The Paired Domain-containing Factor Pax8 and the Homeodomain-containing Factor TTF-1 Directly Interact and Synergistically Activate Transcription

Pax genes encode for transcription factors essential for tissue development in many species. Pax8, the only member of the family expressed in the thyroid tissue, is involved in the morphogenesis of the gland and in the transcriptional regulation of thyroid-specific genes. TTF-1, a homeodomain-containing factor, is also expressed in the thyroid tissue and has been demonstrated to play a role in thyroid-specific gene expression. Despite the presence of Pax8 and TTF-1 also in a few other tissues, the simultaneous expression of the two transcription factors occurs only in the thyroid, supporting the idea that Pax8 and TTF-1 might cooperate to influence thyroid-specific gene expression. In this report, we describe a physical and functional interaction between these two factors. The fusion protein GST-Pax8 is able to bind TTF-1 present in thyroid or in non-thyroid cell extracts, and by using bacterial purified TTF-1 we demonstrate that the interaction is direct. By co-immunoprecipitation, we also show that the interaction between the two proteins occurs in vivo in thyroid cells. Moreover, Pax8 and TTF-1 when co-expressed in HeLa cells synergistically activate Tg gene transcription. The synergism requires the N-terminal activation domain of TTF-1, and deletions of Pax8 indicate that the C-terminal domain of the protein is involved. Our results demonstrate a functional cooperation and a physical interaction between transcription factors of the homeodomain-containing and of the paired domain-containing gene families in the regulation of tissue-specific gene expression

Altered expression of mitochondrial and extracellular matrix genes in the heart of human fetuses with chromosome 21 trisomy

<p>Abstract</p> <p>Background</p> <p>The Down syndrome phenotype has been attributed to overexpression of chromosome 21 (Hsa21) genes. However, the expression profile of Hsa21 genes in trisomic human subjects as well as their effects on genes located on different chromosomes are largely unknown. Using oligonucleotide microarrays we compared the gene expression profiles of hearts of human fetuses with and without Hsa21 trisomy.</p> <p>Results</p> <p>Approximately half of the 15,000 genes examined (87 of the 168 genes on Hsa21) were expressed in the heart at 18–22 weeks of gestation. Hsa21 gene expression was globally upregulated 1.5 fold in trisomic samples. However, not all genes were equally dysregulated and 25 genes were not upregulated at all. Genes located on other chromosomes were also significantly dysregulated. Functional class scoring and gene set enrichment analyses of 473 genes, differentially expressed between trisomic and non-trisomic hearts, revealed downregulation of genes encoding mitochondrial enzymes and upregulation of genes encoding extracellular matrix proteins. There were no significant differences between trisomic fetuses with and without heart defects.</p> <p>Conclusion</p> <p>We conclude that dosage-dependent upregulation of Hsa21 genes causes dysregulation of the genes responsible for mitochondrial function and for the extracellular matrix organization in the fetal heart of trisomic subjects. These alterations might be harbingers of the heart defects associated with Hsa21 trisomy, which could be based on elusive mechanisms involving genetic variability, environmental factors and/or stochastic events.</p

In vivo role of different domains and of phosphorylation in the transcription factor Nkx2-1

Background: The transcription factor Nkx2-1 (also known as TTF-1, Titf1 or T/EBP) contains two apparently redundant activation domains and is post-translationally modified by phosphorylation. We have generated mouse mutant strains to assess the roles of the two activation domains and of phosphorylation in mouse development and differentiation. Results: Mouse strains expressing variants of the transcription factor Nkx2-1 deleted of either activation domain have been constructed. Phenotypic analysis shows for each mutant a distinct set of defects demonstrating that distinct portions of the protein endow diverse developmental functions of Nkx2-1. Furthermore, a mouse strain expressing a Nkx2-1 protein mutated in the phosphorylation sites shows a thyroid gland with deranged follicular organization and gene expression profile demonstrating the functional role of phosphorylation in Nkx2-1. Conclusions: The pleiotropic functions of Nkx2-1 are not all due to the protein as a whole since some of them can be assigned to separate domains of the protein or to specific post-translational modifications. These results have implication for the evolutionary role of mutations in transcription factors. © 2011 Silberschmidt et al; licensee BioMed Central Ltd

