E2F transcription factor 5, a new regulator in adipogenesis to mediate the role of Krüppel-like factor 7 in chicken preadipocyte differentiation and proliferation.

E2F transcription factor 5 (E2F5) gene is a transcription factor, plays an important role in the development of a variety of cells. E2F5 is expressed in human and mouse adipocytes, but its specific function in adipogenesis is unclear. Krüppel-like factor 7 (KLF7) facilitates proliferation and inhibits differentiation in chicken preadipocytes. Our previous KLF7 chromatin immunoprecipitation-sequencing analysis revealed a KLF7-binding peak in the 3' flanking region of the E2F5, indicating a regulatory role of KLF7 in this region. In the present study, we investigated E2F5 potential role, the overexpression and knockdown analyses revealed that E2F5 inhibited the differentiation and promoted the proliferation of chicken preadipocytes. Moreover, we identified enhancer activity in the 3' flanking region (nucleotides +22661/+22900) of E2F5 and found that KLF7 overexpression increased E2F5 expression and luciferase activity in this region. Deleting the putative KLF7-binding site eliminated the promoting effect of KLF7 overexpression on E2F5 expression. Further, E2F5 reversed the KLF7-induced decrease in preadipocyte differentiation and increase in preadipocyte proliferation. Taken together, our findings demonstrate that KLF7 inhibits differentiation and promotes proliferation in preadipocytes by enhancing E2F5 transcription.

Targeting CALM2 Inhibits Hepatocellular Carcinoma Growth and Metastasis by Suppressing E2F5-mediated Cell Cycle Progression.

BACKGROUND/AIM: The aim of this study was to reveal the novel roles of calmodulin 2 (CALM2) in hepatocellular carcinoma (HCC) progression. MATERIALS AND METHODS: The effects of knockdown of CALM2 expression by siRNA were investigated using various experimental approaches in both cellular and molecular levels. RESULTS: Silencing of CALM2 inhibited HCC cell proliferation and colony formation through induction of apoptosis. At the molecular level, CALM2-specific knockdown led to the common dysregulation of 154 genes in HCC cells. Notably, E2F transcription factor 5 (E2F5), which is functionally associated with migration, invasion and proliferation, was generally down-regulated. These functional associations were confirmed in HCC clinical samples. Reflecting the molecular changes, CALM2 knockdown reduced the migration and invasion abilities of HCC cells and abrogated the potency of tumor formation in vivo. CONCLUSION: Targeting CALM2 may be a molecular strategy for both primary HCC treatment and prevention of metastasis or recurrence.

Distinct requirements of E2f4 versus E2f5 activity for multiciliated cell development in the zebrafish embryo.

Multiciliated cells (MCCs) differentiate arrays of motile cilia that beat to drive fluid flow over epithelia. Recent studies have established two Geminin family coiled-coil containing nuclear regulatory proteins, Gmnc and Multicilin (Mci), in the specification and differentiation of the MCCs. Both Gmnc and Mci are devoid of a DNA binding domain: they regulate transcription by associating with E2f family transcription factors, notably E2f4 and E2f5. Here, we have studied the relative contribution of these two E2f factors in MCC development using the zebrafish embryo, which differentiates MCCs within kidney tubules and the nose. We found that while E2f4 is fully dispensable, E2f5 is essential for MCCs to form in the kidney tubules. Moreover, using a variety of double mutant combinations we show that E2f5 has a more prominent role in MCC development in the zebrafish than E2f4. This contrasts with current evidence from the mouse, where E2f4 seems to be more important. Thus, distinct combinatorial activities of the E2f4 and E2f5 proteins regulate the specification and differentiation of MCCs in zebrafish and mice.

E2F5 as an independent prognostic factor in esophageal squamous cell carcinoma.

BACKGROUND: E2F Transcription Factor 5 Protein (E2F5) is considered to act primarily as a transcriptional repressor in the cell cycle. However, its expression and role in esophageal squamous cell carcinoma (ESCC) have not been investigated. We examined whether the expression of E2F5 is related to the clinicopathological features and prognosis of patients with ESCC. MATERIALS AND METHODS: The expression of E2F5 was analyzed by immunohistochemistry in 64 primary tumor samples obtained from patients with ESCC who had undergone curative esophagectomy between 1998 and 2009. According to the expression of E2F5 in tumor cells, cases were divided into E2F5-positive (27 cases) and -negative groups (37 cases). The relationship of various clinicopathological features and prognosis with the E2F5 status, were analyzed. RESULTS: In the clinicopathological analysis, the proportion of poorly-differentiated tumors was significantly higher in the E2F5-positive group than in the E2F5-negative group (p=0.027). The 5-year survival rate of the E2F5-positive group was 39.3%, which was significantly poorer than that of the E2F5-negative group (83.8%) (p=0.006). In multivariate analysis, the expression of E2F5 was one of the most important independent prognostic factors after radical esophagectomy. CONCLUSION: The expression of E2F5 in ESCC may be correlated with a worse prognosis of patients with ESCC.

Human parvovirus B19 causes cell cycle arrest of human erythroid progenitors via deregulation of the E2F family of transcription factors.

