Pax6 Promoter Research Articles

Lactate promotes H3K18 lactylation in human neuroectoderm differentiation

In mammals, early embryonic gastrulation process is high energy demanding. Previous studies showed that, unlike endoderm and mesoderm cells, neuroectoderm differentiated from human embryonic stem cells relied on aerobic glycolysis as the major energy metabolic process, which generates lactate as the final product. Here we explored the function of intracellular lactate during neuroectoderm differentiation. Our results revealed that the intracellular lactate level was elevated in neuroectoderm and exogenous lactate could further promote hESCs differentiation towards neuroectoderm. Changing intracellular lactate levels by sodium lactate or LDHA inhibitors had no obvious effect on BMP or WNT/β-catenin signaling during neuroectoderm differentiation. Notably, histone lactylation, especially H3K18 lactylation was significant upregulated during this process. We further performed CUT&Tag experiments and the results showed that H3K18la is highly enriched at gene promoter regions. By analyzing data from CUT&Tag and RNA-seq experiments, we further identified that four genes, including PAX6, were transcriptionally upregulated by lactate during neuroectoderm differentiation. A H3K18la modification site at PAX6 promoter was verified and exogenous lactate could also rescue the level of PAX6 after shPAX6 inhibition.

Myo-differentiation reporter screen reveals NF-Y as an activator of PAX3–FOXO1 in rhabdomyosarcoma

Recurrent chromosomal rearrangements found in rhabdomyosarcoma (RMS) produce the PAX3-FOXO1 fusion protein, which is an oncogenic driver and a dependency in this disease. One important function of PAX3-FOXO1 is to arrest myogenic differentiation, which is linked to the ability of RMS cells to gain an unlimited proliferation potential. Here, we developed a phenotypic screening strategy for identifying factors that collaborate with PAX3-FOXO1 to block myo-differentiation in RMS. Unlike most genes evaluated in our screen, we found that loss of any of the three subunits of the Nuclear Factor Y (NF-Y) complex leads to a myo-differentiation phenotype that resembles the effect of inactivating PAX3-FOXO1. While the transcriptomes of NF-Y- and PAX3-FOXO1-deficient RMS cells bear remarkable similarity to one another, we found that these two transcription factors occupy nonoverlapping sites along the genome: NF-Y preferentially occupies promoters, whereas PAX3-FOXO1 primarily binds to distal enhancers. By integrating multiple functional approaches, we map the PAX3 promoter as the point of intersection between these two regulators. We show that NF-Y occupies CCAAT motifs present upstream of PAX3 to function as a transcriptional activator of PAX3-FOXO1 expression in RMS. These findings reveal a critical upstream role of NF-Y in the oncogenic PAX3-FOXO1 pathway, highlighting how a broadly essential transcription factor can perform tumor-specific roles in governing cellular state.

Zhx2 maintains islet β-cell mass and function by transcriptionally regulating Pax6

Emerging evidence shows that pancreatic β-cell function and quality are key determinants in the progression of type 2 diabetes (T2D). The transcription factor zinc finger homeobox 2 (Zhx2) is involved in proliferation and development of multiple cells. However, the exact role of Zhx2 in β-cells and T2D remains completely unknown. Here, we report that Zhx2 orchestrates β-cell mass and function by regulating paired box protein pax-6 (Pax6). We found that β-cell-specific knockout Zhx2 (Zhx2BKO) mice showed a decrease in β-cell proliferation and glucose homeostasis. Under prediabetic and diabetic conditions, we discovered glucose intolerance in both Zhx2BKO-HFD mice and Zhx2BKO-db/db mice, with reduced β-cell mass and insulin secretion. Mechanistically, we demonstrated that Zhx2 targeted the Pax6 promoter region (-1740∼-1563; -862∼-559; -251∼+75), enhanced promoter activity. Overall, Zhx2 maintains β-cell function by transcriptionally regulating Pax6, which provides a therapeutic target for diabetes intervention.

