Self-Reporting Transposons Enable Simultaneous Readout of Gene Expression and Transcription Factor Binding in Single Cells.

Cellular heterogeneity confounds in situ assays of transcription factor (TF) binding. Single-cell RNA sequencing (scRNA-seq) deconvolves cell types from gene expression, but no technology links cell identity to TF binding sites (TFBS) in those cell types. We present self-reporting transposons (SRTs) and use them in single-cell calling cards (scCC), a novel assay for simultaneously measuring gene expression and mapping TFBS in single cells. The genomic locations of SRTs are recovered from mRNA, and SRTs deposited by exogenous, TF-transposase fusions can be used to map TFBS. We then present scCC, which map SRTs from scRNA-seq libraries, simultaneously identifying cell types and TFBS in those same cells. We benchmark multiple TFs with this technique. Next, we use scCC to discover BRD4-mediated cell-state transitions in K562 cells. Finally, we map BRD4 binding sites in the mouse cortex at single-cell resolution, establishing a new method for studying TF biology in situ.

Collapse of the hepatic gene regulatory network in the absence of FoxA factors.

The FoxA transcription factors are critical for liver development through their pioneering activity, which initiates a highly complex regulatory network thought to become progressively resistant to the loss of any individual hepatic transcription factor via mutual redundancy. To investigate the dispensability of FoxA factors for maintaining this regulatory network, we ablated all FoxA genes in the adult mouse liver. Remarkably, loss of FoxA caused rapid and massive reduction in the expression of critical liver genes. Activity of these genes was reduced back to the low levels of the fetal prehepatic endoderm stage, leading to necrosis and lethality within days. Mechanistically, we found FoxA proteins to be required for maintaining enhancer activity, chromatin accessibility, nucleosome positioning, and binding of HNF4α. Thus, the FoxA factors act continuously, guarding hepatic enhancer activity throughout adult life.

Foxa2 and H2A.Z mediate nucleosome depletion during embryonic stem cell differentiation.

Nucleosome occupancy is fundamental for establishing chromatin architecture. However, little is known about the relationship between nucleosome dynamics and initial cell lineage specification. Here, we determine the mechanisms that control global nucleosome dynamics during embryonic stem (ES) cell differentiation into endoderm. Both nucleosome depletion and de novo occupation occur during the differentiation process, with higher overall nucleosome density after differentiation. The variant histone H2A.Z and the winged helix transcription factor Foxa2 both act to regulate nucleosome depletion and gene activation, thus promoting ES cell differentiation, whereas DNA methylation promotes nucleosome occupation and suppresses gene expression. Nucleosome depletion during ES cell differentiation is dependent on Nap1l1-coupled SWI/SNF and INO80 chromatin remodeling complexes. Thus, both epigenetic and genetic regulators cooperate to control nucleosome dynamics during ES cell fate decisions.

Foxa1 and Foxa2 are essential for sexual dimorphism in liver cancer.

Hepatocellular carcinoma (HCC) is sexually dimorphic in both rodents and humans, with significantly higher incidence in males, an effect that is dependent on sex hormones. The molecular mechanisms by which estrogens prevent and androgens promote liver cancer remain unclear. Here, we discover that sexually dimorphic HCC is completely reversed in Foxa1- and Foxa2-deficient mice after diethylnitrosamine-induced hepatocarcinogenesis. Coregulation of target genes by Foxa1/a2 and either the estrogen receptor (ERα) or the androgen receptor (AR) was increased during hepatocarcinogenesis in normal female or male mice, respectively, but was lost in Foxa1/2-deficient mice. Thus, both estrogen-dependent resistance to and androgen-mediated facilitation of HCC depend on Foxa1/2. Strikingly, single nucleotide polymorphisms at FOXA2 binding sites reduce binding of both FOXA2 and ERα to their targets in human liver and correlate with HCC development in women. Thus, Foxa factors and their targets are central for the sexual dimorphism of HCC.

Maintenance of CTCF- and Transcription Factor-Mediated Interactions from the Gametes to the Early Mouse Embryo.

