The lincRNA JUNI regulates the stress-dependent induction of c-Jun, cellular migration and survival through the modulation of the DUSP14-JNK axis.

Cancer cells employ adaptive mechanisms to survive various stressors, including genotoxic drugs. Understanding the factors promoting survival is crucial for developing effective treatments. In this study, we unveil a previously unexplored long non-coding RNA, JUNI (JUN-DT, LINC01135), which is upregulated by genotoxic drugs through the activation of stress-activated MAPKs, JNK, and p38 and consequently exerts positive control over the expression of its adjacent gene product c-Jun, a well-known oncoprotein, which transduces signals to multiple transcriptional outputs. JUNI regulates cellular migration and has a crucial role in conferring cellular resistance to chemotherapeutic drugs or UV radiation. Depletion of JUNI markedly increases the sensitivity of cultured cells and spheroids to chemotherapeutic agents. We identified 57 proteins interacting with JUNI. The activity of one of them the MAPK phosphatase and inhibitor, DUSP14, is counteracted by JUNI, thereby, facilitating efficient JNK phosphorylation and c-Jun induction when cells are exposed to UV radiation. The antagonistic interplay with DUSP14 contributes not only to c-Jun induction but also augments the survival of UV-exposed cells. In summary, we introduce JUNI as a novel stress-inducible regulator of c-Jun, positioning it as a potential target for enhancing the sensitivity of cancer cells to chemotherapy.

Correction: The novel long non-coding RNA TALNEC2, regulates tumor cell growth and the stemness and radiation response of glioma stem cells.

[This corrects the article DOI: 10.18632/oncotarget.15991.].

A novel E2F1-regulated lncRNA, LAPAS1, is required for S phase progression and cell proliferation.

The transcription factor E2F1 induces both proliferation and apoptosis and is a critical downstream target of the tumor suppressor RB. Long non-coding RNAs (lncRNAs) are major regulators of many cellular processes, including cell cycle progression and cell proliferation. However, the mode of action as well as the transcriptional regulation of most lncRNAs are only beginning to be understood. Here, we report that a novel human lncRNA, LAPAS1, is an E2F1- regulated lncRNA that affects S phase progression. Inhibition of LAPAS1 expression increases percentage of S phase cells, and its silencing in synchronized cells delays their progression through S phase. In agreement with its suggested role in cell cycle progression, prolonged inhibition of LAPAS1 attenuates proliferation of human cancer cells. Our data demonstrate that LAPAS1 predominantly functions in trans to repress expression of Sphingolipid Transporter 2 (SPNS2). Importantly, knockdown of SPNS2 rescues the effect of LAPAS1 silencing on cell cycle and cell proliferation. Notably, low levels of LAPAS1 are associated with increased survival of kidney cancer patients. Summarily, we identify LAPAS1 as a novel E2F-regulated lncRNA that has a potential role in human cancer and regulates cell-cycle progression and cell proliferation, at least in part, via regulation of SPNS2.

Theileria parasites subvert E2F signaling to stimulate leukocyte proliferation.

Intracellular pathogens have evolved intricate mechanisms to subvert host cell signaling pathways and ensure their own propagation. A lineage of the protozoan parasite genus Theileria infects bovine leukocytes and induces their uncontrolled proliferation causing a leukemia-like disease. Given the importance of E2F transcription factors in mammalian cell cycle regulation, we investigated the role of E2F signaling in Theileria-induced host cell proliferation. Using comparative genomics and surface plasmon resonance, we identified parasite-derived peptides that have the sequence-specific ability to increase E2F signaling by binding E2F negative regulator Retinoblastoma-1 (RB). Using these peptides as a tool to probe host E2F signaling, we show that the disruption of RB complexes ex vivo leads to activation of E2F-driven transcription and increased leukocyte proliferation in an infection-dependent manner. This result is consistent with existing models and, together, they support a critical role of E2F signaling for Theileria-induced host cell proliferation, and its potential direct manipulation by one or more parasite proteins.

In Vitro Evaluation of Chemically Analyzed Hypericum Triquetrifolium Extract Efficacy in Apoptosis Induction and Cell Cycle Arrest of the HCT-116 Colon Cancer Cell Line.