Abstract 3122: Negative correlation of single-cell PAX3:FOXO1 expression with tumorigenicity in rhabdomyosarcoma

Abstract Background: Rhabdomyosarcomas (RMS) are phenotypically and functionally heterogeneous. Expression of the fusion oncogene PAX3:FOXO1 (P3F) was previously shown to differ between individual tumor cells and fluctuate over time. Methods: In mouse Myf6Cre+/-,Pax3:Foxo1+/+,p53-/- RMS tumors, expression of P3F is directed by the Pax3 promoter and coupled to an eYFP fluorescent marker, which is activated as a second cistron downstream from an encephalomyocarditis virus-derived internal ribosome entry site. Low passage Myf6Cre+/-,Pax3:Foxo1+/+,p53-/- mouse RMS cell lines and primary human RMS cultures were used to study the functional impact of variable P3F expression at the cellular level in RMS. Results: The Myf6Cre+/-,Pax3:Foxo1+/+,p53-/- mouse RMS cell pool contains cells expressing different levels of YFP, correlating with variable P3F expression. Single-cell PCR was used to demonstrate substantial cell-to-cell variability in P3F expression in the human RMS cell pool. Myf6Cre+/-,Pax3:Foxo1+/+,p53-/- mouse RMS cells were then sub-fractionated by fluorescence-activated cell sorting (FACS) to discriminate YFPhigh/P3Fhigh and YFPlow/P3Flow cell subsets. YFPlow/P3Flow mouse RMS cells included 87% G0/G1 cells and reorganized their actin cytoskeleton to produce a cellular phenotype characterized by more efficient adhesion and migration. This translated into higher tumor-propagating cell frequencies of YFPlow/P3Flow compared to YFPhigh/P3Fhigh cells after injection into the extremity muscles of immunocompromised mice. We also observed higher clonal activity of YFPlow/P3Flow compared to YFPhigh/P3Fhigh cells in vitro. Both YFPlow/P3Flow and YFPhigh/P3Fhigh cells gave rise to mixed clones in vitro, consistent with fluctuations in P3F expression over time. Finally, exposure to the anti-tropomyosin compound TR100 disrupted the cytoskeleton and reversed enhanced migration and adhesion of YFPlow/P3Flow RMS cells. Conclusions: Our observations indicate that therapies aimed at eliminating P3Fhigh cells by targeting the fusion oncogene may not cure the disease. Moreover, dynamic expression of PAX3:FOXO1 at the single cell level may result in adaptive plasticity, allow RMS cells to adapt to environmental challenges and provide them with a critical advantage during tumor progression. Citation Format: Carla Regina, Geoffroy Andriuex, Sina Angenendt, Michaela Schneider, Manching Ku, Marie Follo, Marco Wachtel, Eugene Ke, Ken Kikuchi, Anton G. Henssen, Beat W. Schäfer, Melanie Boerries, Amy J. Wagers, Charles Keller, Simone Hettmer. Negative correlation of single-cell PAX3:FOXO1 expression with tumorigenicity in rhabdomyosarcoma [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 3122.

Negative correlation of single-cell PAX3:FOXO1 expression with tumorigenicity in rhabdomyosarcoma.

Rhabdomyosarcomas (RMS) are phenotypically and functionally heterogeneous. Both primary human RMS cultures and low-passage Myf6Cre,Pax3:Foxo1,p53 mouse RMS cell lines, which express the fusion oncoprotein Pax3:Foxo1 and lack the tumor suppressor Tp53 (Myf6Cre,Pax3:Foxo1,p53), exhibit marked heterogeneity in PAX3:FOXO1 (P3F) expression at the single cell level. In mouse RMS cells, P3F expression is directed by the Pax3 promoter and coupled to eYFP YFPlow/P3Flow mouse RMS cells included 87% G0/G1 cells and reorganized their actin cytoskeleton to produce a cellular phenotype characterized by more efficient adhesion and migration. This translated into higher tumor-propagating cell frequencies of YFPlow/P3Flow compared with YFPhigh/P3Fhigh cells. Both YFPlow/P3Flow and YFPhigh/P3Fhigh cells gave rise to mixed clones in vitro, consistent with fluctuations in P3F expression over time. Exposure to the anti-tropomyosin compound TR100 disrupted the cytoskeleton and reversed enhanced migration and adhesion of YFPlow/P3Flow RMS cells. Heterogeneous expression of PAX3:FOXO1 at the single cell level may provide a critical advantage during tumor progression.

