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The role of circ_0089153 in breast cancer (BCa) malignancy development was explored. circ_0089153 expression in BCa was analyzed by Gene Expression Omnibus database. Clinical tissues were obtained from 90 BCa patients. Cell counting kit-8 assay, 5-ethnyl-2 deoxyuridine assay and colony formation experiment were applied for proliferation detection. Wound healing assay and Transwell experiment were used for migration and invasion detection. Dual luciferase reporter gene assay, RNA immunoprecipitation assay and RNA pull-down assay were conducted. In vivo growth and metastasis of BCa cells were performed. Quantitative real-time polymerase chain reaction, Western blot and immunohistochemistry were applied for RNAs and proteins expression. The up-modulated circ_0089153 indicated an unfavorable survival of BCa patients. circ_0089153 knockdown attenuated BCa cells proliferation, migration, invasion and epithelial-mesenchymal transition (EMT) (P .01). circ_0089153 was miR-2467-3p sponge. Low miR-2467-3p expression indicated a worse survival of BCa patients. miR-2467-3p overexpression reduced BCa cells proliferation, migration, invasion and EMT (P .05). circ_0089153 enhanced BCa cells proliferation, migration, invasion and EMT by sponging miR-2467-3p (P .05). E2F6 was directly suppressed by miR-2467-3p. E2F6 high expression in BCa patients associated with worse survival. circ_0089153 knockdown suppressed in vivo BCa cells growth and lung metastasis (P .01). circ_0089153 was an oncogene in breast cancer, which enhanced proliferation and metastasis through sponging miR-2467-3p/E2F6. circ_0089153 was suggested to be a potential target for BCa target treatment.

Long noncoding RNA LAMTOR5 antisense RNA 1 (LAMTOR5-AS1) has been certified as a risk predictor and diagnostic biomarker of prostate cancer. However, the expression and exact roles of LAMTOR5-AS1 in non-small cell lung cancer (NSCLC) remain unclear. Thus, we measured LAMTOR5-AS1 expression in NSCLC and gauged its clinical value. The detailed roles and downstream working mechanism of LAMTOR5-AS1 in NSCLC were comprehensively unraveled. qRT-PCR was applied to measure gene expression. Functionally, utilizing small interfering RNA, LAMTOR5-AS1 was ablated, and the functional alterations were addressed by means of different experiments. The targeting activities between LAMTOR5-AS1 and microRNA-506-3p (miR-506-3p) and between miR-506-3p and E2F transcription factor 6 (E2F6) were confirmed by RNA immunoprecipitation and luciferase reporter assays. LAMTOR5-AS1 overexpression in NSCLC was verified in TCGA datasets and our own cohort and manifested an evident relationship with poor prognosis. Interference with LAMTOR5-AS1 led to repression of the proliferation, cloning, and metastasis abilities of NSCLC cells in vitro. We further confirmed an obvious increase in LAMTOR5-AS1-silenced NSCLC cell apoptosis. Furthermore, the absence of LAMTOR5-AS1 restricted tumor growth in vivo. Mechanistically, LAMTOR5-AS1 sponged miR-506-3p in NSCLC cells. Furthermore, E2F6, a downstream target of miR-506-3p, was under the control of LAMTOR5-AS1, which was realized by decoying miR-506-3p. Rescue experiments showed that miR-506-3p suppression or E2F6 reintroduction was capable of remitting LAMTOR5-AS1 deficiency-triggered anticarcinogenic actions in NSCLC. Our study confirmed the exact roles of LAMTOR5-AS1 for the first time and revealed that LAMTOR5-AS1 knockdown disrupts the malignancy of NSCLC by targeting the miR-506-3p/E2F6 axis. Targeting the LAMTOR5-AS1/miR-506-3p/E2F6 pathway may be instrumental for managing patients with NSCLC.

Silencing of a subset of germline genes is dependent upon DNA methylation (DNAme) post-implantation. However, these genes are generally hypomethylated in the blastocyst, implicating alternative repressive pathways before implantation. Indeed, in embryonic stem cells (ESCs), an overlapping set of genes, including germline “genome-defence” (GGD) genes, are upregulated following deletion of the H3K9 methyltransferase SETDB1 or subunits of the non-canonical PRC1 complex PRC1.6. Here, we show that in pre-implantation embryos and naïve ESCs (nESCs), hypomethylated promoters of germline genes bound by the PRC1.6 DNA-binding subunits MGA/MAX/E2F6 are enriched for RING1B-dependent H2AK119ub1 and H3K9me3. Accordingly, repression of these genes in nESCs shows a greater dependence on PRC1.6 than DNAme. In contrast, GGD genes are hypermethylated in epiblast-like cells (EpiLCs) and their silencing is dependent upon SETDB1, PRC1.6/RING1B and DNAme, with H3K9me3 and DNAme establishment dependent upon MGA binding. Thus, GGD genes are initially repressed by PRC1.6, with DNAme subsequently engaged in post-implantation embryos., Germline genes are repressed by DNA methylation and histone marks. Here, the authors show that specific germline genes hypomethylated pre-implantation are enriched for PRC1.6, H2AK119ub1 and H3K9me3, which coordinately repress their expression.

The expression of immunohistochemical markers has been extensively investigated in thymomas to assist in the differential diagnosis. We have studied six select markers to determine their utility in the evaluation of these tumors. A series of 126 thymomas including 33 type A, 27 type AB, 20 type B1, 22 type B2, and 24 type B3, were examined utilizing a tissue microarray (TMA) technique with antibodies to e-cadherin, β-catenin, PAX8, bcl-2, EMA, and MIB-1. Keratin AE1/AE3 and p63 were used for quality control. A significant finding was strong and consistent positivity for bcl-2 in type A (90%) and type AB (88.8%) thymoma, while 100% of B1, B2, and B3 were negative. The distribution of e-cadherin and β-catenin was not useful for differential diagnosis. E-cadherin and β-catenin were expressed in a high proportion of all the tumors (92-100%), except for B2 thymoma which showed only 45% expression. A significant increase in the expression of the MIB-1 proliferation marker (mean: 12.8% nuclear positivity) was also observed in B3 thymoma compared with the other histologic types. Statistical significance was confirmed using Kruskal's non-parameterized test for distribution. EMA was generally negative except for spindle cells in the fibrous septa in types A and AB thymoma. PAX8 showed less consistent nuclear staining than p63 and was only widely expressed in 55.7% of cases. Bcl-2 may serve as a useful marker to separate spindle cell thymomas (Type A and AB) from the other types, and the MIB1 proliferation index may be of use to differentiate type B2 from type B3 thymoma.

