Chromatin-bound RB targets promoters, enhancers, and CTCF-bound loci and is redistributed by cell-cycle progression.

The interaction of RB with chromatin is key to understanding its molecular functions. Here, for first time, we identify the full spectrum of chromatin-bound RB. Rather than exclusively binding promoters, as is often described, RB targets three fundamentally different types of loci (promoters, enhancers, and insulators), which are largely distinguishable by the mutually exclusive presence of E2F1, c-Jun, and CTCF. While E2F/DP facilitates RB association with promoters, AP-1 recruits RB to enhancers. Although phosphorylation in CDK sites is often portrayed as releasing RB from chromatin, we show that the cell cycle redistributes RB so that it enriches at promoters in G1 and at non-promoter sites in cycling cells. RB-bound promoters include the classic E2F-targets and are similar between lineages, but RB-bound enhancers associate with different categories of genes and vary between cell types. Thus, RB has a well-preserved role controlling E2F in G1, and it targets cell-type-specific enhancers and CTCF sites when cells enter S-phase.

Yap1 activation enables bypass of oncogenic Kras addiction in pancreatic cancer.

Activating mutations in KRAS are among the most frequent events in diverse human carcinomas and are particularly prominent in human pancreatic ductal adenocarcinoma (PDAC). An inducible Kras(G12D)-driven mouse model of PDAC has established a critical role for sustained Kras(G12D) expression in tumor maintenance, providing a model to determine the potential for and the underlying mechanisms of Kras(G12D)-independent PDAC recurrence. Here, we show that some tumors undergo spontaneous relapse and are devoid of Kras(G12D) expression and downstream canonical MAPK signaling and instead acquire amplification and overexpression of the transcriptional coactivator Yap1. Functional studies established the role of Yap1 and the transcriptional factor Tead2 in driving Kras(G12D)-independent tumor maintenance. The Yap1/Tead2 complex acts cooperatively with E2F transcription factors to activate a cell cycle and DNA replication program. Our studies, along with corroborating evidence from human PDAC models, portend a novel mechanism of escape from oncogenic Kras addiction in PDAC.

A Code of Mono-phosphorylation Modulates the Function of RB.

Hyper-phosphorylation of RB controls its interaction with E2F and inhibits its tumor suppressor properties. However, during G1 active RB can be mono-phosphorylated on any one of 14 CDK phosphorylation sites. Here, we used quantitative proteomics to profile protein complexes formed by each mono-phosphorylated RB isoform (mP-RB) and identified the associated transcriptional outputs. The results show that the 14 sites of mono-phosphorylation co-ordinate RB's interactions and confer functional specificity. All 14 mP-RBs interact with E2F/DP proteins, but they provide different shades of E2F regulation. RB mono-phosphorylation at S811, for example, alters RB transcriptional activity by promoting its association with NuRD complexes. The greatest functional differences between mP-RBs are evident beyond the cell cycle machinery. RB mono-phosphorylation at S811 or T826 stimulates the expression of oxidative phosphorylation genes, increasing cellular oxygen consumption. These results indicate that RB activation signals are integrated in a phosphorylation code that determines the diversity of RB activity.

Nucleotide deficiency promotes genomic instability in early stages of cancer development.

Chromosomal instability in early cancer stages is caused by stress on DNA replication. The molecular basis for replication perturbation in this context is currently unknown. We studied the replication dynamics in cells in which a regulator of S phase entry and cell proliferation, the Rb-E2F pathway, is aberrantly activated. Aberrant activation of this pathway by HPV-16 E6/E7 or cyclin E oncogenes significantly decreased the cellular nucleotide levels in the newly transformed cells. Exogenously supplied nucleosides rescued the replication stress and DNA damage and dramatically decreased oncogene-induced transformation. Increased transcription of nucleotide biosynthesis genes, mediated by expressing the transcription factor c-myc, increased the nucleotide pool and also rescued the replication-induced DNA damage. Our results suggest a model for early oncogenesis in which uncoordinated activation of factors regulating cell proliferation leads to insufficient nucleotides that fail to support normal replication and genome stability.

E2F regulation of the Phosphoglycerate kinase gene is functionally important in Drosophila development.

