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Arecoline, a major alkaloid within areca nut extract, is recognized as the primary active carcinogen promoting oral squamous cell carcinoma (OSCC) pathological development. Dysregulation of N6‐methyladenosine (m6A) methyltransferase components (e.g., Fat mass and obesity‐associated protein [FTO] and methyltransferase‐like 3 [METTL3]) are closely associated with multiple cancer progression, including oral cancer. However, the biological function role of FTO in arecoline‐induced oral cancer is largely unknown. We identified that FTO was significantly upregulated in OSCC tissues from patients with areca nut chewing habits and chronic arecoline‐treated OSCC cell lines. Depletion of FTO attenuated the arecoline‐promoted stemness, chemoresistance, and oncogenicity of OSCC cells. Finally, we revealed that FTO was negatively regulated by a transcription factor forkhead box protein A2 (FOXA2) in OSCC cells. This study, for the first time, demonstrated that FTO plays an oncogenic role in arecoline‐induced OSCC progression. Thus, developing new therapeutic agents targeting FTO may serve as a promising method to treatment OSCC patients, especially those with areca nut chewing habits., We identified that FTO was significantly upregulated in OSCC tissues from patients with areca nut chewing habits and chronic arecoline‐treated OSCC cell lines. Depletion of FTO attenuated the arecoline‐promoted stemness, chemoresistance, and oncogenicity of OSCC cells. Therapeutic agents targeting FTO may serve as a promising method to treat OSCC patients, especially those with areca nut chewing habits.

O-GlcNAcylation is a posttranslational modification that regulates numerous nuclear and cytoplasmic proteins and is emerging as a key regulator of various biological processes, such as transcription, signal transduction, and cell motility. Although increasing evidence has shown that elevated levels of global O-GlcNAcylation are linked to the metastasis in hepatocellular carcinoma (HCC) cells, the underlying mechanism is still ambiguous. In this study, we demonstrated that forkhead box protein A2 (FOXA2), an essential transcription factor for liver homeostasis and HCC developing, was O-GlcNAcylated by O-GlcNAc transferase (OGT) and regulates HCC cells migration and invasion. Opposite FOXA2 and OGT expression tendency were observed in HCC tissues, and lower FOXA2 levels predicted a poor prognosis in HCC patients. The reduction of FOXA2 in HCC cells was found to be inversely correlated with the cellular O-GlcNAcylation and cell migratory ability. Notably, we found that FOXA2 was modified by O-GlcNAcylation and that O-GlcNAcylation activated the ubiquitination degradation of FOXA2 in highly metastatic HCC cells. Although this modification did not affect FOXA2 nuclear localization capability, O-GlcNAcylation on FOXA2 was key for attenuating FOXA2-mediated transcription. O-GlcNAcylation decreased the transcription of FOXA2 downstream target gene E-cadherin and it ultimately promoted O-GlcNAcylation-mediated HCC cell migration and invasion. The results provide insights into the role of O-GlcNAcylation in regulating FOXA2 activity and suggest its important implications in HCC metastasis.

MRE11, the nuclease component of RAD50/MRE11/NBS1 DNA repair complex which is essential for repair of DNA double-strand-breaks in normal cells, has recently garnered attention as a critical factor in solid tumor development. Herein we report the crucial role of MRE11 in oral cancer progression in a nuclease-independent manner and delineate its key downstream effectors including CXCR4. MRE11 expression in oral cancer samples was positively associated with tumor size, cancer stage and lymph node metastasis, and was predictive of poorer patient survival and radiotherapy resistance. MRE11 promoted cell proliferation/migration/invasion in a nuclease-independent manner but enhanced radioresistance via a nuclease-dependent pathway. The nuclease independent promotion of EMT and metastasis was mediated by RUNX2, CXCR4, AKT, and FOXA2, while CXCR4 neutralizing antibody mitigated these effects in vitro and in vivo. Collectively, MRE11 may serve as a crucial prognostic factor and therapeutic target in oral cancer, displaying dual nuclease dependent and independent roles that permit separate targeting of tumor vulnerabilities in oral cancer treatment.

The oestrogens have been highly implicated in the fertility of female animals. It is widely known that the oestrogens are primarily synthetized by the ovarian granulosa cells (GCs), and the final and essential step of this process is to catalyse the oestrone to the more active oestradiol by the protein coded by hydroxysteroid 17-beta dehydrogenase 1 (HSD17B1) gene. However, the molecular mechanism regarding the transcription of HSD17B1 remains to be fully elucidated in ovarian GCs. In this study, the 5'-deletion, luciferase assay and chromatin immunoprecipitation (ChIP) were utilized to explore the molecular regulation of transcription of HSD17B1 with the porcine ovarian GCs as the cellular model. After the deletions with -2105 to -1754 bp, -1753 to -1429 bp, -1430 to -1081 bp and -1082 to -730 bp, the relative luciferase activity of HSD17B1 promoter did not change significantly, but the deletion of -731 to -332 bp significantly increased the relative luciferase activity of HSD17B1 promoter, and an insertion (GTTT) that might raise the transcription of HSD17B1 was identified at -401 bp of HSD17B1. These findings suggested the region from -731 to +38 bp was the core promoter of HSD17B1, and the region between -731 to -332 bp might be a silence element for HSD17B1. Furthermore, the forkhead box A2 (FoxA2) directly bound at -412 to -401 bp to negatively but p53 bound at -383 to -374 bp to positively regulate the transcription and translation of HSD17B1 in ovarian GCs. These findings will improve our understanding on HSD17B1-mediated oestrogens and provide useful information for further investigations into fertility of females.

Pancreatic cancer (PC) is difficult to defeat due to mechanism (s) driving metastasis and drug resistance. Cancer stemness is a major challenging phenomenon associated with PC metastasis and limiting therapy efficacy. In this study, we evaluated the pre-clinical and clinical significance of eradicating pancreatic cancer stem cells (PCSC) and its components using a pan-EGFR inhibitor afatinib in combination with gemcitabine. Afatinib in combination with gemcitabine significantly reduced KrasG12D/+; Pdx-1 Cre (KC) (P

Mature adipocyte‐derived dedifferentiated fat (DFAT) cells have been identified to possess similar multipotency to mesenchymal stem cells, but a method for converting DFAT cells into hepatocytes was previously unknown. Here, using comprehensive analysis of gene expression profiles, we have extracted three transcription factors, namely Foxa2, Hnf4a and Sall1 (FHS), that can convert DFAT cells into hepatocytes. Hepatogenic induction has converted FHS‐infected DFAT cells into an epithelial‐like morphological state and promoted the expression of hepatocyte‐specific features. Furthermore, the DFAT‐derived hepatocyte‐like (D‐Hep) cells catalyzed the detoxification of several compounds. These results indicate that the transduction of DFAT cells with three genes, which were extracted by comprehensive gene expression analysis, efficiently generated D‐Hep cells with detoxification abilities similar to those of primary hepatocytes. Thus, D‐Hep cells may be useful as a new cell source for surrogate hepatocytes and may be applied to drug discovery studies, such as hepatotoxicity screening and drug metabolism tests., In this study, we used a comprehensive analysis of the global expression profiles of hepatocytes, mature adipocyte‐derived dedifferentiated fat (DFAT) cells, hepatic stem cells and adipocytes to identify three transcription factors—Foxa2, Hnf4a and Sall1—that can specifically induce hepatocyte differentiation in DFAT cells.

