Maintaining quiescent cells in G0 phase is achieved in part through the multiprotein subunit complex known as DREAM, and in human cell lines the transcription factor E2F4 directs this complex to its cell cycle targets. We found that E2F4 binds a highly overlapping set of human genes among three diverse primary tissues and an asynchronous cell line, which suggests that tissue-specific binding partners and chromatin structure have minimal influence on E2F4 targeting. To investigate the conservation of these transcription factor binding events, we identified the mouse genes bound by E2f4 in seven primary mouse tissues and a cell line. E2f4 bound a set of mouse genes that was common among mouse tissues, but largely distinct from the genes bound in human. The evolutionarily conserved set of E2F4 bound genes is highly enriched for functionally relevant regulatory interactions important for maintaining cellular quiescence. In contrast, we found minimal mRNA expression perturbations in this core set of E2f4 bound genes in the liver, kidney, and testes of E2f4 null mice. Thus, the regulatory mechanisms maintaining quiescence are robust even to complete loss of conserved transcription factor binding events.

Cervical cancer is the most common gynecological cancer worldwide and is associated with high morbidity and mortality. Despite improvements in therapeutic strategies, the network regulation mechanism remains unclear and the treatment effect is not satisfactory. Therefore, there is a need to continue studying the mechanism of cervical cancer to explore effective gene targets and precise targeted therapy drugs.

Cervical cancer is one of the major malignancies, the pathophysiology and progression of which remain to be scarcely understood. Long non-coding RNAs (lncRNAs) have been previously implicated in the progression of cervical cancer. Here, the purpose of this study was to identify whether lncRNA heart- and neural crest derivative-expressed 2-antisense RNA 1 (HAND2-AS1) affect the development of cervical cancer through regulation of chromosome 16 open reading frame 74 (C16orf74) by mediating a transcription factor E2F4. RT-qPCR was performed to determine the expression of HAND2-AS1 in cervical cancer cells. Then, cervical cancer cells were treated with HAND2-AS1 or si-E2F4 RNA, or C16orf74, after which the proliferation, colony formation, migration and invasion were detected. Moreover, the binding between HAND2-AS1 and E2F4 or between E2F4 and C16orf74 was explored. Finally, the tumorigenesis of cervical cancer cells was measured in nude mice with altered HAND2-AS1/E2F4/C16orf74 expression. HAND2-AS1 exhibited poor expression in cervical cancer, and HAND2-AS1 overexpression suppressed the proliferation, colony formation, migration and invasion of cervical cancer cells. In addition, HAND2-AS1 was found to recruit transcription factor E2F4 to C16orf74 promoter region and down-regulate C16orf74 expression. Lastly, HAND2-AS1/E2F4/C16orf74 modulated the tumorigenesis of cervical cancer in nude mice. In conclusion, this study provided evidence on the inhibitory effect of HAND2-AS1 on the development of cervical cancer through the suppression of C16orf74 expression by recruiting transcription factor E2F4. This study highlights the potential of lncRNA HAND2-AS1 as a target in the treatment of cervical cancer.

Background: Recent studies have shown that the transcription factor E2F4 is involved in the progression of various tumors, but its expression and influence on immune cell infiltration and biological functions are largely unknown in hepatocellular carcinoma (HCC). Methods: The Cancer Genome Atlas (TCGA) database, the Tumor Immune Estimation Resource (TIMER) and related online tools as well as a tissue microarray (TMA) were used for analyses in our study. Results: E2F4 expression was elevated in HCC tumor tissue compared with adjacent normal tissue at both the mRNA and protein levels. Overexpression of E2F4 was markedly related to a poor prognosis in HCC patients. In addition, positively and negatively correlated significant genes of E2F4 were identified in HCC. Pathway enrichment analyses revealed that the top 100 positively correlated significant genes of E2F4 were closely related to nuclear splicing and degradation-related pathways. Furthermore, nine hub genes correlated with E2F4 expression were validated based on a protein-protein interaction (PPI) network. It was also demonstrated that E2F4 expression was negatively correlated to immune purity and positively correlated to immune cell infiltration. Conclusion: E2F4 could serve as a novel biomarker for HCC diagnosis and prognosis prediction.

