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PAX6 is well known as a transcription factor that drives eye development in animals as widely divergent as flies and mammals. In addition to its localization in eyes, PAX6 expression has been reported in the central nervous system, the pancreas, testes, Merkel cells, nasal epithelium, developing cells of the inner ear, and embryonic submandibular salivary gland. Here we show that PAX6 also appears to be present in the mechanosensory neuromasts of the lateral line system in paedomorphic salamanders of the genus Eurycea. Using immunohistochemistry and confocal microscopy to examine a limited number of larvae of two species, listed by the United States of America's federal government as threatened (E. nana) or endangered (E. rathbuni), we found that anti-PAX6 antibody labeled structures that were extranuclear, and labeling was most intense in the apical appendages of the hair cells of the neuromast. This extranuclear localization raises the possibility of an as yet undescribed function for PAX6 as a cytoskeleton-associated protein., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Dobbins et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Proteasome activity is crucial for cellular integrity, but how tissues adjust proteasome content in response to catabolic stimuli is uncertain. Here, we demonstrate that transcriptional coordination by multiple transcription factors is required to increase proteasome content and activate proteolysis in catabolic states. Using denervated mouse muscle as a model system for accelerated proteolysis in vivo, we reveal that a two-phase transcriptional program activates genes encoding proteasome subunits and assembly chaperones to boost an increase in proteasome content. Initially, gene induction is necessary to maintain basal proteasome levels, and in a more delayed phase (7-10 days after denervation), it stimulates proteasome assembly to meet cellular demand for excessive proteolysis. Intriguingly, the transcription factors PAX4 and α-PALNRF-1 control the expression of proteasome among other genes in a combinatorial manner, driving cellular adaptation to muscle denervation. Consequently, PAX4 and α-PALNRF-1 represent new therapeutic targets to inhibit proteolysis in catabolic diseases (e.g., type-2 diabetes, cancer)., (© 2024 Gilda et al.)

The role extracellular matrix (ECM) in multiple events of morphogenesis has been well described, little is known about its specific role in early eye development. One of the first morphogenic events in lens development is placodal thickening, which converts the presumptive lens ectoderm from cuboidal to pseudostratified epithelium. This process occurs in the anterior pre-placodal ectoderm when the optic vesicle approaches the cephalic ectoderm and is regulated by transcription factor Pax6 and secreted BMP4. Since cells and ECM have a dynamic relationship of interdependence and modulation, we hypothesized that the ECM evolves with cell shape changes during lens placode formation. This study investigates changes in optic ECM including both protein distribution deposition, extracellular gelatinase activity and gene expression patterns during early optic development using chicken and mouse models. In particular, the expression of Timp2, a metalloprotease inhibitor, corresponds with a decrease in gelatinase activity within the optic ECM. Furthermore, we demonstrate that optic ECM remodeling depends on BMP signaling in the placode. Together, our findings suggest that the lens placode plays an active role in remodeling the optic ECM during early eye development., (Copyright © 2024 International Society of Differentiation. Published by Elsevier B.V. All rights reserved.)

Although aberrant methylation of PAX1 is closely associated with cervical cancer (CC), PAX1 methylation (PAX1m) and its role in CC remain to be elucidated. Here, we clarified the biological function of PAX1 in CC. First, PAX1m in ThinPrep cytologic test samples was measured via quantitative methylation-specific PCR. The results showed that PAX1 promoter methylation levels were significantly increased in CC patients (p < 0.001). We also found that PAX1 promoter methylation levels were positively correlated with tumor purity but negatively correlated with immune-infiltration via public databases. Then, CRISPR-based methylation perturbation tools (dCas9-Tet1) were constructed to further demonstrate that DNA methylation participates in the regulation of PAX1 expression directly. Gain- and loss-of-function experiments were used to show that PAX1 overexpression restrained proliferation, migration and improved cisplatin sensitivity by interfering with the WNT/TIMELESS axis in CC cells. Additionally, Co-immunoprecipitation assays further confirmed the interaction between PAX1 and TCF7L2. Taken together, our results suggested that a tumor suppressor role of PAX1 in CC and that CRISPR-based PAX1 demethylation editing might be a promising therapeutic strategy for CC., (© 2024 The Authors. Molecular Carcinogenesis published by Wiley Periodicals LLC.)

Renal cell carcinoma (RCC) is a significant oncological challenge due to its heterogeneous nature and limited treatment options. The PAX developmental gene family encodes nine highly conserved transcription factors that play crucial roles in embryonic development and organogenesis, which have been implicated in the occurrence and development of RCC. This review explores the molecular landscape of RCC, with a specific focus on the role of the PAX gene family in RCC tumorigenesis and disease progression. Of the various RCC subtypes, clear cell renal cell carcinoma (ccRCC) is the most prevalent, characterized by the loss of the von Hippel-Lindau ( VHL ) tumor suppressor gene. Here, we review the published literature on the expression patterns and functional implications of PAX genes, particularly PAX2 and PAX8 , in the three most common RCC subtypes, including ccRCC, papillary RCC (PRCC), and chromophobe RCC (ChRCC). Further, we review the interactions and potential biological mechanisms involving PAX genes and VHL loss in driving the pathogenesis of RCC, including the key signaling pathways mediated by VHL in ccRCC and associated mechanisms implicating PAX . Lastly, concurrent with our update regarding PAX gene research in RCC, we review and comment on the targeting of PAX towards the development of novel RCC therapies.

Background: Diabetes is a chronic metabolic disease that affects many parts of the body. Considering diabetes as a beta cells' defect and loss, the focus is on finding mechanisms and compounds involved in stimulating the function and regeneration of pancreatic β-cells. DNA methylation as an epigenetic mechanism plays a pivotal role in the β-cells' function and development. Considering the regenerative and anti-diabetic effects of Rosa canina extract, this study aimed to assess the methylation levels of Pdx-1, Pax-4, and Ins-1 genes in diabetic rats treated with Rosa Canina extract., Methods and Results: Streptozotocin-induced diabetic rats were used to evaluate the frequency of Pdx-1, Pax-4, and Ins-1 gene methylation. Treatment groups were exposed to Rosa canina as spray-dried and decoction extracts. Following blood glucose measurement, pancreatic DNA was extracted and bisulfited. Genes' methylation was measured using MSP-PCR and qRT-PCR techniques. Oral administration of Rosa canina extracts significantly reduced blood sugar levels in diabetic rats compared to the control group. The methylation levels of the Pdx-1, Pax-4, and Ins-1 genes promoter in streptozotocin-induced diabetic rats increased compared to the control rats while, the treatment of diabetic rats with Rosa canina extracts, spray-dried samples especially, led to a decreased methylation in these genes., Conclusion: The results of this study showed that Rosa canina extract as a spray-dried sample could be effective in treating diabetes by regulating the methylation of genes including Pdx-1, Pax-4, and Ins-1 involved in the activity and regeneration of pancreatic islet cells., (© 2024. The Author(s), under exclusive licence to Springer Nature B.V.)

Paired box (Pax) genes are highly conserved throughout evolution, and the Pax protein is an important transcription factor of embryonic development. The Pax gene Bmgsb is expressed in the silk glands of silkworm, but its biological functions remain unclear. This study aimed to investigate the expression pattern of Bmgsb in the silk gland and explore its functions using RNA interference (RNAi). Here, we identified eight Pax genes in Bombyx mori. Phylogenetic analysis showed that the B. mori Pax genes were highly homologous to the Pax genes in other insects and highly evolutionarily conserved. The tissue expression profile showed that Bmgsb was expressed in the anterior silk gland and anterior part of the middle silk gland (AMSG). RNAi of Bmgsb resulted in defective development of the AMSG, and the larvae were mostly unable to cocoon in the wandering stage. RNA-seq analysis showed that the fibroin genes fib-l, fib-h and p25, cellular heat shock response-related genes and phenol oxidase genes were considerably upregulated upon Bmgsb knockdown. Furthermore, quantitative reverse transcription-PCR results showed that the fibroin genes and ubiquitin proteolytic enzyme-related genes were significantly upregulated in the AMSG after Bmgsb knockdown. This study provides a foundation for future research on the biological functions of B. mori Pax genes. In addition, it demonstrates the important roles of Bmgsb in the transcriptional regulation of fibroin genes and silk gland development., (© 2024 The Royal Entomological Society.)