Neuroendocrine prostate cancer (NEPC) is a more aggressive subtype of castration‐resistant prostate cancer (CRPC). Although it is well established that PHF8 can enhance prostate cancer cell proliferation, whether PHF8 is involved in prostate cancer initiation and progression is relatively unclear. By comparing the transgenic adenocarcinoma of the mouse prostate (TRAMP) mice with or without Phf8 knockout, we systemically examined the role of PHF8 in prostate cancer development. We found that PHF8 plays a minimum role in initiation and progression of adenocarcinoma. However, PHF8 is essential for NEPC because not only is PHF8 highly expressed in NEPC but also animals without Phf8 failed to develop NEPC. Mechanistically, PHF8 transcriptionally upregulates FOXA2 by demethylating and removing the repressive histone markers on the promoter region of the FOXA2 gene, and the upregulated FOXA2 subsequently regulates the expression of genes involved in NEPC development. Since both PHF8 and FOXA2 are highly expressed in NEPC tissues from patients or patient‐derived xenografts, the levels of PHF8 and FOXA2 can either individually or in combination serve as NEPC biomarkers and targeting either PHF8 or FOXA2 could be potential therapeutic strategies for NEPC treatment. © 2020 The Authors. The Journal of Pathology published by John Wiley & Sons, Ltd. on behalf of The Pathological Society of Great Britain and Ireland.

Glands of the uterus are essential for pregnancy establishment. Forkhead box A2 (FOXA2) is expressed specifically in the glands of the uterus and a critical regulator of glandular epithelium (GE) differentiation, development, and function. Mice with a conditional deletion of FOXA2 in the adult uterus, created using the lactotransferrin iCre (Ltf-iCre) model, have a morphologically normal uterus with glands, but lack FOXA2-dependent GE-expressed genes, such as leukemia inhibitory factor (LIF). Adult FOXA2 conditional knockout (cKO; Ltf(iCre/+)Foxa2(f/f)) mice are infertile due to defective embryo implantation arising from a lack of LIF, a critical implantation factor of uterine gland origin. However, intraperitoneal injections of LIF can initiate embryo implantation in the uterus of adult FOXA2 cKO mice with pregnancies maintained to term. Here, we tested the hypothesis that FOXA2-regulated genes in the uterine glands impact development of the decidua, placenta, and fetus. On gestational day 8.5, the antimesometrial and mesometrial decidua transcriptome was noticeably altered in LIF-replaced FOXA2 cKO mice. Viable fetuses were reduced in FOXA2 cKO mice on gestational days 12.5 and 17.5. Sex-dependent differences in fetal weight, placenta histoarchitecture, and the placenta and metrial gland transcriptome were observed between control and FOXA2 cKO mice. The transcriptome of the placenta with a female fetus was considerably more altered than the placenta with a male fetus in FOXA2 cKO dams. These studies reveal previously unrecognized sexually dimorphic effects of FOXA2 and uterine glands on fetoplacental development with potential impacts on offspring health into adulthood.

Lung cancer leads to the highest mortality among all cancer types in the world, and non–small‐cell lung cancer (NSCLC) occupies over 80% of the lung cancer cases. Numerous studies have demonstrated that long non‐coding RNA (lncRNA) is involved in various human diseases including cancer. LncRNA FTX was firstly identified in Xist gene locus and was dysregulated in many human cancers. However, the function of FTX in NSCLC is still unclear. Here, we report that long non‐coding RNA FTX expression level is down‐regulated in NSCLC clinical tissue samples and cell lines. Ectopic expression of FTX inhibits proliferation and metastasis of lung cancer cells in vitro and in vivo. Furthermore, we find that FTX overexpression activates the expression of transcription factor FOXA2, an important regulator in lung cancer progression, and we reveal a novel FTX/miR‐200a‐3p/FOXA2 competing endogenous RNA regulatory axis in lung cancer cells. Our results provide new insights and directions for exploring the function of FTX in lung cancer progression.

Objectives FOXA2 gene methylation links to the progression of cancers, but has not been documented in oral cancer. Herein, we explore the role of FOXA2 in the migration of oral cancer cells. Material and methods Methylation-specific PCR was applied for gene methylation. Wound healing and transwell experiments were tested for cell migration. FOXA2 expression in oral cancer tissues was addressed by immunohistochemistry, followed by statistical analysis of its association with clinical manifestations and patient survival. Results FOXA2 bound to the promoter of CDH1 and enhanced the expression of its gene product E-cadherin, and decreased the cancer cell migration activity. High FOXA2 expression in oral cancer tissues was associated with high E-cadherin expression, decreased lymph node metastasis, and increased patient survival. Conclusion FOXA2-E-cadherin link is involved in regulation of oral cancer cell metastasis and provides a new insight for the tumor suppressor activity of FOXA2 in oral cancer.

TOB1 participates in various kinds of cancers. However, its role in pancreatic cancer has rarely been reported. In this study, we explored the expression and mechanisms of TOB1 in regulating the malignant phenotype of pancreatic cancer cells. TOB1 expression was determined by data mining and immunohistochemistry (IHC), and its localization was observed by immunofluorescence. CCK‐8 cell proliferation, colony formation, flow cytometric, transwell migration, and Western blot (WB) assays were used to examine how it impacts the malignant phenotype of pancreatic cancer. Furthermore, Foxa2 binding to TOB1 was tested by dual‐luciferase reporter assays, and RNA‐Seq was performed to identify signaling pathways. We found TOB1 was downregulated in pancreatic cancer tissues and was mainly located in the cytoplasm. TOB1 overexpression reduced the proliferation of K‐Ras wild‐type pancreatic cancer cells but made no difference to cell migration and invasion. Foxa2 overexpression significantly enhanced TOB1 promoter activity. Moreover, overexpressing TOB1 substantially enriched the calcium pathway in K‐Ras wild‐type pancreatic cancer cells. In conclusion, TOB1 may suppress the proliferation of K‐Ras wild‐type pancreatic cancer cells by regulating calcium pathway genes., Foxa2, as an upstream transcription factor, may regulate TOB1 expression. TOB1 overexpression decreased expression of Cyclin D1, CDK2 and CDK4, and caused proliferation inhibition of K-Ras wild‐type pancreatic cancer cells, at least in part through the calcium pathway.

Metastasis, the development of secondary malignant growths at a distance from a primary tumor, is the cause of death for 90% of cancer patients, but little is known about how metastatic cancer cells adapt to and colonize new tissue environments. Here, using clinical samples, patient-derived xenograft (PDX) samples, PDX cells, and primary/metastatic cell lines, we discovered that liver metastatic colorectal cancer (CRC) cells lose their colon-specific gene transcription program yet gain a liver-specific gene transcription program. We showed that this transcription reprogramming is driven by a reshaped epigenetic landscape of both typical enhancers and super-enhancers. Further, we identified that the liver-specific transcription factors FOXA2 and HNF1A can bind to the gained enhancers and activate the liver-specific gene transcription, thereby driving CRC liver metastasis. Importantly, similar transcription reprogramming can be observed in multiple cancer types. Our data suggest that reprogrammed tissue-specific transcription promotes metastasis and should be targeted therapeutically.

Uterine glands are essential for the establishment of pregnancy and have critical roles in endometrial receptivity to blastocyst implantation, stromal cell decidualization, and placentation. Uterine gland dysfunction is considered a major contributing factor to pregnancy loss, however our understanding of how glands impact embryo survival and stromal cell decidualization is incomplete. Forkhead box A2 (FOXA2) is expressed only in the glandular epithelium and regulates its development and function. Mice with a conditional deletion of FOXA2 in the uterus are infertile due to defective embryo implantation arising from a lack of leukemia inhibitory factor (LIF), a critical factor of uterine gland origin. Here, a glandless FOXA2-deficient mouse model, coupled with LIF repletion to rescue the implantation defect, was used to investigate the roles of uterine glands in embryo survival and decidualization. Studies found that embryo survival and decidualization were compromised in glandless FOXA2-deficient mice on gestational day 6.5, resulting in abrupt pregnancy loss by day 7.5. These findings strongly support the hypothesis that uterine glands secrete factors other than LIF that impact embryo survival and stromal cell decidualization for pregnancy success.