Overexpression of Isl1 under the Pax2 Promoter, Leads to Impaired Sound Processing and Increased Inhibition in the Inferior Colliculus

The LIM homeodomain transcription factor ISL1 is essential for the different aspects of neuronal development and maintenance. In order to study the role of ISL1 in the auditory system, we generated a transgenic mouse (Tg) expressing Isl1 under the Pax2 promoter control. We previously reported a progressive age-related decline in hearing and abnormalities in the inner ear, medial olivocochlear system, and auditory midbrain of these Tg mice. In this study, we investigated how Isl1 overexpression affects sound processing by the neurons of the inferior colliculus (IC). We recorded extracellular neuronal activity and analyzed the responses of IC neurons to broadband noise, clicks, pure tones, two-tone stimulation and frequency-modulated sounds. We found that Tg animals showed a higher inhibition as displayed by two-tone stimulation; they exhibited a wider dynamic range, lower spontaneous firing rate, longer first spike latency and, in the processing of frequency modulated sounds, showed a prevalence of high-frequency inhibition. Functional changes were accompanied by a decreased number of calretinin and parvalbumin positive neurons, and an increased expression of vesicular GABA/glycine transporter and calbindin in the IC of Tg mice, compared to wild type animals. The results further characterize abnormal sound processing in the IC of Tg mice and demonstrate that major changes occur on the side of inhibition.

PAX2 promoter methylation and AIB1 overexpression promote tamoxifen resistance in breast carcinoma patients.

Disease recurrence is an important obstacle in estrogen receptor positive (ER+) tamoxifen treated breast carcinoma patients. Tamoxifen resistance-related molecular mechanisms are not fully understood. Alteration in DNA methylation which contributes to transcriptional regulation of cancer-related genes plays a crucial role in tamoxifen response. In the present study, the contribution of promoter methylation and mRNA expression of PAX2 and AIB1 in the development of breast carcinoma and tamoxifen refractory was assessed. Methylation specific-high resolution melting (MS-HRM) analysis and Real-time quantitative PCR (RT-qPCR) experiment were performed to analyze the promoter methylation and mRNA expression levels of PAX2 and AIB1 genes in 102 breast tumors and adjacent normal breast specimens. We indicated that PAX2 expression is decreased in breast tissues due to hypermethylation in its promoter region. Compared to the adjacent normal tissues, the tumors exhibited significantly lower relative mRNA levels of PAX2 and increased expression of AIB1. Aberrant promoter methylation of PAX2 and overexpression of AIB1 was observed in tamoxifen resistance patients compared to the sensitive ones. Cox regression analysis exhibited that the increased promoter methylation status of PAX2 and overexpression of AIB1 remained as unfavorable identifiers which influence patients' survival independently. Our results revealed that the aberration in PAX2 promoter methylation and AIB1 overexpression are associated with the tamoxifen response in breast carcinoma patients. Further research is needed to demonstrate the potential of using PAX2 and AIB1 expression and their methylation-mediated regulation as predictive or prognostic biomarkers or as a new target therapy for better disease management.

Abstract B12: PAX3 translocations co-opt super enhancers and intrinsically disordered fusion partners in rhabdomyosarcoma

