Transcription Factor Pax6 Research Articles

Synthetic essentiality between PTEN and core dependency factor PAX7 dictates rhabdomyosarcoma identity

PTEN promoter hypermethylation is nearly universal and PTEN copy number loss occurs in ~25% of fusion-negative rhabdomyosarcoma (FN-RMS). Here we show Pten deletion in a mouse model of FN-RMS results in less differentiated tumors more closely resembling human embryonal RMS. PTEN loss activated the PI3K pathway but did not increase mTOR activity. In wild-type tumors, PTEN was expressed in the nucleus suggesting loss of nuclear PTEN functions could account for these phenotypes. Pten deleted tumors had increased expression of transcription factors important in neural and skeletal muscle development including Dbx1 and Pax7. Pax7 deletion completely rescued the effects of Pten loss. Strikingly, these Pten;Pax7 deleted tumors were no longer FN-RMS but displayed smooth muscle differentiation similar to leiomyosarcoma. These data highlight how Pten loss in FN-RMS is connected to a PAX7 lineage-specific transcriptional output that creates a dependency or synthetic essentiality on the transcription factor PAX7 to maintain tumor identity.

A nexus of miR-1271, PAX4 and ALK/RYK influences the cytoskeletal architectures in Alzheimer's Disease and Type 2 Diabetes.

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

Genetic interaction of Pax3 mutation and canonical Wnt signaling modulates neural tube defects and neural crest abnormalities.

Mouse models provide opportunities to investigate genetic interactions that cause or modify the frequency of neural tube defects (NTDs). Mutation of the PAX3 transcription factor prevents neural tube closure, leading to cranial and spinal NTDs whose frequency is responsive to folate status. Canonical Wnt signalling is implicated both in regulation of Pax3 expression and as a target of PAX3. This study investigated potential interactions of Pax3 mutation and canonical Wnt signalling using conditional gain- and loss-of-function models of β-catenin. We found an additive effect of β-catenin gain of function and Pax3 loss of function on NTDs and neural crest defects. β-catenin gain of function in the Pax3 expression domain led to significantly increased frequency of cranial but not spinal NTDs in embryos that are heterozygous for Pax3 mutation, while both cranial and spinal neural tube closure were exacerbated in Pax3 homozygotes. Similarly, deficits of migrating neural crest cells were exacerbated by β-catenin gain of function, with almost complete ablation of spinal neural crest cells and derivatives in Pax3 homozygous mutants. Pax3 expression was not affected by β-catenin gain of function, while we confirmed that loss of function led to reduced Pax3 transcription. In contrast to gain of function, β-catenin knockout in the Pax3 expression domain lowered the frequency of cranial NTDs in Pax3 null embryos. However, loss of function of β-catenin and Pax3 resulted in spinal NTDs, suggesting differential regulation of cranial and spinal neural tube closure. In summary, β-catenin function modulates the frequency of PAX3-related NTDs in the mouse.

Amyloid-β toxicity modulates tau phosphorylation through the PAX6 signalling pathway.

The molecular link between amyloid-β plaques and neurofibrillary tangles, the two pathological hallmarks of Alzheimer's disease, is still unclear. Increasing evidence suggests that amyloid-β peptide activates multiple regulators of cell cycle pathways, including transcription factors CDKs and E2F1, leading to hyperphosphorylation of tau protein. However, the exact pathways downstream of amyloid-β-induced cell cycle imbalance are unknown. Here, we show that PAX6, a transcription factor essential for eye and brain development which is quiescent in adults, is increased in the brains of patients with Alzheimer's disease and in APP transgenic mice, and plays a key role between amyloid-β and tau hyperphosphorylation. Downregulation of PAX6 protects against amyloid-β peptide-induced neuronal death, suggesting that PAX6 is a key executor of the amyloid-β toxicity pathway. Mechanistically, amyloid-β upregulates E2F1, followed by the induction of PAX6 and c-Myb, while Pax6 is a direct target for both E2F1 and its downstream target c-Myb. Furthermore, PAX6 directly regulates transcription of GSK-3β, a kinase involved in tau hyperphosphorylation and neurofibrillary tangles formation, and its phosphorylation of tau at Ser356, Ser396 and Ser404. In conclusion, we show that signalling pathways that include CDK/pRB/E2F1 modulate neuronal death signals by activating downstream transcription factors c-Myb and PAX6, leading to GSK-3β activation and tau pathology, providing novel potential targets for pharmaceutical intervention.