The identification of the molecular mechanisms controlling early cell fate decisions in mammals is of paramount importance as the ability to determine specific lineage differentiation represents a significant opportunity for new therapies. Pancreatic Progenitor Cells (PPCs) constitute a regenerative reserve essential for the maintenance and regeneration of the pancreas. Besides, PPCs represent an excellent model for understanding pathological pancreatic cellular remodeling. Given the lack of valid markers of early endoderm, the identification of new ones is of fundamental importance. Both products of the Ink4a/Arf locus, in addition to being critical cell-cycle regulators, appear to be involved in several disease pathologies. Moreover, the locus’ expression is epigenetically regulated in ES reprogramming processes, thus constituting the ideal candidates to modulate PPCs homeostasis. In this study, starting from mouse embryonic stem cells (mESCs), we analyzed the early stages of pancreatic commitment. By inducing mESCs commitment to the pancreatic lineage, we observed that both products of the Cdkn2a locus, Ink4a and Arf, mark a naïve pancreatic cellular state that resembled PPC-like specification. Treatment with epi-drugs suggests a role for chromatin remodeling in the CDKN2a (Cycline Dependent Kinase Inhibitor 2A) locus regulation in line with previous observations in other cellular systems. Our data considerably improve the comprehension of pancreatic cellular ontogeny, which could be critical for implementing pluripotent stem cells programming and reprogramming toward pancreatic lineage commitment.

AimDespite the enormous efforts to understand Congenital hyperinsulinism (CHI), up to 50% of the patients are genetically unexplained. We aimed to functionally characterize a novel candidate gene in CHI.PatientA 4-month-old boy presented severe hyperinsulinemic hypoglycemia. A routine CHI genetic panel was negative.MethodsA trio-based whole-exome sequencing (WES) was performed. Gene knockout in the RIN-m cell line was established by CRISPR/Cas9. Gene expression was performed using real-time PCR.ResultsHyperinsulinemic hypoglycemia with diffuse beta-cell involvement was demonstrated in the patient, who was diazoxide-responsive. By WES, compound heterozygous variants were identified in the adenylyl cyclase 7, ADCY7 gene p.(Asp439Glu) and p.(Gly1045Arg). ADCY7 is calcium-sensitive, expressed in beta-cells and converts ATP to cAMP. The variants located in the cytoplasmic domains C1 and C2 in a highly conserved and functional amino acid region. RIN-m(-/-Adcy7) cells showed a significant increase in insulin secretion reaching 54% at low, and 49% at high glucose concentrations, compared to wild-type. In genetic expression analysis Adcy7 loss of function led to a 34.1-fold to 362.8-fold increase in mRNA levels of the insulin regulator genes Ins1 and Ins2 (p ≤ 0.0002), as well as increased glucose uptake and sensing indicated by higher mRNA levels of Scl2a2 and Gck via upregulation of Pdx1, and Foxa2 leading to the activation of the glucose stimulated-insulin secretion (GSIS) pathway.ConclusionThis study identified a novel candidate gene, ADCY7, to cause CHI via activation of the GSIS pathway.

Background: Intrahepatic cholangiocarcinoma (ICC) is characterized by a highly aggressive nature and a dismal outcome. FOXA2 is an archetypal transcription factor involved in cholangiocyte proliferation. Results: FOXA2 expression was negatively correlated with tumor stage (p = 0.024). Univariate and multivariate analyses showed that low FoxA2 expression was associated with tumor relapse and survival. At 20 weeks after TAA administration, FoxA2-/- mice displayed significant manifestations of neoplasia, while WT mice did not. RNA sequencing analysis showed that the expression of genes in the MAPK signaling pathway was significantly higher in FoxA2-/- mice. IHC and Western blot results showed that p-ERK1/2, CREB1 and RAS were highly expressed in FoxA2-/- mice. Furthermore, using in vitro experiments with siRNA, we found that low expression of FoxA2 could exacerbate the metastatic potential of ICC. The expression of p-ERK1/2 and RAS, which are key mediators of the MAPK signaling pathway, was significantly increased. Conclusion: Low FOXA2 expression negatively affected the prognosis of patients with ICC. Loss of FoxA2 expression could promote intrahepatic bile duct neoplasia partly via activation of the MAPK signaling pathway. Materials and methods: In all, the data of 85 patients with ICC were retrospectively collected and analyzed. TAA was used to induce ICC in FoxA2-/- mice and WT mice. RNA-sequencing analysis was used to identify the expression of different genes.

Pioneer transcription factors (PTF) can recognize their binding sites on nucleosomal DNA and trigger chromatin opening for recruitment of other non-pioneer transcription factors. However, critical properties of PTFs are still poorly understood, such as how these transcription factors selectively recognize cell type-specific binding sites and under which conditions can they can initiate chromatin remodelling. Here we show that early endoderm binding sites of the paradigm PTF Foxa2 are epigenetically primed by low levels of active chromatin modifications in embryonic stem cells (ESC). Priming of these binding sites is supported by preferential recruitment of Foxa2 to endoderm binding sites compared to lineage-inappropriate binding sites, when ectopically expressed in ESCs. We further show that binding of Foxa2 is required for chromatin opening during endoderm differentiation. However, increased chromatin accessibility was only detected on binding sites which are synergistically bound with other endoderm transcription factors. Thus, our data suggest that binding site selection of PTFs is directed by the chromatin environment and that chromatin opening requires collaboration of PTFs with additional transcription factors.

SUMMARY Transcriptional regulatory mechanisms of lineage priming in embryonic development are largely uncharacterized because of the difficulty of isolating transient progenitor populations. Directed differentiation of human pluripotent stem cells (hPSCs) combined with gene editing provides a powerful system to define precise temporal gene requirements for progressive chromatin changes during cell fate transitions. Here, we map the dynamic chromatin landscape associated with sequential stages of pancreatic differentiation from hPSCs. Our analysis of chromatin accessibility dynamics led us to uncover a requirement for FOXA2, known as a pioneer factor, in human pancreas specification not previously shown from mouse knockout studies. FOXA2 knockout hPSCs formed reduced numbers of pancreatic progenitors accompanied by impaired recruitment of GATA6 to pancreatic enhancers. Furthermore, FOXA2 is required for proper chromatin remodeling and H3K4me1 deposition during enhancer priming. This work highlights the power of combining hPSC differentiation, genome editing, and computational genomics for discovering transcriptional mechanisms during development., In Brief Lee et al. use ATAC-seq to identify key transcriptional factors involved in human pancreatic differentiation. FOXA2 knockout human pluripotent stem cells showed impaired differentiation to pancreatic progenitors, a phenotype not observed in Foxa2 conditional knockout mice. Furthermore, FOXA2 is required for proper chromatin remodeling and H3K4me1 deposition during enhancer priming., Graphical Abstract

Background/Aims: For the clinical application of stem cell therapy, functional enhancement is needed to increase the survival rate and the engraftment rate. The purpose of this study was to investigate functional enhancement of the paracrine effect using stem cells and hepatocyte-like cells and to minimize stem cell homing by using a scaffold system in a liver disease model. Methods: A microporator was used to overexpress Foxa2 in adipose tissue-derived stem cells (ADSCs), which were cultured in a poly(lactic-co-glycolic acid) (PLGA) scaffold. Later, the ADSCs were cultured in hepatic differentiation medium for 2 weeks by a 3-step method. For in vivo experiments, Foxa2-overexpressing ADSCs were loaded in the scaffold, cultured in hepatic differentiation medium and later were implanted in the dorsa of nude mice subjected to acute liver injury (thioacetamide intraperitoneal injection). Results: Foxa2-overexpressing ADSCs showed greater increases in hepatocyte-specific gene markers (alpha fetoprotein [AFP], cytokeratin 18 [CK18], and albumin), cytoplasmic glycogen storage, and cytochrome P450 expression than cells that underwent the conventional differentiation method. In vivo experiments using the nude mouse model showed that 2 weeks after scaffold implantation, the mRNA expression of AFP, CK18, dipeptidyl peptidase 4 (CD26), and connexin 32 (CX32) was higher in the Foxa2-overexpressing ADSCs group than in the ADSCs group. The Foxa2-overexpressing ADSCs scaffold treatment group showed attenuated liver injury without stem cell homing in the thioacetamideinduced acute liver injury model. Conclusions: Foxa2-overexpressing ADSCs applied in a scaffold system enhanced hepatocyte-like differentiation and attenuated acute liver damage in an acute liver injury model without homing effects. (Gut Liver 2019;13:450-460)

