Altered NFE2 activity predisposes to leukemic transformation and myelosarcoma with AML-specific aberrations.

In acute myeloid leukemia (AML), acquired genetic aberrations carry prognostic implications and guide therapeutic decisions. Clinical algorithms have been improved by the incorporation of novel aberrations. Here, we report the presence and functional characterization of mutations in the transcription factor NFE2 in patients with AML and in a patient with myelosarcoma. We previously described NFE2 mutations in patients with myeloproliferative neoplasms and demonstrated that expression of mutant NFE2 in mice causes a myeloproliferative phenotype. Now, we show that, during follow-up, 34% of these mice transform to leukemia presenting with or without concomitant myelosarcomas, or develop isolated myelosarcomas. These myelosarcomas and leukemias acquired AML-specific alterations, including the murine equivalent of trisomy 8, loss of the AML commonly deleted region on chromosome 5q, and mutations in the tumor suppressor Trp53 Our data show that mutations in NFE2 predispose to the acquisition of secondary changes promoting the development of myelosarcoma and/or AML.

CEACAM3 decreases asthma exacerbations and modulates respiratory syncytial virus latent infection in children.

BACKGROUND: Respiratory syncytial virus (RSV) is associated with childhood asthma. Nevertheless, not all children exposed to RSV develop asthma symptoms, possibly because genes modulate the effects of RSV on asthma exacerbations. OBJECTIVE: The purpose of this study was to identify genes that modulate the effect of RSV latent infection on asthma exacerbations. METHODS: We performed a meta-analysis to investigate differentially expressed genes (DEGs) of RSV infection from Gene Expression Omnibus datasets. Expression quantitative trait loci (eQTL) methods were applied to select single nucleotide polymorphisms (SNPs) that were associated with DEGs. Gene-based analysis was used to identify SNPs that were significantly associated with asthma exacerbations in the Taiwanese Consortium of Childhood Asthma Study (TCCAS), and validation was attempted in an independent cohort, the Childhood Asthma Management Program (CAMP). Gene-RSV interaction analyses were performed to investigate the association between the interaction of SNPs and RSV latent infection on asthma exacerbations. RESULTS: A total of 352 significant DEGs were found by meta-analysis of RSV-related genes. We used 38 123 SNPs related to DEGs to investigate the genetic main effects on asthma exacerbations. We found that eight RSV-related genes (GADD45A, GYPB, MS4A3, NFE2, RNASE3, EPB41L3, CEACAM6 and CEACAM3) were significantly associated with asthma exacerbations in TCCAS and also validated in CAMP. In TCCAS, rs7251960 (CEACAM3) significantly modulated the effect of RSV latent infection on asthma exacerbations (false-discovery rate <0.05). The rs7251960 variant was associated with CEACAM3 mRNA expression in lung tissue (p for trend=1.2×10-7). CEACAM3 mRNA was reduced in nasal mucosa from subjects with asthma exacerbations in two independent datasets. CONCLUSIONS: rs7251960 is an eQTL for CEACAM3, and CEACAM3 mRNA expression is reduced in subjects experiencing asthma exacerbations. CEACAM3 may be a modulator of RSV latent infection on asthma exacerbations.

Nuclear factor erythroid 2 (NF-E2) p45-related factor 2 interferes with homeodomain-interacting protein kinase 2/p53 activity to impair solid tumors chemosensitivity.