Conditional Deletion of AP-2β in the Periocular Mesenchyme of Mice Alters Corneal Epithelial Cell Fate and Stratification.

The cornea is an anterior eye structure specialized for vision. The corneal endothelium and stroma are derived from the periocular mesenchyme (POM), which originates from neural crest cells (NCCs), while the stratified corneal epithelium develops from the surface ectoderm. Activating protein-2β (AP-2β) is highly expressed in the POM and important for anterior segment development. Using a mouse model in which AP-2β is conditionally deleted in the NCCs (AP-2β NCC KO), we investigated resulting corneal epithelial abnormalities. Through PAS and IHC staining, we observed structural and phenotypic changes to the epithelium associated with AP-2β deletion. In addition to failure of the mutant epithelium to stratify, we also observed that Keratin-12, a marker of the differentiated epithelium, was absent, and Keratin-15, a limbal and conjunctival marker, was expanded across the central epithelium. Transcription factors PAX6 and P63 were not observed to be differentially expressed between WT and mutant. However, growth factor BMP4 was suppressed in the mutant epithelium. Given the non-NCC origin of the epithelium, we hypothesize that the abnormalities in the AP-2β NCC KO mouse result from changes to regulatory signaling from the POM-derived stroma. Our findings suggest that stromal pathways such as Wnt/β-Catenin signaling may regulate BMP4 expression, which influences cell fate and stratification.

GABAB Receptor-Mediated Impairment of Intermediate Progenitor Maturation During Postnatal Hippocampal Neurogenesis of Newborn Rats.

The neurotransmitter GABA and its receptors assume essential functions during fetal and postnatal brain development. The last trimester of a human pregnancy and early postnatal life involves a vulnerable period of brain development. In the second half of gestation, there is a developmental shift from depolarizing to hyperpolarizing in the GABAergic system, which might be disturbed by preterm birth. Alterations of the postnatal GABA shift are associated with several neurodevelopmental disorders. In this in vivo study, we investigated neurogenesis in the dentate gyrus (DG) in response to daily administration of pharmacological GABAA (DMCM) and GABAB (CGP 35348) receptor inhibitors to newborn rats. Six-day-old Wistar rats (P6) were daily injected (i.p.) to postnatal day 11 (P11) with DMCM, CGP 35348, or vehicle to determine the effects of both antagonists on postnatal neurogenesis. Due to GABAB receptor blockade by CGP 35348, immunohistochemistry revealed a decrease in the number of NeuroD1 positive intermediate progenitor cells and a reduction of proliferative Nestin-positive neuronal stem cells at the DG. The impairment of hippocampal neurogenesis at this stage of differentiation is in line with a significantly decreased RNA expression of the transcription factors Pax6, Ascl1, and NeuroD1. Interestingly, the number of NeuN-positive postmitotic neurons was not affected by GABAB receptor blockade, although strictly associated transcription factors for postmitotic neurons, Tbr1, Prox1, and NeuroD2, displayed reduced expression levels, suggesting impairment by GABAB receptor antagonization at this stage of neurogenesis. Antagonization of GABAB receptors decreased the expression of neurotrophins (BDNF, NT-3, and NGF). In contrast to the GABAB receptor blockade, the GABAA receptor antagonization revealed no significant changes in cell counts, but an increased transcriptional expression of Tbr1 and Tbr2. We conclude that GABAergic signaling via the metabotropic GABAB receptor is crucial for hippocampal neurogenesis at the time of rapid brain growth and of the postnatal GABA shift. Differentiation and proliferation of intermediate progenitor cells are dependent on GABA. These insights become more pertinent in preterm infants whose developing brains are prematurely exposed to spostnatal stress and predisposed to poor neurodevelopmental disorders, possibly as sequelae of early disruption in GABAergic signaling.