Hepatitis B virus (HBV) infection is a major risk factor for hepatocellular carcinoma (HCC), which required developing novel therapies targeting the inhibition of HBV transcription and replication due to current limited treatment options. We explored novel target for the development of novel therapies targeting the inhibition of HBV replication and transcription. The expression of Id1 and E2F4 in HCC cells and tissues was detected by qRT-PCR and western blot. We investigated the Id1 and E2F4-mediated transcription of HBV infection by using HepG2.2.15, HepAD38, HepG2-NTCP cell lines and AAV/HBV-infected mice. Interactions between the two host proteins and viral covalently closed circular DNA (cccDNA) were assessed using subcellular localization, protein-protein interaction, chromatin immunoprecipitation, and luciferase assays. Ectopic Id1 significantly reduced HBV transcription and replication in both HBV-expressing cells and AAV/HBV-infected mice. Id1 and E2F4 could form a heterodimer to prevent E2F4 from promoting HBV transcription and replication. E2F4 could directly bind to cccDNA and activate the HBV core promoter in cell lines. Furthermore, in vitro binding experiments confirmed that the sequence 1758'-TTAAAGGTC-1766', which is highly conserved among HBV genotypes, is the target site of the E2F4 homodimer. The findings suggest that E2F4 function as novel cccDNA-binding protein to directly activate HBV transcription by binding to Cp promoter region. Our results highlight the ability that E2F4 represent a pan-potential therapeutic target against HBV transcription and provide more clues to better understand the life cycle of HBV.

Sirtuin 5 (SIRT5) belongs to the mitochondrial sirtuin family, which constitutes a highly conserved family of nicotinamide adenine dinucleotide NAD+-dependent deacetylases and ADP-ribosyltransferases that play important regulatory roles in stress resistance and metabolic homeostasis. SIRT5 was shown to have deacetylase, desuccinylase, and demalonylase activities. However, the mechanisms regulating SIRT5 transcription remain unclear. To explore the molecular regulation of bovine SIRT5 expression, we obtained a 500-base pair fragment of the 5'-regulatory region of bovine SIRT5 by molecular cloning, which contained a region with 3 CpG islands. Electrophoretic mobility shift assays and luciferase reporter assays revealed the E2F transcription factor 4 (E2F4) and Kruppel-like factor 6 (KLF6) binding sites as transcriptional activators or repressors in the promoter region of SIRT5. We further verified that E2F4 and KLF6 bind to the SIRT5 promoter by chromatin immunoprecipitation assays. Additionally, methylation and luciferase report assays showed that SIRT5 promoter activity was enhanced by demethylation, and transcriptional activation by E2F4 and transcriptional inhibition by KLF6 of SIRT5 expression was strengthened by demethylation during adipocytes differentiation. This study focused on the mechanism underlying the methylation and transcriptional regulation of SIRT5 expression in bovine adipocytes.

Multiciliated cells (MCCs) are specialized epithelial cells that project hundreds of motile cilia. To form these cilia, MCCs differentiate by dramatically expanding centriole number, using assembly factors required for centriole duplication during the cell cycle and multiple, novel assembly sites, called the deuterosome. The small coiled-coil protein, Multicilin, acting in a complex with the E2F proteins can initiate multiciliated cell differentiation, but reportedly only in a limited range of epithelial progenitors. To examine the nature of this restricted activity, we analyzed Multicilin activity in primary mouse embryonic fibroblasts (MEFs), a cell type distant from the epithelial lineages where MCCs normally arise. We show that Multicilin transcriptional activity is markedly attenuated in MEFs, where it induces only limited centriole expansion in a small fraction of cells. We further show that this transcriptional block is largely bypassed by expressing Multicilin along with a form of E2f4 where a generic activation domain from HSV1 VP16 (E2f4VP16) is fused to the carboxy terminus. MEFs respond to Multicilin and E2f4VP16 by undergoing massive centriole expansion via the deuterosome pathway, recapitulating a temporal sequence of organelle biogenesis that occurs in epithelial progenitors during MCC differentiation. These results suggest that the pattern of organelle biogenesis occurring in differentiating MCCs is largely determined by the transcriptional changes induced by Multicilin.