Countless studies have demonstrated that Circular RNAs (circRNAs) exert vital effects in regulating tumorigenesis of various cancers. CircRNA ZNF609 (circ-ZNF609) has been reported as an oncogene in various human cancers. Nevertheless, its regulating effect in cervical cancer (CC) remains to be further explored. RT-qPCR was adopted to measure circ-ZNF609, miR-197-3p and E2F6 levels. CC cell proliferation, migration and invasion were analyzed via CCK-8 and transwell assays. Dual-luciferase reporter assay was adopted to confirm the interaction between miR-197-3p and circ-ZNF609 or E2F6. In the present study, it was found that circ-ZNF609 was elevated in CC tissues and cell lines, and circ-ZNF609 deletion repressed cell viability, migration and invasion in CC. Moreover, circ-ZNF609 was identified to negatively regulate miR-197-3p expression in CC cells. The inhibition of miR-197-3p abrogated the inhibitory effect on CC cell proliferation, migration and invasion induced by circ-ZNF609 knockdown. Additionally, we further demonstrated that circ-ZNF609 upregulated E2F6 by interacting with miR-197-3p. Finally, rescue assays indicated that E2F6 overexpression upended the suppression of CC progression induced by circ-ZNF609 deletion. In conclusion, circ-ZNF609 promoted CC progression through modulating the miR-197-3p/E2F6 axis as an oncogene. This finding offers a unique insight into CC molecular mechanism and suggests a potential target for CC therapy., Graphical abstract

Acute graft-versus-host disease (aGvHD) is the most common complication after allogeneic hematopoietic stem cell transplantation (allo-HSCT) and significantly linked with morbidity and mortality. Although much work has been engaged to investigate aGvHD pathogenesis, the understanding of alloreactive T-cell activation remains incomplete. To address this, we studied transcriptional activation of carbohydrate, nucleotide, tricarboxylic acid (TCA) cycle, and amino acid metabolism of T cells before aGvHD onset by mining the Gene Expression Omnibus (GEO) datasets. Glycolysis had the most extensive correlation with other activated metabolic sub-pathways. Through Pearson correlation analyses, we found that glycolytic activation was positively correlated with activated CD4 memory T-cell subset and T-cell proliferation and migration. T-cell receptor (TCR), mechanistic target of rapamycin complex 1 (mTORC1), myelocytomatosis oncogene (MYC) signaling pathways and E2F6 might be “master regulators” of glycolytic activity. aGvHD predictive model constructed by glycolytic genes (PFKP, ENO3, and GAPDH) through logistic regression showed high predictive and discriminative value. Furthermore, higher expressions of PFKP, ENO3, and GAPDH in alloreactive T cells were confirmed in our pre-aGvHD patient cohort. And the predictive value of the aGvHD risk model was also validated. In summary, our study demonstrated that glycolytic activation might play a pivotal function in alloreactive T-cell activation before aGvHD onset and would be the potential target for aGvHD therapy.

Ovarian cancer remains the sixth most frequently occurring cancer in women worldwide. Long noncoding RNAs (lncRNAs) are capable of regulating gene expression, and thus, participating in a wide range of biological functions and disease processes including cancer development. Our work suggests that lncRNA TMPO antisense RNA 1 (TMPO-AS1) represents an oncogenic lncRNA in ovarian cancer and presents a novel mechanism involving transcription factor E2F transcription factor 6 (E2F6) and lipocalin-2 (LCN2). We identified upregulated lncRNA TMPO-AS1 in ovarian cancer tissues and cells. siRNA-mediated silencing of lncRNA TMPO-AS1 restrained the aggressiveness of ovarian cancer cells and their pro-angiogenic ability, and reduced the expression of LCN2. LncRNA TMPO-AS1 was found to interact with E2F6, a transcriptional repressor that could bind to the promoter region of LCN2 gene. In addition, silencing of E2F6 or overexpression of LCN2 restored the aggressiveness of ovarian cancer cells and their pro-angiogenic ability following siRNA-mediated silencing of lncRNA TMPO-AS1. Taken together, we demonstrated lncRNA TMPO-AS1 could potentially promote LCN2 transcriptional activity by binding to transcription factor E2F6, and thus, stimulated the progression of ovarian cancer. These findings underscore a possible alternative therapeutic strategy for ovarian cancer treatment.

Centromere protein U (CENPU), a centromere-binding protein required for cellular mitosis, has been reported to be closely associated with carcinogenesis in multiple malignancies; however, the role of CENPU in hepatocellular carcinoma (HCC) is still unclear. Herein, we investigated its biological role and molecular mechanism in the development of HCC. High CENPU expression in HCC tissue was observed and correlated positively with a poor prognosis in HCC patients. CENPU knockdown inhibited the proliferation, metastasis, and G1/S transition of HCC cells

Liver cancer is highly heterogeneous, and the tumor tissue harbors a variety of cell types. Liver tumor initiating cells (TICs) well contribute to tumor heterogeneity and account for tumor initiation and metastasis, but the molecular mechanisms of liver TIC self-renewal are elusive. Here, we identified a functional read-through rt-circRNA, termed rtcisE2F, that is highly expressed in liver cancer and liver TICs. rtcisE2F plays essential roles in the self-renewal and activities of liver TICs. rtcisE2F targets E2F6 and E2F3 mRNAs, attenuates mRNA turnover, and increases E2F6/E2F3 expression. Mechanistically, rtcisE2F functions as a scaffold of N-methyladenosine (m

Long noncoding RNA SLC9A3 antisense RNA 1 (SLC9A3-AS1) plays a central role in lung cancer; yet, its functions in nasopharyngeal carcinoma (NPC) have not been elucidated. The present study revealed the roles of SLC9A3-AS1 in NPC and dissected the mechanisms downstream of SLC9A3-AS1. SLC9A3-AS1 levels in NPC were assessed by applying RT-qPCR. The modulatory role of SLC9A3-AS1 interference on NPC cells was examined using numerous functional experiments. High expression of SLC9A3-AS1 was observed in NPC samples. Patients with NPC with a high level of SLC9A3-AS1 experienced a shorter overall survival than those with a low SLC9A3-AS1 level. Loss of SLC9A3-AS1 reduced NPC cell proliferation, colony formation, migration, and invasion but induced cell apoptosis in vitro. Animal experiments further revealed that the depletion of SLC9A3-AS1 hindered NPC tumour growth in vivo. As a competitive endogenous RNA, SLC9A3-AS1 sponged microRNA-486-5p (miR-486-5p), consequently upregulating E2F transcription factor 6 (E2F6). Finally, the effects of SLC9A3-AS1 silencing on NPC cells were reversed by inhibiting miR-486-5p or overexpressing E2F6. In summary, SLC9A3-AS1 exerted carcinogenic effects on NPC cells by adjusting the miR-486-5p/E2F6 axis. Accordingly, the newly identified SLC9A3-AS1/miR-486-5p/E2F6 pathway may offer attractive therapeutic targets for future development.

BACKGROUND It has been shown that circular RNAs (circRNAs) play a vital role in the regulation of neuronal differentiation; however, the precise role of circRNAs in human neuronal differentiation remains largely unexplored. MATERIAL AND METHODS A dual-luciferase reporter assay was carried out to confirm the targets of hsa_circ_0002468, miR-561, E2F8 (E2F transcription factor 8, a protein coding gene), and miR-561. We detected the expression of hsa_circ_0002468, miR-561, and E2F8 by using quantitative real-time polymerase chain reaction (qRT-PCR) analyses. In addition, we performed the functional experiments by using a BrdU (5-bromo-2'-deoxyuridine) assay and qRT-PCR analyses. RESULTS In this study, we showed that hsa_circ_0002468 can act as a sponge of miR-561 to regulate SH-SY5Y proliferation and differentiation. A bioinformatics analysis showed that hsa_circ_0002468 had a binding site that corresponded to miR-561, which was verified by dual-luciferase reporter assay. The expression of hsa_circ_0002468 was increased during SH-SY5Y differentiation and was inversely correlated with miR-561 expression. Using qRT-PCR analysis, we showed that hsa_circ_0002468 negatively regulated miR-561 in SH-SY5Y cells. Intriguingly, the overexpression of hsa_circ_0002468 increased SH-SY5Y differentiation and reduced SH-SY5Y proliferation; the suppression of hsa_circ_0002468 led to decreased SH-SY5Y differentiation levels and increased SH-SY5Y proliferation levels. Additionally, overexpression of miR-561 rescued the SH-SY5Y proliferation deficiency induced by hsa_circ_0002468 overexpression and abolished the SH-SY5Y differentiation promoted by hsa_circ_0002468. Furthermore, E2F8 was validated as a direct target of miR-561. CONCLUSIONS Our data suggested that hsa_circ_0002468 was a novel circRNA that regulated SH-SY5Y cell proliferation and differentiation via targeting the miR-561/E2F8 axis. Therefore, manipulating hsa_circ_0002468 in SH-SY5Y cells could be a novel strategy to develop novel interventions for the treatment of relevant neurological disorders.