During development, neural stem cells in the cerebral cortex, also known as radial glial cells (RGCs), generate excitatory neurons, followed by production of cortical macroglia and inhibitory neurons that migrate to the olfactory bulb (OB). Understanding the mechanisms for this lineage switch is fundamental for unraveling how proper numbers of diverse neuronal and glial cell types are controlled. We and others recently showed that Sonic Hedgehog (Shh) signaling promotes the cortical RGC lineage switch to generate cortical oligodendrocytes and OB interneurons. During this process, cortical RGCs generate intermediate progenitor cells that express critical gliogenesis genes Ascl1 , Egfr, and Olig2 . The increased Ascl1 expression and appearance of Egfr + and Olig2 + cortical progenitors are concurrent with the switch from excitatory neurogenesis to gliogenesis and OB interneuron neurogenesis in the cortex. While Shh signaling promotes Olig2 expression in the developing spinal cord, the exact mechanism for this transcriptional regulation is not known. Furthermore, the transcriptional regulation of Olig2 and Egfr has not been explored. Here, we show that in cortical progenitor cells, multiple regulatory programs, including Pax6 and Gli3, prevent precocious expression of Olig2 , a gene essential for production of cortical oligodendrocytes and astrocytes. We identify multiple enhancers that control Olig2 expression in cortical progenitors and show that the mechanisms for regulating Olig2 expression are conserved between the mouse and human. Our study reveals evolutionarily conserved regulatory logic controlling the lineage switch of cortical neural stem cells., Competing Interests: Competing interests statement:The authors declare no competing interest.

Aim: Type II diabetes (T2D) stems from insulin resistance, with β-cell dysfunction as a hallmark in its progression. Studies reveal that β cells undergo apoptosis or dedifferentiation during T2D development. The transcription factor PAX4 is vital for β differentiation and survival, thus may be a potential enhancer of β-cell function in T2D islets. Materials & methods: Human PAX4 cDNA was delivered into T2D human islets with an adenoviral vector, and its effects on β cells were examined. Results: PAX4 gene delivery significantly improved β-cell survival, and increased β-cell composition in the T2D human islets. Basal insulin and glucose-stimulated insulin secretion in PAX4-expressing islets were substantially higher than untreated or control-treated T2D human islets. Conclusion: Introduced PAX4 expression in T2D human islets improves β-cell function, thus could provide therapeutic benefits for T2D treatment.

Background: Paired box 1 (PAX1) is a transcription factor and essential for the development of pharyngeal pouches-derived tissues, including thymus. PAX1 mutations are identified in Severe Combined Immunodeficiency (SCID) patients with Otofaciocervical Syndrome Type 2 (OTFCS2). However, despite the critical roles of PAX1 in embryonic development and diseases, detailed insights into its molecular mode of action are critically missing., Methods: The repressing roles of PAX1 and SCID associated mutants on Wnt signaling pathway were investigated by luciferase reporter assays, qRT-PCR and in situ hybridization in HEK293FT, HCT116 cells and zebrafish embryos, respectively. Co-immunoprecipitation (co-IP) and western blotting assays were carried out to identify the molecular mechanisms underlying PAX1's role on Wnt signaling pathway. hESC based endoderm differentiation, flow cytometry, high-throughput sequencing data analysis, and qRT-PCR assays were utilized to determine the roles of PAX1 during endoderm differentiation., Results: Here, we show that PAX1 represses canonical Wnt signaling pathway in vertebrate cells. Mechanically, PAX1 competes with SUMO E3 ligase PIASy to bind to TCF7L2, thus perturbing TCF7L2 SUMOylation level, further reducing its transcriptional activity and protein stability. Moreover, we reveal that PAX1 plays dual roles in hESC-derived definitive and foregut/pharyngeal endoderm cells, which give rise to the thymus epithelium, by inhibiting Wnt signaling. Importantly, our data show PAX1 mutations found in SCID patients significantly compromise the suppressing ability of PAX1 on Wnt signaling., Conclusions: Our study presents a novel molecular mode of action of PAX1 in regulation of canonical Wnt signaling and endoderm differentiation, thus providing insights for the molecular basis of PAX1 associated SCID, offering better understanding of the behavior of PAX1 in embryogenesis., (© 2024. The Author(s).)

Fusion-positive rhabdomyosarcoma (FP-RMS) is an aggressive pediatric sarcoma driven primarily by the PAX3-FOXO1 fusion oncogene, for which therapies targeting PAX3-FOXO1 are lacking. Here, we screen 62,643 compounds using an engineered cell line that monitors PAX3-FOXO1 transcriptional activity identifying a hitherto uncharacterized compound, P3FI-63. RNA-seq, ATAC-seq, and docking analyses implicate histone lysine demethylases (KDMs) as its targets. Enzymatic assays confirm the inhibition of multiple KDMs with the highest selectivity for KDM3B. Structural similarity search of P3FI-63 identifies P3FI-90 with improved solubility and potency. Biophysical binding of P3FI-90 to KDM3B is demonstrated using NMR and SPR. P3FI-90 suppresses the growth of FP-RMS in vitro and in vivo through downregulating PAX3-FOXO1 activity, and combined knockdown of KDM3B and KDM1A phenocopies P3FI-90 effects. Thus, we report KDM inhibitors P3FI-63 and P3FI-90 with the highest specificity for KDM3B. Their potent suppression of PAX3-FOXO1 activity indicates a possible therapeutic approach for FP-RMS and other transcriptionally addicted cancers., (© 2024. This is a U.S. Government work and not under copyright protection in the US; foreign copyright protection may apply.)

Oncogenic fusion genes are attractive therapeutic targets because of their tumor-specific expression and central "driver" roles in various human cancers. However, oncogenic fusions involving transcription factors such as PAX3-FOXO1 in alveolar fusion gene-positive rhabdomyosarcoma (FP-RMS) have been difficult to inhibit due to the apparent lack of tractable drug-like binding sites comparable to that recognized by Gleevec (imatinib mesylate) on the BCR-ABL1 tyrosine kinase fusion protein. Toward the identification of novel small molecules that selectively target PAX3-FOXO1, we used CRISPR-Cas9-mediated knock-in to append the pro-luminescent HiBiT tag onto the carboxy terminus of the endogenous PAX3-FOXO1 fusion protein in two human FP-RMS cell lines (RH4 and SCMC). HiBiT is an 11-amino acid peptide derived from the NanoLuc luciferase that produces a luminescence signal which is ~100-fold brighter than firefly or Renilla luciferases through high-affinity binding to a complementary NanoLuc peptide fragment called LgBiT. To facilitate single-cell clonal isolation of knock-ins, the homology-directed repair template encoding HiBiT was followed by a P2A self-cleaving peptide for coexpression of an mCherry fluorescent protein as a fluorescence-activated cell sorter (FACS)-selectable marker. HiBiT tagging thus allows highly sensitive luminescence detection of endogenous PAX3-FOXO1 levels permitting quantitative high-throughput screening of large compound libraries for the discovery of PAX3-FOXO1 inhibitors and degraders., (© 2024. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

The coding variant (p.Arg192His) in the transcription factor PAX4 is associated with an altered risk for type 2 diabetes (T2D) in East Asian populations. In mice, Pax4 is essential for beta cell formation but its role on human beta cell development and/or function is unknown. Participants carrying the PAX4 p.His192 allele exhibited decreased pancreatic beta cell function compared to homozygotes for the p.192Arg allele in a cross-sectional study in which we carried out an intravenous glucose tolerance test and an oral glucose tolerance test. In a pedigree of a patient with young onset diabetes, several members carry a newly identified p.Tyr186X allele. In the human beta cell model, EndoC-βH1, PAX4 knockdown led to impaired insulin secretion, reduced total insulin content, and altered hormone gene expression. Deletion of PAX4 in human induced pluripotent stem cell (hiPSC)-derived islet-like cells resulted in derepression of alpha cell gene expression. In vitro differentiation of hiPSCs carrying PAX4 p.His192 and p.X186 risk alleles exhibited increased polyhormonal endocrine cell formation and reduced insulin content that can be reversed with gene correction. Together, we demonstrate the role of PAX4 in human endocrine cell development, beta cell function, and its contribution to T2D-risk., (© 2023. Springer Nature Limited.)