The pioneer forkhead box (FOX)A2 transcription factor is specifically expressed in the glands of the uterus, which are central to endometrial function and fertility. In mice, FOXA2 is a critical regulator of uterine gland development in the neonate and gland function in the adult. An integrative approach was used here to define the FOXA2 cistrome in the human endometrium. Genome-wide mapping of FOXA2 binding intervals by chromatin immunoprecipitation sequencing was performed using proliferative (P)- and midsecretory (MS)-phase endometrium and integrated with the transcriptome determined by RNA sequencing. Distinctive FOXA2 binding intervals, enriched for different transcription factor binding site motifs, were detected in the P and MS endometrium. Pathway analysis revealed different biologic processes regulated by genes with FOXA2 binding intervals in the P and MS endometrium. Thus, FOXA2 is postulated to regulate gene expression in concert with other transcription factors and impact uterine gland development and function in a cycle phase–dependent manner. Analyses also identified potential FOXA2-regulated genes that influence uterine receptivity, blastocyst implantation, and stromal cell decidualization, which are key events in pregnancy establishment.—Kelleher, A. M., Behura, S. K., Burns, G. W., Young, S. L., DeMayo, F. J., Spencer, T. E. Integrative analysis of the forkhead box A2 (FOXA2) cistrome for the human endometrium.

Transplantation of dopaminergic precursors (DPs) is a promising therapeutic strategy of Parkinson’s disease (PD). However, limited cell source for dopaminergic precursors has become a major obstacle for transplantation therapy. Our group demonstrated previously that mouse fibroblasts can be reprogrammed into induced dopaminergic precursors (iDPs) with high differentiation efficiency. In the current study, we hypothesized that a similar strategy can be applied to generate human iDPs for future cell therapy of PD. We overexpressed transcription factors Brn2, Sox2, and Foxa2 in human fibroblasts and observed formation of neurospheres. Subsequent characterization of the precursor colonies confirmed the generation of human induced dopaminergic precursors (hiDPs). These hiDPs were capable of self-renewal, proliferation, and differentiation. The hiDPs demonstrated high immunoreactivity for neural progenitor markers and high levels of gene expression for ventral mesencephalon-related neural progenitor markers such as Lmx1a, NIKX6.1, Corin, Otx2 and Mash1. Furthermore, the hiDPs could be differentiated into dopaminergic neurons with ˜80% efficiency, which significantly increased major functionally relevant proteins such as TH, DAT, AADC, Lmx1B, and VMAT2 compared to hiDPs. Additionally, hiDPs are more dopaminergic progenitor-restricted compare to those hiDP-like cells reprogrammed only by Brn2 and Sox2. Together, these results suggest that hiDPs with high differentiation efficiency can be generated by direct lineage reprogramming of fibroblasts with transcription factors Brn2, Sox2, and Foxa2. These hiDPs may serve as a safe and effective cell source for transplantation treatment of PD.

SUMMARY Studies in vertebrates have outlined conserved molecular control of definitive endoderm (END) development. However, recent work also shows that key molecular aspects of human END regulation differ even from rodents. Differentiation of human embryonic stem cells (ESCs) to END offers a tractable system to study the molecular basis of normal and defective human-specific END development. Here, we interrogated dynamics in chromatin accessibility during differentiation of ESCs to END, predicting DNA-binding proteins that may drive this cell fate transition. We then combined single-cell RNA-seq with parallel CRISPR perturbations to comprehensively define the loss-of-function phenotype of those factors in END development. Following a few candidates, we revealed distinct impairments in the differentiation trajectories for mediators of TGFβ signaling and expose a role for the FOXA2 transcription factor in priming human END competence for human fore-gut and hepatic END specification. Together, this single-cell functional genomics study provides high-resolution insight on human END development., Graphical Abstract, In Brief Genga et al. utilize a single-cell RNA-sequencing-based CRISPR interference approach to screen transcription factors predicted to have a role in human definitive endoderm differentiation. The perturbation screen identifies an important role of TGFβ signaling-related factors. Follow-up of FOXA2 reveals genome-wide molecular changes and altered differentiation competency in endoderm.

We have previously shown that cooperative, interdependent binding by the pioneer factors FoxO1 and FoxA1/2 is required for recruitment of RNA polymerase II and H3K27 acetylation to the promoters of insulin-regulated genes. However, the underlying mechanisms are unknown. In this study, we demonstrate that, in HepG2 cells, FoxO1 and FoxA2 form a complex on DNA that is disrupted by insulin treatment. Insulin-mediated phosphorylation of FoxO1 and FoxA2 does not impair their cooperative binding to mononucleosome particles assembled from the IGFBP1 promoter, indicating that direct disruption of complex formation by phosphorylation is not responsible for the loss of interdependent FoxO1:FoxA1/2 binding following insulin treatment. Since FoxO1 and FoxA1/2 binding is required for the establishment and maintenance of transcriptionally active chromatin at insulin-regulated genes, we hypothesized that cooperative FoxO1 and FoxA1/2 binding dictates the chromatin remodeling events required for the initial activation of these genes. In support of this idea, we demonstrate that FoxO1 and FoxA2 cooperatively open linker histone compacted chromatin templates containing the IGFBP1 promoter. Taken together, these results provide a mechanism for how interdependent FoxO1:FoxA1/2 binding is negatively impacted by insulin and provide a developmental context for cooperative gene activation by these factors.

A large proportion of the transcriptome is sex biased in a wide range of taxa. Sexually dimorphic genes expression is highly tissue-dependent. Although gastric cancer exhibits sex bias to some extent, sexually dimorphic gene expression in the stomach is yet to be fully understood. The aim of the present study was to compare the expression levels of 12 genes in the gastric tissue between age-matched healthy men and women of different age groups. A total of 70 human antrum gastric tissue samples were obtained by endoscopy. The difference in expression levels of the 12 intended genes between two genders was investigated using quantitative Real-Time PCR, following total RNA extraction. The results indicated that the expression levels of both the GKN2 (7.2-fold, p

The placenta plays a pivotal role in the development of the fetal brain and also influences maternal brain function, but our understanding of communication between the placenta and brain remains limited. Using a gene expression and network analysis approach, we provide evidence that the placenta transcriptome is tightly interconnected with the maternal brain and fetal brain in d 15 pregnant C57BL/6J mice. Activation of serotonergic synapse signaling and inhibition of neurotrophin signaling were identified as potential mediators of crosstalk between the placenta and maternal brain and fetal brain, respectively. Genes encoding specific receptors and ligands were predicted to affect functional interactions between the placenta and brain. Paralogous genes, such as sex comb on midleg homolog 1/scm-like with 4 mbt domains 2 and polycomb group ring finger (Pcgf) 2/ Pcgf5, displayed antagonistic regulation between the placenta and brain. Additionally, conditional ablation of forkhead box a2 ( Foxa2) in the glands of the uterus altered the transcriptome of the d 15 placenta, which provides novel evidence of crosstalk between the uterine glands and placenta. Furthermore, expression of cathepsin 6 and monocyte to macrophage differentiation associated 2 was significantly different in the fetal brain of Foxa2 conditional knockout mice compared with control mice. These findings provide a better understanding of the intricacies of uterus-placenta-brain interactions during pregnancy and provide a foundation and model system for their exploration.-Behura, S. K., Kelleher, A. M., Spencer, T. E. Evidence for functional interactions between the placenta and brain in pregnant mice.