Naturally derived drugs and plant-based products are attractive commodities that are being explored for cancer treatment. This in vitro study aimed to investigate the role of Hypericum triquetrifolium (50% ethanol: 50% water) extract (HTE) treatment on apoptosis, cell cycle modulation, and cell cycle arrest in human colon cancer cell line (HCT-116). HTE induced cell death via an apoptotic process, as assayed by an Annexin V-Cy3 assay. Exposing HCT-116 cells to 0.064, 0.125, 0.25, and 0.5 mg/mL of HTE for 24 h led to 50 ± 9%, 71.6 ± 8%, 85 ± 5%, and 96 ± 1.5% apoptotic cells, respectively. HCT-116 cells treated with 0.25 and 0.5 mg/mL HTE for 3 h resulted in 38.9 ± 1.5% and 57.2 ± 3% cleavage of caspase-3-specific substrate, respectively. RT-PCR analysis revealed that the HTE extract had no effect on mRNA levels of Apaf-1 and NOXA. Moreover, the addition of 0.125 mg/mL and 0.25 mg/mL HTE for 24 h was clearly shown to attenuate the cell cycle progression machinery in HCT-116 cells. GC/MS analysis of the extract identified 21 phytochemicals that are known as apoptosis inducers and cell cycle arrest agents. All the compounds detected are novel in H. triquetrifolium. These results suggest that HTE-induced apoptosis of human colon cells is mediated primarily through the caspase-dependent pathway. Thus, HTE appears to be a potent therapeutic agent for colon cancer treatment.

Targeting LINC00673 expression triggers cellular senescence in lung cancer.

Aberrant expression of noncoding RNAs plays a critical role during tumorigenesis. To uncover novel functions of long non-coding RNA (lncRNA) in lung adenocarcinoma, we used a microarray-based screen identifying LINC00673 with elevated expression in matched tumor versus normal tissue. We report that loss of LINC00673 is sufficient to trigger cellular senescence, a tumor suppressive mechanism associated with permanent cell cycle arrest, both in lung cancer and normal cells in a p53-dependent manner. LINC00673-depleted cells fail to efficiently transit from G1- to S-phase. Using a quantitative proteomics approach, we confirm the modulation of senescence-associated genes as a result of LINC00673 knockdown. In addition, we uncover that depletion of p53 in normal and tumor cells is sufficient to overcome LINC00673-mediated cell cycle arrest and cellular senescence. Furthermore, we report that overexpression of LINC00673 reduces p53 translation and contributes to the bypass of Ras-induced senescence. In summary, our findings highlight LINC00673 as a crucial regulator of proliferation and cellular senescence in lung cancer.

ASK1 (MAP3K5) is transcriptionally upregulated by E2F1 in adipose tissue in obesity, molecularly defining a human dys-metabolic obese phenotype.

OBJECTIVE: Obesity variably disrupts human health, but molecular-based patients' health-risk stratification is limited. Adipose tissue (AT) stresses may link obesity with metabolic dysfunction, but how they signal in humans remains poorly-characterized. We hypothesized that a transcriptional AT stress-signaling cascade involving E2F1 and ASK1 (MAP3K5) molecularly defines high-risk obese subtype. METHODS: ASK1 expression in human AT biopsies was determined by real-time PCR analysis, and chromatin immunoprecipitation (ChIP) adopted to AT explants was used to evaluate the binding of E2F1 to the ASK1 promoter. Dual luciferase assay was used to measure ASK1 promoter activity in HEK293 cells. Effects of E2F1 knockout/knockdown in adipocytes was assessed utilizing mouse-embryonal-fibroblasts (MEF)-derived adipocyte-like cells from WT and E2F1-/- mice and by siRNA, respectively. ASK1 depletion in adipocytes was studied in MEF-derived adipocyte-like cells from WT and adipose tissue-specific ASK1 knockout mice (ASK1-ATKO). RESULTS: Human visceral-AT ASK1 mRNA (N = 436) was associated with parameters of obesity-related cardio-metabolic morbidity. Adjustment for E2F1 expression attenuated the association of ASK1 with fasting glucose, insulin resistance, circulating IL-6, and lipids (triglycerides, HDL-cholesterol), even after adjusting for BMI. Chromatin-immunoprecipitation in human-AT explants revealed BMI-associated increased occupancy of the ASK1 promoter by E2F1 (r2 = 0.847, p < 0.01). In adipocytes, siRNA-mediated E2F1-knockdown, and MEF-derived adipocytes of E2F1-knockout mice, demonstrated decreased ASK1 expression and signaling to JNK. Mutation/truncation of an E2F1 binding site in hASK1 promoter decreased E2F1-induced ASK1 promoter activity, whereas E2F1-mediated sensitization of ASK1 promoter to further activation by TNFα was inhibited by JNK-inhibitor. Finally, MEF-derived adipocytes from adipocyte-specific ASK1-knockout mice exhibited lower leptin and higher adiponectin expression and secretion, and resistance to the effects of TNFα. CONCLUSIONS: AT E2F1 -ASK1 molecularly defines a metabolically-detrimental obese sub-phenotype. Functionally, it may negatively affect AT endocrine function, linking AT stress to whole-body metabolic dysfunction.

