTGTGAAGGTGTGA-3′. We also identified a second consensus sequence comprised of a tandem repeat with a degenerated downstream site. We show that three known human disease-causing TBX1 missense mutations (F148Y, H194Q and G310S) do not alter nuclear localization, or disrupt binding to the tandem repeat consensus sequences, but they reduce transcriptional activity in cell culture reporter assays. To identify Tbx1-downstream genes, we performed an in silico genome wide analysis of potential cis-acting elements in DNA and found strong enrichment of genes required for developmental processes and transcriptional regulation. We found that TBX1 binds to 19 different loci in vitro, which may correspond to putative cis-acting binding sites. In situ hybridization coupled with luciferase gene reporter assays on three gene loci, Fgf8, Bmper, Otog-MyoD, show that these motifs are directly regulated by TBX1 in vitro. Collectively, the present studies establish new insights into molecular aspects of TBX1 binding to DNA. This work lays the groundwork for future in vivo studies, including chromatin immunoprecipitation followed by next generation sequencing (ChIP-Seq) to further elucidate the molecular pathogenesis of VCFS/DGS.

Short communication: Expression of T-box 2 and 3 in the bovine mammary gland

To increase our understanding of the mechanisms by which growth hormone (GH) and insulin-like growth factor (IGF)-I influence bovine mammary gland development, the potential roles of T-box2 (TBX2) and T-box3 (TBX3) were investigated. Although no information regarding expression of either transcription factor in the bovine mammary gland exists, it is known that TBX3 and its closely related family member, TBX2, are required for mammary gland development in humans and mice. Additionally, TBX3 mutations in humans and mice lead to ulnar mammary syndrome. Evidence is present in bone that TBX3 is required for proliferation and its expression is regulated by GH, an important regulator of mammary gland development and milk production. We hypothesized that TBX2 and TBX3 are expressed in the bovine mammary gland and that GH, IGF-I, or both increase TBX2 and TBX3 expression in bovine mammary epithelial cells (MEC). Bovine mammary gland tissue, MAC-T cells, primary MEC, and fibroblasts were obtained and TBX2 and TBX3 expression was determined by real-time reverse transcription PCR. In addition, TBX2 and TBX3 expression was examined in cells treated with 100 or 500ng/mL of GH or 100 or 200ng/mL of IGF-I for 24 or 48h. Both TBX2 and TBX3 were expressed in bovine mammary tissue. Surprisingly, expression of TBX2 was only detected in mammary fibroblast cells, whereas TBX3 was expressed in all 3 cell types. Growth hormone did not alter TBX3 expression in MAC-T cells or MEC. However, IGF-I increased TBX3 expression in MAC-T, but not in primary MEC. We did not observe a change in TBX2 or TBX3 expression in fibroblasts treated with GH and IGF. Therefore, we concluded that (1) TBX2 and TBX3 are expressed in bovine mammary gland, (2) their expression is cell-type specific, and (3) IGF-I stimulates TBX3 expression in MAC-T cells.

TBX3 Regulates Splicing In Vivo: A Novel Molecular Mechanism for Ulnar-Mammary Syndrome

TBX3 is a member of the T-box family of transcription factors with critical roles in development, oncogenesis, cell fate, and tissue homeostasis. TBX3 mutations in humans cause complex congenital malformations and Ulnar-mammary syndrome. Previous investigations into TBX3 function focused on its activity as a transcriptional repressor. We used an unbiased proteomic approach to identify TBX3 interacting proteins in vivo and discovered that TBX3 interacts with multiple mRNA splicing factors and RNA metabolic proteins. We discovered that TBX3 regulates alternative splicing in vivo and can promote or inhibit splicing depending on context and transcript. TBX3 associates with alternatively spliced mRNAs and binds RNA directly. TBX3 binds RNAs containing TBX binding motifs, and these motifs are required for regulation of splicing. Our study reveals that TBX3 mutations seen in humans with UMS disrupt its splicing regulatory function. The pleiotropic effects of TBX3 mutations in humans and mice likely result from disrupting at least two molecular functions of this protein: transcriptional regulation and pre-mRNA splicing.