Resistance to chemotherapy represents a major hurdle to successful cancer treatment. A key role for efficient response to anticancer therapies is played by TP53 oncosuppressor gene that indeed is mutated in 50% of human cancers or inactivated at protein level in the remaining 50%. Homeodomain-interacting protein kinase 2 (HIPK2) is the wild-type p53 (wtp53) apoptotic activator, and its inhibition by hypoxia or hyperglycemia may contribute to tumor chemoresistance mainly by impairing p53 apoptotic activity. Another important molecule able to induce chemoresistance is nuclear factor erythroid 2 (NF-E2) p45-related factor 2 (NRF2) transcription factor, whose activation by oxidative and/or electrophilic stress regulates a transcriptional antioxidant program allowing cancer cells to adapt and survive to stresses. NRF2 may shift from cytoprotective to tumor-promoting function, according to tumor phases. NRF2 may crosstalk with both wtp53 and mutant p53 (mutp53), inhibiting the wtp53 apoptotic function and strengthening the mutp53 oncogenic function. NRF2 has also been shown to induce HIPK2 mRNA expression cooperating in inducing cytoprotection. Although HIPK2, p53, and NRF2 have been individually extensively studied, their interplay has not been clearly addressed yet. On the basis of the background and our results, we aim at hypothesizing the unexpected pro-survival activity played by the NRF2/HIPK2/p53 interplay that can be hijacked by cancer cells to bypass drugs cytotoxicity.

Epigenetic regulation of NFE2 overexpression in myeloproliferative neoplasms.

The transcription factor "nuclear factor erythroid 2" (NFE2) is overexpressed in the majority of patients with myeloproliferative neoplasms (MPNs). In murine models, elevated NFE2 levels cause an MPN phenotype with spontaneous leukemic transformation. However, both the molecular mechanisms leading to NFE2 overexpression and its downstream targets remain incompletely understood. Here, we show that the histone demethylase JMJD1C constitutes a novel NFE2 target gene. JMJD1C levels are significantly elevated in polycythemia vera (PV) and primary myelofibrosis patients; concomitantly, global H3K9me1 and H3K9me2 levels are significantly decreased. JMJD1C binding to the NFE2 promoter is increased in PV patients, decreasing both H3K9me2 levels and binding of the repressive heterochromatin protein-1α (HP1α). Hence, JMJD1C and NFE2 participate in a novel autoregulatory loop. Depleting JMJD1C expression significantly reduced cytokine-independent growth of an MPN cell line. Independently, NFE2 is regulated through the epigenetic JAK2 pathway by phosphorylation of H3Y41. This likewise inhibits HP1α binding. Treatment with decitabine lowered H3Y41ph and augmented H3K9me2 levels at the NFE2 locus in HEL cells, thereby increasing HP1α binding, which normalized NFE2 expression selectively in JAK2V617F-positive cell lines.

Platelet-predominate gene expression and reticulated platelets in nonvalvular atrial fibrillation: Effect of pulmonary veins isolation.

INTRODUCTION: Reticulated platelet (RP) content is increased in nonvalvular atrial fibrillation (NVAF). The purpose of this study was to determine if platelet content, morphology, and RP proportion are modulated by platelet genes. METHODS AND RESULTS: Expression of six platelet-predominate genes impacting platelet formation and release, platelet count, and RP content was assessed in NVAF patients before and 3-4 months after pulmonary veins isolation (PVI) and compared to normal sinus rhythm (NSR) controls. RNA from isolated platelets was reverse-transcribed assayed against selected genes utilizing real-time qPCR, and expressed as mean cycle threshold (ΔCt) using beta-2-microglobulin as endogenous control. RP content was assessed by flow cytometry. A fourfold lower expression of CFL1 gene coding for nonmuscle cofilin (7.8 ± 0.9 vs. 5.7 ± 1.6, P < 0.001) and twofold lower expression of four other genes were associated with similar platelet counts but fourfold higher (28.7+7.0 vs. 6.7+5.4, P < 0.001) RP content (%) in 97 NVAF cases compared to 51 NSR controls. Three to 4 months after PVI, RP decreased by 28%, while CFL1 gene expression increased over twofold but TUBA4A gene expression decreased almost twofold; NFE2 and MYL6 gene expression remained unchanged. CONCLUSIONS: NVAF is associated with notable downregulation of genes directing platelet production and size but increased RP content. PVI impacts the expression of many of these genes, implying a direct relationship between atrial fibrillation and platelet biogenesis.

Mouse prenatal platelet-forming lineages share a core transcriptional program but divergent dependence on MPL.