A novel lncRNA, GASL1, inhibits cell proliferation and restricts E2F1 activity.

The human genome encodes thousands of unique long non-coding RNAs (lncRNAs), many of which are emerging as critical regulators of cell fate. However, their functions as well as their transcriptional regulation are only partially understood. The E2F1 transcription factor induces both proliferation and apoptosis, and is a critical downstream target of the tumor suppressor, RB. Here, we provide evidence that a novel lncRNA named GASL1 is transcriptionally regulated by E2F1; GASL1 levels are elevated upon activation of exogenous E2F1 or endogenous E2Fs. Inhibition of GASL1 expression induced cell cycle progression, and in particular, G1 exit. Moreover, GASL1 silencing enhanced cell proliferation, while, conversely, its ectopic expression inhibited proliferation. Knockdown of GASL1 also enhanced E2F1-induced apoptosis, suggesting the existence of an E2F/GASL1 negative feedback loop. In agreement with this notion, silencing of GASL1 led to increased levels of phosphorylated pRB and loss of Rb impaired the effect of GASL1 silencing on G1 exit. Importantly, xenograft experiments demonstrated that GASL1 deletion enhances tumor growth. Moreover, low levels of GASL1 are associated with decreased survival of liver cancer patients. Taken together, our data identify GASL1 as a novel lncRNA regulator of cell cycle progression and cell proliferation with a potential role in cancer.

The novel long non-coding RNA TALNEC2, regulates tumor cell growth and the stemness and radiation response of glioma stem cells.

Despite advances in novel therapeutic approaches for the treatment of glioblastoma (GBM), the median survival of 12-14 months has not changed significantly. Therefore, there is an imperative need to identify molecular mechanisms that play a role in patient survival. Here, we analyzed the expression and functions of a novel lncRNA, TALNEC2 that was identified using RNA seq of E2F1-regulated lncRNAs. TALNEC2 was localized to the cytosol and its expression was E2F1-regulated and cell-cycle dependent. TALNEC2 was highly expressed in GBM with poor prognosis, in GBM specimens derived from short-term survivors and in glioma cells and glioma stem cells (GSCs). Silencing of TALNEC2 inhibited cell proliferation and arrested the cells in the G1\S phase of the cell cycle in various cancer cell lines. In addition, silencing of TALNEC2 decreased the self-renewal and mesenchymal transformation of GSCs, increased sensitivity of these cells to radiation and prolonged survival of mice bearing GSC-derived xenografts. Using miRNA array analysis, we identified specific miRNAs that were altered in the silenced cells that were associated with cell-cycle progression, proliferation and mesenchymal transformation. Two of the downregulated miRNAs, miR-21 and miR-191, mediated some of TALNEC2 effects on the stemness and mesenchymal transformation of GSCs. In conclusion, we identified a novel E2F1-regulated lncRNA that is highly expressed in GBM and in tumors from patients of short-term survival. The expression of TALNEC2 is associated with the increased tumorigenic potential of GSCs and their resistance to radiation. We conclude that TALNEC2 is an attractive therapeutic target for the treatment of GBM.

Reduced abundance of the E3 ubiquitin ligase E6AP contributes to decreased expression of the INK4/ARF locus in non-small cell lung cancer.