Gaslini's tracheal team: preliminary experience after one year of paediatric airway reconstructive surgery

Background: congenital and acquired airway anomalies represent a relatively common albeit challenging problem in a national tertiary care hospital. In the past, most of these patients were sent to foreign Centres because of the lack of local experience in reconstructive surgery of the paediatric airway. In 2009, a dedicated team was established at our Institute. Gaslini's Tracheal Team includes different professionals, namely anaesthetists, intensive care specialists, neonatologists, pulmonologists, radiologists, and ENT, paediatric, and cardiovascular surgeons. The aim of this project was to provide these multidisciplinary patients, at any time, with intensive care, radiological investigations, diagnostic and operative endoscopy, reconstructive surgery, ECMO or cardiopulmonary bypass. Aim of this study is to present the results of the first year of airway reconstructive surgery activity of the Tracheal Team.Methods: between September 2009 and December 2010, 97 patients were evaluated or treated by our Gaslini Tracheal Team. Most of them were evaluated by both rigid and flexible endoscopy. In this study we included 8 patients who underwent reconstructive surgery of the airways. Four of them were referred to our centre or previously treated surgically or endoscopically without success in other Centres.Results: Eight patients required 9 surgical procedures on the airway: 4 cricotracheal resections, 2 laryngotracheoplasties, 1 tracheal resection, 1 repair of laryngeal cleft and 1 foreign body removal with cardiopulmonary bypass through anterior tracheal opening. Moreover, in 1 case secondary aortopexy was performed. All patients achieved finally good results, but two of them required two surgeries and most required endoscopic manoeuvres after surgery. The most complex cases were the ones who had already been previously treated.Conclusions: The treatment of paediatric airway anomalies requires a dedicated multidisciplinary approach and a single tertiary care Centre providing rapid access to endoscopic and surgical manoeuvres on upper and lower airways and the possibility to start immediately cardiopulmonary bypass or ECMO.The preliminary experience of the Tracheal Team shows that good results can be obtained with this multidisciplinary approach in the treatment of complicated cases. The centralization of all the cases in one or few national Centres should be considered

Identification of Novel Pax8 Targets in FRTL-5 Thyroid Cells by Gene Silencing and Expression Microarray Analysis

The differentiation program of thyroid follicular cells (TFCs), by far the most abundant cell population of the thyroid gland, relies on the interplay between sequence-specific transcription factors and transcriptional coregulators with the basal transcriptional machinery of the cell. However, the molecular mechanisms leading to the fully differentiated thyrocyte are still the object of intense study. The transcription factor Pax8, a member of the Paired-box gene family, has been demonstrated to be a critical regulator required for proper development and differentiation of thyroid follicular cells. Despite being Pax8 well-characterized with respect to its role in regulating genes involved in thyroid differentiation, genomics approaches aiming at the identification of additional Pax8 targets are lacking and the biological pathways controlled by this transcription factor are largely unknown.To identify unique downstream targets of Pax8, we investigated the genome-wide effect of Pax8 silencing comparing the transcriptome of silenced versus normal differentiated FRTL-5 thyroid cells. In total, 2815 genes were found modulated 72 h after Pax8 RNAi, induced or repressed. Genes previously reported to be regulated by Pax8 in FRTL-5 cells were confirmed. In addition, novel targets genes involved in functional processes such as DNA replication, anion transport, kinase activity, apoptosis and cellular processes were newly identified. Transcriptome analysis highlighted that Pax8 is a key molecule for thyroid morphogenesis and differentiation.This is the first large-scale study aimed at the identification of new genes regulated by Pax8, a master regulator of thyroid development and differentiation. The biological pathways and target genes controlled by Pax8 will have considerable importance to understand thyroid disease progression as well as to set up novel therapeutic strategies

Pax8 protein stability is controlled by sumoylation.