Determination of genetic effects and functional SNPs of bovine HTR1B gene on milk fatty acid traits

BackgroundOur previous genome-wide association study (GWAS) on milk fatty acid traits in Chinese Holstein cows revealed, the SNP, BTB-01556197, was significantly associated with C10:0 at genome-wide level (P = 0.0239). It was located in the down-stream of 5-hydroxytryptamine receptor 1B (HTR1B) gene that has been shown to play an important role in the regulation of fatty acid oxidation. Hence, we considered it as a promising candidate gene for milk fatty acids in dairy cattle. In this study, we aimed to investigate whether the HTR1B gene had significant genetic effects on milk fatty acid traits.ResultsWe re-sequenced the entire coding region and 3000 bp of 5′ and 3′ flanking regions of HTR1B gene. A total of 13 SNPs was identified, containing one in 5′ flanking region, two in 5′ untranslated region (UTR), two in exon 1, five in 3′ UTR, and three in 3′ flanking region. By performing genotype-phenotype association analysis with SAS9.2 software, we observed that 13 SNPs were significantly associated with medium-chain saturated fatty acids such as C6:0, C8:0 and C10:0 (P < 0.0001 ~ 0.042). With Haploview 4.1 software, linkage disequilibrium (LD) analysis was performed. Two haplotype blocks formed by two and ten SNPs were observed. Haplotype-based association analysis indicated that both haplotype blocks were strongly associated with C6:0, C8:0 and C10:0 as well (P < 0.0001 ~ 0.0071). With regards to the missense mutation in exon 1 (g.17303383G > T) that reduced amino acid change from alanine to serine, we predicted that it altered the secondary structure of HTR1B protein with SOPMA. In addition, we predicted that three SNPs in promoter region, g.17307103A > T, g.17305206 T > G and g.17303761C > T, altered the binding sites of transcription factors (TFs) HMX2, PAX2, FOXP1ES, MIZ1, CUX2, DREAM, and PPAR-RXR by Genomatix. Of them, luciferase assay experiment further confirmed that the allele T of g.17307103A > T significantly increased the transcriptional activity of HTR1B gene than allele A (P = 0.0007).ConclusionsIn conclusion, our findings provided first evidence that the HTR1B gene had significant genetic effects on milk fatty acids in dairy cattle.

PAX8 plays an essential antiapoptotic role in uterine serous papillary cancer.

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

HMGA2 rs968697 T > C polymorphism is associated with the risk of colorectal cancer

A genetic polymorphism (rs968697 T > C) in the HMGA2 gene has recently been linked to an increased risk of hepatoblastoma. However, no studies have been conducted to investigate the effect of the polymorphism on the risk of colorectal cancer (CRC). The study aimed to explore whether the rs968697 polymorphism had a significant impact on CRC risk. A total of 500 CRC patients and 500 age and gender matched healthy individuals were genotyped by using the SNaPshot method. Quantitative real-time PCR technology was used to detect the relative expression of the HMGA2 gene in 30 pairs of primary CRC and adjacent non-cancerous tissues. Results: HMGA2 rs968697 polymorphism was significantly associated with CRC risk [CC vs. TT: OR = 0.20, 95%CI = 0.06–0.70, P = 0.01; (CC + CT) vs. TT: OR = 0.71, 95%CI = 0.53–0.96, P = 0.02; CC vs. (CT + TT): OR = 0.21, 95%CI = 0.06–0.73, P = 0.01; C vs. T: OR = 0.67, 95%CI = 0.51–0.89, P < 0.01]. The analysis based on tumor stage indicated that the CRC patients with HMGA2 rs968697 C allele were less likely to have high-stage tumors. Furthermore, the genotype-tissue expression analysis revealed that the rs968697 CC genotype was linked to the low expression of HMGA2 gene. The in silico analysis revealed that the rs968697 polymorphism in the promoter region of the HMGA2 gene could influence transcription factor binding, including ATF6, DBP, CDPCR3, DR3, NRSF, PAX8, PPARA, SZF11, TAXCREB and POLR2A. In conclusion, our findings suggested that the HMGA2 rs968697 polymorphism was linked to CRC risk and could be used as a biomarker to detect CRC risk.

Long Term Rescue of the TSH Receptor Knock-Out Mouse - Thyroid Stem Cell Transplantation Restores Thyroid Function.