Ovarian cancer is the most lethal cancer of the female reproductive system. In that regard, several epidemiological studies suggest that long‐term exposure to estrogen could increase ovarian cancer risk, although its precise role remains controversial. To decipher a mechanism for this, we previously generated a mathematical model of how estrogen‐mediated upregulation of the transcription factor, E2F6, upregulates the ovarian cancer stem/initiating cell marker, c‐Kit, by epigenetic silencing the tumor suppressor miR‐193a, and a competing endogenous (ceRNA) mechanism. In this study, we tested that previous mathematical model, showing that estrogen treatment of immortalized ovarian surface epithelial cells upregulated both E2F6 and c‐KIT, but downregulated miR‐193a. Luciferase assays further confirmed that microRNA‐193a targets both E2F6 and c‐Kit. Interestingly, ChIP‐PCR and bisulphite pyrosequencing showed that E2F6 also epigenetically suppresses miR‐193a, through recruitment of EZH2, and by a complex ceRNA mechanism in ovarian cancer cell lines. Importantly, cell line and animal experiments both confirmed that E2F6 promotes ovarian cancer stemness, whereas E2F6 or EZH2 depletion derepressed miR‐193a, which opposes cancer stemness, by alleviating DNA methylation and repressive chromatin. Finally, 118 ovarian cancer patients with miR‐193a promoter hypermethylation had poorer survival than those without hypermethylation. These results suggest that an estrogen‐mediated E2F6 ceRNA network epigenetically and competitively inhibits microRNA‐193a activity, promoting ovarian cancer stemness and tumorigenesis.

Dysregulation of long noncoding RNA (lncRNA) is known to be involved in numerous human diseases, including lung cancer. However, the precise biological functions of most lncRNA remain to be elucidated. Here, we identified a novel up-regulated lncRNA, LINC01436 (RefSeq: NR_110419.1), in non-small cell lung cancer (NSCLC). High expression of LINC01436 was significantly associated with poor overall survival. Notably, LINC01436 expression was transcriptionally repressed by E2F6 under normoxia, and the inhibitory effect was relieved in a hypoxic microenvironment. Gain- and loss-of-function studies revealed that LINC01436 acted as a proto-oncogene by promoting lung cancer cell growth, migration and invasion in vitro. Xenograft tumor assays in nude mice confirmed that LINC01436 promoted tumor growth and metastasis in vivo. Mechanistically, LINC01436 exerted biological functions by acting as a microRNA (miR)-30a-3p sponge to regulate the expression of its target gene EPAS1. Our findings characterize LINC01436 as a new hypoxia-sensitive lncRNA with oncogenic function in NSCLC, suggesting that LINC01436 may be a potential biomarker for prognosis and a potential target for treatment.

In mouse development, long-term silencing by CpG island DNA methylation is specifically targeted to germline genes; however, the molecular mechanisms of this specificity remain unclear. Here, we demonstrate that the transcription factor E2F6, a member of the polycomb repressive complex 1.6 (PRC1.6), is critical to target and initiate epigenetic silencing at germline genes in early embryogenesis. Genome-wide, E2F6 binds preferentially to CpG islands in embryonic cells. E2F6 cooperates with MGA to silence a subgroup of germline genes in mouse embryonic stem cells and in embryos, a function that critically depends on the E2F6 marked box domain. Inactivation of E2f6 leads to a failure to deposit CpG island DNA methylation at these genes during implantation. Furthermore, E2F6 is required to initiate epigenetic silencing in early embryonic cells but becomes dispensable for the maintenance in differentiated cells. Our findings elucidate the mechanisms of epigenetic targeting of germline genes and provide a paradigm for how transient repression signals by DNA-binding factors in early embryonic cells are translated into long-term epigenetic silencing during mouse development., DNA methylation targets CpG island promoters of germline genes to repress their expression in mouse somatic cells. Here the authors show that a transcription factor E2F6 is required to target CpG island DNA methylation and epigenetic silencing to germline genes during early mouse development.

CREB signaling is known for several decades, but how it regulates both positive and negative regulators of cell proliferation is not well understood. On the other hand functions of major epigenetic repressors such as DNMT3B, EZH2 and CUL4B for their repressive epigenetic modifications on chromatin have also been well studied. However, there is very limited information available on how these repressors are regulated at their transcriptional level. Here, using computational tools and molecular techniques including site directed mutagenesis, promoter reporter assay, chromatin immunoprecipitation (ChIP), we identified that CREB acts as a common transcription factor for DNMT3B, EZH2, CUL4B and E2F6. ChIP assay revealed that pCREB binds to promoters of these repressors at CREs and induce their transcription. As expected, the expression of these repressors and their associated repressive marks particularly H3K27me3 and H2AK119ub are increased and decreased upon CREB overexpression and knock-down conditions respectively in the cancer cells indicating that CREB regulates the functions of these repressors by activating their transcription. Since CREB and these epigenetic repressors are overexpressed in various cancer types, our findings showed the molecular relationship between them and indicate that CREB is an important therapeutic target for cancer therapy.

Epstein-Barr virus (EBV) was discovered as the first human tumor virus more than 50 years ago. EBV infects more than 90% of the human population worldwide and is associated with numerous hematologic malignancies and epithelial malignancies. EBV establishes latent infection in B cells, which is the typical program seen in lymphomagenesis. Understanding EBV-mediated transcription regulatory networks is one of the current challenges that will uncover new insights into the mechanism of viral-mediated lymphomagenesis. Here, we describe the regulatory profiles of several cellular factors (E2F6, E2F1, Rb, HDAC1, and HDAC2) together with EBV latent nuclear antigens using next-generation sequencing (NGS) analysis. Our results show that the E2F-Rb-HDAC complex exhibits similar distributions in genomic regions of EBV-positive cells and is associated with oncogenic super-enhancers involving long-range regulatory regions. Furthermore, EBV latent antigens cooperatively hijack this complex to bind at KLFs gene loci and facilitate KLF14 gene expression in lymphoblastoid cell lines (LCLs). These results demonstrate that EBV latent antigens can function as master regulators of this multisubunit repressor complex (E2F-Rb-HDAC) to reverse its suppressive activities and facilitate downstream gene expression that can contribute to viral-induced lymphomagenesis. These results provide novel insights into targets for the development of new therapeutic interventions for treating EBV-associated lymphomas. IMPORTANCE Epstein-Barr virus (EBV), as the first human tumor virus, infects more than 90% of the human population worldwide and is associated with numerous human cancers. Exploring EBV-mediated transcription regulatory networks is critical to understand viral-associated lymphomagenesis. However, the detailed mechanism is not fully explored. Now we describe the regulatory profiles of the E2F-Rb-HDAC complex together with EBV latent antigens, and we found that EBV latent antigens cooperatively facilitate KLF14 expression by antagonizing this multisubunit repressor complex in EBV-positive cells. This provides potential therapeutic targets for the treatment of EBV-associated cancers.