Liver cancer, the second most commonly diagnosed cancer, is associated with high mortality rates. E2F4 is a member of the E2F transcription factor family. There are limited studies on the role of E2F4 in hepatocellular carcinoma (HCC). In this study, the expression of E2F4 in HCC tissue samples and cell lines was analyzed using quantitative real-time polymerase chain reaction. E2F4 expression positively correlated with tumor size in patients with HCC. Additionally, E2F4 expression was greater in HCC cells than in normal LO2 cells. Furthermore, overexpression of E2F4 significantly enhanced the proliferation, migration, and invasion of HCC cells. The results of a luciferase assay revealed that E2F4 upregulated the expression of CDCA3 by binding to its promoter region (1863'-ACGCGCGAGAATG-1875') and consequently promoted proliferation and cell cycle progression of HCC cells. Taken together, these results demonstrated that E2F4 might play a vital role in HCC progression and could serve as a potential biomarker for the diagnosis and as a therapeutic target of HCC.

Alzheimer's disease (AD) has a complex etiology, which requires a multifactorial approach for an efficient treatment. We have focused on E2 factor 4 (E2F4), a transcription factor that regulates cell quiescence and tissue homeostasis, controls gene networks affected in AD, and is upregulated in the brains of Alzheimer's patients and of APPswe/PS1dE9 and 5xFAD transgenic mice. E2F4 contains an evolutionarily conserved Thr-motif that, when phosphorylated, modulates its activity, thus constituting a potential target for intervention. In this study, we generated a knock-in mouse strain with neuronal expression of a mouse E2F4 variant lacking this Thr-motif (E2F4DN), which was mated with 5xFAD mice. Here, we show that neuronal expression of E2F4DN in 5xFAD mice potentiates a transcriptional program consistent with the attenuation of the immune response and brain homeostasis. This correlates with reduced microgliosis and astrogliosis, modulation of amyloid-β peptide proteostasis, and blocking of neuronal tetraploidization. Moreover, E2F4DN prevents cognitive impairment and body weight loss, a known somatic alteration associated with AD. We also show that our finding is significant for AD, since E2F4 is expressed in cortical neurons from Alzheimer patients in association with Thr-specific phosphorylation, as evidenced by an anti-E2F4/anti-phosphoThr proximity ligation assay. We propose E2F4DN-based gene therapy as a promising multifactorial approach against AD.

E2F/RB activity is altered in most human tumors. The retinoblastoma family of proteins plays a key role in regulating the progression of the cell cycle from the G1 to S phases. This is achieved through negative regulation of E2F transcription factors, important positive regulators of cell cycle entry. E2F family members are divided into two groups: activators (E2F1-E2F3a) and repressors (E2F3b-E2F8). E2F4 accounts for a large part of the E2F activity and is a main E2F repressor member in vivo. Perturbations in the balance from quiescence towards proliferation contribute to increased mitotic gene expression levels frequently observed in cancer. We have previously reported that combined Rb1-Rbl1 or Rb1-E2f1 ablation in epidermis produces important alterations in epidermal proliferation and differentiation, leading to tumor development. However, the possible roles of E2F4 in this context are still to be determined. Here, we show the absence of any discernible phenotype in the skin of mice lacking of E2f4. In contrast, the inducible loss of Rb1 in the epidermis of E2F4-null mice produced multiple skin abnormalities including altered differentiation and proliferation, spontaneous wounds, carcinoma in situ development and stem cell perturbations. All these phenotypic alterations are associated with extensive gene expression changes, the induction of c-myc and the Akt activation. Moreover the whole transcriptome analyses in comparison with previous models generated also revealed extensive changes in multiple repressive complexes and in transcription factor activity. These results point to E2F4 as a master regulator in multiple steps of epidermal homeostasis in Rb1 absence.