The epigenetic information present in mammalian gametes and whether it is transmitted to the progeny are relatively unknown. We find that many promoters in mouse sperm are occupied by RNA polymerase II (Pol II) and Mediator. The same promoters are accessible in GV and MII oocytes and preimplantation embryos. Sperm distal ATAC-seq sites containing motifs for various transcription factors are conserved in monkeys and humans. ChIP-seq analyses confirm that Foxa1, ERα, and AR occupy distal enhancers in sperm. Accessible sperm enhancers containing H3.3 and H2A.Z are also accessible in oocytes and preimplantation embryos. Furthermore, their interactions with promoters in the gametes persist during early development. Sperm- or oocyte-specific interactions mediated by CTCF and cohesin are only present in the paternal or maternal chromosomes, respectively, in the zygote and 2-cell stages. These interactions converge in both chromosomes by the 8-cell stage. Thus, mammalian gametes contain complex patterns of 3D interactions that can be transmitted to the zygote after fertilization.

The long noncoding RNA Falcor regulates Foxa2 expression to maintain lung epithelial homeostasis and promote regeneration.

Transcription factors (TFs) are dosage-sensitive master regulators of gene expression, with haploinsufficiency frequently leading to life-threatening disease. Numerous mechanisms have evolved to tightly regulate the expression and activity of TFs at the transcriptional, translational, and posttranslational levels. A subset of long noncoding RNAs (lncRNAs) is spatially correlated with transcription factors in the genome, but the regulatory relationship between these lncRNAs and their neighboring TFs is unclear. We identified a regulatory feedback loop between the TF Foxa2 and a downstream lncRNA, Falcor (Foxa2-adjacent long noncoding RNA). Foxa2 directly represses Falcor expression by binding to its promoter, while Falcor functions in cis to positively regulate the expression of Foxa2. In the lung, loss of Falcor is sufficient to lead to chronic inflammatory changes and defective repair after airway epithelial injury. Moreover, disruption of the Falcor-Foxa2 regulatory feedback loop leads to altered cell adhesion and migration, in turn resulting in chronic peribronchial airway inflammation and goblet cell metaplasia. These data reveal that the lncRNA Falcor functions within a regulatory feedback loop to fine-tune the expression of Foxa2, maintain airway epithelial homeostasis, and promote regeneration.

FOXA2 activates HIF2α expression to promote tumor progression and is regulated by the E3 ubiquitin ligase VHL in renal cell carcinoma.

Renal cell carcinoma (RCC) is a frequent malignancy of the urinary system with high mortality and morbidity. However, the molecular mechanisms underlying RCC progression are still largely unknown. In this study, we identified FOXA2, a pioneer transcription factor, as a driver oncogene for RCC. We show that FOXA2 was commonly upregulated in human RCC samples and promoted RCC proliferation, as evidenced by assays of cell viability, colony formation, migratory and invasive capabilities, and stemness properties. Mechanistically, we found that FOXA2 promoted RCC cell proliferation by transcriptionally activating HIF2α expression in vitro and in vivo. Furthermore, we found that FOXA2 could interact with VHL (von Hippel‒Lindau), which ubiquitinated FOXA2 and controlled its protein stability in RCC cells. We showed that mutation of lysine at position 264 to arginine in FOXA2 could mostly abrogate its ubiquitination, augment its activation effect on HIF2α expression, and promote RCC proliferation in vitro and RCC progression in vivo. Importantly, elevated expression of FOXA2 in patients with RCC positively correlated with the expression of HIF2α and was associated with shorter overall and disease-free survival. Together, these findings reveal a novel role of FOXA2 in RCC development and provide insights into the underlying molecular mechanisms of FOXA2-driven pathological processes in RCC.

Redistribution of lamina-associated domains reshapes binding of pioneer factor FOXA2 in development of nonalcoholic fatty liver disease.