Phenotype of a patient with contiguous deletion of TBX5 and TBX3: Expanding the disease spectrum

The important roles that T-box genes play in the morphogenesis of the heart and its conduction system has long been established, and a number of disorders are linked to mutations in these T-box genes. Holt-Oram syndrome (HOS), the classic heart and hand syndrome, is clinically typified by radial ray upper limb abnormalities and cardiac malformations, and is caused by mutations involving TBX5. Another member of the T-box gene family, TBX3, is found in close proximity to TBX5 on chromosome 12q24. Mutations in TBX3 cause ulnar-mammary syndrome (UMS), which is distinguished by upper limb malformations affecting the ulnar ray, apocrine, and mammary gland hypoplasia, and genital defects. While disorders involving isolated mutations of TBX5 and TBX3 have been well described, contiguous deletions of these T-box genes remain exceptional. We report on a patient with features of both HOS and UMS consisting of bilateral symmetric limb malformations, congenital cardiac defects, and rapidly progressive cardiac conduction disease.

TBX1 Mutation Identified by Exome Sequencing in a Japanese Family with 22q11.2 Deletion Syndrome-Like Craniofacial Features and Hypocalcemia

BackgroundAlthough TBX1 mutations have been identified in patients with 22q11.2 deletion syndrome (22q11.2DS)-like phenotypes including characteristic craniofacial features, cardiovascular anomalies, hypoparathyroidism, and thymic hypoplasia, the frequency of TBX1 mutations remains rare in deletion-negative patients. Thus, it would be reasonable to perform a comprehensive genetic analysis in deletion-negative patients with 22q11.2DS-like phenotypes.Methodology/Principal FindingsWe studied three subjects with craniofacial features and hypocalcemia (group 1), two subjects with craniofacial features alone (group 2), and three subjects with normal phenotype within a single Japanese family. Fluorescence in situ hybridization analysis excluded chromosome 22q11.2 deletion, and genomewide array comparative genomic hybridization analysis revealed no copy number change specific to group 1 or groups 1+2. However, exome sequencing identified a heterozygous TBX1 frameshift mutation (c.1253delA, p.Y418fsX459) specific to groups 1+2, as well as six missense variants and two in-frame microdeletions specific to groups 1+2 and two missense variants specific to group 1. The TBX1 mutation resided at exon 9C and was predicted to produce a non-functional truncated protein missing the nuclear localization signal and most of the transactivation domain.Conclusions/SignificanceClinical features in groups 1+2 are well explained by the TBX1 mutation, while the clinical effects of the remaining variants are largely unknown. Thus, the results exemplify the usefulness of exome sequencing in the identification of disease-causing mutations in familial disorders. Furthermore, the results, in conjunction with the previous data, imply that TBX1 isoform C is the biologically essential variant and that TBX1 mutations are associated with a wide phenotypic spectrum, including most of 22q11.2DS phenotypes.

An interview with Benoit Bruneau

![Figure][1] Benoit Bruneau is a developmental biologist based at the Gladstone Institutes in San Francisco. His lab studies the transcription factors and chromatin remodelling complexes that regulate cardiac organogenesis and differentiation, with the aim of uncovering the basis for

A novel SOX9 H169Q mutation in a family with overlapping phenotype of mild campomelic dysplasia and small patella syndrome

The phenotypic similarities have been demonstrated between non-lethal campomelic dysplasia (CD) and small patella syndrome (SPS), in which different genetic defects have been identified. We report on a familial case of skeletal dysplasia with overlapping phenotype of mild CD and SPS, including defective ischio-pubic ossification, elongated femoral neck, hypoplastic patellae, and increased space between the first and the second toes (sandal gap). Direct sequencing analysis demonstrated a novel missense mutation (p.H169Q) within the coding region of the SOX9 gene and negative for TBX4 mutations. Functional analysis of the p.H169Q mutant revealed reduced but not fully abolished transactivation capacity of the mutated protein. Retained residual SOX9 function might contribute to an extremely mild CD phenotype in the present cases. © 2013 Wiley Periodicals, Inc.