Human parvovirus B19 (B19V) is the only human pathogenic parvovirus. It causes a wide spectrum of human diseases, including fifth disease (erythema infectiosum) in children and pure red cell aplasia in immunocompromised patients. B19V is highly erythrotropic and preferentially replicates in erythroid progenitor cells (EPCs). Current understanding of how B19V interacts with cellular factors to regulate disease progression is limited, due to a lack of permissive cell lines and animal models. Here, we employed a recently developed primary human CD36(+) EPC culture system that is highly permissive for B19V infection to identify cellular factors that lead to cell cycle arrest after B19V infection. We found that B19V exploited the E2F family of transcription factors by downregulating activating E2Fs (E2F1 to E2F3a) and upregulating repressive E2Fs (E2F4 to E2F8) in the primary CD36(+) EPCs. B19V nonstructural protein 1 (NS1) was a key viral factor responsible for altering E2F1-E2F5 expression, but not E2F6-E2F8 expression. Interaction between NS1 and E2F4 or E2F5 enhanced the nuclear import of these repressive E2Fs and induced stable G2 arrest. NS1-induced G2 arrest was independent of p53 activation and increased viral replication. Downstream E2F4/E2F5 targets, which are potentially involved in the progression from G2 into M phase and erythroid differentiation, were identified by microarray analysis. These findings provide new insight into the molecular pathogenesis of B19V in highly permissive erythroid progenitors.

The E2F5 repressor is an activator of E6/E7 transcription and of the S-phase entry in HPV18-associated cells.

High-risk papillomavirus type 18 (HPV18) is one of the less represented HPV types in low-grade lesions of the anogenital tract, whereas it occupies the second place in cervical cancer, where it can be found in 16% of the cases worldwide, after HPV16 present in 54% of them. These epidemiological data indicate that HPV18 infection is more prone to carcinogenic progression. The main oncogenic proteins, E6 and E7 of HPV18, are functionally comparable to the homologous proteins of the other high-risk viruses, including HPV16. In this work, we investigated the possibility that the higher oncogenic potential of HPV18 might be due to transcriptional regulation of the E6/E7 oncogenes. By comparing the E6/E7 promoter and enhancer sequences of the mucosal HPV genomes, we identified E2F binding sites specific for HPV18. The E2F family of transcription factors contains activators (E2F1-3) and repressors (E2F4-8) that regulate the transcription of S-phase and mitotic genes and thereby have a crucial role in cell-cycle progression. Surprisingly, we identified E2F5 as a direct activator of HPV18 E6/E7 transcription by sequential silencing of E2F members in HeLa cells. In addition, we could show that E2F5 positively regulates S-phase entry in HeLa cells and that this activation of the cell cycle by a member of the E2F repressor family is specific for HPV18-expressing cells. Diverting the function of E2F5 from a cell-cycle repressor into an activator might contribute to the higher oncogenic potential of HPV18 when compared with other high-risk HPV types.

E2F5 status significantly improves malignancy diagnosis of epithelial ovarian cancer.

BACKGROUND: Ovarian epithelial cancer (OEC) usually presents in the later stages of the disease. Factors, especially those associated with cell-cycle genes, affecting the genesis and tumour progression for ovarian cancer are largely unknown. We hypothesized that over-expressed transcription factors (TFs), as well as those that are driving the expression of the OEC over-expressed genes, could be the key for OEC genesis and potentially useful tissue and serum markers for malignancy associated with OEC. METHODS: Using a combination of computational (selection of candidate TF markers and malignancy prediction) and experimental approaches (tissue microarray and western blotting on patient samples) we identified and evaluated E2F5 transcription factor involved in cell proliferation, as a promising candidate regulatory target in early stage disease. Our hypothesis was supported by our tissue array experiments that showed E2F5 expression only in OEC samples but not in normal and benign tissues, and by significantly positively biased expression in serum samples done using western blotting studies. RESULTS: Analysis of clinical cases shows that of the E2F5 status is characteristic for a different population group than one covered by CA125, a conventional OEC biomarker. E2F5 used in different combinations with CA125 for distinguishing malignant cyst from benign cyst shows that the presence of CA125 or E2F5 increases sensitivity of OEC detection to 97.9% (an increase from 87.5% if only CA125 is used) and, more importantly, the presence of both CA125 and E2F5 increases specificity of OEC to 72.5% (an increase from 55% if only CA125 is used). This significantly improved accuracy suggests possibility of an improved diagnostics of OEC. Furthermore, detection of malignancy status in 86 cases (38 benign, 48 early and late OEC) shows that the use of E2F5 status in combination with other clinical characteristics allows for an improved detection of malignant cases with sensitivity, specificity, F-measure and accuracy of 97.92%, 97.37%, 97.92% and 97.67%, respectively. CONCLUSIONS: Overall, our findings, in addition to opening a realistic possibility for improved OEC diagnosis, provide an indirect evidence that a cell-cycle regulatory protein E2F5 might play a significant role in OEC pathogenesis.

Making omelets without breaking eggs: E2F-mediated induction of cardiomyoycte cell proliferation without stimulation of apoptosis.

The fundamental role of E2F transcription factors in the regulation of proliferation is well established. According to a widely accepted model, E2F1, E2F2, and E2F3 are classified as "activating" E2Fs since they induce proliferation of quiescent cells whereas E2F4 and E2F5 do not have the power to incite cell cycle progression but are related to differentiation processes and were therefore considered to be "repressive". In addition, it has been postulated that "activating" E2Fs induce apoptosis in a wide variety of cell types depending on their expression level. However, we demonstrated recently that this 'threshold model' does not hold true for cardiomyocytes. In a series of experiments in which we overexpressed individual E2Fs we found that directed expression of E2F2, unlike E2F1, E2F3 and E2F5, did not induce apoptosis but even suppressed expression of several pro-apoptotic genes in primary cardiomyocytes. Furthermore, we established that not only E2F1, E2F2, and E2F3 but also E2F4 was able to induce S-phase entry of primary cardiomyocytes. Our results suggest that it is possible to utilize the proliferation-inducing properties of the E2Fs in cardiomyocytes without activation of potentially harmful pro-apoptotic traits. This finding might open a new access to stimulate regeneration in postmitotic tissues such as the heart.