Nonalcoholic fatty liver disease (NAFLD) is highly prevalent in type 2 diabetes mellitus and the elderly, impacting 40% of individuals over 70. Regulation of heterochromatin at the nuclear lamina has been associated with aging and age-dependent metabolic changes. We previously showed that changes at the lamina in aged hepatocytes and laminopathy models lead to redistribution of lamina-associated domains (LADs), opening of repressed chromatin, and up-regulation of genes regulating lipid synthesis and storage, culminating in fatty liver. Here, we test the hypothesis that change in the expression of lamina-associated proteins and nuclear shape leads to redistribution of LADs, followed by altered binding of pioneer factor FOXA2 and by up-regulation of lipid synthesis and storage, culminating in steatosis in younger NAFLD patients (aged 21-51). Changes in nuclear morphology alter LAD partitioning and reduced lamin B1 signal correlate with increased FOXA2 binding before severe steatosis in young mice placed on a western diet. Nuclear shape is also changed in younger NAFLD patients. LADs are redistrubted and lamin B1 signal decreases similarly in mild and severe steatosis. In contrast, FOXA2 binding is similar in normal and NAFLD patients with moderate steatosis and is repositioned only in NAFLD patients with more severe lipid accumulation. Hence, changes at the nuclear lamina reshape FOXA2 binding with progression of the disease. Our results suggest a role for nuclear lamina in etiology of NAFLD, irrespective of aging, with potential for improved stratification of patients and novel treatments aimed at restoring nuclear lamina function.

FOXA2-initiated transcriptional activation of INHBA induced by methylmalonic acid promotes pancreatic neuroendocrine neoplasm progression.

Pancreatic neuroendocrine neoplasms (PanNENs) are a group of highly heterogeneous neoplasms originating from the endocrine islet cells of the pancreas with characteristic neuroendocrine differentiation, more than 60% of which represent metastases when diagnosis, causing major tumor-related death. Metabolic alterations have been recognized as one of the hallmarks of tumor metastasis, providing attractive therapeutic targets. However, little is known about the molecular mechanism of metabolic changes regulating PanNEN progression. In this study, we first identified methylmalonic acid (MMA) as an oncometabolite for PanNEN progression, based on serum metabolomics of metastatic PanNEN compared with non-metastatic PanNEN patients. One of the key findings was the potentially novel mechanism of epithelial-mesenchymal transition (EMT) triggered by MMA. Inhibin ßA (INHBA) was characterized as a key regulator of MMA-induced PanNEN progression according to transcriptomic analysis, which has been validated in vitro and in vivo. Mechanistically, INHBA was activated by FOXA2, a neuroendocrine (NE) specific transcription factor, which was initiated during MMA-induced progression. In addition, MMA-induced INHBA upregulation activated downstream MITF to regulate EMT-related genes in PanNEN cells. Collectively, these data suggest that activation of INHBA via FOXA2 promotes MITF-mediated EMT during MMA inducing PanNEN progression, which puts forward a novel therapeutic target for PanNENs.

The pioneer transcription factors Foxa1 and Foxa2 regulate alternative RNA splicing during thymocyte positive selection.

During positive selection at the transition from CD4+CD8+ double-positive (DP) to single-positive (SP) thymocyte, TCR signalling results in appropriate MHC restriction and signals for survival and progression. We show that the pioneer transcription factors Foxa1 and Foxa2 are required to regulate RNA splicing during positive selection of mouse T cells and that Foxa1 and Foxa2 have overlapping/compensatory roles. Conditional deletion of both Foxa1 and Foxa2 from DP thymocytes reduced positive selection and development of CD4SP, CD8SP and peripheral naïve CD4+ T cells. Foxa1 and Foxa2 regulated the expression of many genes encoding splicing factors and regulators, including Mbnl1, H1f0, Sf3b1, Hnrnpa1, Rnpc3, Prpf4b, Prpf40b and Snrpd3. Within the positively selecting CD69+DP cells, alternative RNA splicing was dysregulated in the double Foxa1/Foxa2 conditional knockout, leading to >850 differentially used exons. Many genes important for this stage of T-cell development (Ikzf1-3, Ptprc, Stat5a, Stat5b, Cd28, Tcf7) and splicing factors (Hnrnpab, Hnrnpa2b1, Hnrnpu, Hnrnpul1, Prpf8) showed multiple differentially used exons. Thus, Foxa1 and Foxa2 are required during positive selection to regulate alternative splicing of genes essential for T-cell development, and, by also regulating splicing of splicing factors, they exert widespread control of alternative splicing.

