E2F6 inhibits cobalt chloride-mimetic hypoxia-induced apoptosis through E2F1.
ABSTRACT: E2F6, a potent transcriptional repressor, plays important roles in cell cycle regulation. However, roles of E2F6 in hypoxia-induced apoptosis are unknown. Here, we demonstrated biological functions of E2F6 in hypoxia-induced apoptosis and regulatory pathways. During hypoxia (CoCl(2), 800 microM)-induced human embryonic kidney 293 cell apoptosis, E2F6 expression was down-regulated with concurrent increases in E2F1 expression and transactivation. E2F6 overexpression abrogated hypoxia-induced apoptosis and alteration of E2F1. Conversely, specific knockdown of E2F6 by small interfering RNA had opposite effects. Chromatin immunoprecipitation assay confirmed that E2F6 regulated E2F1 expression through the transrepression of E2F1 promoter. Interestingly, E2F1 transactivation and apoptosis induced by hypoxia in cells stably expressing E2F1 were inhibited by E2F6 overexpression, suggesting that the inhibitory effects of E2F6 are not only mediated by the repression of E2F1 promoter. This was confirmed by E2F6-inhibited transactivation of E2F1 and apoptosis via competing with E2F1 for DNA binding sites evidenced by the different behaviors of E2F6DeltaC (C-terminal deletion) and E2F6.E68 (mutant DNA binding site) and by the lack of association of E2f6 with E2F1 protein. Moreover, hypoxia up-regulated expression of E2F1-responsive proapoptotic gene apoptosis protease-activating factor 1 was repressed by E2F6 overexpression. Together, these findings demonstrate a novel role of E2F6 in control of hypoxia-induced apoptosis through regulation of E2F1.
Project description:Epstein-Barr virus (EBV) is considered a ubiquitous herpesvirus with the ability to cause latent infection in humans worldwide. EBV-association is evidently linked to different types of human malignancies, mainly of epithelial and lymphoid origin. Of interest is the EBV nuclear antigen 3C (EBNA3C) which is critical for EBV-mediated immortalization. Recently, EBNA3C was shown to bind the E2F1 transcription regulator. The E2F transcription factors have crucial roles in various cellular functions, including cell cycle, DNA replication, DNA repair, cell mitosis, and cell fate. Specifically, E2F6, one of the unique E2F family members, is known to be a pRb-independent transcription repressor of E2F-target genes. In our current study, we explore the role of EBNA3C in regulating E2F6 activities. We observed that EBNA3C plays an important role in inducing E2F6 expression in LCLs. Our study also shows that EBNA3C physically interacts with E2F6 at its amino and carboxy terminal domains and they form a protein complex in human cells. In addition, EBNA3C stabilizes the E2F6 protein and is co-localized in the nucleus. We also demonstrated that both EBNA3C and E2F6 contribute to reduction in E2F1 transcriptional activity. Moreover, E2F1 forms a protein complex with EBNA3C and E2F6, and EBNA3C competes with E2F1 for E2F6 binding. E2F6 is also recruited by EBNA3C to the E2F1 promoter, which is critical for EBNA3C-mediated cell proliferation. These results demonstrate a critical role for E2F family members in EBV-induced malignancies, and provide new insights for targeting E2F transcription factors in EBV-associated cancers as potential therapeutic intervention strategies.
Project description:Using ChIP-chip assays (employing ENCODE arrays and core promoter arrays), we examined the binding patterns of three members of the E2F family in five cell types. We determined that most E2F1, E2F4, and E2F6 binding sites are located within 2 kb of a transcription start site, in both normal and tumor cells. In fact, the majority of promoters that are active (as defined by TAF1 or POLR2A binding) in GM06990 B lymphocytes and Ntera2 carcinoma cells were also bound by an E2F. This very close relationship between E2F binding sites and binding sites for general transcription factors in both normal and tumor cells suggests that a chromatin-bound E2F may be a signpost for active transcription initiation complexes. In general, we found that several E2Fs bind to a given promoter and that there is only modest cell type specificity of the E2F family. Thus, it is difficult to assess the role of any particular E2F in transcriptional regulation, due to extreme redundancy of target promoters. However, Ntera2 carcinoma cells were exceptional in that a large set of promoters were bound by E2F6, but not by E2F1 or E2F4. It has been proposed that E2F6 contributes to gene silencing by recruiting enzymes involved in methylating histone H3. To test this hypothesis, we created Ntera2 cell lines harboring shRNAs to E2F6. We found that reduction of E2F6 only induced minimal alteration of the transcriptome of Ntera2 transcriptome. Our results support the concept of functional redundancy in the E2F family and suggest that E2F6 is not critical for histone methylation.