The synergistic activation of transcription factors can lead to thyroid progenitor cell speciation. We have previously shown in vitro that mouse or human stem cells, expressing the transcription factors NKx2-1 and Pax8, can differentiate into thyroid neo-follicular structures (TFS). We now show that syngeneic mouse TFS when implanted into hypothyroid TSH receptor knockout (TSHR-KO) mice can ameliorate the hypothyroid state for an extended period. ES cells derived from heterozygous TSHR-KO blastocysts were stably transfected with Nkx2-1-GFP and Pax8-mcherry constructs and purified into 91.8% double positive cells by flow cytometry. After 5 days of activin A treatment these double positive cells were then induced to differentiate into neo-follicles in Matrigel for 21 days in the presence of 500μU/mL of TSH. Differentiated TFS expressing thyroglobulin mRNA were implanted under the kidney capsule of 4-6 weeks old TSHR-KO mice (n=5) as well as hind limb muscle (n=2) and anterior chamber of one eye (n=2). Five of the mice tested after 4 weeks were all rendered euthyroid and all mice remained euthyroid at 20 weeks post implantation. The serum T4 fully recovered (pre-bleed 0.62 ± 0.03 to 8.40 ± 0.57 µg/dL) and the previously elevated TSH became normal or suppressed (pre-bleed 391 ± 7.6 to 4.34 ± 1.25 ng/dL) at the end of the 20 week observation period. The final histology obtained from the implanted kidney tissues showed only rudimentary thyroid follicular structures but which stained positive for thyroglobulin expression. The presence of only rudimentary structures at the site of implant on these extended animals suggested possible migration of cells from the site of implant or an inability of TFCs to maintain proper follicular morphology in these external sites for extended periods. However, there were no signs of tumor formation and no immune infiltration. These preliminary studies show that TSHR-KO mice are a useful model for orthotropic implantation of functional thyroid cells without the need for thyroidectomy, radioiodine ablation or anti thyroid drug control of thyroid function. This approach is also proof of principle that thyroid cells derived from mouse ES cells are capable of surviving as functional neo-follicles in vivo for an extended period of 20 weeks.

Abstract 3022: Synthetic essentiality between PTEN and core dependency factor PAX7 dictates rhabdomyosarcoma indentity

Abstract Rhabdomyosarcoma (RMS) is an embryonal tumor resembling developing skeletal muscle and the most common pediatric soft-tissue sarcoma. RMS is molecularly defined by the presence or absence of a fusion oncoprotein corresponding with the histological subtypes alveolar and embryonal RMS, respectively. Embryonal, or fusion-negative, RMS (FN-RMS) is heterogeneous in its molecular alteration profile; the major exception is the near universal PTEN promoter hypermethylation found in FN-RMS corresponding with decreased PTEN expression. PTEN's functional role in FN-RMS remains unclear as does PTEN's role in defining tumor fate decisions. Organismal cancer models can help elucidate these decisions by defining the potential tumor fate landscape that can occur in transformed multipotent progenitor cells. Our laboratory leverages a highly penetrant, early onset model of FN-RMS driven by the transdifferentiation of endothelial progenitors into skeletal muscle-like RMS cells by Hedgehog pathway activation. Therefore, our model is uniquely primed to empirically determine the core regulatory factors critical in FN-RMS initiation. Here, we conditionally deleted Pten in these cells (ASPcKO). ASPcKO tumors presented earlier than wild-type tumors and more closely resemble human FN-RMS with a less differentiated skeletal muscle state. These were unique characteristics of ASPcKO tumors as mice with homozygous loss of other tumor suppressors - Cdkn2a, Trp53, and Rb1 - did not recapitulate these phenotypes. We further probed the downstream transcriptional outputs of ASPcKO tumors revealing a profound increase in expression of the neural developmental transcription factors Dbx1 and Pax7. These outputs are functionally important as human FN-RMS patient-derived xenografts are dependent on both DBX1 and PAX7. Subsequently, we also show that DBX1 is a downstream transcriptional target of PAX7 highlighting how Pten loss engages a unique transcriptional program for tumor maintenance. PAX7 is also a core FN-RMS regulatory circuit component. To further interrogate the role of PAX7 on tumor initiation, we concomitantly deleted Pten and Pax7 in our FN-RMS model and found not only that Pax7 loss rescues the survival kinetics observed when Pten is lost, but also alters the developmental trajectory of the tumors that do develop. Instead of Smoothened trans-differentiating our aP2-Cre expressing primordial endothelial cell into a skeletal-muscle like FN-RMS, Pten and Pax7 loss dictates these endothelial cells to give rise to tumors with smooth muscle-like differentiation, including human-like leiomyosarcoma. Together, this synthetic essential interaction between Pten and Pax7 in FN-RMS stresses the importance of the bifunctional role of PAX7 in tumor initiation and maintenance and how specific tumor suppressor loss can engage developmental transcriptional programs to alter tumor fate. Citation Format: Casey G. Langdon, Katherine E. Gadek, Matthew R. Garcia, Kristin B. Reed, Madeline Bush, Jason A. Hanna, Catherine J. Drummond, Matthew C. Maguire, Patrick J. Leavey, David Finkelstein, Hongjian Jin, Jerold E. Rehg, Mark E. Hatley. Synthetic essentiality between PTEN and core dependency factor PAX7 dictates rhabdomyosarcoma indentity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 3022.

