Anna Mascia

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 occursin 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.

TAZ is a coactivator for Pax8 and TTF-1, two transcription factors involved in thyroid differentiation.

Pax8 and TTF-1 are transcription factors involved in the morphogenesis of the thyroid gland and in the transcriptional regulation of thyroid-specific genes. Both proteins are expressed in few tissues but their simultaneous presence occurs only in the thyroid where they interact physically and functionally allowing the regulation of genes that are markers of the thyroid differentiated phenotype. TAZ is a transcriptional coactivator that regulates the activity of several transcription factors therefore playing a central role in tissue-specific transcription. The recently demonstrated physical and functional interaction between TAZ and TTF-1 in the lung raised the question of whether TAZ could be an important regulatory molecule also in the thyroid. In this study, we demonstrate the presence of TAZ in thyroid cells and the existence of an important cooperation between TAZ and the transcription factors Pax8 and TTF-1 in the modulation of thyroid gene expression. In addition, we reveal that the three proteins are co-expressed in the nucleus of differentiated thyroid cells and that TAZ interacts with both Pax8 and TTF-1, in vitro and in vivo. More importantly, we show that this interaction leads to a significant enhancement of the transcriptional activity of Pax8 and TTF-1 on the thyroglobulin promoter thus suggesting a role of TAZ in the control of genes involved in thyroid development and differentiation.

Transfection of TTF-1 gene induces thyroglobulin gene expression in undifferentiated FRT cells

The thyroglobulin gene, the substrate for thyroid hormone biosynthesis, is not expressed in the FRT cell line, which, even though it manifests the polarised epithelial phenotype, does not express any of the thyroid functional properties. Two transcription factors, TTF-1 and Pax-8, have been implicated in thyroid specific expression of the thyroglobulin gene. FRT cells contain Pax-8 but they lack TTF-1. In this paper, we show that transfection of TTF-1 expression vectors in FRT cells results in activation of thyroglobulin gene expression. If the expression vector encoded for TTF-1-ER, a fusion gene coding for the entire TTF-1 protein fused to the hormone-binding domain of the steroid receptor, under the control of the RSV promoter, thyroglobulin gene expression was controlled by estrogen. These data provide a direct demonstration that TTF-1 activates the chromosomal thyroglobulin promoter. Since transfection of TTF-1 expression vectors in non-thyroid cell types did not result in thyroglobulin gene expression, it is suggested that Pax-8, in addition, perhaps, to a specific cellular environment, might be required for thyroid specific expression of the thyroglobulin gene.

WBP-2, a WW domain binding protein, interacts with the thyroid-specific transcription factor Pax8.

The Pax gene family encodes transcription factors that are essential in organogenesis and in the differentiation of various organs in higher eukaryotes. Pax proteins have a DNA binding domain at the N-terminus, and a transcriptional activation domain at the C-terminus. How these domains interact with the transcriptional machinery of the cell is still unclear. In the present paper, we describe the identification by means of immunological screening of the WW domain binding protein WBP-2 as a biochemical interactor of Pax8 (a WW domain is a protein-interaction domain containing two conserved tryptophan residues). Pax8 is required for the morphogenesis of the thyroid gland and for the maintenance of the thyroid differentiated cellular phenotype. WBP-2 was identified originally as a WW domain binding protein, and its function is still unknown. WBP-2 binds to Pax8 in vitro in pulldown assays, and in vivo in tissue culture cells in co-immunoprecipitation assays. Interestingly, Pax8 does not contain a WW domain. Our results point to the identification of a new protein-interacting domain that is present in the C-terminal portion of Pax8 and that is required for protein-protein interaction with WBP-2. Our results demonstrate that WBP-2 is not a transcriptional co-activator of Pax8, but rather behaves as an adaptor molecule, as suggested in other studies.

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.

Applying Quality and Project Management methodologies in biomedical research laboratories: a public research network’s case study

Quality principles and Project Management (PM) methodologies have long been ignored in non-regulated scientific research, even though they have been widely used in industrial and business applications, improving management and results and reducing costs. A groundbreaking project named Quality and Project Management OpenLab was implemented by a network of Italian National Research Council institutes, with the aim to realize and disseminate within the scientific community an innovative way to plan and organize research activity, inspired by Quality and PM principles and customized for needs and requisites of biomedical research laboratories. The results show better use of time and project consistency. Our experience of working side by side with Quality consultants clearly shows that the proper and accurate application of Quality and PM methodologies to intellectual and scientific production can facilitate and strengthen research, providing tools to make it faster and more efficient without imposing any undue constraints.