Paired box 4 (Pax4) is a key transcription factor involved in the embryonic development of the pancreatic islets of Langerhans. Consisting of a conserved paired box domain and a homeodomain, this transcription factor plays an essential role in early endocrine progenitor cells, where it is necessary for cell-fate commitment towards the insulin-secreting β cell lineage. Knockout of Pax4 in animal models leads to the absence of β cells, which is accompanied by a significant increase in glucagon-producing α cells, and typically results in lethality within days after birth. Mutations in Pax4 that cause an impaired Pax4 function are associated with diabetes pathogenesis in humans. In adulthood, Pax4 expression is limited to a distinct subset of β cells that possess the ability to proliferate in response to heightened metabolic needs. Upregulation of Pax4 expression is known to promote β cell survival and proliferation. Additionally, ectopic expression of Pax4 in pancreatic islet α cells or δ cells has been found to generate functional β-like cells that can improve blood glucose regulation in experimental diabetes models. Therefore, Pax4 represents a promising therapeutic target for the protection and regeneration of β cells in the treatment of diabetes. The purpose of this review is to provide a thorough and up-to-date overview of the role of Pax4 in pancreatic β cells and its potential as a therapeutic target for diabetes.

Background: Histomorphological differentiation between pancreatic serous cystadenoma (SCA) and clear cell renal cell carcinoma (RCC) can be challenging. We aimed to study Paired box 8 protein (Pax8) expression profile in cytologic and surgical specimens with pancreatic SCA to assess its utility as a differentiating marker from clear cell RCC., Methods: We characterized Pax8 immunohistochemistry in 33 patients with pancreatic SCA (23 surgical resections and 10 cytology specimens). Nine cytology specimens from metastatic clear cell RCC involving pancreas were used as control tissue. Electronic medical records were reviewed to retrieve clinical information., Results: All 10 pancreatic SCA cytology specimens, and 16 of 23 pancreatic SCA surgical resections showed absent Pax8 immunostaining, while the remaining 7 surgical resection specimens showed 1%-2% immunoreactivities. Islet and lymphoid cells adjacent to the pancreatic SCA expressed Pax8. In contrast, the proportion of Pax8 immunoreactivity ranged from 50 to 90% (average of 76%) in nine cases of metastatic clear cell RCC involving pancreas. Using a 5% immunoreactivity cutoff, all cases of pancreatic SCA are interpreted as negative for Pax8 immunostains while all cases of metastatic clear cell RCC involving pancreas are interpreted as positive for Pax8 immunostains., Conclusions: These results suggest that Pax8 immunohistochemistry staining can be a useful adjunct marker to differentiate pancreatic SCA from clear cell RCC in clinical practice. To the best of our knowledge, this is the first large-scale study of Pax8 immunostaining on surgical and cytology specimens with pancreatic SCA., (© 2023. The Author(s).)

Objectives: This hospital-based cohort study evaluated whether ZNF582 and PAX1 methylation levels at baseline can be used as biomarkers to identify lesions with a high potential for malignant transformation in patients with normal mucosa and oral potentially malignant disorders., Patients and Methods: We recruited 171 adult patients with normal mucosa and oral potentially malignant disorders in 2012-2014. They were followed until 2017. Outcomes, including advanced histopathological findings and oral cancer occurrence, were obtained from medical charts, the Taiwan Cancer Registry, and cause-of-death data. Kaplan-Meier analysis and Cox proportional hazards regression models were used to examine the association of ZNF582 and PAX1 methylation levels at baseline with subsequent outcome occurrences., Results: After 260,192 days of follow-up, 11 cases of oral cancer and 4 cases of advanced histopathological progression occurred. Patients with higher ZNF582 and PAX1 methylation levels at baseline had a higher incidence of disease progression. After adjustment for all studied factors using Cox proportional hazards regression models, ZNF582 m level (adjusted hazard ratio, 11.41; 95% CI, 2.05-63.36; p = 0.005) was the only significant and independent predictor of disease progression., Conclusions: ZNF582 hypermethylation can be an effective and noninvasive biomarker for identifying oral lesions with a high potential for malignant transformation., (© 2021 Wiley Periodicals LLC.)

Glyphosate is a pesticide, which contaminates the environment and exposes workers and general population to its residues present in foods and waters. In soil, Glyphosate is degraded in metabolites, amino-methyl-phosphonic acid (AMPA) being the main one. Glyphosate is considered a potential cancerogenic and endocrine-disruptor agent, however its adverse effects on the thyroid were evaluated only in animal models and in vitro data are still lacking. Aim of this study was to investigate whether exposure to Glyphosate could exert adverse effects on thyroid cells in vitro. Two models (adherent-2D and spheroid-3D) derived from the same cell strain Fisher-rat-thyroid-cell line-5 (FRTL-5) were employed. After exposure to Glyphosate at increasing concentrations (0.0, 0.1-0.25- 0.5-1.0-2.0-10.0 mM) we evaluated cell viability by WST-1 (adherent and spheroids), results being confirmed by propidium-iodide staining (only for spheroids). Proliferation of adherent cells was assessed by crystal violet and trypan-blue assays, the increasing volume of spheroids was taken as a measure of proliferation. We also evaluated the ability of cells to form spheroids after Glyphosate exposure. We assessed changes of reactive-oxygen-species (ROS) by the cell-permeant H2DCFDA. Glyphosate-induced changes of mRNAs encoding for thyroid-related genes (TSHR, TPO, TG, NIS, TTF-1 and PAX8) were evaluated by RT-PCR. Glyphosate reduced cell viability and proliferation in both models, even if at different concentrations. Glyphosate at the highest concentration reduced the ability of FRTL-5 to form spheroids. An increased ROS production was found in both models after exposure to Glyphosate. Finally, Glyphosate increased the mRNA levels of some thyroid related genes (TSHR, TPO, TG and TTF-1) in both models, while it increased the mRNAs of PAX8 and NIS only in the adherent model. The present study supports an adverse effect of Glyphosate on cultured thyroid cells. Glyphosate reduced cell viability and proliferation and increased ROS production in thyroid cells., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Background: Anomalies of the FOXO1 gene in alveolar rhabdomyosarcoma are associated with a worse clinical prognosis, which determines the high value of studying the status of this gene when choosing a therapy strategy. The «gold standard» for determining FOXO1 gene rearrangements is currently the fluorescent in situ hybridization (FISH) technique., Objective: Study of the relationship between canonical FOXO1 translocation and immunohistochemical expression of new surrogate markers in alveolar rhabdomyosarcoma to determine their predictive value., Material and Methods: 139 cases of rhabdomyosarcoma were retrospectively studied. The study used tissue matrix technology (TMA). On sections obtained from TMA blocks, the FISH technique was implemented using the locus-specific probe MetaSystems XL FOXO1 Break Apart (Metasystems, Germany). Immunohistochemical studies were performed on similar sections from TMA blocks with OLIG2 (Cell Marque Antibodies, clone 211F1.1) and MUC4 (Cell Marque Antibodies, clone 8G7) antibodies., Results: The final expression analysis and statistical processing using a 2x2 contingency table and Fisher's exact test passed 111 cases (76 without FOXO1 rearrangement and 35 with rearrangement). The specificity of OLIG2 and MUC4 expression for FOXO1-rearranged alveolar rhabdomyosarcoma was 85.53% and 80.26%, respectively ( p <0.01)., Conclusion: The present study confirms the high predictive value of the expression of surrogate markers OLIG2 and MUC4 in determining the genetic status of alveolar rhabdomyosarcoma, which makes it possible to predict with high specificity the detection of the FOXO1 gene rearrangement.