The thrombopoietic environment of the neonate is established during prenatal life; therefore, a comprehensive understanding of platelet-forming cell development during embryogenesis is critical to understanding the etiology of early-onset thrombocytopenia. The recent discovery that the first platelet-forming cells of the conceptus are not megakaryocytes (MKs) but diploid platelet-forming cells (DPFCs) revealed a previously unappreciated complexity in thrombopoiesis. This raises important questions, including the following. When do conventional MKs appear? Do pathogenic genetic lesions of adult MKs affect DPFCs? What role does myeloproliferative leukemia virus (MPL), a key regulator of adult megakaryopoiesis, play in prenatal platelet-forming lineages? We performed a comprehensive study to determine the spatial and temporal appearance of prenatal platelet-forming lineages. We demonstrate that DPFCs originate in the yolk sac and then rapidly migrate to other extra- and intraembryonic tissues. Using gene disruption models of Gata1 and Nfe2, we demonstrate that perturbing essential adult MK genes causes an analogous phenotype in the early embryo before the onset of hematopoietic stem/progenitor cell-driven (definitive) hematopoiesis. Finally, we present the surprising finding that DPFC and MK commitment from their respective precursors is MPL independent in vivo but that completion of MK differentiation and establishment of the prenatal platelet mass is dependent on MPL expression.

RUNX1 and NF-E2 upregulation is not specific for MPNs, but is seen in polycythemic disorders with augmented HIF signaling.

Overexpression of transcription factors runt-related transcription factor 1 (RUNX1) and nuclear factor, erythroid-derived 2 (NF-E2) was reported in granulocytes of patients with polycythemia vera and other myeloproliferative neoplasms (MPNs). Further, a transgenic mouse overexpressing the NF-E2 transgene was reported to be a model of MPN. We hypothesized that increased transcripts of RUNX1 and NF-E2 might characterize other polycythemic states with primary polycythemic features, that is, those with exaggerated erythropoiesis due to augmented erythropoietin (EPO) sensitivity. We tested the expression of RUNX1 and NF-E2 in polycythemic patients of diverse phenotypes and molecular causes. We report that RUNX1 and NF-E2 overexpression is not specific for MPN; these transcripts were also significantly elevated in polycythemias with augmented hypoxia-inducible factor activity whose erythroid progenitors were hypersensitive to EPO. RUNX1 and NF-E2 overexpression was not detected in patients with EPO receptor (EPOR) gain-of-function, suggesting distinct mechanisms by which erythroid progenitors in polycythemias with defects of hypoxia sensing and EPOR mutations exert their EPO hypersensitivity.

Correlation between Conjugated Bisphenol A Concentrations and Efflux Transporter Expression in Human Fetal Livers.

Because of its widespread use in the manufacturing of consumer products over several decades, human exposure to bisphenol A (BPA) has been pervasive. Fetuses are particularly sensitive to BPA exposure, with a number of negative developmental and reproductive outcomes observed in rodent perinatal models. Xenobiotic transporters are one mechanism to extrude conjugated and unconjugated BPA from the liver. In this study, the mRNA expression of xenobiotic transporters and relationships with total, conjugated, and free BPA levels were explored utilizing human fetal liver samples. The mRNA expression of breast cancer resistance protein (BCRP) and multidrug resistance-associated transporter (MRP)4, as well as BCRP and multidrug resistance transporter 1 exhibited the highest degree of correlation, with r(2) values of 0.941 and 0.816 (P < 0.001 for both), respectively. Increasing concentrations of conjugated BPA significantly correlated with high expression of MRP1 (P < 0.001), MRP2 (P < 0.05), and MRP3 (P < 0.05) transporters, in addition to the NF-E2-related factor 2 transcription factor (P < 0.001) and its prototypical target gene, NAD(P)H quinone oxidoreductase 1 (P < 0.001). These data demonstrate that xenobiotic transporters may be coordinately expressed in the human fetal liver. This is also the first report of a relationship between environmentally relevant fetal BPA levels and differences in the expression of transporters that can excrete the parent compound and its metabolites.

Cleaning up after ICH: the role of Nrf2 in modulating microglia function and hematoma clearance.