Recently, abundant evidence has clarified that long noncoding RNAs (lncRNAs) play an oncogenic or anticancer role in the tumorigenesis and development of diverse human cancers. Described as a crucial regulator in some cancers, MIR22HG has not yet been studied in laryngocarcinoma and therefore the underlying regulatory role of MIR22HG in laryngocarcinoma is worth detecting. In this study, MIR22HG expression in laryngocarcinoma cells was confirmed to be downregulated, and upregulated MIR22HG expression led to suppressive effects on laryngocarcinoma cell proliferation and migration. Molecular mechanism assays revealed that MIR22HG sponges miR-5000-3p in laryngocarcinoma cells. Besides, decreased expression of miR-5000-3p suppressed laryngocarcinoma cell proliferation and migration. Moreover, the FBXW7 gene was reported to be a downstream target gene of miR-5000-3p in laryngocarcinoma cells. More importantly, rescue assays verified that FBXW7 depletion or miR-5000-3p upregulation countervailed the repressive effects of MIR22HG overexpression on laryngocarcinoma progression. In addition, E2F6 was proved to be capable of inhibiting MIR22HG transcription in laryngocarcinoma cells. To sum up, E2F6-induced downregulation of MIR22HG promotes laryngocarcinoma progression through the miR-5000-3p/FBXW7 axis.

Cervical cancer (CC) is a prevalent gynecological cancer, and the patients with CC usually suffer from dismal prognosis. Long non-coding RNAs (lncRNAs) are demonstrated to serve as promising biological targets in human cancers. Gastric carcinoma proliferation enhancing transcript 1 (GHET1) has been revealed to function as an oncogene in several cancers, but it has never been investigated in CC. We proposed to examine the biological role of GHET1 in CC and the underlying mechanism and validated the up-regulated expression of GHET1 in CC cell lines. Loss-of-function assays demonstrated that down-regulation of GHET1 inhibited cell growth, migration and epithelial-to-mesenchymal transition (EMT) in CC. Furthermore, we validated that GHET1 down-regulation could inactivate AKT/mTOR and Wnt/β-catenin pathways, and that respective activation of these two pathways abrogated the inhibitive effect of GHET1 knockdown on CC cell growth, migration and EMT. Moreover, we unfolded a preliminary investigation on the modulation of GHET1 on AKT/mTOR and Wnt/β-catenin pathways. We found that GHET1 stabilized E2F6 mRNA through interacting with IGF2BP2, so as to regulate the activity of AKT/mTOR and Wnt/β-catenin pathways. Rescue assays also proved that GHET1 regulated these two pathways and CC cell growth, migration and EMT through E2F6. In conclusion, we revealed that down-regulation of GHET1 suppresses cervical cancer progression through regulating AKT/mTOR and Wnt/β-catenin signaling pathways, indicating GHET1 as a promising molecular biomarker for CC treatment improvement.

MDM2, an E3 ubiquitin ligase, is a potent inhibitor of the p53 tumor suppressor and is elevated in many human cancers that retain wild-type p53. MDM2 SNP309G is a functional polymorphism that results in elevated levels of MDM2 (due to enhanced SP1 binding to the MDM2 promoter) thus decreasing p53 activity. Mdm2SNP309G/G mice are more prone to spontaneous tumor formation than Mdm2SNP309T/T mice, providing direct evidence for the impact of this SNP in tumor development. We asked whether environmental factors impact SNP309G function and show that SNP309G cooperates with ionizing radiation to exacerbate tumor development. Surprisingly, ultraviolet B light or Benzo(a)pyrene exposure of skin shows that SNP309G allele actually protects against squamous cell carcinoma susceptibility. These contrasting differences led us to interrogate the mechanism by which Mdm2 SNP309 regulates tumor susceptibility in a tissue-specific manner. Although basal Mdm2 levels were significantly higher in most tissues in Mdm2SNP309G/G mice compared with Mdm2SNP309T/T mice, they were significantly lower in Mdm2SNP309G/G keratinocytes, the cell-type susceptible to squamous cell carcinoma. The assessment of potential transcriptional regulators in ENCODE ChIP-seq database identified transcriptional repressor E2F6 as a possible negative regulator of MDM2 expression. Our data show that E2F6 suppresses Mdm2 expression in cells harboring the SNP309G allele but not the SNP309T allele. Thus, Mdm2 SNP309G exhibits tissue-specific regulation and differentially impacts cancer risk.

Both type 1 and type 2 diabetes are associated with loss of functional beta cell mass, and strategies to restore beta cells are urgently needed. We reported previously that overexpression of the nuclear receptor TLX induces beta cell proliferation, but the underlying molecular mechanism has not been defined. Here, we identified direct targets of TLX in beta cells at the genome-wide level by ChIP-Seq. These targets include a cadre of regulators that are known to be critical for proliferation. Among these ChIP targets, E2F6 was tightly associated with the cell cycle modules, and thus, we further analyzed E2F6 expression and function in beta cells. We showed that E2F6 is strongly downregulated by TLX, and its expression inhibits beta cell proliferation. Moreover, coexpression of E2F6 with TLX partially abrogated the proliferative effects of TLX. These results strongly suggest that TLX acts through E2F6 to regulate beta cell proliferation. Together, the results of this study reveal a direct interaction between TLX and E2F6 and suggest new targets for the expansion of functional beta cell mass.

Aims To investigate the potential biological role of E2F6 and its underlying molecular mechanism in gastric carcinoma (GC) progression. Main methods The expressions of cancer susceptibility candidate 2 (CASC2), E2F6 and matrix metalloprotein-2 (MMP-2) were measured by quantitative real-time polymerase chain reaction and western blotting. The inhibitory effect of E2F6 on CASC2 was evaluated using luciferase reporter assay. Cell growth was assessed by colony formation assay and cell counting kit-8. Cell invasion and apoptosis were measured by transwell assay and flow cytometry assay, respectively. In vivo tumorigenicity was assessed by tumor xenografts in nude mice. Key findings Our data revealed that CASC2 was downregulated while E2F6 was upregulated in GC tissues and cell lines. Remarkably, lower expression of CASC2 was associated with poor survival in GC patients. E2F6 inhibited the expression of CASC2. Subsequently, reliable data showed that downregulation of E2F6 suppressed the proliferation and invasion, and promoted the apoptosis of GC cells. Furthermore, downregulation of E2F6 decreased the expression of MMP-2 and increased the activity of caspase-3. However, these changes triggered by E2F6 knockdown could be reversed by inhibition of CASC2. Moreover, we also proved that downregulation of CASC2 reverses the effect of E2F6 knockdown on tumor growth in vivo. Significance Our data demonstrated that E2F6 could regulate the proliferation, invasion and apoptosis of GC cells via inhibiting the expression of CASC2, suggesting that E2F6/CASC2 axis is another regulator of GC progression.

Tricholemmal carcinoma is a malignant cutaneous adnexal tumor showing outer root sheath differentiation, thought to be the malignant counterpart of trichilemmoma. Although the real existence of tricholemmal carcinoma continues to be a matter of debate, it has been introduced in the recently published 4th edition of World Health Organization classification of skin tumors. Herein, we evaluated whether immunohistochemistry (EMA, CK7, CK5/14, p63, p16, and Ber-EP4) supports tricholemmal carcinoma as a separate entity and whether it could be useful in this differential diagnosis. A total of 9 cases, 3 tricholemmal carcinomas and 6 clear-cell squamous cell carcinomas were evaluated on the basis of histological criteria suggested by the WHO. In our opinion, although these results need to be validated in larger series, they support tricholemmal carcinoma as a separate entity and suggest an immunohistochemical profile (clear-cell squamous cell carcinomas: EMA diffusely positive, CK7 negative; tricholemmal carcinoma: EMA negative, CK7 patchy or moderately positive) that could be useful for this differential diagnosis.