Development of the Xenopus pronephros relies on renal precursors grouped at neurula stage into a specific region of dorso-lateral mesoderm called the kidney field. Formation of the kidney field at early neurula stage is dependent on retinoic (RA) signaling acting upstream of renal master transcriptional regulators such as pax8 or lhx1. Although lhx1 might be a direct target of RA-mediated transcriptional activation in the kidney field, how RA controls the emergence of the kidney field remains poorly understood. In order to better understand RA control of renal specification of the kidney field, we have performed a transcriptomic profiling of genes affected by RA disruption in lateral mesoderm explants isolated prior to the emergence of the kidney field and cultured at different time points until early neurula stage. Besides genes directly involved in pronephric development (pax8, lhx1, osr2, mecom), hox (hoxa1, a3, b3, b4, c5 and d1) and the hox co-factor meis3 appear as a prominent group of genes encoding transcription factors (TFs) downstream of RA. Supporting the idea of a role of meis3 in the kidney field, we have observed that meis3 depletion results in a severe inhibition of pax8 expression in the kidney field. Meis3 depletion only marginally affects expression of lhx1 and aldh1a2 suggesting that meis3 principally acts upstream of pax8. Further arguing for a role of meis3 and hox in the control of pax8, expression of a combination of meis3, hoxb4 and pbx1 in animal caps induces pax8 expression, but not that of lhx1. The same combination of TFs is also able to transactivate a previously identified pax8 enhancer, Pax8-CNS1. Mutagenesis of potential PBX-Hox binding motifs present in Pax8-CNS1 further allows to identify two of them that are necessary for transactivation. Finally, we have tested deletions of regulatory sequences in reporter assays with a previously characterized transgene encompassing 36.5 ​kb of the X. tropicalis pax8 gene that allows expression of a truncated pax8-GFP fusion protein recapitulating endogenous pax8 expression. This transgene includes three conserved pax8 enhancers, Pax8-CNS1, Pax8-CNS2 and Pax8-CNS3. Deletion of Pax8-CNS1 alone does not affect reporter expression, but deletion of a 3.5 ​kb region encompassing Pax8-CNS1 and Pax8-CNS2 results in a severe inhibition of reporter expression both in the otic placode and kidney field domains., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Tweetable abstract Zinc finger proteins control the transcription of downstream genes that are implicated in migration, invasion, cell death and proliferation. More mechanistic research on ZNF582 is needed to ascertain how this protein's methylation regulates the inflammatory pathway in oral cancer.

Rhabdomyosarcoma (RMS) is the most common soft tissue sarcoma of childhood and adolescence that includes FP-RMS, harboring the fusion oncoprotein PAX3/7-FOXO1 and FN-RMS, often mutant in the RAS pathway. Risk stratifications of RMS patients determine different prognostic groups and related therapeutic treatment. Current multimodal therapeutic strategies involve surgery, chemotherapy (CHT) and radiotherapy (RT), but despite the deeper knowledge of response mechanisms underpinning CHT treatment and the technological improvements that characterize RT, local failures and recurrence frequently occur. This review sums up the RMS classification and the management of RMS patients, with special attention to RT treatment and possible radiosensitizing strategies for RMS tumors. Indeed, RMS radioresistance is a clinical problem and further studies aimed at dissecting radioresistant molecular mechanisms are needed to identify specific targets to hit, thus improving RT-induced cytotoxicity.

Rhabdomyosarcoma is a soft tissue cancer that arises in skeletal muscle due to mutations in myogenic progenitors that lead to ineffective differentiation and malignant transformation. The transcription factors Pax3 and Pax7 and their downstream target genes are tightly linked with the fusion positive alveolar subtype, whereas the RAS pathway is usually involved in the embryonal, fusion negative variant. Here, we analyse the role of Pax3 in a fusion negative context, by linking alterations in gene expression in pax3a/pax3b double mutant zebrafish with tumour progression in kRAS-induced rhabdomyosarcoma tumours. Several genes in the RAS/MAPK signalling pathway were significantly down-regulated in pax3a/pax3b double mutant zebrafish. Progression of rhabdomyosarcoma tumours was also delayed in the pax3a/pax3b double mutant zebrafish indicating that Pax3 transcription factors have an unappreciated role in mediating malignancy in fusion negative rhabdomyosarcoma., (© 2022. The Author(s).)

The development of stable specialized cell types in multicellular organisms relies on mechanisms controlling inductive intercellular signals and the competence of cells to respond to such signals. In developing cerebral cortex, progenitors generate only glutamatergic excitatory neurons despite being exposed to signals with the potential to initiate the production of other neuronal types, suggesting that their competence is limited. Here, we tested the hypothesis that this limitation is due to their expression of transcription factor Pax6. We used bulk and single-cell RNAseq to show that conditional cortex-specific Pax6 deletion from the onset of cortical neurogenesis allowed some progenitors to generate abnormal lineages resembling those normally found outside the cortex. Analysis of selected gene expression showed that the changes occurred in specific spatiotemporal patterns. We then compared the responses of control and Pax6-deleted cortical cells to in vivo and in vitro manipulations of extracellular signals. We found that Pax6 loss increased cortical progenitors' competence to generate inappropriate lineages in response to extracellular factors normally present in developing cortex, including the morphogens Shh and Bmp4. Regional variation in the levels of these factors could explain spatiotemporal patterns of fate change following Pax6 deletion in vivo. We propose that Pax6's main role in developing cortical cells is to minimize the risk of their development being derailed by the potential side effects of morphogens engaged contemporaneously in other essential functions., Competing Interests: The authors have declared that no competing interests exist.

We report a 10-month-old girl with familial congenital hypothyroidism harboring a novel heterozygous pathogenic variant in the paired DNA-binding domain of PAX8 (NM&#95;003466:c.110T>C:p.Leu37Pro). Genotype-phenotype correlation revealed complete penetrance of this PAX8 defect in this family, in which the affected father and half-brother carry the same mutation. This deleterious variant has not been reported in any of the available databases [MAFgnomAD = 0, dbSNP (-)], and the amino acid leucine at position 37 is highly conserved across species. Establishing the molecular diagnosis expands our knowledge on the cause of thyroid dysgenesis and provides a guide for counseling and early treatment.

Chimeric transcription factors drive lineage-specific oncogenesis but are notoriously difficult to target. Alveolar rhabdomyosarcoma (RMS) is an aggressive childhood soft tissue sarcoma transformed by the pathognomonic Paired Box 3-Forkhead Box O1 (PAX3-FOXO1) fusion protein, which governs a core regulatory circuitry transcription factor network. Here, we show that the histone lysine demethylase 4B (KDM4B) is a therapeutic vulnerability for PAX3-FOXO1 + RMS. Genetic and pharmacologic inhibition of KDM4B substantially delayed tumor growth. Suppression of KDM4 proteins inhibited the expression of core oncogenic transcription factors and caused epigenetic alterations of PAX3-FOXO1-governed superenhancers. Combining KDM4 inhibition with cytotoxic chemotherapy led to tumor regression in preclinical PAX3-FOXO1 + RMS subcutaneous xenograft models. In summary, we identified a targetable mechanism required for maintenance of the PAX3-FOXO1-related transcription factor network, which may translate to a therapeutic approach for fusion-positive RMS.