Embryonic taste bud primordia are specified as taste placodes on the tongue surface and differentiate into the first taste receptor cells (TRCs) at birth. Throughout adult life, TRCs are continually regenerated from epithelial progenitors. Sonic hedgehog (SHH) signaling regulates TRC development and renewal, repressing taste fate embryonically, but promoting TRC differentiation in adults. Here, using mouse models, we show TRC renewal initiates at birth and coincides with onset of SHHs pro-taste function. Using transcriptional profiling to explore molecular regulators of renewal, we identified Foxa1 and Foxa2 as potential SHH target genes in lingual progenitors at birth and show that SHH overexpression in vivo alters FoxA1 and FoxA2 expression relevant to taste buds. We further bioinformatically identify genes relevant to cell adhesion and cell locomotion likely regulated by FOXA1;FOXA2 and show that expression of these candidates is also altered by forced SHH expression. We present a new model where SHH promotes TRC differentiation by regulating changes in epithelial cell adhesion and migration.

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., Summary: The pioneer transcription factors Foxa1 and Foxa2 are required for positive selection during T-cell development in the thymus for regulation of RNA splicing.

BACKGROUND The aim of the present work was to evaluate FOXA2 expression in ovarian cancer and to use integrated bioinformatics analysis to correlate it with patient prognosis. MATERIAL AND METHODS FOXA2 expression was evaluated in multiple cancers in The Cancer Genome Atlas database. A protein-protein interaction (PPI) network relevant to FOXA2 was constructed using the Search Tool for Retrieval of Interacting Genes/Proteins (STRIN). Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathway enrichment analyses were performed of FOXA2 and relevant genes. Correlations between overall survival (OS), disease-free survival, and FOXA2 expression were evaluated. An immunohistochemical assay (IHC) was used to test for FOXA2 protein expression in 79 ovarian cancer specimens. RESULTS FOXA2 mRNA was upregulated in colorectal, stomach, liver, and endometrial cancers. In the PPI network, 21 protein nodes and 533 edges were constructed with a local clustering coefficient of 0.698, which indicated significant PPI enrichment (P

(1) Background: Transcription factors (TFs) are main regulators of eukaryotic gene expression. The cooperative binding to genomic DNA of at least two TFs is the widespread mechanism of transcription regulation. Cooperating TFs can be revealed through the analysis of co-occurrence of their motifs. (2) Methods: We applied the motifs co-occurrence tool (MCOT) that predicted pairs of spaced or overlapped motifs (composite elements, CEs) for a single ChIP-seq dataset. We improved MCOT capability for the prediction of asymmetric CEs with one of the participating motifs possessing higher conservation than another does. (3) Results: Analysis of 119 ChIP-seq datasets for 45 human TFs revealed that almost for all families of TFs the co-occurrence with an overlap between motifs of target TFs and more conserved partner motifs was significantly higher than that for less conserved partner motifs. The asymmetry toward partner TFs was the most clear for partner motifs of TFs from the ETS (E26 Transformation Specific) family. (4) Conclusion: Co-occurrence with an overlap of less conserved motif of a target TF and more conserved motifs of partner TFs explained a substantial portion of ChIP-seq data lacking conserved motifs of target TFs. Among other TF families, conservative motifs of TFs from ETS family were the most prone to mediate interaction of target TFs with its weak motifs in ChIP-seq.

The differentiated functions of the human airway epithelium are coordinated by a complex network of transcription factors. These include the pioneer factors Forkhead box A1 and A2 (FOXA1 and FOXA2), which are well studied in several tissues, but their role in airway epithelial cells is poorly characterized. Here, we define the cistrome of FOXA1 and FOXA2 in primary human bronchial epithelial (HBE) cells by chromatin immunoprecipitation with deep-sequencing (ChIP-seq). Next, siRNA-mediated depletion of each factor is used to investigate their transcriptome by RNA-seq. We found that, as predicted from their DNA-binding motifs, genome-wide occupancy of the two factors showed substantial overlap; however, their global impact on gene expression differed. FOXA1 is an abundant transcript in HBE cells, while FOXA2 is expressed at low levels, and both these factors likely exhibit autoregulation and cross-regulation. FOXA1 regulated loci are involved in cell adhesion and the maintenance of epithelial cell identity, particularly through repression of genes associated with epithelial to mesenchymal transition (EMT). FOXA1 also directly targets other transcription factors with a known role in the airway epithelium such as SAM-pointed domain-containing Ets-like factor (SPDEF). The intersection of the cistrome and transcriptome for FOXA1 revealed enrichment of genes involved in epithelial development and tissue morphogenesis. Moreover, depletion of FOXA1 was shown to reduce the transepithelial resistance of HBE cells, confirming the role of this factor in maintaining epithelial barrier integrity.

Pulmonary mycoses are difficult to treat and detrimental to patients. Fungal infections modulate the lung immune response, induce goblet cell hyperplasia and metaplasia, and mucus hypersecretion in the airways. Excessive mucus clogs small airways and reduces pulmonary function by decreasing oxygen exchange, leading to respiratory distress. The forkhead box protein A2 (FOXA2) is a transcription factor that regulates mucus homeostasis in the airways. However, little is known whether pulmonary mycosis modulates FOXA2 function. Herein, we investigated whether Blastomyces dermatitidis and Histoplasma capsulatum-infected canine and feline lungs and airway epithelial cells could serve as higher animal models to examine the relationships between fungal pneumonia and FOXA2-regulated airway mucus homeostasis. The results indicate that fungal infection down-regulated FOXA2 expression in airway epithelial cells, with concomitant overexpression of mucin 5AC (MUC5AC) and mucin 5B (MUC5B) mucins. Mechanistic studies reveal that B. dermatitidis infection, as well as β-glucan exposure, activated the Dectin-1-SYK-epidermal growth factor receptor-AKT/extracellular signal-regulated kinase 1/2 signaling pathway that inhibits the expression of FOXA2, resulting in overexpression of MUC5AC and MUC5B in canine airway cells. Further understanding of the role of FOXA2 in mucus hypersecretion may lead to novel therapeutics against excessive mucus in both human and veterinary patients with pulmonary mycosis.

Forkhead box (FOX) proteins are transcriptional factors that regulate various cellular processes. This minireview provides an overview of FOXA2 functions, with a special emphasis on the regulation airway mucus homeostasis in both healthy and diseased lungs. FOXA2 plays crucial roles during lung morphogenesis, surfactant protein production, goblet cell differentiation and mucin expression. In healthy airways, FOXA2 exerts a tight control over goblet cell development and mucin biosynthesis. However, in diseased airways, microbial infections and proinflammatory responses deplete FOXA2 expression, resulting in uncontrolled goblet cell hyperplasia and metaplasia, mucus hypersecretion, and impaired mucociliary clearance of pathogens. Furthermore, accumulated mucus clogs the airways and creates a niche environment for persistent microbial colonization and infection, leading to acute exacerbation and deterioration of pulmonary function in patients with chronic lung diseases. Various studies have shown that FOXA2 inhibition is mediated through induction of antagonistic EGFR and IL-13R-STAT6 signaling pathways as well as through posttranslational modifications induced by microbial infections. An improved understanding of how bacterial pathogens inactivate FOXA2 may pave the way for developing therapeutics that preserve the protein's function, which in turn, will improve the mucus status and mucociliary clearance of pathogens, reduce microbial-mediated acute exacerbation and restore lung function in patients with chronic lung diseases.