Abstract 1316: PAX8-directed nanotherapeutics for high-grade serous ovarian cancer

Abstract Objective: High-grade serous ovarian cancer (HGSOC) is the most common epithelial ovarian cancer subtype. Targeted therapy for HGSOC is challenging as the disease is characterized by copy number variation and not somatic mutations. HGSOC displays well-conserved features such as expression of transcription factor PAX8. We propose employing therapies that capitalize on interactions between PAX8 and specific DNA-binding sites to develop therapeutic approaches to target HGSOC. Our hypothesis is to use a biomimetic approach wherein PAX8 is induced to transcribe an ectopically transfected CRISPR-base editor (BE)-guide RNA (gRNA) plasmid containing a PAX8 binding site in its promoter. The gRNA will impart a dominant-negative allele of ataxia telangiectasia and Rad3-related protein (ATR), which has been shown to be synthetically lethal with mutant p53 in cancer cells. In the presence of PAX8, the plasmid will be transcribed, while in the absence of PAX8, the plasmid is degraded, with minimal toxicity observed in healthy cells. Methods: PAX8-expressing ovarian cancer cell lines including COV362, Kuramochi, OVCAR8 and OVSAHO or PAX8-null cell lines including MCF-7 and HeLa were transfected with polyplexes bearing the plasmid of interest. Polyplex diameter, polydispersity indices and surface zeta potentials were measured on a Malvern Zetasizer. Polyplexes were imaged for uptake and functionality using fluorescence microscopy and Amnis ImageStream. Polyplex toxicity was assessed in vitro using cell viability assays. Preliminary in vivo experiments to demonstrate luciferase plasmid-bearing polyplexes have been performed in NSG mice. To monitor in vivo transfection efficiencies, we will deliver fluorescently labeled plasmids or luciferase-encoding plasmids to test bio-distribution and pharmacokinetics. Results: We have synthesized sub-200nm (diameter) polyplexes using the polyamine PPLG-g-azidopropylamine (PPLG) and BE or gRNA plasmids. Using -/- ATR gRNA, we observed that the BE and the gRNA can be efficiently packaged into polymeric nanoplexes and delivered to ovarian cancer cells or control cells to impart a dominant-negative mutant of ATR. Ongoing work includes cell cycle analysis using flow cytometry to monitor checkpoint activation, quantifying percent of total sequencing reads with target base pair conversions and testing gRNAs against other targets for efficacy in ovarian cancer targeting. Conclusion: Combining nanotechnology and gene editing can improve specificity toward ovarian cancer cells as well as incorporate precision therapy aimed at the underlying genetic architecture of this disease. Due to the synthetic lethality nature of ATR and p53, addition of a combinatorial PARP inhibitor to the proposed therapy will reduce tumor burden. While resistance can develop to ATR inhibitors, ovarian cancer cells are unable to downregulate PAX8, thus ensuring continued response to the PAX8-directed therapy. Citation Format: Akshaya Chandrasekaran, Mariam Ahmed, Nicole Holub, Wade Wang, Paula T. Hammond, Kevin M. Elias. PAX8-directed nanotherapeutics for high-grade serous ovarian cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1316.

Pax7 pioneer factor action requires both paired and homeo DNA binding domains.

The pioneer transcription factor Pax7 contains two DNA binding domains (DBD), a paired and a homeo domain. Previous work on Pax7 and the related Pax3 showed that each DBD binds a cognate DNA sequence, thus defining two targets of binding and possibly modalities of action. Genomic targets of Pax7 pioneer action leading to chromatin opening are enriched for composite DNA target sites containing juxtaposed sites for both paired and homeo domains. The present work investigated the implication of the DBDs in pioneer action. We show that the composite sequence is a higher affinity binding site and that efficient binding to this site involves both DBDs of the same Pax7 molecule. This binding is not sensitive to cytosine methylation of the DNA sites consistent with pioneer action within nucleosomal heterochromatin. Introduction of single amino acid mutations in either paired or homeo domain that impair binding to cognate DNA sequences showed that both DBDs must be intact for pioneer action. In contrast, only the paired domain is required for low affinity binding of heterochromatin sites. Thus, Pax7 pioneer action on heterochromatin requires unique protein:DNA interactions that are more complex compared to its simpler DNA binding modalities at accessible enhancer target sites.