Tbx1 regulates brain vascularization

The transcription factor TBX1 is the major gene involved in 22q11.2 deletion syndrome (22q11.2DS). Using mouse models of these diseases, we have previously shown that TBX1 activates VEGFR3 in endothelial cells (EC), and that this interaction is critical for the development of the lymphatic vasculature. In this study, we show that TBX1 regulates brain angiogenesis. Using loss-of-function genetics and molecular approaches, we show that TBX1 regulates the VEGFR3 and DLL4 genes in brain ECs. In mice, loss of TBX1 causes global brain vascular defects, comprising brain vessel hyperplasia, enhanced angiogenic sprouting and vessel network disorganization. This phenotype is recapitulated in EC-specific Tbx1 conditional mutants and in an EC-only 3-dimensional cell culture system (matrigel), indicating that the brain vascular phenotype is cell autonomous. Furthermore, EC-specific conditional Tbx1 mutants have poorly perfused brain vessels and brain hypoxia, indicating that the expanded vascular network is functionally impaired. In EC-matrigel cultures, a Notch1 agonist is able to partially rescue microtubule hyperbranching induced by TBX1 knockdown. Thus, we have identified a novel transcriptional regulator of angiogenesis that exerts its effect in brain by negatively regulating angiogenesis through the DLL4/Notch1-VEGFR3 regulatory axis. Given the similarity of the phenotypic consequences of TBX1 mutation in humans and mice, this unexpected role of TBX1 in murine brain vascularization should stimulate clinicians to search for brain microvascular anomalies in 22q11.2DS patients and to evaluate whether some of the anatomical and functional brain anomalies in patients may have a microvascular origin.

Lack of Genetic Interaction between Tbx20 and Tbx3 in Early Mouse Heart Development

Members of the T-box family of transcription factors are important regulators orchestrating the complex regionalization of the developing mammalian heart. Individual mutations in Tbx20 and Tbx3 cause distinct congenital heart abnormalities in the mouse: Tbx20 mutations result in failure of heart looping, developmental arrest and lack of chamber differentiation, while hearts of Tbx3 mutants progress further, loop normally but show atrioventricular convergence and outflow tract defects. The two genes have overlapping areas of expression in the atrioventricular canal and outflow tract of the heart but their potential genetic interaction has not been previously investigated. In this study we produced compound mutants to investigate potential genetic interactions at the earliest stages of heart development. We find that Tbx20; Tbx3 double heterozygous mice are viable and fertile with no apparent abnormalities, while double homozygous mutants are embryonic lethal by midgestation. Double homozygous mutant embryos display abnormal cardiac morphogenesis, lack of heart looping, expression patterns of cardiac genes and time of death that are indistinguishable from Tbx20 homozygous mutants. Prior to death, the double homozygotes show an overall developmental delay similar to Tbx3 homozygous mutants. Thus the effects of Tbx20 are epistatic to Tbx3 in the heart but Tbx3 is epistatic to Tbx20 with respect to developmental delay.

TBX4 mutations (small patella syndrome) are associated with childhood-onset pulmonary arterial hypertension

BackgroundChildhood-onset pulmonary arterial hypertension (PAH) is rare and differs from adult-onset disease in clinical presentation, with often unexplained mental retardation and dysmorphic features (MR/DF). Mutations in the major PAH gene,...

Mouse Tbx3 Mutants Suggest Novel Molecular Mechanisms for Ulnar-Mammary Syndrome

The transcription factor TBX3 plays critical roles in development and TBX3 mutations in humans cause Ulnar-mammary syndrome. Efforts to understand how altered TBX3 dosage and function disrupt the development of numerous structures have been hampered by embryonic lethality of mice bearing presumed null alleles. We generated a novel conditional null allele of Tbx3: after Cre-mediated recombination, no mRNA or protein is detectable. In contrast, a putative null allele in which exons 1-3 are deleted produces a truncated protein that is abnormally located in the cytoplasm. Heterozygotes and homozygotes for this allele have different phenotypes than their counterparts bearing a true null allele. Our observations with these alleles in mice, and the different types of TBX3 mutations observed in human ulnar-mammary syndrome, suggest that not all mutations observed in humans generate functionally null alleles. The possibility that mechanisms in addition to TBX3 haploinsufficiency may cause UMS or other malformations merits investigation in the human UMS population.

The T-box Transcription Factors TBX2 and TBX3 in Mammary Gland Development and Breast Cancer

TBX2 and TBX3, closely related members of the T-box family of transcription factor genes, are expressed in mammary tissue in both humans and mice. Ulnar mammary syndrome (UMS), an autosomal dominant disorder caused by mutations in TBX3, underscores the importance of TBX3 in human breast development, while abnormal mammary gland development in Tbx2 or Tbx3 mutant mice provides models for experimental investigation. In addition to their roles in mammary development, aberrant expression of TBX2 and TBX3 is associated with breast cancer. TBX2 is preferentially amplified in BRCA1/2-associated breast cancers and TBX3 overexpression has been associated with advanced stage disease and estrogen-receptor-positive breast tumors. The regulation of Tbx2 and Tbx3 and the downstream targets of these genes in development and disease are not as yet fully elucidated. However, it is clear that the two genes play unique, context-dependent roles both in mammary gland development and in mammary tumorigenesis.