Rab7 Regulates CDH1 Endocytosis, Circular Dorsal Ruffles Genesis, and Thyroglobulin Internalization in a Thyroid Cell Line

Rab7 regulates the biogenesis of late endosomes, lysosomes, and autophagosomes. It has been proposed that a functional and physical interaction exists between Rab7 and Rac1 GTPases in CDH1 endocytosis and ruffled border formation. In FRT cells over‐expressing Rab7, increased expression and activity of Rac1 was observed, whereas a reduction of Rab7 expression by RNAi resulted in reduced Rac1 activity, as measured by PAK1 phosphorylation. We found that CDH1 endocytosis was extremely reduced only in Rab7 over‐expressing cells but was unchanged in Rab7 silenced cells. In Rab7 under or over‐expressing cells, Rab7 and LC3B‐II co‐localized and co‐localization in large circular structures occurred only in Rab7 over‐expressing cells. These large circular structures occurred in about 10% of the cell population; some of them (61%) showed co‐localization of Rab7 with cortactin and f‐actin and were identified as circular dorsal ruffles (CDRs), the others as mature autophagosomes. We propose that the over‐expression of Rab7 is sufficient to induce CDRs. Furthermore, in FRT cells, we found that the expression of the insoluble/active form of Rab7, rather than Rab5, or Rab8, was inducible by cAMP and that cAMP‐stimulated FRT cells showed increased PAK1 phosphorylation and were no longer able to endocytose CDH1. Finally, we demonstrated that Rab7 over‐expressing cells are able to endocytose exogenous thyroglobulin via pinocytosis/CDRs more efficiently than control cells. We propose that the major thyroglobulin endocytosis described in thyroid autonomous adenomas due to Rab7 increased expression, occurs via CDRs. J. Cell. Physiol. 231: 1695–1708, 2016.

Quality-based model for Life Sciences research guidelines

Nowadays, the requirement to define adequate standards and to identify and validate general guidelines for scientific activity is becoming increasingly apparent also in non-regulated scientific research. Guidelines are fundamental tools to provide valid indications for proper conduct in a research laboratory, the correct use of equipment and procedures, as well as for aligning and standardizing the procedures used in different scientific contexts. The identification, dissemination and application of common guidelines can improve significantly the reproducibility of the scientific results and the exchange of materials and data in the context of scientific consortia, comprising also industrial partners and meta-analysis projects. A Quality and Project Management OpenLab research network was formed in 2012 to develop and apply Total Quality Management models to Life Sciences research laboratories. One of the main tasks of the network has been the definition of a model for the drafting of guidelines, firmly based on quality principles and documentation management. The outcome is an operational flow describing all the phases of the process, which has been validated by four different drafting groups through the production of 13 guidelines ranging from research activity to equipment and facility management. Different institutes of the National Research Council are currently following these guidelines; some of them have also been used to define the procedures included in a certified Quality Management System for a research laboratory. Our experience shows that the model for guidelines we have developed makes drafting guidelines easier and more immediate, and significantly, it is applicable to different scientific contexts and disciplines, including both non-regulated research and technology transfer-oriented research, and also the Quality Management System of a scientific laboratory.

Applying Design of Experiments Methodology to PEI Toxicity Assay on Neural Progenitor Cells

Design of Experiments (DoE) statistical methodology permits the simultaneous evaluation of the effects of different factors on experimental performance and the analysis of their interactions in order to identify their optimal combinations. Compared to classical approaches based on changing only one factor at a time (OFAT), DoE facilitates the exploration of a broader range of parameters combinations, as well as providing the possibility to select a limited number of combinations covering the whole frame. The advantage of DoE is to maximise the amount of information provided and to save both time and money. DoE has been primarily used in industry to maximise process robustness, but recently it has also been applied in biomedical research to different types of multivariable analyses, from determination of the best cell media composition to the optimisation of entire multi-step laboratory protocols such as cell transfection.