Abstract Chromosomal translocations drive many types of childhood cancer. Fusion-positive rhabdomyosarcoma (FP-RMS) tumors are found most frequently with a fusion between PAX3 and FOXO1, but less frequent translocation partners have been discovered, including INO80D and NCOA1. We hypothesized that all FP-RMS translocations are selecting simultaneously for (1) enhancers active in a myoblast-like epigenome and (2) protein partners with high-levels of intrinsic disorder to give PAX3 enhanced transcriptional strength. Analysis of primary tumors revealed these diverse PAX3 fusions recapitulate a near-identical transcriptome, suggesting uniform underlying molecular mechanisms. ChIP-seq evidence from cell lines and primary tumors suggested that in all FP-RMS tumors, large super-enhancer (SE) elements were present near each chosen translocation partner (distal to FOXO1, INO80D, or NCOA1). Using tools to determine the 3-D folding of chromatin (3C, 4C-seq, and HiChIP), we discovered an extensive network of hijacked FOXO1 enhancers and a SE that physically interact together and with the PAX3 promoter, only in PAX3-FOXO1 positive cells. Furthermore, pooled CRISPR tiling of cis-regulatory elements revealed special dependence on the FOXO1 SE, or certain CTCF boundary elements that facilitate enhancer interactions. ChIP-seq paired to short-term CRISPR experiments shows PAX3-FOXO1 transcription depends on an extended network of related enhancers distal to FOXO1. We find these enhancers are unique to early myoblast stages of differentiation and are bound by myogenic TFs in RMS, suggesting miswiring of normal myogenic enhancer logic. While many SE-driven genes exist in FP-RMS, most are never selected for translocation partners. We found that FOXO1, INO80D, and NCOA1 are all highly disordered proteins, a theme they hold in common despite having no amino acid sequence homology. Initial evidence suggests that PAX3-FOXO1 enables the formation of phase condensates in the nucleus that recruit high-levels of transcriptional machinery such as BRD4. Ongoing work is exploring the mechanistic chemical determinants of these interactions, and how phase condensates are manipulated by drugs that block PAX3-FOXO1’s transcriptional output. Together our studies are illuminating new paradigms for understanding how fusion transcription factors drive cancer. Citation Format: Berkley E. Gryder, Marco Wachtel, Winston Ewert, Kenneth Chang, Osama El Demerdash, Young Song, Beat W Schäfer, Christopher R. Vakoc, Javed Khan. PAX3 translocations co-opt super enhancers and intrinsically disordered fusion partners in rhabdomyosarcoma [abstract]. In: Proceedings of the AACR Special Conference on the Advances in Pediatric Cancer Research; 2019 Sep 17-20; Montreal, QC, Canada. Philadelphia (PA): AACR; Cancer Res 2020;80(14 Suppl):Abstract nr B12.

TET1 Deficiency Impairs Morphogen-free Differentiation of Human Embryonic Stem Cells to Neuroectoderm

The TET family of 5-methylcytosine (5mC) dioxygenases plays critical roles in development by modifying DNA methylation. Using CRISPR, we inactivated the TET1 gene in H9 human embryonic stem cells (hESCs). Mutant H9 hESCs remained pluripotent, even though the level of hydroxymethylcytosine (5hmC) decreased to 30% of that in wild-type cells. Neural differentiation induced by dual SMAD inhibitors was not significantly affected by loss of TET1 activity. However, in a morphogen-free condition, TET1 deficiency significantly reduced the generation of NESTIN+SOX1+ neuroectoderm cells from 70% in wild-type cells to 20% in mutant cells. This was accompanied by a 20-fold reduction in the expression level of PAX6 and a significant decrease in the amount of 5hmC on the PAX6 promoter. Overexpression of the TET1 catalytic domain in TET1-deficient hESCs significantly increased 5hmC levels and elevated PAX6 expression during differentiation. Consistent with these in vitro data, PAX6 expression was significantly decreased in teratomas formed by TET1-deficient hESCs. However, TET1 deficiency did not prevent the formation of neural tube-like structures in teratomas. Our results suggest that TET1 deficiency impairs the intrinsic ability of hESCs to differentiate to neuroectoderm, presumably by decreasing the expression of PAX6, a key regulator in the development of human neuroectoderm.

Miswired Enhancer Logic Drives a Cancer of the Muscle Lineage.