CK1α deficiency impairs mouse uterine adenogenesis by inducing epithelial cell apoptosis through GSK3ß pathway and inhibiting Foxa2 expression through p53 pathway†.

Uterine glands and their secretions are crucial for conceptus survival and implantation in rodents and humans. In mice, the development of uterine gland known as adenogenesis occurs after birth, whereas the adenogenesis in humans initiates from fetal life and completed at puberty. Uterine adenogenesis involves dynamic epithelial cell proliferation, differentiation, and apoptosis. However, it is largely unexplored about the mechanisms governing adenogenesis. CK1α plays important roles in regulating cell division, differentiation, and death, but it is unknown whether CK1α affects adenogenesis. In the current study, uterus-specific CK1α knockout female mice (Csnk1a1d/d) were infertile resulted from lack of uterine glands. Subsequent analysis revealed that CK1α deletion induced massive apoptosis in uterine epithelium by activating GSK3ß, which was confirmed by injections of GSK3ß inhibitor SB216763 to Csnk1a1d/d females, and the co-treatment of SB216763 and CK1 inhibitor d4476 on cultured epithelial cells. Another important finding was that our results revealed CK1α deficiency activated p53, which then blocked the expression of Foxa2, an important factor for glandular epithelium development and function. This was confirmed by that Foxa2 expression level was elevated in p53 inhibitor pifithrin-α injected Csnk1a1d/d mouse uterus and in vitro dual-luciferase reporter assay between p53 and Foxa2. Collectively, these studies reveal that CK1α is a novel factor regulating uterine adenogenesis by inhibiting epithelial cell apoptosis through GSK3ß pathway and regulating Foxa2 expression through p53 pathway. Uncovering the mechanisms of uterine adenogenesis is expected to improve pregnancy success in humans and other mammals.

FoxA2 represses ERß-mediated pyroptosis in endometriosis by transcriptionally inhibiting IGF2BP1.

BACKGROUND: Endometriosis is a severe disease which is associated with excessive activation of pyroptosis. Our present research aimed to investigate the function of Forkhead Box A2 (FoxA2) in regulating pyroptosis in endometriosis. METHODS: IL-1ß and IL-18 concentrations were assessed using ELISA. Cell pyroptosis was analyzed using flow cytometry. TUNEL staining was performed to determine human endometrial stromal cells (HESC) death. Moreover, ERß mRNA stability was assessed using RNA degradation assay. Finally, the binding relationships between FoxA2, IGF2BP1 and ERß were verified by dual-luciferase reporter system, ChIP, RIP and RNA pull-down assays. RESULTS: Our results revealed that IGF2BP1 and ERß were significantly upregulated in ectopic endometrium (EC) tissues of endometriosis patients compared to that in eutopic endometrium (EU) tissues as well as IL-18 and IL-1ß levels. Loss-of-function experiments subsequently demonstrated that either IGF2BP1 knockdown or ERß knockdown could repress HESC pyroptosis. In addition, IGF2BP1 upregulation promoted the pyroptosis in endometriosis by binding to ERß and promoting ERß mRNA stability. Our further research displayed that FoxA2 upregulation suppressed HESC pyroptosis by interacting with IGF2BP1 promoter. CONCLUSION: Our research proved that FoxA2 upregulation downregulated ERß by transcriptionally inhibiting IGF2BP1, thereby repressing pyroptosis in endometriosis.

The Pioneer Transcription Factor FoxA Maintains an Accessible Nucleosome Configuration at Enhancers for Tissue-Specific Gene Activation.