Abnormal development of multiciliated cells is a hallmark of a variety of human conditions associated with chronic airway diseases, hydrocephalus and infertility. Multiciliogenesis requires both activation of a specialized transcriptional program and assembly of cytoplasmic structures for large-scale centriole amplification that generates basal bodies. It remains unclear, however, what mechanism initiates formation of these multiprotein complexes in epithelial progenitors. Here we show that this is triggered by nucleocytoplasmic translocation of the transcription factor E2f4. After inducing a transcriptional program of centriole biogenesis, E2f4 forms apical cytoplasmic organizing centres for assembly and nucleation of deuterosomes. Using genetically altered mice and E2F4 mutant proteins we demonstrate that centriole amplification is crucially dependent on these organizing centres and that, without cytoplasmic E2f4, deuterosomes are not assembled, halting multiciliogenesis. Thus, E2f4 integrates nuclear and previously unsuspected cytoplasmic events of centriole amplification, providing new perspectives for the understanding of normal ciliogenesis, ciliopathies and cancer.

Replicative stress promotes genomic instability and tumorigenesis but also presents an effective therapeutic endpoint, rationalizing detailed analysis of pathways that control DNA replication. We show here that the transcription factor E2f4 recruits the DNA helicase Recql to facilitate progression of DNA replication forks upon drug- or oncogene-induced replicative stress. In unperturbed cells, the Trim33 ubiquitin ligase targets E2f4 for degradation, limiting its genomic binding and interactions with Recql. Replicative stress blunts Trim33-dependent ubiquitination of E2f4, which stimulates transient Recql recruitment to chromatin and facilitates recovery of DNA synthesis. In contrast, deletion of Trim33 induces chronic genome-wide recruitment of Recql and strongly accelerates DNA replication under stress, compromising checkpoint signaling and DNA repair. Depletion of Trim33 in Myc-overexpressing cells leads to accumulation of replication-associated DNA damage and delays Myc-driven tumorigenesis. We propose that the Trim33-E2f4-Recql axis controls progression of DNA replication forks along transcriptionally active chromatin to maintain genome integrity.

E2F4 is an important basal transcription factor with the potential to promote tumor growth. Its upregulation in various types of cancer has been linked to numerous genetic factors; however, the nature of the involvement of epigenetic mechanisms, including DNA methylation, remains elusive. In the present study, E2F4 expression profiles were determined in 100 paired breast tumor and control samples, through RT-qPCR using the SYBR® green method. Furthermore, the E2F4 promoter methylation status in each of these samples was assessed using methylation specific PCR, in order to evaluate its impact on gene expression. A two-fold increase in E2F4 gene expression was observed in the breast tumors compared with in their respective controls (P=0.022); of these tumors, ~72% were under-methylated. The change in methylation status was also significantly higher (P<0.001) in the tumor samples. Methylation status was negatively correlated (r=-30) with E2F4 expression profiles, indicating that a decrease in methylation may promote higher expression of E2F4. The two study cohorts (>45 and ≤45 years) had comparable methylation profiles, though they had significantly decreased methylation status compared with controls. Various histo-pathological types also have different methylation profiles, indicating the presence of a tissue specific methylation signature. The results of the present study demonstrated that E2F4 methylation status can have a notable influence on its expression, and that it may have prognostic value in breast carcinogenesis.