Summary Polycomb repressive complexes 1 and 2 (PRC1 and PRC2) control cell identity by establishing facultative heterochromatin repressive domains at common sets of target genes. PRC1, which deposits H2Aub1 through the E3 ligases RING1A/B, forms six biochemically distinct subcomplexes depending on the assembled PCGF protein (PCGF1–PCGF6); however, it is yet unclear whether these subcomplexes have also specific activities. Here we show that PCGF1 and PCGF2 largely compensate for each other, while other PCGF proteins have high levels of specificity for distinct target genes. PCGF2 associates with transcription repression, whereas PCGF3 and PCGF6 associate with actively transcribed genes. Notably, PCGF3 and PCGF6 complexes can assemble and be recruited to several active sites independently of RING1A/B activity (therefore, of PRC1). For chromatin recruitment, the PCGF6 complex requires the combinatorial activities of its MGA-MAX and E2F6-DP1 subunits, while PCGF3 requires an interaction with the USF1 DNA binding transcription factor., Graphical Abstract, Highlights • PRC1 complexes retain high target specificity with little compensatory functions • PCGF2 defines repressive signatures and PCGF3 and PCGF6 transcription active states • RING1A/B is dispensable for the assembly and recruitment of PCGF3 and PCGF6 complexes • PCGF3 and PCGF6 complexes bind target genes through specific DNA binding activities, PRC1 encompasses different sub-complexes with distinct biochemical properties. Scelfo et al. dissect these activities, highlighting their little redundancy and high functional specificity. PRC1 sub-complexes associate specifically with transcriptional repression or activation, can function independently of the core RING1A/B enzymes, and can be recruited to chromatin by specific DNA binding activities.

To investigate the effect of miR-425 on the proliferation and apoptosis of clear cell renal carcinoma (ccRCA) cells, and to explore the underlying mechanism.A total of 80 pairs of human clear cell renal carcinoma (ccRCA) and cancer-adjacent normal tissue samples were collected in this study. Human ccRCA cell line (786-O) and normal human kidney cell line (HK-2) were used in cellular research. The expression level of miR-425 was detected in ccRCA tissues and cells, respectively. Target genes of miR-425 were predicted by bioinformatics and verified by luciferase reporter gene assay. Moreover, the role of miR-425 in regulating E2F6 as well as its effect on the proliferation and apoptosis of ccRCA cells were detected.Quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR) results showed that the expression of miR-425 was significantly decreased in ccRCA tissues and cells. The proliferation ability and cell cycle of 786-O cells were significantly inhibited after miR-425 overexpression. The percentage of cells in G0/G1 phase was remarkably increased, while the percentage of cells in S and G2/M phases was significantly decreased. Besides, the number of apoptotic cells was significantly increased in the miR-425 intervention group. On-line target gene prediction software indicated that E2F6 was the potential downstream target gene of miR-425. RT-PCR, Western blotting and luciferase reporter gene assay demonstrated that the expression of E2F6 was negatively regulated by miR-425. In addition, subsequent experiments showed that the up-regulation of E2F6 could suppress the inhibitory effect of miR-425 on the proliferation and apoptosis of ccRCA cells.Our research demonstrated the inhibitory function of miR-425 in ccRCA. Therefore, the miR-425/E2F6 axis was expected to be one of the targets of ccRCA targeted therapy.

Background Pancreatic cancer characterizes high recurrence and poor prognosis. In clinical practice, radiotherapy is widely used for pancreatic cancer treatment. However, the outcome remains undesirable due to tumor repopulation and following recurrence and metastasis after radiation. So, it is highly needed to explore the underlying molecular mechanisms and accordingly develop therapeutic strategies. Our previous studies revealed that dying cells from chemoradiation could stimulate repopulation of surviving pancreatic cancer cells. However, we still knew little how dying cells provoke pancreatic cancer cell repopulation. We herein would explore the significance of TGF-β2 changes and investigate the modulation of microRNA-193a (miR-193a), and identify their contributions to pancreatic cancer repopulation and metastasis. Methods In vitro and in vivo repopulation models were established to mimic the biological processes of pancreatic cancer after radiation. Western blot, real-time PCR and dual-luciferase reporter assays were accordingly used to detect miR-193a and TGF-β2/TGF-βRIII signalings at the level of molecular, cellular and experimental animal model, respectively. Flow cytometry analysis, wound healing and transwell assay, vascular endothelial cell penetration experiment, and bioluminescence imaging were employed to assessthe biological behaviors of pancreatic cancer after different treatments. Patient-derived tumor xenograft (PDX) mice models were established to evaluate the therapeutic potential of miR-193a antagonist on pancreatic cancer repopulation and metastasis after radiation. Results miR-193a was highly expressed in the irradiated pancreatic cancer dying cells, accordingly elevated the level of miR-193a in surviving cells, and further promoted pancreatic cancer repopulation and metastasis in vitro and in vivo. miR-193a accelerated pancreatic cancer cell cycle and stimulated cell proliferation and repopulation through inhibiting TGF-β2/TGF-βRIII/SMADs/E2F6/c-Myc signaling, and even destroyed normal intercellular junctions and promoted metastasis via repressing TGF-β2/TGF-βRIII/ARHGEF15/ABL2 pathway. Knockdown of miR-193a or restoration of TGF-β2/TGF-βRIII signaling in pancreatic cancer cells was found to block pancreatic cancer repopulation and metastasis after radiation. In PDX models, the treatment in combination with miR-193a antagonist and radiation was found to dramatically inhibit pancreatic cancer cell repopulation and metastasis, and further improved the survival after radiation. Conclusions Our findings demonstrated that miR-193a stimulated pancreatic cancer cell repopulation and metastasis through modulating TGF-β2/TGF-βRIII signalings, and miR-193a might be a potential therapeutic target for pancreatic cancer repopulation and metastasis.

The E2F/Pocket protein (Rb) pathway regulates cell growth, differentiation, and death by modulating gene expression. We previously examined this pathway in the myocardium via manipulation of the unique E2F repressor, E2F6, which is believed to repress gene activity independently of Rb. Mice with targeted expression of E2F6 in postnatal myocardium developed dilated cardiomyopathy (DCM) without hypertrophic growth. We assessed the mechanisms of the apparent failure of compensatory hypertrophic growth as well as their response to the β-adrenergic agonist isoproterenol. As early as 2 weeks, E2F6 transgenic (Tg) mice present with dilated thinner left ventricles and significantly reduced ejection fraction and fractional shortening which persists at 6 weeks of age, but with no apparent increase in left ventricle weight: body weight (LVW:BW). E2F6-Tg mice treated with isoproterenol (6.1 mg/kg/day) show double the increase in LVW:BW than their Wt counterparts (32% vs 16%, p-value: 0.007). Western blot analysis revealed the activation of the adrenergic pathway in Tg heart tissue under basal conditions with ~2-fold increase in the level of β2-adrenergic receptors (p-value: 8.9E-05), protein kinase A catalytic subunit (PKA-C) (p-value: 0.0176), activated c-Src tyrosine-protein kinase (p-value: 0.0002), extracellular receptor kinase 2 (ERK2) (p-value: 0.0005), and induction of the anti-apoptotic protein Bcl2 (p-value 0. 0.00001). In contrast, a ~60% decrease in the cardiac growth regulator: AKT1 (p-value 0.0001) and a ~four fold increase in cyclic AMP dependent phosphodiesterase 4D (PDE4D), the negative regulator of PKA activity, were evident in the myocardium of E2F6-Tg mice. The expression of E2F3 was down-regulated by E2F6, but was restored by isoproterenol. Further, Rb expression was down-regulated in Tg mice in response to isoproterenol implying a net activation of the E2F pathway. Thus the unique regulation of E2F activity by E2F6 renders the myocardium hypersensitive to adrenergic stimulus resulting in robust hypertrophic growth. These data reveal a novel interplay between the E2F pathway, β2-adrenergic/PKA/PDE4D, and ERK/c-Src axis in fine tuning the pathological hypertrophic growth response. E2F6 deregulates E2F3 such that pro-hypertrophic growth and survival are enhanced via β2-adrenergic signaling however this response is outweighed by the induction of anti-hypertrophic signals so that left ventricle dilation proceeds without any increase in muscle mass.