The canonical role of the transcription factor E2F is to control the expression of cell cycle genes by binding to the E2F sites in their promoters. However, the list of putative E2F target genes is extensive and includes many metabolic genes, yet the significance of E2F in controlling the expression of these genes remains largely unknown. Here, we used the CRISPR/Cas9 technology to introduce point mutations in the E2F sites upstream of five endogenous metabolic genes in Drosophila melanogaster. We found that the impact of these mutations on both the recruitment of E2F and the expression of the target genes varied, with the glycolytic gene, Phosphoglycerate kinase (Pgk), being mostly affected. The loss of E2F regulation on the Pgk gene led to a decrease in glycolytic flux, tricarboxylic acid cycle intermediates levels, adenosine triphosphate (ATP) content, and an abnormal mitochondrial morphology. Remarkably, chromatin accessibility was significantly reduced at multiple genomic regions in PgkΔE2F mutants. These regions contained hundreds of genes, including metabolic genes that were downregulated in PgkΔE2F mutants. Moreover, PgkΔE2F animals had shortened life span and exhibited defects in high-energy consuming organs, such as ovaries and muscles. Collectively, our results illustrate how the pleiotropic effects on metabolism, gene expression, and development in the PgkΔE2F animals underscore the importance of E2F regulation on a single E2F target, Pgk.

Genetic modifiers of the BRD4-NUT dependency of NUT midline carcinoma uncovers a synergism between BETis and CDK4/6is.

Bromodomain and extraterminal (BET) domain inhibitors (BETis) show efficacy on NUT midline carcinoma (NMC). However, not all NMC patients respond, and responders eventually develop resistance and relapse. Using CRISPR and ORF expression screens, we systematically examined the ability of cancer drivers to mediate resistance of NMC to BETis and uncovered six general classes/pathways mediating resistance. Among these, we showed that RRAS2 attenuated the effect of JQ1 in part by sustaining ERK pathway function during BRD4 inhibition. Furthermore, overexpression of Kruppel-like factor 4 (KLF4), mediated BETi resistance in NMC cells through restoration of the E2F and MYC gene expression program. Finally, we found that expression of cyclin D1 or an oncogenic cyclin D3 mutant or RB1 loss protected NMC cells from BETi-induced cell cycle arrest. Consistent with these findings, cyclin-dependent kinase 4/6 (CDK4/6) inhibitors showed synergistic effects with BETis on NMC in vitro as well as in vivo, thereby establishing a potential two-drug therapy for NMC.

Role of E2F transcription factor in oral cancer: Recent insight and advancements.

The family of mammalian E2F transcription factors (E2Fs) comprise of 8 members (E2F1-E2F8) classified as activators (E2F1-E2F3) and repressors (E2F4-E2F8) primarily regulating the expression of several genes related to cell proliferation, apoptosis and differentiation, mainly in a cell cycle-dependent manner. E2F activity is frequently controlled via the retinoblastoma protein (pRb), cyclins, p53 and the ubiquitin-proteasome pathway. Additionally, genetic or epigenetic changes result in the deregulation of E2F family genes expression altering S phase entry and apoptosis, an important hallmark for the onset and development of cancer. Although studies reveal E2Fs to be involved in several human malignancies, the mechanisms underlying the role of E2Fs in oral cancer lies nascent and needs further investigations. This review focuses on the role of E2Fs in oral cancer and the etiological factors regulating E2Fs activity, which in turn transcriptionally control the expression of their target genes, thus contributing to cell proliferation, metastasis, and drug/therapy resistance. Further, we will discuss therapeutic strategies for E2Fs, which may prevent oral tumor growth, metastasis, and drug resistance.

Genome-wide characterization and expression profiling of E2F/DP gene family members in response to abiotic stress in tomato (Solanum lycopersicum L.).