Nuclear DNA wraps around core histones to form nucleosomes, which restricts the binding of transcription factors to gene regulatory sequences. Pioneer transcription factors can bind DNA sites on nucleosomes and initiate gene regulatory events, often leading to the local opening of chromatin. However, the nucleosomal configuration of open chromatin and the basis for its regulation is unclear. We combined low and high levels of micrococcal nuclease (MNase) digestion along with core histone mapping to assess the nucleosomal configuration at enhancers and promoters in mouse liver. We find that MNase-accessible nucleosomes, bound by transcription factors, are retained more at liver-specific enhancers than at promoters and ubiquitous enhancers. The pioneer factor FoxA displaces linker histone H1, thereby keeping enhancer nucleosomes accessible in chromatin and allowing other liver-specific transcription factors to bind and stimulate transcription. Thus, nucleosomes are not exclusively repressive to gene regulation when they are retained with, and exposed by, pioneer factors.

FOXA2 suppresses gallbladder carcinoma cell migration, invasion, and epithelial-mesenchymal transition by targeting SERPINB5.

BACKGROUND: Gallbladder cancer (GBC), a highly malignant gastrointestinal tumor, lacks effective therapies. Foxhead box A2 (FOXA2) is a tumor suppressor that is poorly expressed in various human malignancies. This study aimed to ascertain FOXA2 expression in GBC and its relevance to tumor metastasis, and to elucidate its regulatory mechanism with epithelial-mesenchymal transition (EMT) as an entry point, in the hope of providing a potential therapeutic target for GBC. METHODS: FOXA2 expression in GBC tissues was first detected using immunohistochemistry (IHC), followed by correlation analysis with clinicopathological characteristics and survival prognosis. Subsequently, the effects of FOXA2 on GBC cell migration and invasion, as well as EMT induction, were evaluated by scratch, Transwell, RT-PCR, and Western blot assays, together with animal experimentation. Ultimately, mRNA sequencing was carried out to identify the key downstream target genes of FOXA2 in controlling the EMT process in GBC cells, and dual-luciferase reporter and chromatin immunoprecipitation assays were used to determine its regulatory mechanism. RESULTS: FOXA2 was underexpressed in GBC tissues and inversely correlated with tumor node metastasis stage, lymph node metastasis, and poor patient prognosis. FOXA2 exerts suppressive effects on EMT and metastasis of GBC in vivo and in vitro. FOXA2 can impede GBC cell migratory and invasive functions and EMT by positively mediating serine protein kinase inhibitor B5 (SERPINB5) expression. CONCLUSION: FOXA2 directly binds to the SERPINB5 promoter region to stimulate its transcription, thereby modulating the migration and invasion behaviors of GBC cells as well as the EMT process, which might be an effective therapeutic target against GBC.

PIAS1 upregulation confers protection against Cerulein-induced acute pancreatitis via FTO downregulation by enhancing sumoylation of Foxa2.

OBJECTIVE: This research discussed the specific mechanism by which PIAS1 affects acute pancreatitis (AP). METHODS: PIAS1, Foxa2, and FTO expression was assessed in Cerulein-induced AR42J cells and mice. Loss- and gain-of-function assays and Cerulein induction were conducted in AR42J cells and mice for analysis. The relationship among PIAS1, Foxa2, and FTO was tested. Cell experiments run in triplicate, and eight mice for each animal group. RESULTS: Cerulein-induced AP cells and mice had low PIAS1 and Foxa2 and high FTO. Cerulein induced pancreatic injury in mice and inflammation and oxidative stress in pancreatic tissues, which could be reversed by PIAS1 or Foxa2 upregulation or FTO downregulation. PIAS1 elevated SUMO modification of Foxa2 to repress FTO transcription. FTO upregulation neutralized the ameliorative effects of PIAS1 or Foxa2 upregulation on Cerulein-induced AR42J cell injury, inflammation, and oxidative stress. CONCLUSION: PIAS1 upregulation diminished FTO transcription by increasing Foxa2 SUMO modification, thereby ameliorating Cerulein-induced AP.

FOXA2 prevents hyperbilirubinaemia in acute liver failure by maintaining apical MRP2 expression.