The tumor suppressor p16INK4a, one protein encoded by the INK4/ARF locus, is frequently absent in multiple cancers, including non-small cell lung cancer (NSCLC). Whereas increased methylation of the encoding gene (CDKN2A) accounts for its loss in a third of patients, no molecular explanation exists for the remainder. We unraveled an alternative mechanism for the silencing of the INK4/ARF locus involving the E3 ubiquitin ligase and transcriptional cofactor E6AP (also known as UBE3A). We found that the expression of three tumor suppressor genes encoded in the INK4/ARF locus (p15INK4b, p16INK4a, and p19ARF) was decreased in E6AP-/- mouse embryo fibroblasts. E6AP induced the expression of the INK4/ARF locus at the transcriptional level by inhibiting CDC6 transcription, a gene encoding a key repressor of the locus. Luciferase assays revealed that E6AP inhibited CDC6 expression by reducing its E2F1-dependent transcription. Chromatin immunoprecipitation analysis indicated that E6AP reduced the amount of E2F1 at the CDC6 promoter. In a subset of NSCLC samples, an E6AP-low/CDC6-high/p16INK4a-low protein abundance profile correlated with low methylation of the gene encoding p16INK4a (CDKN2A) and poor patient prognosis. These findings define a previously unrecognized tumor-suppressive role for E6AP in NSCLC, reveal an alternative silencing mechanism of the INK4/ARF locus, and reveal E6AP as a potential prognostic marker in NSCLC.

LncRNA Khps1 Regulates Expression of the Proto-oncogene SPHK1 via Triplex-Mediated Changes in Chromatin Structure.

Although thousands of long noncoding RNAs (lncRNAs) have been discovered, very little is known about their mode of action. Here we functionally characterize an E2F1-regulated lncRNA named Khps1, which is transcribed in antisense orientation to the proto-oncogene SPHK1. Khps1 activates SPHK1 expression by recruiting the histone acetyltransferase p300/CBP to the SPHK1 promoter, which leads to local changes of the chromatin structure that ensures E2F1 binding and enhances transcription. Mechanistically, this is achieved by direct association of Khps1 with a homopurine stretch upstream of the transcription start site of SPHK1, which forms a DNA-RNA triplex that anchors the lncRNA and associated effector proteins to the gene promoter. The results reveal an lncRNA- and E2F1-driven regulatory loop in which E2F1-dependent induction of antisense RNA leads to changes in chromatin structure, facilitating E2F1-dependent expression of SPHK1 and restriction of E2F1-induced apoptosis.

A novel mitosis-associated lncRNA, MA-linc1, is required for cell cycle progression and sensitizes cancer cells to Paclitaxel.

Long noncoding RNAs (lncRNAs) are major regulators of many cellular processes including cell cycle progression and tumorigenesis. In this study, we identify a novel lncRNA, MA-linc1, and reveal its effects on cell cycle progression and cancer growth. Inhibition of MA-linc1 expression alters cell cycle distribution, leading to a decrease in the number of G1 cells and a concomitant increase in all other stages of the cell cycle, and in particular G2/M, suggesting its involvement in the regulation of M phase. Accordingly, knock down of MA-linc1 inhibits M phase exit upon release from a mitotic block. We further demonstrate that MA-linc1 predominantly functions in cis to repress expression of its neighboring gene, Purα, which is often deleted in human cancers and whose ectopic expression inhibits cell cycle progression. Knock down of Purα partially rescues the MA-linc1 dependent inhibition of M phase exit. In agreement with its suggested role in M phase, inhibition of MA-linc1 enhances apoptotic cell death induced by the antimitotic drug, Paclitaxel and this enhancement of apoptosis is rescued by Purα knockdown. Furthermore, high levels of MA-linc1 are associated with reduced survival in human breast and lung cancer patients.Taken together, our data identify MA-linc1 as a novel lncRNA regulator of cell cycle and demonstrate its potential role in cancer progression and treatment.

Elevated autophagy gene expression in adipose tissue of obese humans: A potential non-cell-cycle-dependent function of E2F1.