As a consequence of intracerebral hemorrhage (ICH), blood components enter brain parenchyma causing progressive damage to the surrounding brain. Unless hematoma is cleared, the reservoirs of blood continue to inflict injury to neurovascular structures and blunt the brain repair processes. Microglia/macrophages (MMΦ) represent the primary phagocytic system that mediates the cleanup of hematoma. Thus, the efficacy of phagocytic function by MMΦ is an essential step in limiting ICH-mediated damage. Using primary microglia to model red blood cell (main component of hematoma) clearance, we studied the role of transcription factor nuclear factor-erythroid 2 p45-related factor 2 (Nrf2), a master-regulator of antioxidative defense, in the hematoma clearance process. We showed that in cultured microglia, activators of Nrf2 (i) induce antioxidative defense components, (ii) reduce peroxide formation, (iii) up-regulate phagocytosis-mediating scavenger receptor CD36, and (iv) enhance red blood cells (RBC) phagocytosis. Through inhibiting Nrf2 or CD36 in microglia, by DNA decoy or neutralizing antibody, we documented the important role of Nrf2 and CD36 in RBC phagocytosis. Using autologous blood injection ICH model to measure hematoma resolution, we showed that Nrf2 activator, sulforaphane, injected to animals after the onset of ICH, induced CD36 expression in ICH-affected brain and improved hematoma clearance in rats and wild-type mice, but expectedly not in Nrf2 knockout (KO) mice. Normal hematoma clearance was impaired in Nrf2-KO mice. Our experiments suggest that Nrf2 in microglia play an important role in augmenting the antioxidative capacity, phagocytosis, and hematoma clearance after ICH.

A high concentration of triiodothyronine attenuates the stimulatory effect on hemin-induced erythroid differentiation of human erythroleukemia K562 cells.

Although thyroid hormone is a known stimulator of erythropoietic differentiation, severe anemia is sometimes observed in patients with hyperthyroidism and this mechanism is not fully understood. The aim of this study was to investigate the effect of triiodothyronine (T3) on hemin-induced erythropoiesis. Human erythroleukemia K562 cells were used as an erythroid differentiation model. Cell differentiation was induced by hemin and the effect of pre-incubation with T3 (0.1 to 100 nM) was analyzed by measuring the benzidine-positive rate, hemoglobin content, CD71 expression (transferrin receptor), and mRNA expression for transcription factors related to erythropoiesis and thyroid hormone receptors (TRs). Hemin, a promoter of erythroid differentiation, increased the levels of mRNAs for TRα, TRß, and retinoid X receptor α (RXRα), as well as those for nuclear factor-erythroid 2 (NFE2), GATA-binding protein 1 (GATA1) and GATA-binding protein 2 (GATA2). Lower concentrations of T3 had a stimulatory effect on hemin-induced hemoglobin production (1 and 10 nM), CD71 expression (0.1 nM), and α-globin mRNA expression (1 nM), while a higher concentration of T3 (100 nM) abrogated the stimulatory effect on these parameters. T3 at 100 nM did not affect cell viability and proliferation, suggesting that the abrogation of erythropoiesis enhancement was not due to toxicity. T3 at 100 nM also significantly inhibited expression of GATA2 and RXRα mRNA, compared to 1 nM T3. We conclude that a high concentration of T3 attenuates the classical stimulatory effect on erythropoiesis exerted by a low concentration of T3 in hemin-induced K562 cells.

Interferon-γ suppresses activin A/NF-E2 induction of erythroid gene expression through the NF-κB/c-Jun pathway.