Transrepression activity of T-box1 in a gene regulation network in mouse cells

T-box1 (TBX1) has been identified as a candidate disease-causing gene in DiGeorge syndrome/conotruncal anomaly face syndrome (DGS/CAFS). Tbx1 can function as a transcriptional transactivator as well as a transrepressor. Although the transactivating role of Tbx1 has been the focus of a number of published studies, its transrepression activity has been largely unexplored. Thus, this study centers on the identification of potential transrepressed targets of Tbx1. By subtractive hybridization, we compared the expression profiles of control mouse P19 cells and P19 cells depleted of Tbx1 via RNA interference. We identified 127 genes that were potentially transrepressed by Tbx1. Of the transrepressed genes, we focused on Ywhae and C1qbp and carried out promoter assays. The results showed that Tbx1 potentially transrepresses the promoter activities of these genes via palindromic sequences, including 5′-CCACAG-3′ and 5′-(C/G)TGTG(C/G)-3′, harbored within the promoters. Electromobility shift assays also showed that Tbx1 specifically interacts with certain portions of these promoter sequences. Moreover, the construction of Tbx1 mutants containing known human TBX1 mutations showed that these mutations result in the loss of Tbx1 transrepression activity. These results indicate that Tbx1 functions as a transrepressor in a gene regulation network, wherein Ywhae and C1qbp are 2 of the targets transrepressed by Tbx1 via T-box binding elements. Hence, the loss of TBX1 transrepression activity could be associated with the disease phenotypes of patients with DGS/CAFS.

A novel mutation in the SHH long‐range regulator (ZRS) is associated with preaxial polydactyly, triphalangeal thumb, and severe radial ray deficiency

Sonic Hedgehog (SHH) within the posteriorly located zone of polarizing activity is the main controller of the antero-posterior axis of limb development. The ZRS (zone of polarizing activity regulatory sequence) is a long-range limb-specific SHH enhancer. Several point mutations in the ZRS have been described in humans. These mutations cause enhanced SHH activity and ectopic anterior expression of SHH and a variable phenotype of preaxial polydactyly and triphalangeal thumb. Absent thumb or radius has not been reported with ZRS mutations. Here, we report on a family with a variable phenotype of preaxial polydactyly as well as absent thumb and radius, with kidney and cardiac defects. The family was screened for SALL1, SALL4, and TBX5 mutations, but all were normal. Finally, they were screened for ZRS mutations, which showed a novel point mutation within the ZRS, NG_009240.1: g.106954C>T (traditional nomenclature: ZRS619C>T) in the five affected members. This mutation was not previously reported in any public domain database, and was not found in our healthy and ethnically matched control individuals or unaffected family members. We hypothesize that interactions of SHH and SALL1 explain the overlapping features of the family described here and patients with Townes-Brocks syndrome.

Interaction between Tbx1 and HoxD10 and connection with TGFβ-BMP signal pathway during kidney development

Renal malformations are commonly found among patients carrying a 22q11 deletion which renders loss of Tbx1 gene, an important transcriptional factor implicated in a number of developmental processes. Smad1 is known to interact with Tbx1, but the exact mechanism remains unknown. In this study, we have measured the expression of Tbx1 in both murine and human tissues using RT-PCR, and analyzed its protein product and protein-protein interactions with Western blotting and immunoprecipitation assays. Precipitated proteins were verified with mass spectrometry. As discovered, Tbx1 binds with Hoxd10. Tbx1 and Hoxd10 genes also have similar expression profiles during murine kidney development. Based on homology between mouse and human, we hypothesized that such interaction also exists in human. Through a RNA interference experiment using a human embryonic kidney HEK293 cell line, we demonstrated that TBX1 can alter TGF-β/BMP, an important signaling pathway, through interacting with HOXD10. Above findings may shed light on the mechanism of TBX1 mutations leading to renal malformations found in patients carrying a 22q11 deletion.