After decades of unfruitful work, no effective therapies are available for Alzheimer's disease (AD), likely due to its complex etiology that requires a multifactorial therapeutic approach. We have recently shown using transgenic mice that E2 factor 4 (E2F4), a transcription factor that regulates cell quiescence and tissue homeostasis, and controls gene networks affected in AD, represents a good candidate for a multifactorial targeting of AD. Here we show that the expression of a dominant negative form of human E2F4 (hE2F4DN), unable to become phosphorylated in a Thr-conserved motif known to modulate E2F4 activity, is an effective and safe AD multifactorial therapeutic agent. Neuronal expression of hE2F4DN in homozygous 5xFAD (h5xFAD) mice after systemic administration of an AAV.PHP.B-hSyn1.hE2F4DN vector reduced the production and accumulation of Aβ in the hippocampus, attenuated reactive astrocytosis and microgliosis, abolished neuronal tetraploidization, and prevented cognitive impairment evaluated by Y-maze and Morris water maze, without triggering side effects. This treatment also reversed other alterations observed in h5xFAD mice such as paw-clasping behavior and body weight loss. Our results indicate that E2F4DN-based gene therapy is a promising therapeutic approach against AD.

The p16INK4a-RB pathway plays a critical role in preventing inappropriate cell proliferation and is often targeted by viral oncoproteins during immortalization. Latent membrane protein 1 (LMP1) of Epstein-Barr virus (EBV) is often present in EBV-associated proliferative diseases and is critical for the immortalizing and transforming activity of EBV. Unlike other DNA tumor virus oncoproteins, which possess immortalizing activity, LMP1 does not bind to retinoblastoma tumor suppressor protein, but instead blocks the expression of p16INK4a tumor suppressor gene. However, it has been unclear how LMP1 represses the p16INK4a gene expression. Here, we report that LMP1 promotes the CRM1-dependent nuclear export of Ets2, which is an important transcription factor for p16INK4a gene expression, thereby reducing the level of p16INK4a expression. We further demonstrate that LMP1 also blocks the function of E2F4 and E2F5 (E2F4/5) transcription factors through promoting their nuclear export in a CRM1-dependent manner. As E2F4/5 are essential downstream mediators for a p16INK4a-induced cell cycle arrest, these results indicate that the action of LMP1 on nuclear export has two effects on the p16INK4a-RB pathway: (1) repression of p16INK4a expression and (2) blocking the downstream mediator of the p16INK4a-RB pathway. These results reveal a novel activity of LMP1 and increase an understanding of how viral oncoproteins perturb the p16INK4a-RB pathway.

The prognosis of high-grade gliomas, such as glioblastoma multiforme (GBM), is extremely poor due to the highly invasive nature of these aggressive cancers. Previous work has demonstrated that TNF-weak like factor (TWEAK) induction of the noncanonical NF-κB pathway promotes the invasiveness of GBM cells in an NF-κB-inducing kinase (NIK)-dependent manner. While NIK activity is predominantly regulated at the posttranslational level, we show here that NIK (MAP3K14) is upregulated at the transcriptional level in invading cell populations, with the highest NIK expression observed in the most invasive cells. GBM cells with high induction of NIK gene expression demonstrate characteristics of collective invasion, facilitating invasion of neighboring cells. Furthermore, we demonstrate that the E2F transcription factors E2F4 and E2F5 directly regulate NIK transcription and are required to promote GBM cell invasion in response to TWEAK. Overall, our findings demonstrate that transcriptional induction of NIK facilitates collective cell migration and invasion, thereby promoting GBM pathogenesis.