Transcription growth factor β (TGF-β) signaling-triggered epithelial-to-mesenchymal transition (EMT) process is associated with tumor stemness, metastasis, and chemotherapy resistance. However, the epigenomic basis for TGF-β-induced EMT remains largely unknown. Here we reveal that HDAC1-mediated global histone deacetylation and the gain of specific histone H3 lysine 27 acetylation (H3K27ac)-marked enhancers are essential for the TGF-β-induced EMT process. Enhancers gained upon TGF-β treatment are linked to gene activation of EMT markers and cancer metastasis. Notably, dynamic enhancer gain or loss mainly occurs within pre-existing topologically associated domains (TADs) in epithelial cells, with minimal three-dimensional (3D) genome architecture reorganization. Through motif enrichment analysis of enhancers that are lost or gained upon TGF-β stimulation, we identify FOXA2 as a key factor to activate epithelial-specific enhancer activity, and we also find that TEAD4 forms a complex with SMAD2/3 to mediate TGF-β signaling-triggered mesenchymal enhancer reprogramming. Together, our results implicate that key transcription-factor (TF)-mediated enhancer reprogramming modulates the developmental transition in TGF-β signaling-associated cancer metastasis.

Summary Human glial progenitor cells (hGPCs) are promising cellular substrates to explore for the in situ production of new neurons for brain repair. Proof of concept for direct neuronal reprogramming of glial progenitors has been obtained in mouse models in vivo, but conversion using human cells has not yet been demonstrated. Such studies have been difficult to perform since hGPCs are born late during human fetal development, with limited accessibility for in vitro culture. In this study, we show proof of concept of hGPC conversion using fetal cells and also establish a renewable and reproducible stem cell-based hGPC system for direct neural conversion in vitro. Using this system, we have identified optimal combinations of fate determinants for the efficient dopaminergic (DA) conversion of hGPCs, thereby yielding a therapeutically relevant cell type that selectively degenerates in Parkinson's disease. The induced DA neurons show a progressive, subtype-specific phenotypic maturation and acquire functional electrophysiological properties indicative of DA phenotype., Graphical Abstract, Highlights • Human glial progenitors (hGPCs) can be directly converted into functional neurons • Specific transcription factor combinations result in dopaminergic conversion • Reprogrammed neurons show subtype-specific and functional maturation over time, In this article, Parmar and colleagues show proof of concept that human glial progenitors (hGPCs) sourced from fetal brains can be converted into functional neurons. To allow for large-scale studies, a stem cell-based model system that mimics the fetal cell conversion was established and used to identify optimal combinations of fate determinants for the efficient dopaminergic conversion of hGPCs.

microRNA (miR)-141-3p has context-dependent effects on tumor progression. In this study, we attempted to explore the expression and function of miR-141-3p in cervical cancer. We found that miR-141-3p expression was significantly increased in cervical cancer specimens relative to normal cervical tissues. Moreover, miR-141-3p levels were associated with tumor size and lymph node metastasis status. Ectopic expression of miR-141-3p significantly increased cervical cancer cell proliferation, colony formation, invasion, and epithelial to mesenchymal transition, whereas depletion of miR-141-3p suppressed cervical cancer cell proliferation and invasion. FOXA2 was identified to be a target of miR-141-3p. Overexpression of miR-141-3p led to a marked inhibition of endogenous FOXA2 in cervical cancer cells. FOXA2 silencing phenocopied the effects of miR-141-3p overexpression on cervical cancer cell proliferation and invasion. Enforced expression of FOXA2 blocked the effects of miR-141-3p on cervical cancer cell proliferation and invasion. miR-141-3p overexpression significantly accelerated the growth of xenograft tumors, which was accompanied by a striking reduction in FOXA2 expression. miR-141-3p acts as an oncogene in cervical cancer largely through repression of FOXA2. Targeting miR-141-3p may represent a potential therapeutic strategy for cervical cancer.

Studying the profile of micro RNAs (miRs) elucidated the highest expressed miRs in hepatic differentiation. In this study, we investigated to clarify the role of three embryonic overexpressed miRs (miR-106a, miR-574-3p and miR-451) during hepatic differentiation of human umbilical cord derived mesenchymal stem cells (UC-MSCs). We furthermore, aimed to explore whether overexpression of any of these miRs alone is sufficient to induce the differentiation of the UC-MSCs into hepatocyte-like cells. UC-MSCs were transfected either alone or together with miR-106a, miR-574-3p and miR-451 and their potential hepatic differentiation and alteration in gene expression profile, morphological changes and albumin secretion ability were investigated. We found that up-regulation of any of these three miRs alone cannot induce expression of all hepatic specific genes. Transfection of each miR alone, led to Sox17, FoxA2 expression that are related to initiation step of hepatic differentiation. However, concurrent ectopic overexpression of three miRs together can induce UC-MSCs differentiation into functionally mature hepatocytes. These results show that miRs have the capability to directly convert UC-MSCs to a hepatocyte phenotype in vitro.

Forkhead box A2 (FOXA2) is a pioneer transcription factor involved in organ development, function, and cancer. In the uterus, FOXA2 is essential for pregnancy and expressed specifically in the glands of the endometrium. Loss of FOXA2 function occurs during development of endometrial cancer in humans. The current study describes the development of a mouse model for conditional expression of mouse FOXA2. Using a system consisting of a minigene located at the Rosa26 locus, we generated a CAG-S-mFOXA2 allele in embryonic stem cells and subsequently in mice; before activation, the minigene is silent because of a floxed stop cassette inserted between the promoter and the transgene. To validate functionality, mice with the CAG-S-mFOXA2 allele were crossed with progesterone receptor (Pgr)–Cre mice and lactotransferrin (Ltf)-iCre mice that express Cre in the immature and adult uterus, respectively. In immature Pgr-Cre-CAG-S-mFoxa2 mice, FOXA2 protein was expressed in the luminal epithelium (LE), glandular epithelium (GE), stroma, and inner layer of the myometrium. Interestingly, FOXA2 protein was not observed in most of the LE of uteri from adult Pgr-Cre-CAG-S-mFoxa2 mice, although FOXA2 was maintained in the stroma, GE, and myometrium. The adult Pgr-Cre-CAG-S-mFoxa2 females were completely infertile. In contrast, Ltf-iCre-CAG-S-mFoxa2 mice were fertile with no detectable histological differences in the uterus. The adult uterus of Pgr-Cre-CAG-S-mFoxa2 mice was smaller, contained few endometrial glands, and displayed areas of partially stratified LE and GE. This transgenic mouse line is a valuable resource to elucidating and exploring FOXA2 function.

Transcription factors (TFs) direct developmental transitions by binding to target DNA sequences, influencing gene expression and establishing complex gene-regultory networks. To systematically determine the molecular components that enable or constrain TF activity, we investigated the genomic occupancy of FOXA2, GATA4 and OCT4 in several cell types. Despite their classification as pioneer factors, all three TFs exhibit cell-type-specific binding, even when supraphysiologically and ectopically expressed. However, FOXA2 and GATA4 can be distinguished by low enrichment at loci that are highly occupied by these factors in alternative cell types. We find that expression of additional cofactors increases enrichment at a subset of these sites. Finally, FOXA2 occupancy and changes to DNA accessibility can occur in G1-arrested cells, but subsequent loss of DNA methylation requires DNA replication.

Transplantation of neural progenitor cells (NPCs) is a potential therapy for treating neurodegenerative disorders, but this approach has faced many challenges and limited success, primarily because of inhospitable host brain environments that interfere with enriched neuron engraftment and function. Astrocytes play neurotrophic roles in the developing and adult brain, making them potential candidates for helping with modification of hostile brain environments. In this study, we examined whether astrocytic function could be utilized to overcome the current limitations of cell-based therapies in a murine model of Parkinson's disease (PD) that is characterized by dopamine (DA) neuron degeneration in the midbrain. We show here that cografting astrocytes, especially those derived from the midbrain, remarkably enhanced NPC-based cell therapeutic outcomes along with robust DA neuron engraftment in PD rats for at least 6 months after transplantation. We further show that engineering of donor astrocytes with Nurr1 and Foxa2, transcription factors that were recently reported to polarize harmful immunogenic glia into the neuroprotective form, further promoted the neurotrophic actions of grafted astrocytes in the cell therapeutic approach. Collectively, these findings suggest that cografting astrocytes could be a potential strategy for successful cell therapeutic outcomes in neurodegenerative disorders.