Project description:Our previous study explored the roles of microRNA-424 (miR-424) in the development of endometrial carcinoma (EC) and analyzed the miR-424/E2F7 axis in EC cell growth. In this study, we investigated the status of miR-424 in human endometrial cancer tissues, which were collected from a cohort of Zunyi patients. We found that the expression level of miR-424 was associated with clinical tumor stage, cell differentiation, lymph node metastasis and cell migration ability. Cell function experiments demonstrated that miR-424 overexpression suppressed the invasion and migration abilities of endometrial carcinoma cells in vitro. Bioinformatic predictions and dual-luciferase reporter assays suggested E2F6 as a possible target of miR-424. RT-PCR and western blot assays demonstrated that miR-424 transfection reduced the expression level of E2F6, while inhibiting miR-424 with ASO-miR-424 (antisense oligonucleotides of miR-424) increased the expression level of E2F6. Cell function experiments indicated that E2F6 transfection rescued the EC cell phenotype induced by miR-424. In addition, we also found that E2F6 negatively regulated miR-424 expression in EC cells. In summary, our results demonstrated that the miR-424/E2F6 feedback loop modulates cell invasion, migration and EMT in EC and that the miR-424/E2Fs regulation network may serve as a new and potentially important therapeutic target in EC.
Project description:The human cytochrome c(1) promoter is strongly activated in transfected Drosophila SL2 cells expressing exogenous human E2F1. Transfection-deletion experiments, DNase I protection by E2F1 and gel mobility-shift experiments locate E2F1 activation sites to two regions on either side of the transcription start site. Deletion of either region prevents E2F1 activation in transfected SL2 cells, suggesting a co-operative interaction between them. E2F6, a member of the E2F family that lacks transactivation domains but contains specific suppressor domains, inhibits cytochrome c(1) promoter activity when co-transfected into HeLa cells, indicating that the E2F proteins modulate the cytochrome c(1) promoter in mammalian cells. However, E2F is not a general regulator of oxidative phosphorylation genes since three additional nuclear-encoded mitochondrial genes were unaffected by E2F1 or E2F6.
Project description:E2F6 is widely expressed in human tissues and cell lines. Recent studies have demonstrated its involvement in developmental patterning and in the regulation of various genes implicated in chromatin remodelling. Despite a growing number of studies, nothing is really known concerning the E2F6 expression regulation. To understand how cells control E2F6 expression, we analysed the activity of the previously cloned promoter region of the human E2F6 gene. DNase I footprinting, gel electrophoreticmobility shift, transient transfection and site-directed mutagenesis experiments allowed the identification of two functional NRF-1/alpha-PAL (nuclear respiratory factor-1/alpha-palindrome-binding protein)-binding sites within the human E2F6 core promoter region, which are conserved in the mouse and rat E2F6 promoter region. Moreover, ChIP (chromatin immunoprecipitation) analysis demonstrated that overexpressed NRF-1/alpha-PAL is associated in vivo with the E2F6 promoter. Furthermore, overexpression of full-length NRF-1/alpha-PAL enhanced E2F6 promoter activity, whereas expression of its dominant-negative form reduced the promoter activity. Our results indicate that NRF-1/alpha-PAL is implicated in the regulation of basal E2F6 gene expression.
Project description:During gametogenesis, germ cells must undergo meiosis in order to become viable haploid gametes. Successful completion of this process is dependent upon the expression of genes whose protein products function specifically in meiosis. Failure to express these genes in meiotic cells often results in infertility, whereas aberrant expression in somatic cells may lead to mitotic catastrophe. The mechanisms responsible for regulating the timely expression of meiosis-specific genes have not been fully elucidated. Here we demonstrate that E2F6, a member of the E2F family of transcription factors, is essential for the repression of the newly identified meiosis-specific gene, Slc25a31 (also known as Ant4, Aac4), in somatic cells. This discovery, along with previous studies, prompted us to investigate the role of E2F6 in the regulation of meiosis-specific genes in general. Interestingly, the core E2F6-binding element (TCCCGC) was highly conserved in the proximal promoter regions of 19 out of 24 (79.2%) meiosis-specific genes. This was significantly higher than the frequency found in the promoters of all mouse genes (15.4%). In the absence of E2F6, only a portion of these meiosis-specific genes was derepressed in somatic cells. However, endogenous E2F6 bound to the promoters of these meiosis-specific genes regardless of whether they required E2F6 for their repression in somatic cells. Further, E2F6 overexpression was capable of reducing their transcription. These findings indicate that E2F6 possesses a broad ability to bind to and regulate the meiosis-specific gene population.
Project description:The E2F6 protein functions as an Rb-independent repressor of gene transcription. We have previously provided evidence suggesting a role for E2F6 in repression of E2F-responsive genes at S phase. Here, we have identified BRG1, the ATPase subunit of the SWI/SNF chromatin-remodeling complex, as an E2F6 interacting protein. Immunoprecipitation experiments demonstrate that BRG1 binds specifically to E2F6 and E2F4 but not the activator E2Fs. E2F6 was also able to interact with BAF155, a BRG1-associated factor, in the SWI/SNF complex. Chromatin immunoprecipitation assays demonstrate the binding of BRG1 coincident with E2F6 on G1/S gene promoters during S phase. Collectively, our studies suggest that E2F6 may recruit BRG1 in transcriptional regulation of genes important for G1/S phase transition of the cell cycle.