Autophagy genes' expression is upregulated in visceral fat in human obesity, associating with obesity-related cardio-metabolic risk. E2F1 (E2F transcription factor 1) was shown in cancer cells to transcriptionally regulate autophagy. We hypothesize that E2F1 regulates adipocyte autophagy in obesity, associating with endocrine/metabolic dysfunction, thereby, representing non-cell-cycle function of this transcription factor. E2F1 protein (N=69) and mRNA (N=437) were elevated in visceral fat of obese humans, correlating with increased expression of ATG5 (autophagy-related 5), MAP1LC3B/LC3B (microtubule-associated protein 1 light chain 3 ß), but not with proliferation/cell-cycle markers. Elevated E2F1 mainly characterized the adipocyte fraction, whereas MKI67 (marker of proliferation Ki-67) was elevated in the stromal-vascular fraction of adipose tissue. In human visceral fat explants, chromatin-immunoprecipitation revealed body mass index (BMI)-correlated increase in E2F1 binding to the promoter of MAP1LC3B, but not to the classical cell cycle E2F1 target, CCND1 (cyclin D1). Clinically, omental fat E2F1 expression correlated with insulin resistance, circulating free-fatty-acids (FFA), and with decreased circulating ADIPOQ/adiponectin, associations attenuated by adjustment for autophagy genes. Overexpression of E2F1 in HEK293 cells enhanced promoter activity of several autophagy genes and autophagic flux, and sensitized to further activation of autophagy by TNF. Conversely, mouse embryonic fibroblast (MEF)-derived adipocytes from e2f1 knockout mice (e2f1-/-) exhibited lower autophagy gene expression and flux, were more insulin sensitive, and secreted more ADIPOQ. Furthermore, e2f1-/- MEF-derived adipocytes, and autophagy-deficient (by Atg7 siRNA) adipocytes were resistant to cytokines-induced decrease in ADIPOQ secretion. Jointly, upregulated E2F1 sensitizes adipose tissue autophagy to inflammatory stimuli, linking visceral obesity to adipose and systemic metabolic-endocrine dysfunction.

TODRA, a lncRNA at the RAD51 Locus, Is Oppositely Regulated to RAD51, and Enhances RAD51-Dependent DSB (Double Strand Break) Repair.

Expression of RAD51, a crucial player in homologous recombination (HR) and DNA double-strand break (DSB) repair, is dysregulated in human tumors, and can contribute to genomic instability and tumor progression. To further understand RAD51 regulation we functionally characterized a long non-coding (lnc) RNA, dubbed TODRA (Transcribed in the Opposite Direction of RAD51), transcribed 69bp upstream to RAD51, in the opposite direction. We demonstrate that TODRA is an expressed transcript and that the RAD51 promoter region is bidirectional, supporting TODRA expression (7-fold higher than RAD51 in this assay, p = 0.003). TODRA overexpression in HeLa cells induced expression of TPIP, a member of the TPTE family which includes PTEN. Similar to PTEN, we found that TPIP co-activates E2F1 induction of RAD51. Analysis of E2F1's effect on the bidirectional promoter showed that E2F1 binding to the same site that promotes RAD51 expression, results in downregulation of TODRA. Moreover, TODRA overexpression induces HR in a RAD51-dependent DSB repair assay, and increases formation of DNA damage-induced RAD51-positive foci. Importantly, gene expression in breast tumors supports our finding that E2F1 oppositely regulates RAD51 and TODRA: increased RAD51 expression, which is associated with an aggressive tumor phenotype (e.g. negative correlation with positive ER (r = -0.22, p = 0.02) and positive PR status (r = -0.27, p<0.001); positive correlation with ki67 status (r = 0.36, p = 0.005) and HER2 amplification (r = 0.41, p = 0.001)), correlates as expected with lower TODRA and higher E2F1 expression. However, although E2F1 induction resulted in TPIP downregulation in cell lines, we find that TPIP expression in tumors is not reduced despite higher E2F1 expression, perhaps contributing to increased RAD51 expression. Our results identify TPIP as a novel E2F1 co-activator, suggest a similar role for other TPTEs, and indicate that the TODRA lncRNA affects RAD51 dysregulation and RAD51-dependent DSB repair in malignancy. Importantly, gene expression in breast tumors supports our finding that E2F1 oppositely regulates RAD51 and TODRA: increased RAD51 expression, which is associated with an aggressive tumor phenotype (e.g. negative correlation with positive ER (r = -0.22, p = 0.02) and positive PR status (r = -0.27, p<0.001); positive correlation with ki67 status (r = 0.36, p = 0.005) and HER2 amplification (r = 0.41, p = 0.001)), correlates as expected with lower TODRA and higher E2F1 expression. However, although E2F1 induction resulted in TPIP downregulation in cell lines, we find that TPIP expression in tumors is not reduced despite higher E2F1 expression, perhaps contributing to increased RAD51 expression. Our results identify TPIP as a novel E2F1 co-activator, suggest a similar role for other TPTEs, and indicate that the TODRA lncRNA affects RAD51 dysregulation and RAD51-dependent DSB repair in malignancy.

