Comparative genomics reveals a functional thyroid-specific element in the far upstream region of the PAX8 gene

<p>Abstract</p> <p>Background</p> <p>The molecular mechanisms leading to a fully differentiated thyrocite are still object of intense study even if it is well known that thyroglobulin, thyroperoxidase, NIS and TSHr are the marker genes of thyroid differentiation. It is also well known that Pax8, TTF-1, Foxe1 and Hhex are the thyroid-enriched transcription factors responsible for the expression of the above genes, thus are responsible for the differentiated thyroid phenotype. In particular, the role of Pax8 in the fully developed thyroid gland was studied in depth and it was established that it plays a key role in thyroid development and differentiation. However, to date the bases for the thyroid-enriched expression of this transcription factor have not been unraveled yet. Here, we report the identification and characterization of a functional thyroid-specific enhancer element located far upstream of the <it>Pax8 </it>gene.</p> <p>Results</p> <p>We hypothesized that regulatory cis-acting elements are conserved among mammalian genes. Comparison of a genomic region extending for about 100 kb at the 5'-flanking region of the mouse and human <it>Pax8 </it>gene revealed several conserved regions that were tested for enhancer activity in thyroid and non-thyroid cells. Using this approach we identified one putative thyroid-specific regulatory element located 84.6 kb upstream of the <it>Pax8 </it>transcription start site. The <it>in silico </it>data were verified by promoter-reporter assays in thyroid and non-thyroid cells. Interestingly, the identified far upstream element manifested a very high transcriptional activity in the thyroid cell line PC Cl3, but showed no activity in HeLa cells. In addition, the data here reported indicate that the thyroid-enriched transcription factor TTF-1 is able to bind <it>in vitro </it>and <it>in vivo </it>the Pax8 far upstream element, and is capable to activate transcription from it.</p> <p>Conclusions</p> <p>Results of this study reveal the presence of a thyroid-specific regulatory element in the 5' upstream region of the <it>Pax8 </it>gene. The identification of this regulatory element represents the first step in the investigation of upstream regulatory mechanisms that control <it>Pax8 </it>transcription during thyroid differentiation and are relevant to further studies on <it>Pax8 </it>as a candidate gene for thyroid dysgenesis.</p

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

In Search of Double Materiality in Non-Financial Reports: First Empirical Evidence

Materiality is the key principle that drives the selection of issues that companies must report on. The European Union regulation on companies&rsquo; non-financial disclosure coined a special meaning of materiality that holistically combines the two perspectives of financial and impact materiality into an overall &ldquo;double materiality&rdquo; (DM). The contrast detected between the early debate and the low level of empirical knowledge on DM provided by the literature on materiality disclosure gave rise to our research aim, which was to map the pioneering experiences of DM. In order to achieve this aim, we carried out an exploratory analysis on the non-financial reports of 58 companies, both European and non-European, operating in various industries (period 2019&ndash;2021). The results reveal &ldquo;traces&rdquo; of DM in the reports of few companies, mainly European ones. The aspects we examined, both with atomistic and summative perspectives of inquiry, highlight variety in both double materiality assessments and adoption disclosures, as well as related criticalities. This foreshadows a fragmented landscape of materiality analysis disclosure over the next few years that presently requires great attention and increased operational guidance by the international standard setters involved. The article closes by proposing implications, limitations and research perspectives

In Search of Double Materiality in Non-Financial Reports: First Empirical Evidence

Materiality is the key principle that drives the selection of issues that companies must report on. The European Union regulation on companies’ non-financial disclosure coined a special meaning of materiality that holistically combines the two perspectives of financial and impact materiality into an overall “double materiality” (DM). The contrast detected between the early debate and the low level of empirical knowledge on DM provided by the literature on materiality disclosure gave rise to our research aim, which was to map the pioneering experiences of DM. In order to achieve this aim, we carried out an exploratory analysis on the non-financial reports of 58 companies, both European and non-European, operating in various industries (period 2019–2021). The results reveal “traces” of DM in the reports of few companies, mainly European ones. The aspects we examined, both with atomistic and summative perspectives of inquiry, highlight variety in both double materiality assessments and adoption disclosures, as well as related criticalities. This foreshadows a fragmented landscape of materiality analysis disclosure over the next few years that presently requires great attention and increased operational guidance by the international standard setters involved. The article closes by proposing implications, limitations and research perspectives

Rise and Fall of the Materiality Matrix: Lessons from a Missed Takeoff

After a long period of the inclusion of materiality matrices within standard setters documents and non-financial reports, the Global Reporting Initiative officially abandoned the materiality matrix in 2021 after the GRI 3 standard release. To bridge the detected gaps in the literature, this article aims to investigate approaches to and arguments for the matrix until the issuance of GRI 3. The two-step research strategy adopted gives the same level of attention to the opposite positions found. Phase 1 (approach-oriented) reviews the materiality matrix presentation in the 2014–2020 non-financial reports of a sample of worldwide sustainability-oriented companies. Phase 2 (argument-oriented) performs qualitative content analysis on feedback for the GRI 3 preparatory works. The findings show that, besides the staunch adopters, a core of non-adopters persisted and prevented the takeoff of the matrix. Moreover, further insights into possible drivers both in favour of and against these approaches are provided. The final discussion both considers the lessons learnt, overlapping with policy implications, and suggests future research avenues