Alpha-fetoprotein (AFP)-producing adenocarcinoma is a high-malignant variant of adenocarcinoma with a hepatic or fetal-intestinal phenotype. The number of cases of AFP-producing adenocarcinomas is increasing, but the molecular mechanism underlying the aberrant production of AFP is unclear. Here we sought to assess the role of Forkhead box A (FoxA)2, which is a pioneer transcription factor in the differentiation of hepatoblasts. FoxA2 expression was investigated in five cases of AFP-producing gastric adenocarcinomas by immunohistochemistry, and all cases showed FoxA2 expression. Chromatin immunoprecipitation revealed the DNA binding of FoxA2 on the regulatory element of AFP gene in AFP-producing adenocarcinoma cells. The inhibition of FoxA2 expression with siRNA reduced the mRNA expression of liver-specific proteins, including AFP, albumin, and transferrin. The inhibition of FoxA2 also reduced the expressions of liver-enriched nuclear factors, i.e., hepatocyte nuclear factor (HNF) 4α and HNF6, although the expressions of HNF1α and HNF1β were not affected. The same effect as FoxA2 knockdown in AFP producing adenocarcinoma cells was also observed in hepatocellular carcinoma cells. Our results suggest that FoxA2 plays a key role in the expression of hepatic phenotype of AFP-producing adenocarcinomas.

Successful pregnancy relies on dynamic control of cell signaling to achieve uterine receptivity and the necessary biological changes required for endometrial decidualization, embryo implantation, and fetal development. Glucocorticoids are master regulators of intracellular signaling and can directly regulate embryo implantation and endometrial remodeling during murine pregnancy. In immortalized human uterine cells, we have shown that glucocorticoids and estradiol (E2) coregulate thousands of genes. Recently, glucocorticoids and E2 were shown to coregulate the expression of Left-right determination factor 1 (LEFTY1), previously implicated in the regulation of decidualization. To elucidate the molecular mechanism by which glucocorticoids and E2 regulate the expression of LEFTY1, immortalized and primary human endometrial cells were evaluated for gene expression and receptor recruitment to regulatory regions of the LEFTY1 gene. Glucocorticoid administration induced expression of LEFTY1 messenger RNA and protein and recruitment of the glucocorticoid receptor (GR) and activated polymerase 2 to the promoter of LEFTY1. Glucocorticoid-mediated recruitment of GR was dependent on pioneer factors FOXA1 and FOXA2. E2 was found to antagonize glucocorticoid-mediated induction of LEFTY1 by reducing recruitment of GR, FOXA1, FOXA2, and activated polymerase 2 to the LEFTY1 promoter. Gene expression analysis identified several genes whose glucocorticoid-dependent induction required FOXA1 and FOXA2 in endometrial cells. These results suggest a molecular mechanism by which E2 antagonizes GR-dependent induction of specific genes by preventing the recruitment of the pioneer factors FOXA1 and FOXA2 in a physiologically relevant model., Glucocorticoids selectively regulate human endometrial gene expression through cooperation with the pioneer factors FOXA1 and FOXA2, which can be modulated through estrogen receptor activity.

J. Yao et al. demonstrate that loss of MGP, a BMP inhibitor, causes abnormal hepatic differentiation in lungs. They find that interactions between endothelium and epithelium separate pulmonary from hepatic differentiation during development. Lack of MGP triggers hepatic differentiation in the pulmonary epithelium, as regulated by the endothelium., The vascular endothelium is critical for induction of appropriate lineage differentiation in organogenesis. In this study, we report that dysfunctional pulmonary endothelium, resulting from the loss of matrix Gla protein (MGP), causes ectopic hepatic differentiation in the pulmonary epithelium. We demonstrate uncontrolled induction of the hepatic growth factor (HGF) caused by dysregulated cross talk between pulmonary endothelium and epithelium in Mgp-null lungs. Elevated HGF induced hepatocyte nuclear factor 4 α (Hnf4a), which competed with NK2 homeobox 1 (Nkx2.1) for binding to forkhead box A2 (Foxa2) to drive hepatic differentiation in Mgp-null airway progenitor cells. Limiting endothelial HGF reduced Hnf4a, abolished interference of Hnf4a with Foxa2, and reduced hepatic differentiation in Mgp-null lungs. Together, our results suggest that endothelial–epithelial interactions, maintained by MGP, are essential in pulmonary cell differentiation.

Extraembryonic endoderm (XEN) cells are stem cell lines derived from primitive endoderm cells of inner cell mass in blastocysts. These cells have self-renewal properties and differentiate into visceral endoderm (VE) and parietal endoderm (PE) of the yolk sac. Recently, it has been reported that XEN cells can contribute to fetal embryonic endoderm, and their unique potency has been evaluated. In this study, we have described the induction and characterization of new canine stem cell lines that closely resemble to XEN cells. These cells, which we designated canine induced XEN (ciXEN)-like cells, were induced from canine embryonic fibroblasts by introducing four transgenes. ciXEN-like cells expressed XEN markers, which could be maintained over 50 passages in N2B27 medium supplemented with inhibitors of mitogen-activated protein kinase p38 and transforming growth factor-beta 1. Our ciXEN-like cells were maintained without transgene expression and exhibited upregulated expression of VE and PE markers in feeder-free conditions. The cells differentiated from ciXEN-like cells using a coculture system showed multiple nuclei and expressed albumin protein, similar to characteristics of hepatocytes. Furthermore, these cells expressed the adult hepatocyte marker, CYP3A4. Interestingly, these cells also formed a net structure expressing the bile epithelium capillary marker, multidrug resistance-associated protein 2. Thus, we have demonstrated the induction of a new canine stem cell line, ciXEN-like cells, which could form an embryonic endodermal cell layer. Our ciXEN-like cells may be a helpful tool to study the canine embryo development and represent a promising cell source for proceeding human and canine regenerative medicine.

FOXA2, a member of the forkhead family of transcription factors, plays essential roles in liver development and bile acid homeostasis. In this study, we report a 2.8 Å co-crystal structure of the FOXA2 DNA-binding domain (FOXA2-DBD) bound to a DNA duplex containing a forkhead consensus binding site (GTAAACA). FOXA2-DBD adopts the canonical winged-helix fold, with helix H3 and wing 1 regions mainly mediating the DNA recognition. Although the wing 2 region was not defined in the structure, isothermal titration calorimetry (ITC) assays suggested that this region was required for optimal DNA binding. Structure comparison with the FOXA3-DBD bound to DNA revealed more major groove contacts and less minor groove contacts in the FOXA2 structure compared to the FOXA3 structure. Structure comparison with the FOXO1-DBD bound to DNA showed that different forkhead proteins could induce different DNA conformations upon binding to identical DNA sequences. Our findings provide the structural basis for FOXA2 protein binding to a consensus forkhead site and elucidate how members of the forkhead protein family bind different DNA sites.