SummaryCore regulatory transcription factors (CR TFs) establish enhancers with logical ordering during embryogenesis and development. Here we report that in fusion-positive rhabdomyosarcoma, a cancer of the muscle lineage, the chief oncogene PAX3-FOXO1 is driven by a translocated FOXO1 super enhancer (SE) restricted to a late stage of myogenesis. Using chromatin conformation capture techniques, we demonstrate that the extensive FOXO1 cis-regulatory domain interacts with PAX3. Furthermore, RNA sequencing and chromatin immunoprecipitation sequencing data in tumors bearing rare PAX translocations implicate enhancer miswiring across all fusion-positive tumors. HiChIP of H3K27ac showed connectivity between the FOXO1 SE, additional intra-domain enhancers, and the PAX3 promoter. We show that PAX3-FOXO1 transcription is diminished when this network of enhancers is ablated by CRISPR. Our data reveal a hijacked enhancer network that disrupts the stepwise CR TF logic of normal skeletal muscle development (PAX3 to MYOD to MYOG), replacing it with an “infinite loop” enhancer logic that locks rhabdomyosarcoma in an undifferentiated stage.

Aberrant DNA methylation in the PAX2 promoter is associated with Müllerian duct anomalies.

Abnormalities during Müllerian duct and female reproductive tract formation during embryonic development result in Müllerian duct anomalies (MDA). Previous studies have identified a role for mutations in related genes and DNA copy number variation (CNV). However, the correlation between gene methylation and MDA remains to be understood. Endometrial tissues were collected from patients with septate (n = 23) or normal uterus (n = 28). We detected the methylation status of CpG sites and mRNA levels of nine candidate genes, including HOXA10, EMX2, TP63, ITGB3, PAX2, LHX1, GSC, WNT4, and H19, using MethyTarget and quantitative real-time polynucleotide chain reaction (qRT-PCR), respectively RESULTS: Compared with healthy controls, we detected three hypomethylated CpG sites (P < 0.05) and increased mRNA levels of PAX2 (P < 0.05) in individuals with MDA. HOXA10, EMX2, TP63, ITGB3, LHX1, and GSC had 1, 1, 2, 1, 5, and 2 differentially methylated CpG sites (P < 0.05), respectively, but there were no significant differences in their mRNA levels (P > 0.05). WNT4 and H19 did not show differences in methylation (P > 0.05) and mRNA levels (P > 0.05). Aberrant DNA methylation within the promoter of PAX2 may contribute to the development of MDA by regulating its gene expression. However, the methylation status of HOXA10, EMX2, TP63, ITGB3, LHX1, GSC, WNT4, and H19, may not contribute to the development of MDA.

Activating β-catenin/Pax6 axis negatively regulates osteoclastogenesis by selectively inhibiting phosphorylation of p38/MAPK.

Balance of osteoclast formation is regulated by the receptor activator of NF-κB ligand and extracellular negative regulators such as IFN-γ and IFN-β. However, very little is known about the intrinsic negative regulatory factors of osteoclast differentiation. Recently, the paired-box homeodomain transcription factor Pax6 was shown to negatively regulate receptor activator of NF-κB ligand-mediated osteoclast differentiation. However, the mechanism underlying this regulation is still unclear. In this study, we show that a p38 inhibitor (VX-745) up-regulates the expression of Pax6 during osteoclast differentiation. Subsequently, we found that β-catenin could bind to the proximal region of Pax6 promoter to induce its expression, and this action could be impaired by p38-induced ubiquitin-mediated degradation of β-catenin. Our results suggest that Pax6 is regulated by a novel p38/β-catenin pathway. Pax6 can further regulate the nuclear translocation of NF of activated T cells, cytoplasmic 1. Our study indicates that this novel p38/β-catenin/Pax6 axis contributes to negative regulation of osteoclastogenesis. In addition, our study proposes a novel approach to treat osteoclast-related diseases through the use of VX-745 complemented with the β-catenin activator SKL2001.-Jie, Z., Shen, S., Zhao, X., Xu, W., Zhang, X., Huang, B., Tang, P., Qin, A., Fan, S., Xie, Z. Activating β-catenin/Pax6 axis negatively regulates osteoclastogenesis by selectively inhibiting phosphorylation of p38/MAPK.