Project description:The E2f6 transcriptional repressor is an E2F-family member essential for the silencing of a group of meiosis-specific genes in somatic tissues. Although E2f6 has been shown to associate with both polycomb repressive complexes (PRC) and the methyltransferase Dnmt3b, the cross-talk between these repressive machineries during E2f6-mediated gene silencing has not been clearly demonstrated yet. In particular, it remains largely undetermined when and how E2f6 establishes repression of meiotic genes during embryonic development. We demonstrate here that the inactivation of a group of E2f6 targeted genes, including Stag3 and Smc1?, first occurs at the transition from mouse embryonic stem cells (ESCs) to epiblast stem cells (EpiSCs), which represent pre- and post-implantation stages, respectively. This process was accompanied by de novo methylation of their promoters. Of interest, despite a clear difference in DNA methylation status, E2f6 was similarly bound to the proximal promoter regions both in ESCs and EpiSCs. Neither E2f6 nor Dnmt3b overexpression in ESCs decreased meiotic gene expression or increased DNA methylation, indicating that additional factors are required for E2f6-mediated repression during the transition. When the SET domain of Ezh2, a core subunit of the PRC2 complex, was deleted, however, repression of Stag3 and Smc1? during embryoid body differentiation was largely impaired, indicating that the event required the enzymatic activity of Ezh2. In addition, repression of Stag3 and Smc1? occurred in the absence of Dnmt3b. The data presented here suggest a primary role of PRC2 in E2f6-mediated gene silencing of the meiotic genes.
Project description:<h4>Rationale</h4>The E2F pathway plays a critical role in cardiac growth and development, yet its role in cardiac metabolism remains to be defined. Metabolic changes play important roles in human heart failure and studies imply the ketogenic enzyme ?-hydroxybutyrate dehydrogenase I (BDH1) is a potential biomarker.<h4>Objective</h4>To define the role of the E2F pathway in cardiac metabolism and dilated cardiomyopathy (DCM) with a focus on BDH1.<h4>Methods and results</h4>We previously developed transgenic (Tg) mice expressing the transcriptional repressor, E2F6, to interfere with the E2F/Rb pathway in post-natal myocardium. These Tg mice present with an E2F6 dose dependent DCM and deregulated connexin-43 (CX-43) levels in myocardium. Using the Seahorse platform, a 22% decrease in glycolysis was noted in neonatal cardiomyocytes isolated from E2F6-Tg hearts. This was associated with a 39% reduction in the glucose transporter GLUT4 and 50% less activation of the regulator of glucose metabolism AKT2. The specific reduction of cyclin B1 (70%) in Tg myocardium implicates its importance in supporting glycolysis in the postnatal heart. No changes in cyclin D expression (known to regulate mitochondrial activity) were noted and lipid metabolism remained unchanged in neonatal cardiomyocytes from Tg hearts. However, E2F6 induced a 40-fold increase of the Bdh1 transcript and 890% increase in its protein levels in hearts from Tg pups implying a potential impact on ketolysis. By contrast, BDH1 expression is not activated until adulthood in normal myocardium. Neonatal cardiomyocytes from Wt hearts incubated with the ketone ?-hydroxybutyrate (?-OHB) showed a 100% increase in CX-43 protein levels, implying a role for ketone signaling in gap junction biology. Neonatal cardiomyocyte cultures from Tg hearts exhibited enhanced levels of BDH1 and CX-43 and were not responsive to ?-OHB.<h4>Conclusions</h4>The data reveal a novel role for the E2F pathway in regulating glycolysis in the developing myocardium through a mechanism involving cyclin B1. We reveal BDH1 expression as an early biomarker of heart failure and its potential impact, through ketone signaling, on CX-43 levels in E2F6-induced DCM.
Project description:Hypoxia complicates islet isolation for transplantation and may contribute to pancreatic ?-cell failure in type 2 diabetes. Pancreatic ?-cells are susceptible to hypoxia-induced apoptosis. Severe hypoxic conditions during the immediate post-transplantation period are a main non-immune factor leading to ?-cell death and islet graft failure. In this study, we identified the transcription factor Ets-1 (v-ets erythroblastosis virus E26 oncogene homolog 1) as an early response gene against hypoxia-induced apoptosis in pancreatic ?-cells. Hypoxia regulates Ets-1 at multiple levels according to the degree of ?-cell oxygen deprivation. Moderate hypoxia promotes Ets-1 gene transcription, whereas severe hypoxia promotes its transactivation activity, as well as its ubiquitin-proteasome mediated degradation. This degradation causes a relative insufficiency of Ets-1 activity, and limits the transactivation effect of Ets-1 on downstream hypoxic-inducible genes and its anti-apoptotic function. Overexpression of ectopic Ets-1 in MIN6 and INS-1 cells protects them from severe hypoxia-induced apoptosis in a mitochondria-dependent manner, confirming that a sufficient amount of Ets-1 activity is critical for protection of pancreatic ?-cells against hypoxic injury. Targeting Ets-1 expression may be a useful strategy for islet graft protection during the immediate post-transplantation period.