BACKGROUND: E2F/DP (Eukaryotic 2 transcription factor/dimerization partner) family proteins play an essential function in the cell cycle development of higher organisms. E2F/DP family genes have been reported only in a few plant species. However, comprehensive genome-wide characterization analysis of the E2F/DP gene family of Solanum lycopersicum has not been reported so far. RESULTS: This study identified eight nonredundant SlE2F/DP genes that were classified into seven groups in the phylogenetic analysis. All eight genes had a single E2F-TDP domain and few genes had additional domains. Two segmental duplication gene pairs were observed within tomato, in addition to cis-regulatory elements, miRNA target sites and phosphorylation sites which play an important role in plant development and stress response in tomato. To explore the three-dimensional (3D) models and gene ontology (GO) annotations of SlE2F/DP proteins, we pointed to their putative transporter activity and their interaction with several putative ligands. The localization of SlE2F/DP-GFP fused proteins in the nucleus and endoplasmic reticulum suggested that they may act in other biological functions. Expression studies revealed the differential expression pattern of most of the SlE2F/DP genes in various organs. Moreover, the expression of E2F/DP genes against abiotic stress, particularly SlE2F/DP2 and/or SlE2F/DP7, was upregulated in response to heat, salt, cold and ABA treatment. Furthermore, the co-expression analysis of SlE2F/DP genes with multiple metabolic pathways was co-expressed with defence genes, transcription factors and so on, suggested their crucial role in various biological processes. CONCLUSIONS: Overall, our findings provide a way to understand the structure and function of SlE2F/DP genes; it might be helpful to improve fruit development and tolerance against abiotic stress through marker-assisted selection or transgenic approaches.

The Oncogenic Protein Kinase/ATPase RIOK1 Is Up-Regulated via the c-myc/E2F Transcription Factor Axis in Prostate Cancer.

The atypical protein kinase/ATPase RIO kinase (RIOK)-1 is involved in pre-40S ribosomal subunit production, cell-cycle progression, and protein arginine N-methyltransferase 5 methylosome substrate recruitment. RIOK1 overexpression is a characteristic of several malignancies and is correlated with cancer stage, therapy resistance, poor patient survival, and other prognostic factors. However, its role in prostate cancer (PCa) is unknown. In this study, the expression, regulation, and therapeutic potential of RIOK1 in PCa were examined. RIOK1 mRNA and protein expression were elevated in PCa tissue samples and correlated with proliferative and protein homeostasis-related pathways. RIOK1 was identified as a downstream target gene of the c-myc/E2F transcription factors. Proliferation of PCa cells was significantly reduced with RIOK1 knockdown and overexpression of the dominant-negative RIOK1-D324A mutant. Biochemical inhibition of RIOK1 with toyocamycin led to strong antiproliferative effects in androgen receptor-negative and -positive PCa cell lines with EC50 values of 3.5 to 8.8 nmol/L. Rapid decreases in RIOK1 protein expression and total rRNA content, and a shift in the 28S/18S rRNA ratio, were found with toyocamycin treatment. Apoptosis was induced with toyocamycin treatment at a level similar to that with the chemotherapeutic drug docetaxel used in clinical practice. In summary, the current study indicates that RIOK1 is a part of the MYC oncogene network, and as such, could be considered for future treatment of patients with PCa.

The N-Terminal Phosphorylation of RB by p38 Bypasses Its Inactivation by CDKs and Prevents Proliferation in Cancer Cells.

Control of the G1/S phase transition by the Retinoblastoma (RB) tumor suppressor is critical for the proliferation of normal cells in tissues, and its inactivation is one of the most fundamental events leading to cancer. Cyclin-dependent kinase (CDK) phosphorylation inactivates RB to promote cell cycle-regulated gene expression. Here we show that, upon stress, the p38 stress-activated protein kinase (SAPK) maximizes cell survival by downregulating E2F gene expression through the targeting of RB. RB undergoes selective phosphorylation by p38 in its N terminus; these phosphorylations render RB insensitive to the inactivation by CDKs. p38 phosphorylation of RB increases its affinity toward the E2F transcription factor, represses gene expression, and delays cell-cycle progression. Remarkably, introduction of a RB phosphomimetic mutant in cancer cells reduces colony formation and decreases their proliferative and tumorigenic potential in mice.

The p53 transcriptional response across tumor types reveals core and senescence-specific signatures modulated by long noncoding RNAs.