Methylation status of the putative Pax6 promoter in olive ridley sea turtle embryos with eye defects: An initial approach

Normal development involves the interplay of genetic and epigenetic regulatory mechanisms. Pax6 is an eye-selector factor responsible for initiating the regulatory cascade for the development of the eyes. For the olive ridley sea turtle (Lepidochelys olivacea), a threatened species, eye malformations have been reported. In order to study the DNA methylation status of the putative promoter of the Pax6 gene in embryos with ocular malformations, an exploratory study was carried out in which DNA was isolated from embryos with anophthalmia, microphthalmia, and cyclopia, as well as from their normal counterparts. The 5′-flanking region from the Pax6 gene was isolated, showing two CpG islands (CGIs). The methylation status of CGIs in malformed embryos was compared with that of normal embryos by bisulfite sequencing. Putative transcription factor binding sites and regulatory features were identified. Methylation patterns were observed in both CpG and non-CpG contexts, and were unique for each malformed embryo; in the CpG context, an embryo with cyclopia showed a methylated cytosine upstream the CGI-1 not present in other embryos, an embryo with left anophthalmia presented two methylated cytosines in the CGI-1, whereas an embryo with left anophthalmia and right microphthalmia showed two methylated cytosines in the CGI-2. Normal embryos did not show methylated cytosines in the CGI-1, but one of them showed one methylcytosine in the CGI-2. Methylated transcription factor-binding sites may affect Pax6 expression associated to the cellular response to environmental compounds and hypoxia, signal transduction, cell cycle, lens physiology and development, as well as the transcription rate. Although preliminary, these results suggest that embryos with ocular malformations present unique DNA methylation patterns in the putative promoter of the Pax6 gene in L. olivacea, and probably those subtle, random changes in the methylation status can cause (at least in part) the aberrant phenotypes observed in these embryos.

Functional characteristics of novel pancreatic Pax6 regulatory elements.

Complex diseases, such as diabetes, are influenced by comprehensive transcriptional networks. Genome-wide association studies have revealed that variants located in regulatory elements for pancreatic transcription factors are linked to diabetes, including those functionally linked to the paired box transcription factor Pax6. Pax6 deletions in adult mice cause rapid onset of classic diabetes, but the full spectrum of pancreatic Pax6 regulators is unknown. Using a regulatory element discovery approach, we identified two novel Pax6 pancreatic cis-regulatory elements in a poorly characterized regulatory desert. Both new elements, Pax6 pancreas cis-regulatory element 3 (PE3) and PE4, are located 50 and 100 kb upstream and interact with different parts of the Pax6 promoter and nearby non-coding RNAs. They drive expression in the developing pancreas and brain and code for multiple pancreas-related transcription factor-binding sites. PE3 binds CCCTC-binding factor (CTCF) and is marked by stem cell identity markers in embryonic stem cells, whilst a common variant located in the PE4 element affects binding of Pax4, a known pancreatic regulator, altering Pax6 gene expression. To determine the ability of these elements to regulate gene expression, synthetic transcriptional activators and repressors were targeted to PE3 and PE4, modulating Pax6 gene expression, as well as influencing neighbouring genes and long non-coding RNAs, implicating the Pax6 locus in pancreas function and diabetes.

SMAD proteins directly suppress PAX2 transcription downstream of transforming growth factor-beta 1 (TGF-β1) signalling in renal cell carcinoma.

Canonical TGF-β1 signalling promotes tumor progression by facilitating invasion and metastasis, whereby release of TGF-β1, by (for example) infiltrating immune cells, induces epithelial to mesenchymal transition (EMT). PAX2, a member of the Paired box family of transcriptional regulators, is normally expressed during embryonic development, including in the kidney, where it promotes mesenchymal to epithelial transition (MET). PAX2 expression is silenced in many normal adult tissues. However, in contrast, PAX2 is expressed in several cancer types, including kidney, prostate, breast, and ovarian cancer. While multiple studies have implicated TGF-β superfamily members in modulating expression of Pax genes during embryonic development, few have investigated direct regulation of Pax gene expression by TGF-β1. Here we have investigated direct regulation of PAX2 expression by TGF-β1 in clear cell renal cell carcinoma (CC-RCC) cell lines. Treatment of PAX2-expressing 786-O and A498 CC-RCC cell lines with TGF-β1 resulted in inhibition of endogenous PAX2 mRNA and protein expression, as well as expression from transiently transfected PAX2 promoter constructs; this inhibition was abolished in the presence of expression of the inhibitory SMAD, SMAD7. Using ChIP-PCR we showed TGF-β1 treatment induced SMAD3 protein phosphorylation in 786-O cells, and direct SMAD3 binding to the human PAX2 promoter, which was inhibited by SMAD7 over-expression. Overall, these data suggest that canonical TGF-β signalling suppresses PAX2 transcription in CC-RCC cells due to the direct binding of SMAD proteins to the PAX2 promoter. These studies improve our understanding of tumor progression and epithelial to mesenchyme transition (EMT) in CC-RCC and in other PAX2-expressing cancer types.