Perchlorate and Nitrate Treatments Disrupt the Endoderm and Thyroid Development Through Epigenetic Mechanisms

Nitrate and perchlorate competitively inhibit iodide uptake in thyrocytes and disrupt thyroid function in rodents and humans. Our previous data indicated that the intrauterine exposure to perchlorate or nitrate induced thyroid dysfunction in the offspring rats during adult life. Therefore, this study aimed to investigate the effects of these endocrine disruptors during the embryonic period on the endoderm and thyroid development. Additionally, it was also investigated the role of epigenetic modifications in the programming of gene expression of the evaluated tissues. For this purpose, CD1 pregnant female mice received filtered water (control) or filtered water supplemented with sodium perchlorate (0.3 or 1 ppm) or sodium nitrate (20 or 50 ppm). At gestational day 16.5 (GD16.5), the embryonic thyroid lobes were collected and processed for molecular analysis. Besides the in vivo model, the effect of these thyroid disruptors was also evaluated during the differentiation of mouse embryonic stem cells (mESc) into endoderm and thyroid cells. Endoderm cells differentiation was achieved through the treatment of mESc with several growth factors. During the entire protocol the cells were exposed or not to sodium perchlorate or sodium nitrate (10-5 or 10-7 M). The effects of perchlorate and nitrate were also evaluated during the differentiation of mESC into thyroid cells. For this purpose, mESC-derived endoderm cells were transiently transfected with Pax8/Nkx2.1 expressing vectors. During the endoderm-to-thyrocytes differentiation protocol, the cells were also exposed or not to perchlorate or nitrate (10-5 or 10-7 M). The results demonstrated that both thyroid disruptors reduced the mRNA and protein expression of several endoderm markers (Foxa1, Gata4, Sox17) in the mESc-derived endoderm cells. Moreover, perchlorate or nitrate treatment also reduced the expression of thyroid transcription factors (Pax8, Nkx2.1, Foxe1) and thyroid differentiation markers (Slc5a5, Tpo, Tshr, Tg) both in the embryonic thyroid lobes and in the mESc-derived thyrocytes. Epigenetic mechanisms related to transcription repression seem to be involved in the gene expression downregulation both in vivo and in vitro, since perchlorate and nitrate increased the mRNA expression of Dnmt1, Dnmt3, Hdac and reduced the expression of Hat. Additionally, the methylation of histone H3 was increased, and the acetylation status of this histone was decreased in perchlorate- or nitrate-exposed thyroid lobes and mESc-derived endoderm/thyroid cells. In conclusion, our data strongly suggest that the programming of thyroid dysfunction induced by intrauterine exposure to perchlorate or nitrate involve the disruption of the endoderm and thyroid development during embryonic life through epigenetic mechanisms.

Transcription factor overexpression drives reliable differentiation of retinal pigment epithelium from human induced pluripotent stem cells

Age-related macular degeneration and genetic forms of blindness such as Best Disease and Retinitis Pigmentosa can be caused by degeneration of the Retinal Pigment Epithelium (RPE). RPE generated from patient-derived induced pluripotent stem cells (iPSCs) is valuable for both the study of disease mechanisms and development of therapeutic strategies. However, protocols to produce iPSC-derived RPE in vitro are often inefficient, labor-intensive, low-throughput, and highly variable between cell lines and within batches. Here, we report a robust, scalable method to generate iPSC-RPE using doxycycline-inducible expression of eye field transcription factors OTX2, PAX6 and MITF paired with RPE-permissive culture media. Doxycycline addition induces exogenous expression of these transcription factors in Best Disease patient- and wildtype iPSCs to efficiently produce monolayers of RPE with characteristic morphology and gene expression. Further, these RPE monolayers display functionality features including light absorption via pigmentation, polarity-driven fluid transport, and phagocytosis. With this method, we achieve a highly efficient and easily scalable differentiation without the need for mechanical isolation or enrichment methods, generating RPE cultures applicable for in vitro studies.