Rhabdomyosarcoma (RMS) is an aggressive pediatric soft tissue sarcoma characterized by a very poor prognosis when relapses occur after front-line therapy. Therefore, a major challenge for patients' management remains the identification of markers associated with refractory and progressive disease. In this context, cancer autoantibodies are natural markers of disease onset and progression, useful to unveil novel therapeutic targets. Herein, we matched autoantibody profiling of alveolar RMS (ARMS) patients with genes under regulatory control of PAX3-FOXO1 transcription factor and revealed fibroblast growth factor 8 (FGF8) as a novel ARMS tumor antigen of diagnostic, prognostic, and therapeutic potential. We demonstrated that high levels of FGF8 autoantibodies distinguished ARMS patients from healthy subjects and represented an independent prognostic factor of better event-free survival. FGF8 was overexpressed in ARMS tumors compared to other types of pediatric soft tissue sarcomas, acting as a positive regulator of cell signaling. Indeed, FGF8 was capable of stimulating ARMS cells migration and expression of pro-angiogenic and metastasis-related factors, throughout MAPK signaling activation. Of note, FGF8 was found to increase in recurrent tumors, independently of PAX3-FOXO1 expression dynamics. Risk of recurrence correlated positively with FGF8 expression levels at diagnosis and reduced FGF8 autoantibodies titer, almost as if to suggest a failure of the immune response to control tumor growth in recurring patients. This study provides evidence about the crucial role of FGF8 in ARMS and the protective function of natural autoantibodies, giving new insights into ARMS biology and laying the foundations for the development of new therapeutic strategies., Competing Interests: The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results., (© 2022 The Author(s). Published with license by Taylor & Francis Group, LLC.)

Normal processes of embryonic development and abnormal transformation to cancer have many parallels, and in fact many aberrant cancer cell capabilities are embryonic traits restored in a distorted, unorganized way. Some of these capabilities are cell autonomous, such as proliferation and resisting apoptosis, while others involve a complex interplay with other cells that drives significant changes in neighboring cells. The correlation between embryonic development and cancer is driven by shared proteins. Some embryonic proteins disappear after embryogenesis in adult differentiated cells and are restored in cancer, while others are retained in adult cells, acquiring new functions upon transformation to cancer. Many embryonic factors embraced by cancer cells are transcription factors; some are master regulators that play a major role in determining cell fate. The paired box (PAX) domain family of developmental transcription factors includes nine members involved in differentiation of various organs. All paired box domain proteins are involved in different cancer types carrying pro-tumorigenic or anti-tumorigenic roles. This review focuses on PAX8, a master regulator of transcription in embryonic development of the thyroid, kidney, and male and female genital tracts. We detail the role of PAX8 in each of these organ systems, describe its role during development and in the adult if known, and highlight its pro-tumorigenic role in cancers that emerge from PAX8 expressing organs.

Rhabdomyosarcoma (RMS) is one of the most common pediatric soft-tissue cancer. Previously, we discovered a gene fusion, MARS-AVIL formed by chromosomal inversion in RMS. Suspecting that forming a fusion with a housekeeping gene may be one of the mechanisms to dysregulate an oncogene, we investigated AVIL expression and its role in RMS. We first showed that MARS-AVIL translates into an in-frame fusion protein, which is critical for RMS cell tumorigenesis. Besides forming a gene fusion with the housekeeping gene, MARS , the AVIL locus is often amplified, and its RNA and protein expression are overexpressed in the majority of RMSs. Tumors with AVIL dysregulation exhibit evidence of oncogene addiction: Silencing MARS-AVIL in cells harboring the fusion, or silencing AVIL in cells with AVIL overexpression, nearly eradicated the cells in culture, as well as inhibited in vivo xenograft growth in mice. Conversely, gain-of-function manipulations of AVIL led to increased cell growth and migration, enhanced foci formation in mouse fibroblasts, and most importantly transformed mesenchymal stem cells in vitro and in vivo. Mechanistically, AVIL seems to serve as a converging node functioning upstream of two oncogenic pathways, PAX3-FOXO1 and RAS, thus connecting two types of RMS associated with these pathways. Interestingly, AVIL is overexpressed in other sarcoma cells as well, and its expression correlates with clinical outcomes, with higher levels of AVIL expression being associated with worse prognosis. AVIL is a bona fide oncogene in RMS, and RMS cells are addicted to its activity., Competing Interests: The authors declare no competing interest.

Pax6 is a sequence-specific DNA binding transcription factor that positively and negatively regulates transcription and is expressed in multiple cell types in the developing and adult central nervous system (CNS). As indicated by the morphological and functional abnormalities in spontaneous Pax6 mutant rodents, Pax6 plays pivotal roles in various biological processes in the CNS. At the initial stage of CNS development, Pax6 is responsible for brain patterning along the anteroposterior and dorsoventral axes of the telencephalon. Regarding the anteroposterior axis, Pax6 is expressed inversely to Emx2 and Coup-TF1, and Pax6 mutant mice exhibit a rostral shift, resulting in an alteration of the size of certain cortical areas. Pax6 and its downstream genes play important roles in balancing the proliferation and differentiation of neural stem cells. The Pax6 gene was originally identified in mice and humans 30 years ago via genetic analyses of the eye phenotypes. The human PAX6 gene was discovered in patients who suffer from WAGR syndrome (i.e., Wilms tumor, aniridia, genital ridge defects, mental retardation). Mutations of the human PAX6 gene have also been reported to be associated with autism spectrum disorder (ASD) and intellectual disability. Rodents that lack the Pax6 gene exhibit diverse neural phenotypes, which might lead to a better understanding of human pathology and neurodevelopmental disorders. This review describes the expression and function of Pax6 during brain development, and their implications for neuropathology.

Type 1 diabetes results from the autoimmune-mediated loss of insulin-producing beta-cells. Accordingly, important research efforts aim at regenerating these lost beta-cells by converting pre-existing endogenous cells. Following up on previous results demonstrating the conversion of pancreatic somatostatin delta-cells into beta-like cells upon Pax4 misexpression and acknowledging that somatostatin-expressing cells are highly represented in the gastrointestinal tract, one could wonder whether this Pax4 -mediated conversion could also occur in the GI tract. We made use of transgenic mice misexpressing Pax4 in somatostatin cells (SSTCrePOE) to evaluate a putative Pax4 -mediated D-to-beta-like cell conversion. Additionally, we implemented an ex vivo approach based on mice-derived gut organoids to assess the functionality of these neo-generated beta-like cells. Our results outlined the presence of insulin + cells expressing several beta-cell markers in gastrointestinal tissues of SSTCrePOE animals. Further, using lineage tracing, we established that these cells arose from D cells. Lastly, functional tests on mice-derived gut organoids established the ability of neo-generated beta-like cells to release insulin upon stimulation. From this study, we conclude that the misexpression of Pax4 in D cells appears sufficient to convert these into functional beta-like cells, thus opening new research avenues in the context of diabetes research., Competing Interests: Author SB was employed by PlantaCorp GmbH. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Garrido-Utrilla, Ayachi, Friano, Atlija, Balaji, Napolitano, Silvano, Druelle and Collombat.)