Objective The transcription factors (TF) Foxa2 and Pdx1 are key regulators of beta-cell (β-cell) development and function. Mutations of these TFs or their respective cis-regulatory consensus binding sites have been linked to maturity diabetes of the young (MODY), pancreas agenesis, or diabetes susceptibility in human. Although Foxa2 has been shown to directly regulate Pdx1 expression during mouse embryonic development, the impact of this gene regulatory interaction on postnatal β-cell maturation remains obscure. Methods In order to easily monitor the expression domains of Foxa2 and Pdx1 and analyze their functional interconnection, we generated a novel double knock-in homozygous (FVFPBFDHom) fluorescent reporter mouse model by crossing the previously described Foxa2-Venus fusion (FVF) with the newly generated Pdx1-BFP (blue fluorescent protein) fusion (PBF) mice. Results Although adult PBF homozygous animals exhibited a reduction in expression levels of Pdx1, they are normoglycemic. On the contrary, despite normal pancreas and endocrine development, the FVFPBFDHom reporter male animals developed hyperglycemia at weaning age and displayed a reduction in Pdx1 levels in islets, which coincided with alterations in β-cell number and islet architecture. The failure to establish mature β-cells resulted in loss of β-cell identity and trans-differentiation towards other endocrine cell fates. Further analysis suggested that Foxa2 and Pdx1 genetically and functionally cooperate to regulate maturation of adult β-cells. Conclusions Our data show that the maturation of pancreatic β-cells requires the cooperative function of Foxa2 and Pdx1. Understanding the postnatal gene regulatory network of β-cell maturation will help to decipher pathomechanisms of diabetes and identify triggers to regenerate dedifferentiated β-cell mass., Highlights • Fusion of fluorescent proteins to Foxa2 and Pdx1 induce hyperglycemia at weaning age. • Double knock-in (FVFPBFDHom) reporter animals exhibit low expression levels of Pdx1 protein. • FVFPBFDHom reporter male mice show impairment in β-cell maturation and function. • Cooperative function of Foxa2 and Pdx1 regulates postnatal maturation of β-cells.

The extracellular matrix (ECM) microenvironment is involved in the regulation of hepatocyte phenotype and function. Recently, the cell‐derived extracellular matrix has been proposed to represent the bioactive and biocompatible materials of the native ECM. Here, we show that the endothelial cell‐derived matrix (EC matrix) promotes the metabolic maturation of human adipose stem cell‐derived hepatocyte‐like cells (hASC‐HLCs) through the activation of the transcription factor forkhead box protein A2 (FOXA2) and the nuclear receptors hepatocyte nuclear factor 4 alpha (HNF4α) and pregnane X receptor (PXR). Reducing the fibronectin content in the EC matrix or silencing the expression of α5 integrin in the hASC‐HLCs inhibited the effect of the EC matrix on Src phosphorylation and hepatocyte maturation. The inhibition of Src phosphorylation using the inhibitor PP2 or silencing the expression of Src in hASC‐HLCs also attenuated the up‐regulation of the metabolic function of hASC‐HLCs in a nuclear receptor‐dependent manner. These data elucidate integrin‐Src signalling linking the extrinsic EC matrix signals and metabolic functional maturation of hepatocyte. This study provides a model for studying the interaction between hepatocytes and non‐parenchymal cell‐derived matrix.

The forkhead box A2 (FOXA2) is the key transcriptional factor that plays an important role in tumorigenesis. However, until now the expression pattern and role of FOXA2 in glioma have yet to be elucidated. Therefore, the aim of this study was to evaluate the expression of FOXA2 in glioma and investigate its role in glioma cells. Our data showed that FOXA2 was significantly downregulated in human glioma cell lines. Forced expression of FOXA2 suppressed the ability of glioma cells to proliferate, migrate, and invade and influenced the expression level of EMT-associated proteins. In addition, forced expression of FOXA2 attenuated tumor growth of glioma in a nude mouse xenograft model. Mechanistically, we disclosed that forced expression of FOXA2 greatly downregulated the expression of β-catenin, cyclin D1, and c-Myc in glioma cells. Taken together, these results show that FOXA2 may play an important role in proliferation, invasion, and tumorigenesis in glioma cells. Thus, FOXA2 may be a potential therapeutic target for the treatment of glioma.

Lipoprotein lipase (LPL) plays a major role in the hydrolysis of triglycerides (TG) from circulating TG-rich lipoproteins. The role of LPL in the liver has been controversial but recent studies in mice with liver LPL overexpression or deficiency have revealed important new roles of the enzyme in glucose and lipid metabolism. The objective of this study was to identify regulatory elements and factors that control the transcription of the human LPL gene in hepatocytes. Deletion analysis of the human LPL promoter revealed that the proximal region which harbors a binding site for the forkhead box transcription factor FOXA2/HNF-3β at position -47/-40 is important for its hepatic cell activity. Silencing of FOXA2 in HepG2 cells reduced the LPL mRNA and protein levels. Direct binding of FOXA2 to the novel binding site was established in vitro and ex vivo. Mutagenesis of the FOXA2 site reduced the basal activity and abolished the FOXA2-mediated transactivation of the LPL promoter. Ιnsulin decreased LPL mRNA levels in HepG2 cells and this was associated with phosphorylation of AKT and nuclear export of FOXA2. In summary, the data of the present study combined with previous findings on the role of FOXA2 in HDL metabolism and gluconeogenesis, suggest that FOXA2 is a key regulator of lipid and glucose homeostasis in the adult liver. Understanding the mechanisms by which FOXA2 exerts its functions in hepatocytes may open the way to novel therapeutic strategies for patients with metabolic diseases such as dyslipidemia, diabetes and the metabolic syndrome.

So far, the understanding of germ cell cancer (GCC) pathogenesis is based on a model, where seminomas and non-seminomas represent distinct entities although originating from a common precursor termed germ cell neoplasia in situ (GCNIS). Embryonal carcinomas (ECs), the stem cell population of the non-seminomas, is pluri- to totipotent and able to differentiate into cells of all three germ layers, giving rise to teratomas or tumours mimicking extraembryonic tissues (yolk sac tumours, choriocarcinomas). With regard to gene expression, (epi)genetics and histology, seminomas are highly similar to GCNIS and primordial germ cells, but limited in development. It remains elusive, whether this block in differentiation is controlled by cell intrinsic mechanisms or by signals from the surrounding microenvironment. Here, we reviewed the recent literature emphasizing the plasticity of GCCs, especially of seminomas. We propose that this plasticity is controlled by the microenvironment, allowing seminomas to transit into an EC or mixed non-seminoma and vice versa. We discuss several mechanisms and routes of reprogramming that might be responsible for this change in the cell fate. We finally integrate this plasticity into a new model of GCC pathogenesis, allowing for an alternative view on the dynamics of GCC development and progression.

miRNAs are small non-coding RNA sequences of 18-25 nucleotides. They can regulate different cellular pathways by acting on tumor suppressors, oncogenes, or both. miRNAs are mostly tissue-specific, and their expression varies depending on the cancer or the tissue in which they are found. hsa-miR-590-3p was found to be involved in several types of cancers. In this study, we identified potential downstream target genes of hsa-miR-590-3p computationally. Several bioinformatics tools and more than one approach were used to identify potential downstream target genes of hsa-miR-590-3p. CX3CL1, SOX2, N-cadherin, E-cadherin, and FOXA2 were utilized as potential downstream target genes of hsa-miR-590-3p. SNU449 and HepG2, hepatocellular carcinoma cell lines, were used to carry out various molecular techniques to further validate our in silico results. mRNA and protein expression levels of these genes were detected using RT-PCR and western blotting, respectively. Co-localization of hsa-miR-590-3p and its candidate downstream target gene, SOX2, was carried out using a miRNA in situ hybridization combined with immunohistochemistry staining through anti-SOX2. The results show that there is an inverse correlation between hsa-miR-590-3p expression and SOX2 protein expression in SNU449. Subsequently, we suggest that SOX2 can be a direct downstream target of has-miR-590-3p indicating that it may have a role in the self-renewal and self-maintenance of cancer cells. We also suggest that CX3CL1, E-cadherin, N-cadherin, and FOXA2 show a lot of potential as downstream target genes of hsa-miR-590-3p signifying its role in epithelial-mesenchymal transition. Studying the expression of hsa-miR-590-3p downstream targets can enrich our understanding of the cancer pathogenesis and how it can be used as a therapeutic tool.