A role for PAX8 in the tumorigenic phenotype of ovarian cancer cells

Background: PAX8 is a member of the paired box (Pax) multigene family of transcription factors, which are involved in the developmental and tissue-specific control of the expression of several genes in both vertebrates and invertebrates. Previously, several studies reported that PAX8 is expressed at high levels in specific types of tumors. In particular, PAX8 has been recently reported to be conspicuously expressed in human ovarian cancer, but the functional role of PAX8 in the carcinogenesis of this type of tumor has not been addressed. In this study, we investigated the contribution of PAX8 in ovarian cancer progression. Methods: Stable PAX8 depleted ovarian cancer cells were generated using short hairpin RNA (shRNA) constructs. PAX8 mRNA and protein were detected by RT-PCR, immunoblot and immunofluorescence. Cell proliferation, motility and invasion potential of PAX8 silenced cells were analyzed by means of growth curves, wound healing and Matrigel assays. In addition, PAX8 knockdown and control cells were injected into nude mice for xenograft tumorigenicity assays. Finally, qPCR was used to detect the expression levels of EMT markers in PAX8-overexpressing and control cells. Results: Here, we show that PAX8 plays a critical role in the migration, invasion and tumorigenic ability of ovarian cancer cells. Our results show that RNA interference-mediated knockdown of PAX8 expression in SKOV-3 ovarian cancer cells produces a significant reduction of cell proliferation, migration ability and invasion activity compared with control parental SKOV-3 cells. Moreover, PAX8 silencing strongly suppresses anchorage-independent growth in vitro. Notably, tumorigenesis in vivo in a nude mouse xenograft model is also significantly inhibited. Conclusions: Overall, our results indicate that PAX8 plays an important role in the tumorigenic phenotype of ovarian cancer cells and identifies PAX8 as a potential new target for the treatment of ovarian cancer

An essential role for Pax8 in the transcriptional regulation of Cadherin-16 in thyroid cells.

Cadherin-16 was originally identified as a tissue-specific cadherin present exclusively in kidney. Only recently, Cadherin-16 has been detected also on the plasma membrane of mouse thyrocytes. This last finding prompted us to note that the expression profile of Cadherin-16 resembles that of the transcription factor Pax8, a member of the Pax (paired-box) gene family, predominantly expressed in the developing and adult kidney and thyroid. Pax8 has been extensively characterized in the thyroid and shown to be a master gene for thyroid development and differentiation. In this study, we determined the role of the transcription factor Pax8 in the regulation of Cadherin-16 expression. We demonstrate that the Cadherin-16 minimal promoter is transcriptionally active in thyroid cells as well as in kidney cells, that Pax8 is able to activate transcription from a Cadherin-16 promoter reporter construct, and more importantly, that indeed Pax8 is able to bind in vivo the Cadherin-16 promoter region. In addition, by means of Pax8 RNA interference in thyroid cells and by analyzing Pax8 null mice, we demonstrate that Pax8 regulates also in vivo the expression of Cadherin-16. Finally, we reveal that the expression of Cadherin-16 is TSH dependent in FRTL-5 thyroid cells and significantly reduced in mouse thyroid carcinomas. Therefore, we conclude that Cadherin-16 is a novel downstream target of the transcription factor Pax8, likely since the early steps of thyroid development, and that its expression is associated with the fully differentiated state of the thyroid cell

Candidate genes and pathways downstream of PAX8 involved in ovarian high-grade serous carcinoma

Understanding the biology and molecular pathogenesis of ovarian epithelial cancer (EOC) is key to developing improved diagnostic and prognostic indicators and effective therapies. Although research has traditionally focused on the hypothesis that high-grade serous carcinoma (HGSC) arises from the ovarian surface epithelium (OSE), recent studies suggest that additional sites of origin exist and a substantial proportion of cases may arise from precursor lesions located in the Fallopian tubal epithelium (FTE). In FTE cells, the transcription factor PAX8 is a marker of the secretory cell lineage and its expression is retained in 96% of EOC. We have recently reported that PAX8 is involved in the tumorigenic phenotype of ovarian cancer cells. In this study, to uncover genes and pathways downstream of PAX8 involved in ovarian carcinoma we have determined the molecular profiles of ovarian cancer cells and in parallel of Fallopian tube epithelial cells by means of a silencing approach followed by an RNA-seq analysis. Interestingly, we highlighted the involvement of pathways like WNT signaling, epithelial-mesenchymal transition, p53 and apoptosis. We believe that our analysis has led to the identification of candidate genes and pathways regulated by PAX8 that could be additional targets for the therapy of ovarian carcinom

Long Non-Coding RNA MAGI2-AS3 is a New Player with a Tumor Suppressive Role in High Grade Serous Ovarian Carcinoma

High-Grade Serous Ovarian Carcinoma (HGSC) is the most incidental and lethal subtype of epithelial ovarian cancer (EOC) with a high mortality rate of nearly 65%. Recent findings aimed at understanding the pathogenesis of HGSC have attributed its principal source as the Fallopian Tube (FT). To further comprehend the exact mechanism of carcinogenesis, which is still less known, we performed a transcriptome analysis comparing FT and HGSC. Our study aims at exploring new players involved in the development of HGSC from FT, along with their signaling network, and we chose to focus on non-coding RNAs. Non-coding RNAs (ncRNAs) are increasingly observed to be the major regulators of several cellular processes and could have key functions as biological markers, as well as even a therapeutic approach. The most physiologically relevant and significantly dysregulated non-coding RNAs were identified bioinformatically. After analyzing the trend in HGSC and other cancers, MAGI2-AS3 was observed to be an important player in EOC. We assessed its tumor-suppressive role in EOC by means of various assays. Further, we mapped its signaling pathway using its role as a miRNA sponge to predict the miRNAs binding to MAGI2AS3 and showed it experimentally. We conclude that MAGI2-AS3 acts as a tumor suppressor in EOC, specifically in HGSC by sponging miR-15-5p, miR-374a-5p and miR-374b-5p, and altering downstream signaling of certain mRNAs through a ceRNA network