Thyroid dysfunctions are associated with liver diseases ranging, in severity, from insulin resistance (IR) to hepatocellular carcinoma. The pathogenic mechanisms appear complex and are not attributable, exclusively, to the impaired thyroid hormone (TH) signalling. Using a mouse model of human congenital hypothyroidism, young double heterozygote for both NK2 homeobox 1 (Nkx2-1)- and Paired box 8 (Pax8)-null mutations (DHTP) mice, and single heterozygous Pax8+/- and Nkx2-1+/- mice, we studied the liver pathways, the endocrine and metabolic factors affected in conditions of different dysthyroidisms. Young Nkx2-1+/- females displayed a slight hyperthyroidism and, in liver, increased TH signalling (i.e. increased expression of Dio1 and Trβ1) and lipogenic gene expression, with triglycerides accumulation. Hypothyroid DHTP and euthyroid Pax8+/- females shared liver and skeletal muscle IR and hepatic hypothyroidism (i.e. reduced expression of Mct8, Dio1 and TRβ1), activation of AKT and increased expression of glutathione peroxidase 4. Oxidative stress and reduced mitochondrial COX activity were observed in DHTP mice only. Pax8+/- females, but, unexpectedly, not DHTP ones, displayed transcriptional activation of the hepatic (and renal) gluconeogenic pathway, hypercortisolemia, fasting hyperglycaemia and hyperinsulinemia, reduced serum β-hydroxybutyrate, associated with hepatic AMPK activation. DHTP mice showed hypercholesterolemia and activation of mTOR. Collectively, the data indicate that heterozygote mutations of Pax8 and Nkx2-1 genes may produce multiple dysmetabolisms, even under systemic euthyroidism. Differential liver pathways and multiple hormonal axes are affected with implications for energy and nutrient homeostasis. The identified players may be specific target in the management of thyroid dysfunction-associated dysmetabolisms in terms of prevention/counteraction of IR, type 2 diabetes and related comorbidities.

Background: Pax6, a multifunctional protein and a transcriptional regulator is critical for optimal functioning of neuronal cells. It is known that alternatively spliced Pax6 isoforms and co-expressed interacting proteins mediate cell/tissue specific autoregulation of Pax6, however, underlying mechanism(s) are poorly understood., Methods and Results: We used Neuro-2a cells to explore the mechanism of autoregulation of Pax6 in neuronal cells whereas NIH/3T3 cells were used as control. We first studied the transcript expression of the three Pax6 isoforms: Pax6, Pax6(5a), and Pax6(ΔPD); and the two co-expressed Pax6-interacting partners: SPARC and p53 in normal and overexpressed conditions, through the semi-quantitative RT-PCR. Further, we used the luciferase reporter assay to study the binding and transactivation of the three Pax6 isoforms: Pax6, Pax6(5a), and Pax6(ΔPD) to their respective promoters: P0, P1, and Pα; followed by that of the two co-expressed Pax6-interacting partners: SPARC and p53 to the Pax6-P1 promoter. Expression and distribution of Pax6, Pax6(5a) and Pax6(ΔPD), their binding to Pax6-promoters (P0, P1, and Pα) and transactivation were modulated in transfected Neuro-2a cells., Conclusion: Our results suggest that autoregulation of Pax6 in neuronal cells is driven by a promoter dependent mechanism which is mediated by spliced variants [Pax6(5a) and Pax6(ΔPD)] and interacting proteins (SPARC and p53) of Pax6., (© 2022. The Author(s), under exclusive licence to Springer Nature B.V.)

Pax6 is a well-known regulator of early neuroepithelial progenitor development. Its constitutive loss has a particularly strong effect on the developing prethalamus, causing it to become extremely hypoplastic. To overcome this difficulty in studying the long-term consequences of Pax6 loss for prethalamic development, we used conditional mutagenesis to delete Pax6 at the onset of neurogenesis and studied the developmental potential of the mutant prethalamic neurons in vitro. We found that Pax6 loss affected their rates of neurite elongation, the location and length of their axon initial segments, and their electrophysiological properties. Our results broaden our understanding of the long-term consequences of Pax6 deletion in the developing mouse forebrain, suggesting that it can have cell-autonomous effects on the structural and functional development of some neurons., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2022. Published by The Company of Biologists Ltd.)

The cetacean hindlimb skeleton massively decreased to only vestigial limb elements as cetaceans evolved from land to aquatic lifestyles; however, the molecular mechanism underlying this major morphological transition remains unclear. In this study, four deletions and specific substitutions were detected in cetacean hindlimb enhancer A (HLEA), an enhancer that can regulate Tbx4 expression in hindlimb tissues to control hindlimb development. Transcriptional activation of HLEA was significantly weaker in bottlenose dolphin than mice, and this was found to be closely associated with cetacean-specific deletions. Furthermore, deletions in cetacean HLEA might disrupt HOX and PITX1 binding sites, which are required for enhancer activation. The ancestral state of these deletions was investigated, and all four specific deletions were found to have occurred after the species diverged from their common ancestor, suggesting that the deletion occurred recently, during a secondary aquatic adaptation. Taking these findings together, we suggest that cetacean-specific sequence changes reduced the Tbx4 gene expression pattern, and consequently drove the gradual loss of hindlimb in cetaceans., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Context: Primary hyperparathyroidism (PHPT) results from the hypersecretion of parathyroid hormone from parathyroid tumors. A transcription factor, namely Paired box1 (PAX1), is active in parathyroid gland development., Objective: We aimed to study potential epigenetic-mediated mechanism of PAX1 gene in sporadic parathyroid adenomas., Methods: In parathyroid adenomas tissues, we analyzed the DNA methylation via bisulfite-specific polymerase chain reaction (BSP) and histone modifications via chromatin immunoprecipitation in regulating the differential expression of PAX1., Results: The results showed that mRNA and protein expression of PAX1 was significantly reduced in parathyroid adenomas. Bisulfite sequencing demonstrated hypermethylation in the promoter region of PAX1 (35%; 14/40) and lower levels of histone 3 lysine 9 acetylation (H3K9ac) were observed on the promoter region of PAX1 (6-fold; P < .004) in parathyroid adenomas. Furthermore, upon treatment with a pharmacologic inhibitor, namely 5'aza-2 deoxycytidine, in rat parathyroid continuous cells, we found re-expression of PAX1 gene., Conclusion: Our study not only reveals expression of PAX1 is epigenetically deregulated but also paves a way for clinical and therapeutic implications in patients with PHPT., (© The Author(s) 2021. Published by Oxford University Press on behalf of the Endocrine Society. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Background: A low-frequency electromagnetic field (LF-EMF) exerts important biological effects on the human body., Objective: We previously studied the immunity and atrophy of gastrocnemius muscles in rats with spinal cord injuries and found that LF-EMF with a magnetic flux density of 1.5 mT exerted excellent therapeutic and preventive effects on reducing myotubes and increasing spatium intermusculare. However, the effects of LF-EMF on all stages of skeletal myogenesis, such as activation, proliferation, differentiation, and fusion of satellite cells to myotubes as stimulated by myogenic regulatoryfactors (MRFs), have not been fully elucidated., Methods: This study investigated the optimal LF-EMF magnetic flux density that exerted maximal effects on all stages of C2C12 cell skeletal myogenesis as well as its impact on regulatory MRFs., Results: The results showed that an LF-EMF with a magnetic flux density of 2.0 mT could activate C2C12 cells and upregulate the proliferation-promoting transcription factor PAX7. On the other hand, 1.5 mT EMF could upregulate the expression of MyoD and myogenin., Conclusion: LF-EMF could prevent the disappearance of myotubes, with different magnetic flux densities of LF-EMF exerting independent and positive effects on skeletal myogenesis such as satellite cell activation and proliferation, muscle cell differentiation, and myocyte fusion.