The p53 pathway is a universal tumor suppressor mechanism that limits tumor progression by triggering apoptosis or permanent cell cycle arrest, called senescence. In recent years, efforts to reactivate p53 function in cancer have proven to be a successful therapeutic strategy in murine models and have gained traction with the development of a range of small molecules targeting mutant p53. However, knowledge of the downstream mediators of p53 reactivation in different oncogenic contexts has been limited. Here, we utilized a panel of murine cancer cell lines from three distinct tumor types susceptible to alternative outcomes following p53 restoration to define unique and shared p53 transcriptional signatures. While we found that the majority of p53-bound sites and p53-responsive transcripts are tumor-type specific, analysis of shared targets identified a core signature of genes activated by p53 across all contexts. Furthermore, we identified repression of E2F and Myc target genes as a key feature of senescence. Characterization of p53-induced transcripts revealed core and senescence-specific long noncoding RNAs (lncRNAs) that are predominantly chromatin associated and whose production is coupled to cis-regulatory activities. Functional investigation of the contributions of p53-induced lncRNAs to p53-dependent outcomes highlighted Pvt1b, the p53-dependent isoform of Pvt1, as a mediator of p53-dependent senescence via Myc repression. Inhibition of Pvt1b led to decreased activation of senescence markers and increased levels of markers of proliferation. These findings shed light on the core and outcome-specific p53 restoration signatures across different oncogenic contexts and underscore the key role of the p53-Pvt1b-Myc regulatory axis in mediating proliferative arrest.

RB depletion is required for the continuous growth of tumors initiated by loss of RB.

The retinoblastoma (RB) tumor suppressor is functionally inactivated in a wide range of human tumors where this inactivation promotes tumorigenesis in part by allowing uncontrolled proliferation. RB has been extensively studied, but its mechanisms of action in normal and cancer cells remain only partly understood. Here, we describe a new mouse model to investigate the consequences of RB depletion and its re-activation in vivo. In these mice, induction of shRNA molecules targeting RB for knock-down results in the development of phenotypes similar to Rb knock-out mice, including the development of pituitary and thyroid tumors. Re-expression of RB leads to cell cycle arrest in cancer cells and repression of transcriptional programs driven by E2F activity. Thus, continuous RB loss is required for the maintenance of tumor phenotypes initiated by loss of RB, and this new mouse model will provide a new platform to investigate RB function in vivo.

Oncogenic HPV promotes the expression of the long noncoding RNA lnc-FANCI-2 through E7 and YY1.

Long noncoding RNAs (lncRNAs) play diverse roles in biological processes, but their expression profiles and functions in cervical carcinogenesis remain unknown. By RNA-sequencing (RNA-seq) analyses of 18 clinical specimens and selective validation by RT-qPCR analyses of 72 clinical samples, we provide evidence that, relative to normal cervical tissues, 194 lncRNAs are differentially regulated in high-risk (HR)-HPV infection along with cervical lesion progression. One such lncRNA, lnc-FANCI-2, is extensively characterized because it is expressed from a genomic locus adjacent to the FANCI gene encoding an important DNA repair factor. Both genes are up-regulated in HPV lesions and in in vitro model systems of HR-HPV18 infection. We observe a moderate reciprocal regulation of lnc-FANCI-2 and FANCI in cervical cancer CaSki cells. In these cells, lnc-FANCI-2 is transcribed from two alternative promoters, alternatively spliced, and polyadenylated at one of two alternative poly(A) sites. About 10 copies of lnc-FANCI-2 per cell are detected preferentially in the cytoplasm. Mechanistically, HR-HPVs, but not low-risk (LR)-HPV oncogenes induce lnc-FANCI-2 in primary and immortalized human keratinocytes. The induction is mediated primarily by E7, and to a lesser extent by E6, mostly independent of p53/E6AP and pRb/E2F. We show that YY1 interacts with an E7 CR3 core motif and transactivates the promoter of lnc-FANCI-2 by binding to two critical YY1-binding motifs. Moreover, HPV18 increases YY1 expression by reducing miR-29a, which targets the 3' untranslated region of YY1 mRNA. These data have provided insights into the mechanisms of how HR-HPV infections contribute to cervical carcinogenesis.

E2F coregulates an essential HSF developmental program that is distinct from the heat-shock response.

Heat-shock factor (HSF) is the master transcriptional regulator of the heat-shock response (HSR) and is essential for stress resilience. HSF is also required for metazoan development; however, its function and regulation in this process are poorly understood. Here, we characterize the genomic distribution and transcriptional activity of Caenorhabditis elegans HSF-1 during larval development and show that the developmental HSF-1 transcriptional program is distinct from the HSR. HSF-1 developmental activation requires binding of E2F/DP to a GC-rich motif that facilitates HSF-1 binding to a heat-shock element (HSE) that is degenerate from the consensus HSE sequence and adjacent to the E2F-binding site at promoters. In contrast, induction of the HSR is independent of these promoter elements or E2F/DP and instead requires a distinct set of tandem canonical HSEs. Together, E2F and HSF-1 directly regulate a gene network, including a specific subset of chaperones, to promote protein biogenesis and anabolic metabolism, which are essential in development.