BACKGROUND Pancreatic adenocarcinoma (PAAD) is one of the deadliest types of cancer. In the early stages, patients often have atypical symptoms, making diagnosis difficult. The prognosis of diagnosed patients is very poor and treating PAAD is challenging. Therefore, determining reliable risk factors related to PAAD development is critical for improving patient prognosis. E2F family transcription factors (TFs) are essential regulators of DNA synthesis and cell cycle progression in eukaryotic cells, and they have been identified as prognostic biomarkers associated with multiple cancer types. However, further research is necessary to establish the prognostic relevance of these TFs in PAAD patients. MATERIAL AND METHODS We assessed PAAD patient transcriptional and outcome data using the TIMER, ONCOMINE, STRING, GEPIA, cBioPortal, Kaplan-Meier Plotter, GSCALite, and starBase databases. RESULTS PAAD tumor tissues exhibited increased expression of E2F1/3/5/7/8 relative to that in normal tissues, while the expression of E2F2/3/6/8 was associated with a more advanced tumor stage. Survival analyses indicated that PAAD patients expressing higher levels of E2F1/2/3/7/8 exhibited shorter overall survival (OS) and disease-free survival (DFS) than patients expressing lower levels of these TFs. In addition, E2F4 and E2F6 overexpression was associated with poorer DFS and OS, respectively. We also found that the expression of E2Fs was significantly correlated with immune infiltrates, including CD8+ T cells, CD4+ T cells, B cells, dendritic cells, neutrophils, and macrophages. CONCLUSIONS Our study may provide new insights into the optimal choice of immunotherapy and promising novel targets for therapeutic intervention in PAAD patients.

Purpose: Colorectal cancer (CRC) is the 3rd most prevalent malignant tumour globally. Although significant strides have been made in diagnosis and treatment, its prognosis at the moment remains unpromising. Therefore, there is an urgent and desperate need to identify novel biomarkers of CRC and evaluate its mechanism of tumourigenesis and development. Methods: JASPAR and RNAinter databases are used to analyze target genes associated with colorectal cancer. Western blotting, q-PCR and immunohistochemistry et, al. were used to detect the level of MNX1 in patients with colorectal cancer, and Chip-PCR was used to detect the targeted binding ability of E2F4 and MNX1. The cells and animal models overexpressed MNX1 and E2F4 were constructed by shRNA transfection. Results: Herein, MNX1 was highly expressed and linked to favourable overall survival curves in colorectal cancer. The functional assay revealed that MNX1 overexpression could promote proliferation, migration, and invasion of CRC cells. Based on the prediction of the JASPAR and RNAinter databases, the transcription factor, E2F4, was bound to the MNX1 promoter region. The Chromatin Immunoprecipitation (ChIP) assay verified the interactions between MNX1 and E2F4 in CRC. Additionally, we found that sh-E2F4 markedly downregulated the MNX1 levels and reduced CRC progression in vivo and in vitro, which reversed MNX1 overexpression. Conclusion: Therefore, our research discovered that E2F4-mediated abnormal MNX1 expression promotes CRC progression and could become a novel diagnostic or therapeutic target of CRC.

The transcription factor E2F4 controls proliferation of normal and cancerous intestinal epithelial cells. E2F4 localization in normal human intestinal epithelial cells (HIEC) is cell cycle-dependent, being cytoplasmic in quiescent differentiated cells but nuclear in proliferative cells. However, the intracellular signaling mechanisms regulating such E2F4 localization remain unknown.

The prognosis of high-grade gliomas, such as glioblastoma multiforme (GBM), is extremely poor due to the highly invasive nature of these aggressive cancers. Previous work has demonstrated that TNF-weak like factor (TWEAK) induction of the noncanonical NF-κB pathway increases the invasiveness of glioma cells in an NF-κB-inducing kinase (NIK)-dependent manner. While NIK activity is predominantly regulated at the posttranslational level, we show here that NIK ( MAP3K14 ) is upregulated at the transcriptional level in invading cell populations, with the highest expression observed in the most invasive cells. Glioma cells with high induction of NIK gene expression demonstrate characteristics of collective invasion, facilitating invasion of neighboring cells. Furthermore, we demonstrate that the E2F transcription factors E2F4 and E2F5 directly regulate NIK transcription and are required to promote glioma cell invasion in response to TWEAK. Overall, our findings demonstrate that transcriptional induction of NIK facilitates collective cell migration and invasion, thereby promoting glioma pathogenesis.