Background: Type 2 diabetes mellitus (T2DM) has been shown to increase the risks of cognitive decline and dementia. Paired box gene 4 (PAX4), a transcription factor for beta cell development and function, has recently been implicated in pathways intersecting Alzheimer's disease and T2DM., Objective: In this report, we evaluated the association of the ethnic-specific PAX4 R192H variant, a T2DM risk factor for East Asians which contributes to earlier diabetes onset, and cognitive function of Chinese T2DM patients., Methods: 590 Chinese patients aged 45-86 from the SMART2D study were genotyped for PAX4 R192H variation using Illumina OmniExpress-24 Array. The Repeatable Battery for the Assessment of Neuropsychological Status (RBANS) which had been validated in the Singapore population was administered to assess five cognitive domains: immediate memory, visuospatial/constructional, language, attention, and delayed memory. Multiple linear regression was used to assess the association of the R192H risk allele and cognitive domains., Results: Patients with two PAX4 R192H risk alleles showed significantly lower attention index score (β= -8.46, 95% CI [-13.71, -3.21], p = 0.002) than patients with wild-type alleles after adjusting for age, gender, diabetes onset age, HbA1c, body-mass index, renal function, lipid profiles, systolic blood pressure, metformin usage, smoking history, education level, Geriatric Depression Scale score, and presence of APOEɛ4 allele., Conclusion: Ethnic-specific R192H variation in PAX4 is associated with attention-specific cognitive impairment in Chinese with T2DM. Pending further validation studies, determining PAX4 R192H genotype may be helpful for early risk assessment of early-onset T2DM and cognitive impairment to improve diabetes care.

Developmental genes are frequently controlled by multiple enhancers sharing similar specificities. As a result, deletions of such regulatory elements have often failed to reveal their full function. Here, we use the Pitx1 testbed locus to characterize in detail the regulatory and cellular identity alterations following the deletion of one of its enhancers (Pen). By combining single cell transcriptomics and an in-embryo cell tracing approach, we observe an increased fraction of Pitx1 non/low-expressing cells and a decreased fraction of Pitx1 high-expressing cells. We find that the over-representation of Pitx1 non/low-expressing cells originates from a failure of the Pitx1 locus to coordinate enhancer activities and 3D chromatin changes. This locus mis-activation induces a localized heterochrony and a concurrent loss of irregular connective tissue, eventually leading to a clubfoot phenotype. This data suggests that, in some cases, redundant enhancers may be used to locally enforce a robust activation of their host regulatory landscapes., (© 2021. The Author(s).)

Rhabdomyosarcoma (RMS) is a pediatric malignancy of skeletal muscle lineage. The aggressive alveolar subtype is characterized by t(2;13) or t(1;13) translocations encoding for PAX3- or PAX7-FOXO1 chimeric transcription factors, respectively, and are referred to as fusion positive RMS (FP-RMS). The fusion gene alters the myogenic program and maintains the proliferative state while blocking terminal differentiation. Here, we investigated the contributions of chromatin regulatory complexes to FP-RMS tumor maintenance. We define the mSWI/SNF functional repertoire in FP-RMS. We find that SMARCA4 (encoding BRG1) is overexpressed in this malignancy compared to skeletal muscle and is essential for cell proliferation. Proteomic studies suggest proximity between PAX3-FOXO1 and BAF complexes, which is further supported by genome-wide binding profiles revealing enhancer colocalization of BAF with core regulatory transcription factors. Further, mSWI/SNF complexes localize to sites of de novo histone acetylation. Phenotypically, interference with mSWI/SNF complex function induces transcriptional activation of the skeletal muscle differentiation program associated with MYCN enhancer invasion at myogenic target genes, which is recapitulated by BRG1 targeting compounds. We conclude that inhibition of BRG1 overcomes the differentiation blockade of FP-RMS cells and may provide a therapeutic strategy for this lethal childhood tumor., (© 2021. This is a U.S. Government work and not under copyright protection in the US; foreign copyright protection may apply.)

Paired-like homeodomain transcription factor 1 (PITX1) is involved in numerous biological processes, including cell growth, progression, and invasion in various malignant tumors. Nevertheless, the relationship between PITX1 and kidney renal clear cell carcinoma (KIRC) remains unclear. The clinical role and functions of PITX1 were analyzed by integrating multiple open-access online datasets. Further experimental verification was performed via quantitative real-time PCR (qRT-PCR) to detect the expression of PITX1 in 10 pairs of KIRC tissues. Our results revealed that PITX1 mRNA was overexpressed in tumor tissues compared with normal tissues in the TCGA-KIRC database ( p < 0.001) and numerous independent cohorts ( p < 0.05). Further, high expression of PITX1 mRNA was detected in KIRC tissues compared with adjacent normal tissues in our center by qRT-PCR ( N = 10, p < 0.05). Logistic regression analysis demonstrated that the PITX1 level was positively associated with KIRC patients, T and M stages, histologic grade, and pathologic stage (all p < 0.05). Survival analysis showed that upregulation of PITX1 mRNA was associated with poor overall survival (OS), disease-free survival (DFS), and disease-specific survival (DSS) (all p < 0.05). Univariate/multivariate Cox hazard regression analysis revealed that PITX1 was an independent risk factor for OS in patients with KIRC (HR = 1.998, p = 0.003). Accordingly, the time-independent receiver operating characteristic (ROC) curve confirmed that PITX1 had good predictive efficacy for OS and DSS. Meanwhile, a prediction model constructed by nomogram was used to predict the OS of KIRC patients, and the calibration plot indicated this model shows high accuracy. We also revealed some downstream target genes of PITX1-related signaling pathways. Our finding suggested that high PITX1 mRNA expression may act as an independent predictive factor of poor prognosis in patients with KIRC. The prognostic model based on the nomogram would be instrumental in evaluating the survival rate in KIRC patients., Competing Interests: The authors declare that they have no conflict of interest., (Copyright © 2021 Yinglang Zhang et al.)

We dissect genetically a gene regulatory network that involves the transcription factors Tbx4, Pitx1 and Isl1 acting cooperatively to establish the hindlimb bud, and identify key differences in the pathways that initiate formation of the hindlimb and forelimb. Using live image analysis of murine limb mesenchyme cells undergoing chondrogenesis in micromass culture, we distinguish a series of changes in cellular behaviours and cohesiveness that are required for chondrogenic precursors to undergo differentiation. Furthermore, we provide evidence that the proximal hindlimb defects observed in Tbx4 mutant mice result from a failure in the early differentiation step of chondroprogenitors into chondrocytes, providing an explanation for the origins of proximally biased limb defects., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2021. Published by The Company of Biologists Ltd.)

Alzheimer's Disease (AD) and Type 2 Diabetes (T2D) share a common hallmark of insulin resistance. Reportedly, two non-canonical Receptor Tyrosine Kinases (RTKs), ALK and RYK, both targets of the same micro RNA miR-1271, exhibit significant and consistent functional down-regulation in post-mortem AD and T2D tissues. Incidentally, both have Grb2 as a common downstream adapter and NOX4 as a common ROS producing factor. Here we show that Grb2 and NOX4 play critical roles in reducing the severity of both the diseases. The study demonstrates that the abundance of Grb2 in degenerative conditions, in conjunction with NOX4, reverse cytoskeletal degradation by counterbalancing the network of small GTPases. PAX4, a transcription factor for both Grb2 and NOX4, emerges as the key link between the common pathways of AD and T2D. Down-regulation of both ALK and RYK through miR-1271, elevates the PAX4 level by reducing its suppressor ARX via Wnt/β-Catenin signaling. For the first time, this study brings together RTKs beyond Insulin Receptor (IR) family, transcription factor PAX4 and both AD and T2D pathologies on a common regulatory platform., (© 2021 The Author(s).)