The transcription factor Pax8 is involved in the morphogenesis of the thyroid gland and in the maintenance of the differentiated thyroid phenotype. Despite the critical role played by Pax8 during thyroid development and differentiation, very little is known of its post-translational modifications and how these modifications may regulate its activity. We focused our attention on the study of a specific post-translational modification, i.e., sumoylation. Sumoylation is a dynamic and reversible process regulating gene expression by altering transcription factor stability, protein-protein interaction and subcellular localization of target proteins. The analysis of Pax8 protein sequence revealed the presence of one sumoylation consensus motif (psiKxE), strongly conserved among mammals, amphibians, and fish. We demonstrated that Pax8 is sumoylated by the addition of a single small ubiquitin-like modifier (SUMO) molecule on its lysine residue 309 and that Pax8(K309R), a substitution mutant in which the candidate lysine is replaced with an arginine, is no longer modified by SUMO. In addition, we analyzed whether protein inhibitor of activated signal transducers and activators of transcription (PIASy), a member of the PIAS STAT family of proteins, could function as a SUMO ligase and we demonstrated that indeed PIASy is able to increase the fraction of sumoylated Pax8. Interestingly, we show that Pax8 is targeted in the SUMO nuclear bodies, which are structures that regulate the nucleoplasmic concentration of transcription factors by SUMO trapping. Finally, we report here that the steady-state protein level of Pax8 is controlled by sumoylation

The paired domain-containing factor Pax8 and the homeodomain-containing factor TTF-1 directly interact and synergistically activate transcription.

Pax genes encode for transcription factors essential for tissue development in many species. Pax8, the only member of the family expressed in the thyroid tissue, is involved in the morphogenesis of the gland and in the transcriptional regulation of thyroid-specific genes. TTF-1, a homeodomain-containing factor, is also expressed in the thyroid tissue and has been demonstrated to play a role in thyroid-specific gene expression. Despite the presence of Pax8 and TTF-1 also in a few other tissues, the simultaneous expression of the two transcription factors occurs only in the thyroid, supporting the idea that Pax8 and TTF-1 might cooperate to influence thyroid-specific gene expression. In this report, we describe a physical and functional interaction between these two factors. The fusion protein GST-Pax8 is able to bind TTF-1 present in thyroid or in non-thyroid cell extracts, and by using bacterial purified TTF-1 we demonstrate that the interaction is direct. By co-immunoprecipitation, we also show that the interaction between the two proteins occurs in vivo in thyroid cells. Moreover, Pax8 and TTF-1 when co-expressed in HeLa cells synergistically activate Tg gene transcription. The synergism requires the N-terminal activation domain of TTF-1, and deletions of Pax8 indicate that the C-terminal domain of the protein is involved. Our results demonstrate a functional cooperation and a physical interaction between transcription factors of the homeodomain-containing and of the paired domain-containing gene families in the regulation of tissue-specific gene expression

Identification of Novel Pax8 Targets in FRTL-5 Thyroid Cells by Gene Silencing and Expression Microarray Analysis

Background: The differentiation program of thyroid follicular cells (TFCs), by far the most abundant cell population of the thyroid gland, relies on the interplay between sequence-specific transcription factors and transcriptional coregulators with the basal transcriptional machinery of the cell. However, the molecular mechanisms leading to the fully differentiated thyrocyte are still the object of intense study. The transcription factor Pax8, a member of the Paired-box gene family, has been demonstrated to be a critical regulator required for proper development and differentiation of thyroid follicular cells. Despite being Pax8 well-characterized with respect to its role in regulating genes involved in thyroid differentiation, genomics approaches aiming at the identification of additional Pax8 targets are lacking and the biological pathways controlled by this transcription factor are largely unknown. Methodology/Principal Findings: To identify unique downstream targets of Pax8, we investigated the genome-wide effect of Pax8 silencing comparing the transcriptome of silenced versus normal differentiated FRTL-5 thyroid cells. In total, 2815 genes were found modulated 72 h after Pax8 RNAi, induced or repressed. Genes previously reported to be regulated by Pax8 in FRTL-5 cells were confirmed. In addition, novel targets genes involved in functional processes such as DNA replication, anion transport, kinase activity, apoptosis and cellular processes were newly identified. Transcriptome analysis highlighted that Pax8 is a key molecule for thyroid morphogenesis and differentiation. Conclusions/Significance: This is the first large-scale study aimed at the identification of new genes regulated by Pax8, a master regulator of thyroid development and differentiation. The biological pathways and target genes controlled by Pax8 will have considerable importance to understand thyroid disease progression as well as to set up novel therapeutic strategies