Embryonic stem (ES) cells, derived from the inner cell mass of blastocysts, have a characteristic cell cycle with truncated G1 and G2 phases. Recent findings that suppression of Oct3/4 expression results in a reduced proliferation rate of ES cells suggest the involvement of Oct3/4 in the regulation of ES cell growth, although the underlying molecular mechanism remains unclear. In the present study, we identified E2F3a as a direct target gene of Oct3/4 in ES cells. Oct3/4 directly bound to the promoter region of the E2F3a gene and positively regulated expression of E2F3a in mouse ES cells. Suppression of E2F3a activity by E2F6 overexpression led to the reduced proliferation in ES cells, which was relieved by co-expression of E2F3a. Furthermore, cell growth retardation caused by loss of Oct3/4 was rescued by E2F3a expression. These results suggest that Oct3/4 upregulates E2F3a expression to promote ES cell growth.

Epithelioid sarcoma (ES) is a rare malignant soft tissue tumor occurring in the distal extremities of young adults and is characterized histologically by nodules of epithelioid cells showing central necrosis. Intra-articular ES is extremely rare; only four cases have been reported, but their radiologic and histologic documentation of intra-articular origin have been imprecise. We report the first radiologically and histologically well-documented case of primary synovial ES. A 59-year-old woman presented with pain followed by swelling of her right knee for 6 months. MRI revealed an entirely intra-articular nodular synovial mass in the lateral part of the right knee joint in a background of diffusely thickened synovium. Synovectomy was performed under the clinical impression of pigmented villonodular synovitis (PVNS), a diagnosis erroneously confirmed by the reporting pathologist. The tumor rapidly recurred 3 months afterward and the diagnosis of primary synovial ES was made. Despite above-knee amputation, the tumor continued to spread proximally to the retroperitoneum. She developed multiple lung metastases and died 20 months after initial presentation. The nodular aggregates of tumor cells with central necrosis resulted in diffuse polypoid synovial thickening mimicking tuberculous synovitis and PVNS. The tumor cells showed positive staining for EMA, CK19, CD34, and complete loss of INI1 staining, establishing the diagnosis of primary synovial ES. The ES spread from the synovium to and along the joint capsule, and then extra-articularly into the soft tissue surrounding the knee joint, with lymphovascular permeation. Such pattern of spread calls for radical surgical excision as the treatment of choice.

The E2F/Rb pathway regulates cell growth, differentiation, and death. In particular, E2F1 promotes apoptosis in all cells including those of the heart. E2F6, which represses E2F activity, was found to induce dilated cardiomyopathy in the absence of apoptosis in murine post-natal heart. Here we evaluate the anti-apoptotic potential of E2F6 in neonatal cardiomyocytes (NCM) from E2F6-Tg hearts which showed significantly less caspase-3 cleavage, a lower Bax/Bcl2 ratio, and improved cell viability in response to CoCl2 exposure. This correlated with a decrease in the pro-apoptotic E2F3 protein levels. In contrast, no difference in apoptotic markers or cell viability was observed in response to Doxorubicin (Dox) treatment between Wt and Tg-NCM. Dox caused a rapid and dramatic loss of the E2F6 protein in Tg-NCM within 6h and was undetectable after 12h. The level of e2f6 transcript was unchanged in Wt NCM, but was dramatically decreased in Tg cells in response to both Dox and CoCl2. This was related to an impact of the drugs on the α-myosin heavy chain promoter used to drive the E2F6 transgene. By comparison in HeLa, Dox induced apoptosis through upregulation of endogenous E2F1 involving post-transcriptional mechanisms, while E2F6 was down regulated with induction of the Checkpoint kinase-1 and proteasome degradation. These data imply that E2F6 serves to modulate E2F activity and protect cells including cardiomyocytes from apoptosis and improve survival. Strategies to modulate E2F6 levels may be therapeutically useful to mitigate cell death associated disorders.

Processing of microRNAs (miRNAs) from their precursors to their biologically active mature forms is regulated during development and cancer. We show that mouse pri- or pre-miR-151 can bind to and compete with mature miR-151-5p and miR-151-3p for binding sites contained within the complementary regions of the E2f6 mRNA 3' untranslated region (UTR). E2f6 mRNA levels were directly regulated by pri- or pre-miR-151. Conversely, miR-151-mediated repression of ARHGDIA mRNA was dependent on the level of mature miR-151 because only the mature miRNA binds the 3' UTR. Thus, processing of miR-151 can have different effects on separate mRNA targets within a cell. A bioinformatics pipeline revealed additional candidate regions where precursor miRNAs can compete with their mature miRNA counterparts. We validated this experimentally for miR-124 and the SNAI2 3' UTR. Hence, miRNA precursors can serve as post-transcriptional regulators of miRNA activity and are not mere biogenesis intermediates.

The Th2 cytokine IL-13 plays a key role in allergy, by regulating IgE, airway hyper secretion, eosinophils and mast cells. In this study, we aimed to identify novel transcription factors (TFs) that potentially regulated IL-13. We analyzed Th2 polarized naïve T cells from four different blood donors with gene expression microarrays to find clusters of genes that were correlated or anti-correlated with IL13. These clusters were further filtered, by selecting genes that were functionally related. In these clusters, we identified three transcription factors (TFs) that were predicted to regulate the expression of IL13, namely CEBPB, E2F6 and AHR. siRNA mediated knockdowns of these TFs in naïve polarized T cells showed significant increases of IL13, following knockdown of CEBPB and E2F6, but not AHR. This suggested an inhibitory role of CEBPB and E2F6 in the regulation of IL13 and allergy. This was supported by analysis of E2F6, but not CEBPB, in allergen-challenged CD4+ T cells from six allergic patients and six healthy controls, which showed decreased expression of E2F6 in patients. In summary, our findings indicate an inhibitory role of E2F6 in the regulation of IL-13 and allergy. The analytical approach may be generally applicable to elucidate the complex regulatory patterns in Th2 cell polarization and allergy.