OBJECTIVE: Multidrug resistance protein 2 (MRP2) is a bottleneck in bilirubin excretion. Its loss is sufficient to induce hyperbilirubinaemia, a prevailing characteristic of acute liver failure (ALF) that is closely associated with clinical outcome. This study scrutinises the transcriptional regulation of MRP2 under different pathophysiological conditions. DESIGN: Hepatic MRP2, farnesoid X receptor (FXR) and Forkhead box A2 (FOXA2) expression and clinicopathologic associations were examined by immunohistochemistry in 14 patients with cirrhosis and 22 patients with ALF. MRP2 regulatory mechanisms were investigated in primary hepatocytes, Fxr -/- mice and lipopolysaccharide (LPS)-treated mice. RESULTS: Physiologically, homeostatic MRP2 transcription is mediated by the nuclear receptor FXR/retinoid X receptor complex. Fxr-/- mice lack apical MRP2 expression and rapidly progress into hyperbilirubinaemia. In patients with ALF, hepatic FXR expression is undetectable, however, patients without infection maintain apical MRP2 expression and do not suffer from hyperbilirubinaemia. These patients express FOXA2 in hepatocytes. FOXA2 upregulates MRP2 transcription through binding to its promoter. Physiologically, nuclear FOXA2 translocation is inhibited by insulin. In ALF, high levels of glucagon and tumour necrosis factor α induce FOXA2 expression and nuclear translocation in hepatocytes. Impressively, ALF patients with sepsis express low levels of FOXA2, lose MRP2 expression and develop severe hyperbilirubinaemia. In this case, LPS inhibits FXR expression, induces FOXA2 nuclear exclusion and thus abrogates the compensatory MRP2 upregulation. In both Fxr -/- and LPS-treated mice, ectopic FOXA2 expression restored apical MRP2 expression and normalised serum bilirubin levels. CONCLUSION: FOXA2 replaces FXR to maintain MRP2 expression in ALF without sepsis. Ectopic FOXA2 expression to maintain MRP2 represents a potential strategy to prevent hyperbilirubinaemia in septic ALF.

FOXA2 controls the cis-regulatory networks of pancreatic cancer cells in a differentiation grade-specific manner.

Differentiation of normal and tumor cells is controlled by regulatory networks enforced by lineage-determining transcription factors (TFs). Among them, TFs such as FOXA1/2 bind naïve chromatin and induce its accessibility, thus establishing new gene regulatory networks. Pancreatic ductal adenocarcinoma (PDAC) is characterized by the coexistence of well- and poorly differentiated cells at all stages of disease. How the transcriptional networks determining such massive cellular heterogeneity are established remains to be determined. We found that FOXA2, a TF controlling pancreas specification, broadly contributed to the cis-regulatory networks of PDACs. Despite being expressed in both well- and poorly differentiated PDAC cells, FOXA2 displayed extensively different genomic distributions and controlled distinct gene expression programs. Grade-specific functions of FOXA2 depended on its partnership with TFs whose expression varied depending on the differentiation grade. These data suggest that FOXA2 contributes to the regulatory networks of heterogeneous PDAC cells via interactions with alternative partner TFs.

Upregulation of FOXA2 in uterine luminal epithelium and vaginal basal epithelium of epiERα-/- (Esr1fl/flWnt7aCre/+) mice†.