Aucubin Promotes Differentiation of Neural Precursor Cells into GABAergic Neurons.

Aucubin is a small compound naturally found in traditional medicinal herbs with primarily anti-inflammatory and protective effects. In the nervous system, aucubin is reported to be neuroprotective by enhancing neuronal survival and inhibiting apoptotic cell death in cultures and disease models. Our previous data, however, suggest that aucubin facilitates neurite elongation in cultured hippocampal neurons and axonal regrowth in regenerating sciatic nerves. Here, we investigated whether aucubin facilitates the differentiation of neural precursor cells (NPCs) into specific types of neurons. In NPCs cultured primarily from the rat embryonic hippocampus, aucubin significantly elevated the number of GAD65/67 immunoreactive cells and the expression of GAD65/67 proteins was upregulated dramatically by more than three-fold at relatively low concentrations of aucubin (0.01 µM to 10 µM). The expression of both NeuN and vGluT1 of NPCs, the markers for neurons and glutamatergic cells, respectively, and the number of vGluT1 immunoreactive cells also increased with higher concentrations of aucubin (1 µM and 10 µM), but the ratio of the increases was largely lower than GAD expression and GAD immunoreactive cells. The GABAergic differentiation of pax6-expressing late NPCs into GABA-producing cells was further supported in cortical NPCs primarily cultured from transgenic mouse brains, which express recombinant GFP under the control of pax6 promoter. The results suggest that aucubin can be developed as a therapeutic candidate for neurodegenerative disorders caused by the loss of inhibitory GABAergic neurons.

PAX6 Promoter Methylation Correlates with MDA-MB-231 Cell Migration, and Expression of MMP2 and MMP9

Objective:Breast cancer is a heterogeneous disease characterized by an accumulation of genetic and epigenetic alterations that lead tumor cells to acquire characteristics like the capacity for invasion and metastasis. Metastasis remains a major challenge in cancer management and understanding of its molecular basis should result in improved prevention, diagnosis, and treatment of breast cancer patients. The aim of this study was to investigate how promoter DNA methylation regulates PAX6 gene expression and influences breast carcinoma cell migration.Methods:PAX6 promoter methylation was detected by Methyl Specific-Multiplex Ligation Probe Amplification (MS-MLPA). Gene expression was evaluated using qRT-PCR, while the effect of PAX6 on migration was ssessed by wound healing assay. In addition, MMP2 and MMP9 genes were studied using different bioinformatic tools.Results:The PAX6 promoter is methylated in breast cancer cell lines and methylation in this region impacts on its expression. Migration assays revealed that PAX6 overexpression promotes cell migration, while PAX6 inhibition decreases it. More importantly, we found that migration is affected by PAX6 methylation status. Employing bioinformatic analysis, binding sites for PAX6 on the regulatory regions of the MMP2 and MMP9 genes were established, PAX6 overexpression increasing MMP2 and MMP9 expression at the mRNA level.Conclusion:Our study provides novel insights into epigenetic events that regulate PAX6 expression and molecular mechanisms by which PAX6 modifies the migration capacity of breast cancer cells.

Genomic and epigenetic signatures associated with survival rate in oral squamous cell carcinoma patients.