RB1: a prototype tumor suppressor and an enigma.

The retinoblastoma susceptibility gene (RB1) was the first tumor suppressor gene to be molecularly defined. RB1 mutations occur in almost all familial and sporadic forms of retinoblastoma, and this gene is mutated at variable frequencies in a variety of other human cancers. Because of its early discovery, the recessive nature of RB1 mutations, and its frequency of inactivation, RB1 is often described as a prototype for the class of tumor suppressor genes. Its gene product (pRB) regulates transcription and is a negative regulator of cell proliferation. Although these general features are well established, a precise description of pRB's mechanism of action has remained elusive. Indeed, in many regards, pRB remains an enigma. This review summarizes some recent developments in pRB research and focuses on progress toward answers for the three fundamental questions that sit at the heart of the pRB literature: What does pRB do? How does the inactivation of RB change the cell? How can our knowledge of RB function be exploited to provide better treatment for cancer patients?

E2Fb and E2Fa transcription factors independently regulate the DNA damage response after ultraviolet B exposure in Arabidopsis.

Ultraviolet (UV)B radiation affects plant growth inhibiting cell proliferation. This inhibition is in part controlled by the activity of transcription factors from the E2F family. In particular, the participation of E2Fc and E2Fe in UV-B responses in Arabidopsis plants was previously reported. However, the E2Fa and E2Fb contribution to these processes has still not been investigated. Thus, in this work, we provide evidence that, in Arabidopsis, both E2Fa and E2Fb control leaf size under UV-B conditions without participating in the repair of cyclobutane pyrimidine dimers in the DNA. Nevertheless, in UV-B-exposed seedlings, E2Fa, but not E2Fb, regulates primary root elongation, cell proliferation, and programmed cell death in the meristematic zone. Using e2fa mutants that overexpress E2Fb, we showed that the role of E2Fa in the roots could not be replaced by E2Fb. Finally, our results show that E2Fa and E2Fb differentially regulate the expression of genes that activate the DNA damage response and cell cycle progression, both under conditions without UV-B and after exposure. Overall, we showed that both E2Fa and E2Fb have different and non-redundant roles in developmental and DNA damage responses in Arabidopsis plants exposed to UV-B.

E2F transcription regulation: an orphan cyclin enters the stage.

Marked cyclin protein oscillations over the cell cycle ensure tight regulation of all cell cycle transitions. Despite expression patterns closely mirroring those of cyclin A, cyclin F has long been regarded as an odd outlier within the cyclin family. Constituting part of an E3 ubiquitin ligase, its main role was seen as highly restricted to timely degradation of very few key substrates to ensure termination of one error-free round of replication. Now, a recent series of studies suggests that cyclin F has very similar roles as its closest relatives, merely mediated through a very different mechanism.

The TFDP1 gene coding for DP1, the heterodimeric partner of the transcription factor E2F, is a target of deregulated E2F.

The TFDP1 gene codes for the heterodimeric partner DP1 of the transcription factor E2F. E2F, principal target of the tumor suppressor pRB, plays central roles in cell proliferation by activating a group of growth-related genes. E2F also mediates tumor suppression by activating tumor suppressor genes such as ARF, an upstream activator of the tumor suppressor p53, when deregulated from pRB upon oncogenic changes. Among 8 E2F family members (E2F1∼E2F8), expression of activator E2Fs (E2F1∼E2F3a) is induced at the G1/S boundary of the cell cycle after growth stimulation by E2F itself. However, mechanisms regulating DP1 expression are not known. We show here that over-expression of E2F1 and forced inactivation of pRB, by adenovirus E1a, induced TFDP1 gene expression in human normal fibroblast HFFs, suggesting that the TFDP1 gene is a target of E2F. Serum stimulation of HFFs also induced TFDP1 gene expression, but with different kinetics from that of the CDC6 gene, a typical growth-related E2F target. Both over-expression of E2F1 and serum stimulation activated the TFDP1 promoter. We searched for E2F1-responsive regions by 5' and 3' deletion of the TFDP1 promoter and by introducing point mutations in putative E2F1-responsive elements. Promoter analysis identified several GC-rich elements, mutation of which reduced E2F1-responsiveness but not serum-responsiveness. ChIP assays showed that the GC-rich elements bound deregulated E2F1 but not physiological E2F1 induced by serum stimulation. These results suggest that the TFDP1 gene is a target of deregulated E2F. In addition, knockdown of DP1 expression by shRNA enhanced ARF gene expression, which is specifically induced by deregulated E2F activity, suggesting that activation of the TFDP1 gene by deregulated E2F may function as a failsafe feedback mechanism to suppress deregulated E2F and maintain normal cell growth in the event that DP1 expression is insufficient relative to that of its partner activator E2Fs. a maximum of 6 keywords: E2F, DP1, TFDP1 gene, pRB, gene expression.