Forkhead box protein A2 (FOXA2) is a pioneer transcription factor important for epithelial budding and morphogenesis in different organs. It has been used as a specific marker for uterine glandular epithelial cells (GE). FOXA2 has close interactions with estrogen receptor α (ERα). ERα binding to Foxa2 gene in the uterus indicates its regulation of Foxa2. The intimate interactions between ERα and FOXA2 and their essential roles in early pregnancy led us to investigate the expression of FOXA2 in the female reproductive tract of pre-implantation epiERα-/- (Esr1fl/flWnt7aCre/+) mice, in which ERα is conditionally deleted in the epithelium of reproductive tract. In the oviduct, FOXA2 is detected in the ciliated epithelial cells of ampulla but absent in the isthmus of day 3.5 post-coitum (D3.5) Esr1fl/fl control and epiERα-/- mice. In the uterus, FOXA2 expression in the GE appears to be comparable between Esr1fl/fl and epiERα-/- mice. However, FOXA2 is upregulated in the D0.5 and D3.5 but not PND25-28 epiERα-/- uterine luminal epithelial cells (LE). In the vagina, FOXA2 expression is low in the basal layer and increases toward the superficial layer of the D3.5 Esr1fl/fl vaginal epithelium, but FOXA2 is detected in the basal, intermediate, and superficial layers, with the strongest FOXA2 expression in the intermediate layers of the D3.5 epiERα-/- vaginal epithelium. This study demonstrates that loss of ERα in LE and vaginal basal layer upregulates FOXA2 expression in these epithelial cells during early pregnancy. The mechanisms for epithelial cell-type specific regulation of FOXA2 by ERα remain to be elucidated.

Updating germ cell tumour pathogenesis - the ability of seminomas for FOXA2-driven extra-embryonic differentiation.

AIMS: Testicular germ cell tumours are the most common solid malignancies in young men of age 14-44 years. It is generally accepted that both seminomas and non-seminomas arise from a common precursor, the germ cell neoplasia in-situ, which itself is the result of a defective (primordial) germ cell development. The stem cell-like population of the non-seminomas, the embryonal carcinoma, is capable of the differentiation of all three germ layers (teratomas) and extra-embryonic tissues (yolk-sac tumours, choriocarcioma) into cells. In contrast, seminomas are thought to have a limited differentiation potential. Nevertheless, several studies have highlighted their ability to undergo reprogramming to an embryonal carcinoma or differentiation into other non-seminomatous entities. Here, we demonstrate that in approximately 5% of seminomas, the yolk-sac tumour driver gene FOXA2 is detectable at the protein level, indicative of an occult yolk-sac tumour subpopulation that putatively arose from seminoma cells, as the presence of other GCT entities could be excluded. The presence of these subpopulations might render the tumour more aggressive and argue for an adjustment of the therapeutic concept. We used our data to update the model of germ cell tumour pathogenesis, especially regarding the developmental potential of seminomas. Additionally, we suggest to include detection of FOXA2 into standard routine diagnosis of seminomas.

Genome-wide differential expression profiling of long non-coding RNAs in FOXA2 knockout iPSC-derived pancreatic cells.

BACKGROUND: Our recent studies have demonstrated the crucial involvement of FOXA2 in the development of human pancreas. Reduction of FOXA2 expression during the differentiation of induced pluripotent stem cells (iPSCs) into pancreatic islets has been found to reduce α-and ß-cell masses. However, the extent to which such changes are linked to alterations in the expression profile of long non-coding RNAs (lncRNAs) remains unraveled. METHODS: Here, we employed our recently established FOXA2-deficient iPSCs (FOXA2-/- iPSCs) to investigate changes in lncRNA profiles and their correlation with dysregulated mRNAs during the pancreatic progenitor (PP) and pancreatic islet stages. Furthermore, we constructed co-expression networks linking significantly downregulated lncRNAs with differentially expressed pancreatic mRNAs. RESULTS: Our results showed that 442 lncRNAs were downregulated, and 114 lncRNAs were upregulated in PPs lacking FOXA2 compared to controls. Similarly, 177 lncRNAs were downregulated, and 59 lncRNAs were upregulated in islet cells lacking FOXA2 compared to controls. At both stages, we observed a strong correlation between lncRNAs and several crucial pancreatic genes and TFs during pancreatic differentiation. Correlation analysis revealed 12 DE-lncRNAs that strongly correlated with key downregulated pancreatic genes in both PPs and islet cell stages. Selected DE-lncRNAs were validated using RT-qPCR. CONCLUSIONS: Our data indicate that the observed defects in pancreatic islet development due to the FOXA2 loss is associated with significant alterations in the expression profile of lncRNAs. Therefore, our findings provide novel insights into the role of lncRNA and mRNA networks in regulating pancreatic islet development, which warrants further investigations. Video Abstract.