Purpose: Although oral squamous cell carcinoma (OSCC) presents great mortality and morbidity worldwide, the mechanisms behind its clinical behavior remain unclear. Biomarkers are needed to forecast patients' survival and, among those patients undergoing curative therapy, which are more likely to develop tumor recurrence/metastasis. Demonstrating clinical relevance of these biomarkers could be crucial both for surveillance and in helping to establish adjuvant therapy strategies. We aimed to identify genomic and epigenetic biomarkers of OSCC prognosis as well as to explore a noninvasive strategy to perform its detection.Methods: OSCC tumor and non-tumor tissue samples and cells scrapped from the tumor surface were genomic and epigenetically evaluated by Methylation-Specific Multiplex Ligation-dependent Probe Amplification technique.Results: Copy number alterations in ATM, CASR, TP73, CADM1, RARB, CDH13, PAX5, RB1 genes and GATA5, PAX6, CADM1 and CHFR promoter methylation were shown to be associated with worse OSCC patients' survival. Copy number alterations in BRCA1, CDKN2A, CHFR, GATA5, PYCARD, STK11, TP53, VHL genes and GATA5, CADM1, KLLN, MSH6, PAX5, WT1 promoter methylation were shown to be associated with development of metastasis/relapses during or after OSCC patients' treatment. We also found a good agreement in the status of CDKN2A promoter methylation evaluated noninvasively or in the tumor tissue.Conclusions: Genomic and epigenetic signatures were validated in a larger and geographically separate cohort, from TCGA database, which reinforce their clinical applicability. Noninvasive methodologies for detection of these signatures require further studies before translation in to clinical practice.

TET proteins safeguard bivalent promoters from de novo methylation in human embryonic stem cells.

The TET enzymes oxidize 5-methylcytosine to 5-hydroxymethylcytosine, which can lead to DNA demethylation. However, direct connections between TET-mediated DNA demethylation and transcriptional output are difficult to establish due to challenges of distinguishing global versus locus-specific effects. Here we show that TET1/2/3 triple knockout (TKO) human embryonic stem cells (hESCs) exhibit prominent bivalent promoter hypermethylation without an overall corresponding gene expression decrease in the undifferentiated state. Focusing on the bivalent PAX6 locus, we find increased DNMT3B binding is associated with promoter hypermethylation, which precipitates a neural differentiation defect and failure of PAX6 induction during differentiation. dCas9-mediated locus-specific demethylation and global inactivation of DNMT3B in TKO hESCs partially reverses the hypermethylation at the PAX6 promoter and improves differentiation to neuroectoderm. Taken together with further genome-wide methylation and TET1 and DNMT3B ChIP-Seq analysis, we conclude that the TET proteins safeguard bivalent promoters from de novo methylation to ensure robust lineage-specific transcription upon differentiation.

Regulatory landscape fusion in rhabdomyosarcoma through interactions between the PAX3 promoter and FOXO1 regulatory elements

BackgroundThe organisation of vertebrate genomes into topologically associating domains (TADs) is believed to facilitate the regulation of the genes located within them. A remaining question is whether TAD organisation is achieved through the interactions of the regulatory elements within them or if these interactions are favoured by the pre-existence of TADs. If the latter is true, the fusion of two independent TADs should result in the rewiring of the transcriptional landscape and the generation of ectopic contacts.ResultsWe show that interactions within the PAX3 and FOXO1 domains are restricted to their respective TADs in normal conditions, while in a patient-derived alveolar rhabdomyosarcoma cell line, harbouring the diagnostic t(2;13)(q35;q14) translocation that brings together the PAX3 and FOXO1 genes, the PAX3 promoter interacts ectopically with FOXO1 sequences. Using a combination of 4C-seq datasets, we have modelled the three-dimensional organisation of the fused landscape in alveolar rhabdomyosarcoma.ConclusionsThe chromosomal translocation that leads to alveolar rhabdomyosarcoma development generates a novel TAD that is likely to favour ectopic PAX3:FOXO1 oncogene activation in non-PAX3 territories. Rhabdomyosarcomas may therefore arise from cells which do not normally express PAX3. The borders of this novel TAD correspond to the original 5'- and 3'- borders of the PAX3 and FOXO1 TADs, respectively, suggesting that TAD organisation precedes the formation of regulatory long-range interactions. Our results demonstrate that, upon translocation, novel regulatory landscapes are formed allowing new intra-TAD interactions between the original loci involved.