The inhibitory ability of miR-424 on the proliferation of renal carcinoma cell and the migration and invasion of cancer cells has been widely explored and demonstrated. However, the effects of miR-424 on non-small cell lung cancer (NSCLC) have not been systematically examined. In this study, detected the growth and invasion effect of miR-424 in NSCLC A549 cell. The migration and molecular mechanism of this cell are also detected.NSCLC A549 cell was transfected with miR-424 and its inhibitor. After transfection, the proliferation ability of A549 cell was detectedby CCK8 assay. Then, the migration ability in A549 cell was detected by migration assays. Furthermore, the expression level of MMP2 and MMP9 in A549 was detected by Western blot and immune fluorescence. The 3'UTR of E2F6 was cloned into luciferase reporter vector and its enzymatic activitywas detected to verify whether miR-424 can target E2F6. The expression level of E2F6 in a549 cell after transfecing with miR-424 was detected by Western blot.After transfection of miR-424, the proliferation and migration abilities were remarkably decreased and the expression level of MMP-2 and MMP-9 were down-regulated in A549. Moreover, MiR-424 inhibited the enzymatic activity of luviferase reporter vector of E2F6. Specifically, the expression level of E2F6 was down-regulated in A549.miR-424 can inhibit the proliferation and migration abilities of A549 by negatively regulating the expression of E2F6. .背景与目的 已有的研究表明miR-424可抑制肾癌细胞增殖，抑制宫颈鳞癌细胞的迁移和侵袭能力，而其对非小细胞肺癌（non-small cell lung cancer, NSCLC）细胞的影响目前尚无系统研究。本研究探讨miR-424对NSCLC A549细胞生长和侵袭迁移能力的影响并进一步研讨其分子机制。方法 应用CCK8检测过表达及抑制miR-424的表达对A549细胞增殖的影响。应用Transwell检测过表达及抑制miR-424的表达对A549细胞侵袭能力的影响。应用Western blot检测过表达及抑制miR-424的表达对A549细胞中MMP9和MMP2蛋白水平的影响。构建E2F6 3’UTR区的荧光素酶报告载体，验证miR-424对E2F6的靶向作用。采用Western blot检测过表达及抑制miR-424的表达后，A549细胞中E2F6的表达。结果 过表达miR-424后，A549的生长和侵袭能力显著降低。过表达miR-424后，A549细胞的MMP-2和MMP-9表达下降。荧光素酶活性检测表明miR-424能够抑制E2F6的荧光素酶活性。过表达miR-424后，细胞内E2F6的表达降低。结论 miR-424能够通过调控E2F6而抑制A549的生长和侵袭能力。.

Aberrant activation of Wnt and base excision repair (BER) signaling pathways are implicated in tumor progression and chemotherapy resistance in gastric adenocarcinoma. This study was conducted to clarify the role of E2F6 and RhoA, components of the Wnt signaling pathway, and SMUG1, a component of the BER pathway in gastric adenocarcinoma. Expression levels and clinicopathological significance of three biomarkers, namely E2F6, RhoA, and SMUG1, as potential signaling molecules involved in tumorigenesis and aggressive behavior, were examined using tissue microarray. Our analysis showed a relative increase in the expression of E2F6 in gastric adenocarcinoma with no lymph node metastasis (χ 2, P = 0.04 and OR, P = 0.08), while overexpression of RhoA and SMUG1 was found more often in the diffuse subtype of gastric adenocarcinoma as compared to the intestinal subtype (χ 2, P = 0.05, OR, P = 0.08 and χ 2, P = 0.001, OR, P = 0.009, respectively). Higher expression of RhoA was frequently seen in tumors with vascular invasion (χ 2, P = 0.01 and OR, P = 0.01). In addition, increased expression of SMUG1 was found more often in poorly differentiated tumors (χ 2, P = 0.01 and OR, P = 0.01). The distinct phenotype of E2F6Low/SMUG1High was more common in poorly differentiated tumors (P = 0.04) and with omental involvement (P = 0.01). The RhoAHigh/SMUG1High expression pattern was significantly more often found in diffuse subtype compared to the intestinal subtype (P = 0.001) as well as in poorly differentiated tumors (P = 0.004). The E2F6Low/SMUG1High and RhoAHigh/SMUG1High phenotypes can be considered as aggressive phenotypes of gastric adenocarcinoma. Our findings also demonstrated the synergistic effect of RhoA and SMUG1 in conferring tumor aggressiveness in diffuse subtype of gastric adenocarcinoma.

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., Author Summary EBV is associated with a broad range of human cancers. EBV-encoded nuclear antigen 3C (EBNA3C) is one of the essential latent antigens important for deregulating the functions of numerous host transcription factors which play vital roles in B-cell immortalization. The family of E2F transcription factors are involved in diverse cellular functions. More specifically, E2F6 is one of the E2F family members with a unique property of transcriptional repression. Our current study now demonstrates that EBNA3C can enhance E2F6 repressive functions, and is also responsible for increased E2F6 protein expression in EBV-transformed LCLs. EBNA3C directly interacts with E2F6 at its amino and carboxy terminal domains. Additionally, E2F6 was stabilized by EBNA3C and co-localized in nuclear compartments. Our study also demonstrated that EBNA3C and E2F6 expression resulted in decreased transcriptional activity of E2F1, and that EBNA3C, E2F6 and E2F1 can form a protein complex, and EBNA3C competes with E2F1 for E2F6 binding. The recruitment of E2F6 by EBNA3C was also shown to be important for its related cell proliferation. These results showed a crucial role for EBNA3C-mediated deregulation of E2F6 and its impact on the activities of other E2F family members. Our findings also provide new insights for targeting these E2F transcription factors as potential therapeutic intervention strategies in EBV-associated cancers.

Primary intraosseous myoepithelial tumours, including carcinomas are rare tumours. The concept of histopathological spectrum of these tumours is evolving. We describe clinicopathological and immunohistochemical features of five myoepithelial carcinomas, including molecular cytogenetic results in one case. There were five male patients within age-range of 8-40 years (median = 26). Four tumours occurred in the long bones, including two tumours, each, in the femur and fibula, respectively, while a single tumour occurred in the proximal phalanges. Tumour size (n = 3 cases) varied from 5.6 to 8.6 cm. On radiological imaging, most tumours appeared as expansile, lytic and destructive lesions. Two tumours appeared as sclerotic lesions. Two cases were referred with diagnoses of chondrosarcomas and a single case was referred with two different diagnoses, including an adamantinoma and an osteosarcoma. Histopathological examination in all these cases showed multinodular tumours comprising mostly polygonal cells, exhibiting moderate nuclear atypia and interspersed mitotic figures within a stroma containing variable amount of myxoid, chondroid, hyalinised and osteoid-like material. Three tumours revealed prominent squamous differentiation. By immunohistochemistry, tumour cells were positive for EMA (5/5), pan CK (AE1/AE3) (3/3), CK5/6 (4/4), CK MNF116 (1/1), S100 protein (5/5) and GFAP (3/5). The first tumour revealed EWSR1 rearrangement. The first patient, 10 months after tumour resection and a simultaneous lung metastatectomy, is free-of-disease (FOD). The second patient, 11 months after tumour resection is FOD. The third and fourth patients underwent wide resections and are on follow-up. The fifth patient underwent resections, including a lung metastatectomy. Primary intraosseous myoepithelial carcinomas are rare and mimic conventional primary bone tumours. Some primary intraosseous myoepithelial carcinomas display EWSR1 rearrangement. Squamous differentiation may be considered as an addition to their evolving histopathological spectrum. Immunohistochemical stains constitute as a necessary tool for arriving at the correct diagnosis in such cases, which has treatment implications. Surgical resection remains the treatment mainstay.