The long non-coding RNA ERIC is regulated by E2F and modulates the cellular response to DNA damage.

BACKGROUND: The human genome encodes thousands of unique long non-coding RNAs (lncRNAs), and these transcripts are emerging as critical regulators of gene expression and cell fate. However, the transcriptional regulation of their expression is not fully understood. The pivotal transcription factor E2F1 which can induce both proliferation and cell death, is a critical downstream target of the tumor suppressor, RB. The retinoblastoma pathway is often inactivated in human tumors resulting in deregulated E2F activity. RESULTS: Here, we report that lncRNA XLOC 006942, which we named ERIC, is regulated by E2F1 and, most probably, also E2F3. We show that expression levels of ERIC were elevated upon activation of exogenous E2F1, E2F3 or endogenous E2Fs. Moreover, knockdown of either E2F1 or E2F3 reduced ERIC levels and endogenous E2F1 binds ERIC's promoter. Expression of ERIC was cell cycle regulated and peaked in G1 in an E2F1-dependent manner. Inhibition of ERIC expression increased E2F1-mediated apoptosis, suggesting that E2F1 and ERIC constitute a negative feedback loop that modulates E2F1 activity. Furthermore, ERIC levels were increased following DNA damage by the chemotherapeutic drug Etoposide, and inhibition of ERIC expression enhanced Etoposide -induced apoptosis. CONCLUSIONS: Our data identify ERIC as a novel lncRNA that is transcriptionally regulated by E2Fs, and restricts apoptosis induced by E2F1, as well as by DNA damage.

RBM38 is a direct transcriptional target of E2F1 that limits E2F1-induced proliferation.

The E2F family of transcription factors plays a pivotal role in the regulation of cell proliferation in higher eukaryotes and is a critical downstream target of the tumor suppressor pRB. The pRB/E2F pathway is defective in most human tumors, resulting in deregulated E2F activity that induces uncontrolled cell proliferation, a hallmark of tumor cells. The RNA-binding protein RBM38, also named RNPC1, induces cell-cycle arrest in G(1), at least in part, via binding to and stabilizing the mRNA of the cyclin-dependent kinase inhibitor p21. RBM38 levels are altered in human cancer. Generally, RBM38 is overexpressed in various tumors; however, RBM38 mRNA levels are reduced in some breast tumors due to increased methylation of its promoter region. We show here that expression of RBM38 is regulated by E2F1. Specifically, RBM38 mRNA and protein levels are elevated upon activation of either exogenous E2F1 or endogenous E2Fs. Moreover, endogenous E2F1 binds the human RBM38 promoter and E2F1 knockdown reduces RBM38 levels. Our data raise the possibility that E2F1 together with E2F1-regulated RBM38 constitute a negative feedback loop that modulates E2F1 activity. In support of this, inhibition of RBM38 expression increases E2F1-mediated cell-cycle progression. Moreover, in human ovarian cancer, high correlation between expression of E2F1 and RBM38 is associated with increased survival. Overall, our data identify RBM38 as novel transcriptional target of E2F1 that restricts E2F1-induced proliferation. Furthermore, this negative feedback loop seems to restrict tumor aggressiveness, thereby promoting survival of patients with cancer.

Regulation of modular Cyclin and CDK feedback loops by an E2F transcription oscillator in the mammalian cell cycle.