PAX8 and MECOM are interaction partners driving ovarian cancer

The transcription factor PAX8 is critical for the development of the thyroid and urogenital system. Comprehensive genomic screens furthermore indicate an additional oncogenic role for PAX8 in renal and ovarian cancers. While a plethora of PAX8-regulated genes in different contexts have been proposed, we still lack a mechanistic understanding of how PAX8 engages molecular complexes to drive disease-relevant oncogenic transcriptional programs. Here we show that protein isoforms originating from the MECOM locus form a complex with PAX8. These include MDS1-EVI1 (also called PRDM3) for which we map its interaction with PAX8 in vitro and in vivo. We show that PAX8 binds a large number of genomic sites and forms transcriptional hubs. At a subset of these, PAX8 together with PRDM3 regulates a specific gene expression module involved in adhesion and extracellular matrix. This gene module correlates with PAX8 and MECOM expression in large scale profiling of cell lines, patient-derived xenografts (PDXs) and clinical cases and stratifies gynecological cancer cases with worse prognosis. PRDM3 is amplified in ovarian cancers and we show that the MECOM locus and PAX8 sustain in vivo tumor growth, further supporting that the identified function of the MECOM locus underlies PAX8-driven oncogenic functions in ovarian cancer.

BAX -248 G>A and BCL2 -938 C>A Variant Lowers the Survival in Patients with Nasopharyngeal Carcinoma and Could be Associated with Tissue-Specific Malignancies: A Multi-Method Approach.

Background:The association of BAX -248 G>A and BCL2 -938 C>A with different cancers created conflicts. We studied the correlation and the effect of these polymorphisms in patients with Nasopharyngeal Carcinoma (NPC). Methods:PCR-RFLP and Sanger sequencing were used to detect polymorphisms. Statistical analysis including forest plot and Kaplan-Meier Log-rank test was conducted to investigate the association and effect of these SNPs on the NPC patients’ survival. The computational study was performed to investigate the possible regulatory role between these polymorphisms and the poor survival of NPC patients. Meta-analysis was executed to check the tissue-specific association of these polymorphisms in the context of global cancer prognosis. Results:We observed an increased and significant association of BAX -248 G>A [GA:OR=5.29, 95%CI=1.67,16.67, P=0.004; GA+AA:OR=5.71, 95%CI=1.82,17.90, P =0.002; A:OR=5.33, 95%CI=1.76,16.13, P=0.003], and BCL2 -938 C>A [CA:OR=2.26, 95%CI=1.03,4.96, P=0.04; AA:OR=3.56, 95%CI=0.97,13.05, P=0.05; CA+AA:OR=3.10, 95%CI=1.51,6.35, P=0.002; A:OR=2.90, 95% CI=1.59,5.29, P=0.0005] with the risk of NPC. Also, these SNPs were strongly correlated with poor survival in NPC patients (lower estimated survival mean, lower estimated proportion surviving at 5 years with p<0.05). The computational study showed that these SNPs altered the binding affinity of transcription factors HIF1, SP1, PAX3, PAX9 and CREB towards promoter (Lower p indicates strong affinity). The meta-analysis revealed the tissue-specific association of these polymorphisms. BAX -248 G>A showed a significant correlation with carcinomas [A vs G:OR=1.60, 95%CI=1.09,2.34, P=0.01; AA vs GG:OR=2.61, 95%CI=1.68,4.06, P<0.001; AA+GA vs GG:OR=1.53,95%CI=1.04,2.25, P=0.02); AA vs GG+GA:OR=2.53, 95%CI=1.65,3.87, P<0.001], and BCL2 -938 C>A with other malignancies [A vs C:OR=1.45, 95%CI=1.26,1.66, P<0.001; AA vs CC:OR=2.07, 95%CI: 1.15,3.72, P=0.01; AA+CA vs CC:OR=1.42, 95%CI=1.18,1.72, P<0.001; AA vs CC+CA:OR=1.89, 95%CI=1.02,3.50, P=0.04]. Conclusions:BAX -248 G>A and BCL2 -938 C>A was associated with poor survival in NPC patients. It may increase cancer susceptibility through transcriptional regulation. Moreover, these SNPs’ effects could be tissue-specific.