International audience; Methylation of cytosine residues within the CpG dinucleotide in mammalian cells is an important mediator of gene expression, genome stability, X-chromosome inactivation, genomic imprinting, chromatin structure, and embryonic development. The majority of CpG sites in mammalian cells is methylated in a nonrandom fashion, raising the question of how DNA methylation is distributed along the genome. Here, we focused on the functions of DNA methyltransferase-3b (Dnmt3b), of which deregulated activity is linked to several human pathologies. We generated Dnmt3b hypomorphic mutant mice with reduced catalytic activity, which first revealed a deregulation of Hox genes expression, consistent with the observed homeotic transformations of the posterior axis. In addition, analysis of deregulated expression programs in Dnmt3b mutant embryos, using DNA microarrays, highlighted illegitimate activation of several germ-line genes in somatic tissues that appeared to be linked directly to their hypomethylation in mutant embryos. We provide evidence that these genes are direct targets of Dnmt3b. Moreover, the recruitment of Dnmt3b to their proximal promoter is dependant on the binding of the E2F6 transcriptional repressor, which emerges as a common hallmark in the promoters of genes found to be up-regulated as a consequence of impaired Dnmt3b activity. Therefore, our results unraveled a coordinated regulation of genes involved in meiosis, through E2F6-dependant methylation and transcriptional silencing in somatic tissues. DNA methylation | immunodeficiency | centromeric instability | facial anomalies | E2F family | hypomorphic mutation | hox genes

Diverse Polycomb repressive complexes 1 (PRC1) play essential roles in gene regulation, differentiation and development. Six major groups of PRC1 complexes that differ in their subunit composition have been identified in mammals. How the different PRC1 complexes are recruited to specific genomic sites is poorly understood. The Polycomb Ring finger protein PCGF6, the transcription factors MGA and E2F6, and the histone-binding protein L3MBTL2 are specific components of the non-canonical PRC1.6 complex. In this study, we have investigated their role in genomic targeting of PRC1.6. ChIP-seq analysis revealed colocalization of MGA, L3MBTL2, E2F6 and PCGF6 genome-wide. Ablation of MGA in a human cell line by CRISPR/Cas resulted in complete loss of PRC1.6 binding. Rescue experiments revealed that MGA recruits PRC1.6 to specific loci both by DNA binding-dependent and by DNA binding-independent mechanisms. Depletion of L3MBTL2 and E2F6 but not of PCGF6 resulted in differential, locus-specific loss of PRC1.6 binding illustrating that different subunits mediate PRC1.6 loading to distinct sets of promoters. Mga, L3mbtl2 and Pcgf6 colocalize also in mouse embryonic stem cells, where PRC1.6 has been linked to repression of germ cell-related genes. Our findings unveil strikingly different genomic recruitment mechanisms of the non-canonical PRC1.6 complex, which specify its cell type- and context-specific regulatory functions., Author summary Polycomb group proteins assemble in two major repressive multi-subunit complexes (PRC1 and PRC2), which play important roles in many physiological processes, including stem cell maintenance, differentiation, cell cycle control and cancer. In mammals, six different groups of PRC1 complexes exist (PRC1.1 to PRC1.6), which differ in their subunit composition. The mechanisms that target the different PRC1 complexes to specific genomic sites appear diverse and are poorly understood. In this study, we have investigated the genomic targeting mechanisms of the non-canonical PRC1.6 complex. In PRC1.6, the defining subunit PCGF6 is specifically associated with several proteins including the transcription factors MGA and E2F6, and the histone-binding protein L3MBTL2. We found that MGA is absolutely essential for targeting PRC1.6. MGA executes recruitment of PRC1.6 to its target sites through two distinct functions. On the one hand it acts as a sequence-specific DNA-binding factor; on the other hand it has a scaffolding function, which is independent of its DNA binding capacity. E2F6 and L3MBTL2 are also important in genomic targeting of PRC1.6 as they promote binding of PRC1.6 to different sets of genes associated with distinct functions. Our finding that different components specify loading of PRC1.6 to distinct sets of genes could establish a paradigm for other chromatin-associated complexes.

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 (CoCl2, 800 μM)-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 E2F6ΔC (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.

In the last few years, microRNAs (miRNA) have started a revolution in molecular biology and emerged as key players in the carcinogenesis. They have been identified in various tumor types, showing that different sets of miRNAs are usually deregulated in different cancers. To identify the miRNA signature that was specific for oral squamous cell carcinoma (OSCC), we first examined expression profiles of 148 miRNAs in a panel of 18 OSCC cell lines and the immortalized oral keratinocyte line RT7 as a control. Compared with RT7, the expression of 54 miRNAs (36.5%) was frequently down-regulated in OSCC lines (

Bmi1 is a Polycomb Group protein that functions as a component of Polycomb Repressive Complex 1 (PRC1) to control axial skeleton development through Hox gene repression. Bmi1 also represses transcription of the Ink4a-Arf locus and is consequently required to maintain the proliferative and self-renewal properties of hematopoietic and neural stem cells. Previously, one E2F family member, E2F6, has been shown to interact with Bmi1 and other known PRC1 components. However, the biological relevance of this interaction is unknown. In this study, we use mouse models to investigate the interplay between E2F6 and Bmi1. This analysis shows that E2f6 and Bmi1 cooperate in the regulation of Hox genes, and consequently axial skeleton development, but not in the repression of the Ink4a-Arf locus. These findings underscore the significance of the E2F6-Bmi1 interaction in vivo and suggest that the Hox and Ink4a-Arf loci are regulated by somewhat different mechanisms.

E2F6 is believed to repress E2F-responsive genes and therefore plays an important role in cell-cycle regulation. However, the role of E2F6 in the control of apoptosis remains unknown. We show here that the expression of E2F6 was downregulated with a concurrent increase in BRCA1 mRNA and cleaved protein during ultraviolet (UV)-induced apoptosis in human embryonic kidney 293 cells. Moreover, E2F6 overexpression distinctly inhibited UV-induced apoptosis as well as UV-induced increases in BRCA1 expression and cleavage, accompanied with increases of the full-length BRCA1 and BRCA1 nuclear foci. In contrast, knockdown of E2F6 by small interfering RNA had opposite effects. Furthermore, these effects of E2F6 on BRCA1 depended upon the association of E2F6 with BRCA1 via its C-terminus in a UV-triggered manner and upon the transcriptional repression by E2F6 on the BRCA1 promoter. These findings provide the first demonstration of the important role for E2F6 in the control of apoptosis via targeting of BRCA1.

E2F6 is believed to repress E2F-responsive genes and therefore serve a role in cell cycle regulation. Analysis of the human E2F6 promoter region revealed the presence of two putative E2F binding sites, both of which were found to be functionally critical because deletion or mutations of these sites abolished promoter activity. Ectopic expression of E2F1 protein was found to increase E2F6 mRNA levels and significantly upregulate E2F6 promoter activity. Deletion or mutation of the putative E2F binding sites nullified the effects of E2F1 on the E2F6 promoter activity. Studies on the temporal induction of E2F family members demonstrated that the activating E2Fs, and most notably E2F1, were upregulated before E2F6 during cell cycle progression at the G1/S phase, and this coincided with the time course of induction experienced by the E2F6 promoter during the course of the cell cycle. EMSAs indicated the specific binding of nuclear complexes to the E2F6 promoter that contained E2F1-related species whose binding was specifically competed by the consensus E2F binding site. Chromatin immunoprecipitation assays with anti-E2Fs demonstrated the association of E2F family members with the E2F6 promoter in vivo. These data indicate that the expression of the E2F6 repressor is influenced at the transcriptional level by E2F family members and suggest that interplay among these transcriptional regulators, especially E2F1, may be critical for cell cycle regulation.