Interferon (IFN)-γ is a proinflammatory cytokine that is linked to erythropoiesis inhibition and may contribute to anemia. However, the mechanism of IFN-γ-inhibited erythropoiesis is unknown. Activin A, a member of the transforming growth factor (TGF)-ß superfamily, induces the erythropoiesis of hematopoietic progenitor cells. In this study, a luciferase reporter assay showed that IFN-γ suppressed activin A-induced ζ-globin promoter activation in K562 erythroblast cells in a dose-dependent manner. Activin A reversed the suppressive effect of IFN-γ on the luciferase activity of ζ-globin promoter in a dose-dependent manner. IFN-γ also suppressed the activation of activin A-induced α-globin promoter. IFN-γ reduced the mRNA expression of α-globin, ζ-globin, NF-E2p45, and GATA-1 induced by activin A. The results also showed that IFN-γ induced c-Jun expression when NF-κBp65 and c-Jun bound to two AP-1-binding sites on the c-Jun promoter. The luciferase activity of α-globin and ζ-globin promoters were enhanced by wild-type c-Jun and eliminated by dominant-negative (DN) c-Jun. The suppressive effects of IFN-γ on the mRNA expression of α-globin and ζ-globin were absent in cells expressing DN c-Jun. The ability of NF-E2 to enhance activin A-induced ζ-globin promoter activation decreased when c-Jun was present, and IFN-γ treatment further enhanced the decreasing effect of c-Jun. Chromatin immunoprecipitation revealed that NF-E2p45 bound to the upstream regulatory element (HS-40) of the α-globin gene cluster in response to activin A, whereas c-Jun eliminated this binding. These results suggest that IFN-γ modulates NF-κB/c-Jun to antagonize activin A-mediated NF-E2 transcriptional activity on globin gene expression.

Rho kinase inhibition drives megakaryocyte polyploidization and proplatelet formation through MYC and NFE2 downregulation.

The processes of megakaryocyte polyploidization and demarcation membrane system (DMS) formation are crucial for platelet production, but the mechanisms controlling these processes are not fully determined. Inhibition of Rho kinase (ROCK) signalling leads to increased polyploidization in umbilical cord blood-derived megakaryocytes. To extend these findings we determined the effect of ROCK inhibition on development of the DMS and on proplatelet formation. The underlying mechanisms were explored by analysing the effect of ROCK inhibition on the expression of MYC and NFE2, which encode two transcription factors critical for megakaryocyte development. ROCK inhibition promoted DMS formation, and increased proplatelet formation and platelet release. Rho kinase inhibition also downregulated MYC and NFE2 expression in mature megakaryocytes, and this down-regulation correlated with increased proplatelet formation. Our findings suggest a model whereby ROCK inhibition drives polyploidization, DMS growth and proplatelet formation late in megakaryocyte maturation through downregulation of MYC and NFE2 expression.

p45NF-E2 represses Gcm1 in trophoblast cells to regulate syncytium formation, placental vascularization and embryonic growth.

Absence of the leucine zipper transcription factor p45NF-E2 results in thrombocytopenia, impaired placental vascularization and intrauterine growth restriction (IUGR) in mice. The mechanism underlying the p45NF-E2-dependent placental defect and IUGR remains unknown. Here, we show that the placental defect and IUGR of p45NF-E2 (Nfe2) null mouse embryos is unrelated to thrombocytopenia, establishing that embryonic platelets and platelet-released mediators are dispensable for placentation. Rather, p45NF-E2, which was hitherto thought to be specific to hematopoietic cells, is expressed in trophoblast cells, where it is required for normal syncytiotrophoblast formation, placental vascularization and embryonic growth. Expression of p45NF-E2 in labyrinthine trophoblast cells colocalizes with that of Gcm1, a transcription factor crucial for syncytiotrophoblast formation. In the absence of p45NF-E2, the width of syncytiotrophoblast layer 2 and the expression of Gcm1 and Gcm1-dependent genes (Synb and Cebpa) are increased. In vitro, p45NF-E2 deficiency results in spontaneous syncytiotrophoblast formation, which can be reversed by Gcm1 knockdown. Increased Gcm1 expression in the absence of p45NF-E2 is dependent on enhanced protein acetylation, including post-translational modification of Gcm1. Increasing and inhibiting acetylation in the placenta of wild-type control embryos phenocopies and corrects, respectively, the changes observed in p45NF-E2-deficient embryos. These studies identify a novel function of p45NF-E2 during placental development: in trophoblast cells, p45NF-E2 represses Gcm1 and syncytiotrophoblast formation via acetylation.