Melanoma is one of the most aggressive types of cancer wherein resistance to treatment prevails. Therefore, it is important to discover novel molecular targets of melanoma progression as potential treatments. Here we show that paired-like homeodomain transcription factor 1 (PITX1) plays a crucial role in the inhibition of melanoma progression through regulation of SRY-box transcription factors (SOX) gene family mRNA transcription. Overexpression of PITX1 in melanoma cell lines resulted in a reduction in cell proliferation and an increase in apoptosis. Additionally, analysis of protein levels revealed an antagonistic cross-regulation between SOX9 and SOX10. Interestingly, PITX1 binds to the SOX9 promoter region as a positive regulatory transcription factor; PITX1 mRNA expression levels were positively correlated with SOX9 expression, and negatively correlated with SOX10 expression in melanoma tissues. Furthermore, transcription of the long noncoding RNA (lncRNA), survival-associated mitochondrial melanoma-specific oncogenic noncoding RNA (SAMMSON), was decreased in PITX1-overexpressing cells. Taken together, the findings in this study indicate that PITX1 may act as a negative regulatory factor in the development and progression of melanoma via direct targeting of the SOX signaling., (© 2021. The Author(s).)

Aim: We aimed to explore the application of PAX1 methylation and human papillomavirus (HPV) E6/E7 mRNA detection in cervical cancer screening and to compare the efficacy with high-risk (HR)-HPV detection., Patients and Methods: The cervical exfoliative cytology samples of 337 patients were collected, including 70 cases of cervical inflammation, 72 cases of low-grade squamous intraepithelial lesions, 97 cases of high-grade squamous intraepithelial lesions, and 98 cases of cervical carcinoma. The PAX1 gene methylation (PAX1) status was detected by multiple quantitative PCR, HPV E6/E7 mRNA (E6/E7) was detected by QuantiVirus detection, and HR-HPV (HPV) was detected by the Cobas 4800 detection system. The sensitivities, specificities, and accuracies were validated in the testing set., Results: The sensitivities of the HPV, HPV E6/E7, and PAX1 testing were 89.23%, 84.10%, and 86.67%, respectively, which all maintained a high level. In contrast, the specificities of the HPV, E6/E7, and PAX1 testing were only 19.10%, 37.32%, and 97.18% (in pairwise comparisons, p = 0.000). The AUC of PAX1 (0.919) was significantly larger than that of HPV (0.541) and E6/E7 detection (0.607) (p < 0.0001). In addition, the AUC areas of all combined parallel testing were lower than that of single PAX1 test (p < 0.05)., Conclusion: The diagnostic efficacy of E6/E7 detection and PAX1 detection was better than that of HPV detection, especially for PAX1 detection., (© 2021 Japan Society of Obstetrics and Gynecology.)

Endometrial carcinoma (EC) is the fourth-most common cancer in women in the United States, and generally carries a favorable prognosis. However, about 10% of EC patients have a rare and aggressive form, uterine serous papillary carcinoma (USPC), which carries a much higher mortality rate. The developmental transcription factor PAX8 is expressed in nearly 100% of USPCs. We show that PAX8 plays a critical antiapoptotic role in USPC and this role is established via transcriptional activation of two aberrant signaling pathways. First, PAX8 positively regulates mutated p53, and missense p53 mutations have an oncogenic gain of function effect. Second, PAX8 directly transcriptionally regulates p21, in a p53-independent manner, and p21 acquires a growth promoting role that is mediated via cytoplasmic localization of the protein. We propose that mutated p53 and cytoplasmic p21 can independently mediate the pro-proliferative role of PAX8 in USPC. In addition, we performed a genome-wide transcriptome analysis to detect pathways that are regulated by PAX8, and propose that metabolism and HIF-1alpha -related pathways are potential candidates for mediating the role of PAX8 in USPC. Taken together our findings demonstrate for the first time that PAX8 is an essential lineage marker in USPC, and suggest its mechanism of action., (© 2021. The Author(s), under exclusive licence to Springer Nature Limited.)

Background: The heterogeneous subtypes and stages of epithelial ovarian cancer (EOC) differ in their biological features, invasiveness, and response to chemotherapy, but the transcriptional regulators causing their differences remain nebulous., Methods: In this study, we compared high-grade serous ovarian cancers (HGSOCs) to low malignant potential or serous borderline tumors (SBTs). Our aim was to discover new regulatory factors causing distinct biological properties of HGSOCs and SBTs., Results: In a discovery dataset, we identified 11 differentially expressed genes (DEGs) between SBTs and HGSOCs. Their expression correctly classified 95% of 267 validation samples. Two of the DEGs, TMEM30B and TSPAN1, were significantly associated with worse overall survival in patients with HGSOC. We also identified 17 DEGs that distinguished stage II vs. III HGSOC. In these two DEG promoter sets, we identified significant enrichment of predicted transcription factor binding sites, including those of RARA, FOXF1, BHLHE41, and PITX1. Using published ChIP-seq data acquired from multiple non-ovarian cell types, we showed additional regulatory factors, including AP2-gamma/TFAP2C, FOXA1, and BHLHE40, bound at the majority of DEG promoters. Several of the factors are known to cooperate with and predict the presence of nuclear hormone receptor estrogen receptor alpha (ER-alpha). We experimentally confirmed ER-alpha and PITX1 presence at the DEGs by performing ChIP-seq analysis using the ovarian cancer cell line PEO4. Finally, RNA-seq analysis identified recurrent gene fusion events in our EOC tumor set. Some of these fusions were significantly associated with survival in HGSOC patients; however, the fusion genes are not regulated by the transcription factors identified for the DEGs., Conclusions: These data implicate an estrogen-responsive regulatory network in the differential gene expression between ovarian cancer subtypes and stages, which includes PITX1. Importantly, the transcription factors associated with our DEG promoters are known to form the MegaTrans complex in breast cancer. This is the first study to implicate the MegaTrans complex in contributing to the distinct biological trajectories of malignant and indolent ovarian cancer subtypes.

Rhabdomyosarcomas (RMS) are phenotypically and functionally heterogeneous. Both primary human RMS cultures and low-passage Myf6Cre,Pax3:Foxo1,p53 mouse RMS cell lines, which express the fusion oncoprotein Pax3:Foxo1 and lack the tumor suppressor Tp53 ( Myf6Cre,Pax3:Foxo1,p53 ), exhibit marked heterogeneity in PAX3:FOXO1 ( P3F ) expression at the single cell level. In mouse RMS cells, P3F expression is directed by the Pax3 promoter and coupled to eYFP YFP low /P3F low mouse RMS cells included 87% G0/G1 cells and reorganized their actin cytoskeleton to produce a cellular phenotype characterized by more efficient adhesion and migration. This translated into higher tumor-propagating cell frequencies of YFP low /P3F low compared with YFP high /P3F high cells. Both YFP low /P3F low and YFP high /P3F high cells gave rise to mixed clones in vitro, consistent with fluctuations in P3F expression over time. Exposure to the anti-tropomyosin compound TR100 disrupted the cytoskeleton and reversed enhanced migration and adhesion of YFP low /P3F low RMS cells. Heterogeneous expression of PAX3:FOXO1 at the single cell level may provide a critical advantage during tumor progression., (© 2021 Regina et al.)

Gastric cancer (GC) is one of the most common aggressive cancers. The discovery of an effective biomarker is necessary for GC diagnosis. In this study, we confirmed that Paired box gene 4 (PAX4) is up-regulated in GC tissues and cells via quantitative real time polymerase chain reaction (qRT-PCR), western blot and immunohistochemical staining. It was also identified that PAX4 contributed to GC cell proliferation, migration and invasion through Cell Counting Kit-8, BrdU, flow cytometry assay, colony formation assay, transwell assays, and wound healing assay. miR-27b-3p was confirmed with the binding site with PAX4 using ChIP assay and served as a tumor suppressor that inhibiting GC cell growth and metastasis, and reversed the effect of PAX4. Bioinformatics prediction and dual luciferase assay results demonstrated that miR-27b-3p targeted Grb2, which could alter the function of miR-27b-3p. Furthermore, the transcriptional control of PAX4-regulated miR-27b-3p activated the Ras-ERK pathway. Taken together, the PAX4/miR-27b-3p/Grb2 loop is known to be involved in GC cell promotion, and can be seen as a promising target for GC therapy.