The cell cycle is regulated by a large number of enzymes and transcription factors. We have developed a modular description of the cell cycle, based on a set of interleaved modular feedback loops, each leading to a cyclic behavior. The slowest loop is the E2F transcription and ubiquitination, which determines the cycling frequency of the entire cell cycle. Faster feedback loops describe the dynamics of each Cyclin by itself. Our model shows that the cell cycle progression as well as the checkpoints of the cell cycle can be understood through the interactions between the main E2F feedback loop and the driven Cyclin feedback loops. Multiple models were proposed for the cell cycle dynamics; each with differing basic mechanisms. We here propose a new generic formalism. In contrast with existing models, the proposed formalism allows a straightforward analysis and understanding of the dynamics, neglecting the details of each interaction. This model is not sensitive to small changes in the parameters used and it reproduces the observed behavior of the transcription factor E2F and different Cyclins in continuous or regulated cycling conditions. The modular description of the cell cycle resolves the gap between cyclic models, solely based on protein-protein reactions and transcription reactions based models. Beyond the explanation of existing observations, this model suggests the existence of unknown interactions, such as the need for a functional interaction between Cyclin B and retinoblastoma protein (Rb) de-phosphorylation.

MiR-15 and miR-16 are direct transcriptional targets of E2F1 that limit E2F-induced proliferation by targeting cyclin E.

MicroRNAs (miR) are small noncoding RNA molecules that have recently emerged as critical regulators of gene expression and are often deregulated in cancer. In particular, miRs encoded by the miR-15a, miR-16-1 cluster seem to act as tumor suppressors. Here, we evidence that the miR-15a, miR-16-1 cluster and related miR-15b, miR-16-2 cluster comprise miRs regulated by E2F1, a pivotal transcription factor that can induce both proliferation and cell death. E2F1 is a critical downstream target of the tumor suppressor retinoblastoma (RB). The RB pathway is often inactivated in human tumors resulting in deregulated E2F activity. We show that expression levels of the 4 mature miRs, miR-15a, miR-16-1 and miR-15b, miR-16-2, as well as their precursor pri-miRNAs, are elevated upon activation of ectopic E2F1. Moreover, activation of endogenous E2Fs upregulates expression of these miRs and endogenous E2F1 binds their respective promoters. Importantly, we corroborate that miR-15a/b inhibits expression of cyclin E, the latter a key direct transcriptional target of E2F pivotal for the G(1)/S transition, raising the possibility that E2F1, miR-15, and cyclin E constitute a feed-forward loop that modulates E2F activity and cell-cycle progression. In support of this, ectopic expression of miR-15 inhibits the G(1)/S transition, and, conversely, inhibition of miR-15 expression enhances E2F1-induced upregulation of cyclin E1 levels. Furthermore, inhibition of both miR-15 and miR-16 enhances E2F1-induced G(1)/S transition. In summary, our data identify the miR-15 and miR-16 families as novel transcriptional targets of E2F, which, in turn, modulates E2F activity.

JNK activation is regulated by E2F and promotes E2F1-induced apoptosis.

Members of the E2F transcription factor family are critical downstream targets of the tumor suppressor RB and are often deregulated and hyperactive in human tumors. E2F regulates a diverse array of cellular functions including cell proliferation and apoptosis. Recent studies indicate that E2F also regulates expression of upstream components of pivotal signal transduction pathways, thereby modulating the activity of these pathways. We show here that E2F modulates the activity of the JNK pathway via E2F-induced upregulation of JNK phosphorylation. Accordingly, downregulating E2F1and E2F3 inhibits sustained UV-induced JNK phosphorylation and ectopic expression of E2F1 or E2F3 induces JNK phosphorylation and activation. The mechanism by which E2F modulates JNK phosphorylation involves transcriptional induction of the kinase GCK, a MAP4K that can activate JNK indirectly. Hence, inhibition of GCK expression impairs E2F1-induced JNK phosphorylation. The JNK pathway is an important mediator of stress-induced apoptosis and we show here that inhibition of JNK expression or activity significantly hinders E2F1-induced apoptosis. Overall, our data identify the kinase GCK as a novel E2F-regulated gene and reveal a functional link between a central signaling pathway, namely the JNK pathway, and the transcription factor E2F.