Silencing PTEN in the fallopian tube promotes enrichment of cancer stem cell-like function through loss of PAX2

High-grade serous ovarian cancer (HGSOC) is the most lethal gynecological malignancy that is primarily detected at the metastatic stage. Most HGSOC originates from the fallopian tube epithelium (FTE) and metastasizes to the ovary before invading the peritoneum; therefore, it is crucial to study disease initiation and progression using FTE-derived models. We previously demonstrated that loss of PTEN from the FTE leads to ovarian cancer. In the present study, loss of PTEN in FTE led to the enrichment of cancer stem cell markers such as LGR5, WNT4, ALDH1, CD44. Interestingly, loss of the transcription factor PAX2, which is a common and early alteration in HGSOC, played a pivotal role in the expression of cancer stem-like cells (CSC) markers and cell function. In addition, loss of PTEN led to the generation of two distinct subpopulations of cells with different CSC marker expression, tumorigenicity, and chemoresistance profiles. Taken together, these data suggest that loss of PTEN induces reprogramming of the FTE cells into a more stem-like phenotype due to loss of PAX2 and provides a model to study early events during the FTE-driven ovarian cancer tumor formation.

Maternal Vitamin and Mineral Supplementation and Rate of Maternal Weight Gain Affects Placental Expression of Energy Metabolism and Transport-Related Genes

Maternal nutrients are essential for proper fetal and placental development and function. However, the effects of vitamin and mineral supplementation under two rates of maternal weight gain on placental genome-wide gene expression have not been investigated so far. Furthermore, biological processes and pathways in the placenta that act in response to early maternal nutrition are yet to be elucidated. Herein, we examined the impact of maternal vitamin and mineral supplementation (from pre-breeding to day 83 post-breeding) and two rates of gain during the first 83 days of pregnancy on the gene expression of placental caruncles (CAR; maternal placenta) and cotyledons (COT; fetal placenta) of crossbred Angus beef heifers. We identified 267 unique differentially expressed genes (DEG). Among the DEGs from CAR, we identified ACAT2, SREBF2, and HMGCCS1 that underlie the cholesterol biosynthesis pathway. Furthermore, the transcription factors PAX2 and PAX8 were over-represented in biological processes related to kidney organogenesis. The DEGs from COT included SLC2A1, SLC2A3, SLC27A4, and INSIG1. Our over-representation analysis retrieved biological processes related to nutrient transport and ion homeostasis, whereas the pathways included insulin secretion, PPAR signaling, and biosynthesis of amino acids. Vitamin and mineral supplementation and rate of gain were associated with changes in gene expression, biological processes, and KEGG pathways in beef cattle placental tissues.

Suppressing Hippo signaling in the stem cell niche promotes skeletal muscle regeneration.

Lack of blood flow to the lower extremities in peripheral arterial disease causes oxygen and nutrient deprivation in ischemic skeletal muscles, leading to functional impairment. Treatment options for muscle regeneration in this scenario are lacking. Here, we selectively targeted the Hippo pathway in myofibers, which provide architectural support for muscle stem cell niches, to facilitate functional muscle recovery in ischemic extremities by promoting angiogenesis, neovascularization, and myogenesis. We knocked down the core Hippo pathway component, Salvador (SAV1), by using an adeno-associated virus 9 (AAV9) vector expressing a miR30-based triple short-hairpin RNA (shRNA), controlled by a muscle-specific promoter. In a mouse hindlimb-ischemia model, AAV9 SAV1 shRNA administration in ischemic muscles induced nuclear localization of the Hippo effector YAP, accelerated perfusion restoration, and increased exercise endurance. Intravascular lectin labeling of the vasculature revealed enhanced angiogenesis. Using 5-ethynyl-2'-deoxyuridine to label replicating cellular DNA in vivo, we found SAV1 knockdown concurrently increased paired box transcription factor Pax7+ muscle satellite cell and CD31+ endothelial cell proliferation in ischemic muscles. To further study Hippo suppression in skeletal muscle regeneration, we used a cardiotoxin-induced muscle damage model in adult (12-15 weeks old) and aged mice (26-month old). Two weeks after delivery of AAV9 SAV1 shRNA into injured muscles, the distribution of regenerative myofibers shifted toward a larger cross-sectional area and increased capillary density compared with mice receiving AAV9 control. Together, these findings suggest our approach may have clinical promise in regenerative therapy for leg ischemia and muscle injury.