Rb-E2F-HDAC Repressor Complexes Control Interferon-Induced Repression of Adenovirus To Promote Persistent Infection.

Interferons (IFNs) are cytokines that induce a global change in the cell to establish antiviral immunity. We previously demonstrated that human adenovirus (HAdV) exploits IFN-induced viral repression to persist in infected cells. Although this in vitro persistence model has been described, the mechanism behind how persistent HAdV infection is established is not well understood. In this study, we demonstrate that IFN signaling is essential for viral repression and promoting persistent infection. Cyclin-dependent kinase 4 (CDK4), an antagonist of retinoblastoma (Rb) family proteins, was shown to disrupt the viral repression induced by IFNs. Consistent with this result, knockout of the Rb family proteins pRb, p107, and/or p130 drastically reduced the effect of IFNs on viral replication. The pRb protein specifically contributed the greatest effect to IFN inhibition of viral replication. Interestingly, IFNs did not impact pRb through direct changes in protein or phosphorylation levels. Cells treated with IFNs continued to cycle normally, consistent with observations that persistently infected cells remain for long periods of time in the host and in our in vitro persistent infection model. Finally, we observed that histone deacetylase (HDAC) inhibitors activated productive viral replication in persistently infected cells in the presence of IFN. Thus, HDACs, specifically class I HDACs, which are commonly associated with Rb family proteins, play a major role in the maintenance of persistent HAdV infection in vitro. This study uncovers the critical role of pRb and class I HDACs in the IFN-induced formation of a repressor complex that promotes persistent HAdV infections. IMPORTANCE Adenoviruses are ubiquitous viruses infecting more than 90% of the human population. HAdVs cause persistent infections that may lead to serious complications in immunocompromised patients. Therefore, exploring how HAdVs establish persistent infections is critical for understanding viral reactivation in immunosuppressed individuals. The mechanism underlying HAdV persistence has not been fully explored. Here, we provide insight into the contributions of the host cell to IFN-mediated persistent HAdV infection. We found that HAdV-C5 productive infection is inhibited by an Rb-E2F-HDAC repressor complex. Treatment with HDAC inhibitors converted a persistent infection to a lytic infection. Our results suggest that this process involves the noncanonical regulation of Rb-E2F signaling. This study provides insight into a highly prevalent human pathogen, bringing a new level of complexity and understanding to the replicative cycle.

SNF1-related protein kinase 1 represses Arabidopsis growth through post-translational modification of E2Fa in response to energy stress.

Cellular sugar starvation and/or energy deprivation serves as an important signaling cue for the live cells to trigger the necessary stress adaptation response. When exposed to cellular energy stress (ES) conditions, the plants reconfigure metabolic pathways and rebalance energy status while restricting vegetative organ growth. Despite the vital importance of this ES-induced growth restriction, the regulatory mechanism underlying the response remains largely elusive in plants. Using plant cell- and whole plant-based functional analyses coupled with extended genetic validation, we show that cellular ES-activated SNF1-related protein kinase 1 (SnRK1.1) directly interacts with and phosphorylates E2Fa transcription factor, a critical cell cycle regulator. Phosphorylation of E2Fa by SnRK1.1 leads to its proteasome-mediated protein degradation, resulting in S-phase repression and organ growth restriction. Our findings show that ES-dependently activated SnRK1.1 adjusts cell proliferation and vegetative growth for plants to cope with constantly fluctuating environments.