A tissue-specific chromatin loop activates the erythroid ankyrin-1 promoter.

The human ankyrin-1 gene (ANK1) contains 3 tissue-specific alternative promoters. We have shown previously that the erythroid-specific ankyrin 1 (ANK1E) core promoter contains a 5' DNase I hypersensitive site (HS) with barrier insulator function that prevents gene silencing in vitro and in vivo. Mutations in the ANK1E barrier region lead to decreased ANK1 mRNA levels and hereditary spherocytosis. In this report, we demonstrate a second ANK1E regulatory element located in an adjacent pair of DNase I HS located 5.6 kb 3' of the ANK1E promoter at the 3' boundary of an erythroid-specific DNase I-sensitive chromatin domain. The 3' regulatory element exhibits enhancer activity in vitro and in transgenic mice, and it has the histone modifications associated with an enhancer element. One of the ANK1E 3'HS contains an NF-E2 binding site that is required for enhancer function. We show that a chromatin loop brings the 3' enhancer and NF-E2 into proximity with the 5' barrier region including the ANK1E core promoter. These observations demonstrate a model for the tissue-specific activation of alternative promoters that may be applicable to the ∼ 30% of mammalian genes with alternative promoters that exhibit distinct expression patterns.

[An association between NF-E2-related factor 2-617 C/A gene polymorphisms and sepsis in Chinese Han population].

OBJECTIVE: To investigate the association between the single nucleotide polymorphisms (SNPs) in NF-E2-related factor 2-617 (NRF2-617) promoter region with the susceptibility to the risk of sepsis. METHODS: In this case-control association study, 203 healthy controls and 174 patients with sepsis in Wenzhou Han population were enrolled and genotyped by DNA direct sequencing. RESULTS: (1) The (CA+AA) genotype frequency was significantly higher in the sepsis group than in the control group (59.2% vs 46.3%, P = 0.012). (2) Compared with the general sepsis group, higher (CA+AA) genotype frequency was found in the severe sepsis group (47.5% vs 65.5%, P = 0.033) . However, no significant difference was shown in the (CA+AA) genotype frequency between the shock group and the non-shock group as well as between the death group and the non-death group (61.8% vs 57.1%, P = 0.221; 56.8% vs 66.7%, P = 0.258) . (3) The unconditional logistic regression analysis showed that the mutation of C to A at the gene promoter locus of Nrf2-617 was associated with the increased onset risk of sepsis (OR = 1.584, 95%CI 1.025-2.447, P = 0.038) and the severity of sepsis (OR = 0.453, 95%CI 0.233-0.878, P = 0.019). CONCLUSION: The mutation of C to A at the gene promoter locus of Nrf2-617 may increase the onset risk of sepsis and organ failure in sepsis patients, while not associated with the incidence of shock and the prognosis of sepsis.

Zebrafish globin switching occurs in two developmental stages and is controlled by the LCR.

Globin gene switching is a complex, highly regulated process allowing expression of distinct globin genes at specific developmental stages. Here, for the first time, we have characterized all of the zebrafish globins based on the completed genomic sequence. Two distinct chromosomal loci, termed major (chromosome 3) and minor (chromosome 12), harbor the globin genes containing α/ß pairs in a 5'-3' to 3'-5' orientation. Both these loci share synteny with the mammalian α-globin locus. Zebrafish globin expression was assayed during development and demonstrated two globin switches, similar to human development. A conserved regulatory element, the locus control region (LCR), was revealed by analyzing DNase I hypersensitive sites, H3K4 trimethylation marks and GATA1 binding sites. Surprisingly, the position of these sites with relation to the globin genes is evolutionarily conserved, despite a lack of overall sequence conservation. Motifs within the zebrafish LCR include CACCC, GATA, and NFE2 sites, suggesting functional interactions with known transcription factors but not the same LCR architecture. Functional homology to the mammalian α-LCR MCS-R2 region was confirmed by robust and specific reporter expression in erythrocytes of transgenic zebrafish. Our studies provide a comprehensive characterization of the zebrafish globin loci and clarify the regulation of globin switching.