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CBP activates the dhfr promoter in an E2F1 transactivator bypass experiment. Activation of the DHFRGal4 reporter construct by Gal4 fusion proteins containing the p62 subunit of TFIIH, CBP, and TBP was examined in transiently transfected NIH 3T3 cells, as described in the legend to Fig. 2. -Fold activation was calculated by dividing the luciferase levels conferred to DHFRGal4 by the specified Gal4 fusion protein by the luciferase levels conferred by the Gal4 DNA binding domain alone. Bars represent standard errors of the means.
Source publication
The E2F family of heterodimeric transcription factors plays an important role in the regulation of gene expression at the
G1/S phase transition of the mammalian cell cycle. Previously, we have demonstrated that cell cycle regulation of murine dihydrofolate
reductase (dhfr) expression requires E2F-mediated activation of the dhfr promoter in S phase....
Citations
... The mechanism by which E2F1 and RB remodel chromatin at DNA breaks shares similarities with how they regulate gene transcription. In fact, E2F1 and RB recruit some of the same chromatin modifying enzymes, e.g., the histone acetyltransferases p300 and CBP, and the chromatin remodeler BRG1, to both DSBs and gene promoters [60,61,95,99,[102][103][104][105][106][107]. Recruitment of p300 and CBP to DSBs involves an interaction between the bromodomains of p300 and CBP and a motif on E2F1 that is acetylated in response to DNA damage ( Figure 3) [99]. ...
Simple Summary
Retinoblastoma (RB) proteins and E2F transcription factors partner together to regulate the cell cycle in many eukaryotic organisms. In organisms that lack one or both of these proteins, other proteins have taken on the essential function of cell cycle regulation. RB and E2F also have important functions outside of the cell cycle, including DNA repair. This review summarizes the non-canonical functions of RB and E2F in maintaining genome integrity and raises the question of whether such functions have always been present or have evolved more recently.
Abstract
Members of the E2F transcription factor family regulate the expression of genes important for DNA replication and mitotic cell division in most eukaryotes. Homologs of the retinoblastoma (RB) tumor suppressor inhibit the activity of E2F factors, thus controlling cell cycle progression. Organisms such as budding and fission yeast have lost genes encoding E2F and RB, but have gained genes encoding other proteins that take on E2F and RB cell cycle-related functions. In addition to regulating cell proliferation, E2F and RB homologs have non-canonical functions outside the mitotic cell cycle in a variety of eukaryotes. For example, in both mammals and plants, E2F and RB homologs localize to DNA double-strand breaks (DSBs) and directly promote repair by homologous recombination (HR). Here, we discuss the parallels between mammalian E2F1 and RB and their Arabidopsis homologs, E2FA and RB-related (RBR), with respect to their recruitment to sites of DNA damage and how they help recruit repair factors important for DNA end resection. We also explore the question of whether this role in DNA repair is a conserved ancient function of the E2F and RB homologs in the last eukaryotic common ancestor or whether this function evolved independently in mammals and plants.
... Findings presented here are consistent with those previous studies and now establish the molecular mechanism by which p300 and CBP are recruited to sites of DSBs dependent on RB and E2F1 posttranslational modifications. This role for E2F1 in recruiting histone acetyltransferases to regulate DNA repair is reminiscent of its function in activating transcription, although the mechanisms by which E2F1 localizes to sites of DNA damage and target gene promoters are different [42][43][44][45] . ...
E2F1 and retinoblastoma (RB) tumor-suppressor protein not only regulate the periodic expression of genes important for cell proliferation, but also localize to DNA double-strand breaks (DSBs) to promote repair. E2F1 is acetylated in response to DNA damage but the role this plays in DNA repair is unknown. Here we demonstrate that E2F1 acetylation creates a binding motif for the bromodomains of the p300/KAT3B and CBP/KAT3A acetyltransferases and that this interaction is required for the recruitment of p300 and CBP to DSBs and the induction of histone acetylation at sites of damage. A knock-in mutation that blocks E2F1 acetylation abolishes the recruitment of p300 and CBP to DSBs and also the accumulation of other chromatin modifying activities and repair factors, including Tip60, BRG1 and NBS1, and renders mice hypersensitive to ionizing radiation (IR). These findings reveal an important role for E2F1 acetylation in orchestrating the remodeling of chromatin structure at DSBs to facilitate repair.
... Our sequence analysis shows that PDK1 expression, possibly, is linked to RB-E2F pathway axis, and targeting PDK1 might result in sustained therapeutic response. In few earlier studies, the role of E2F transcription factors in the regulation of metabolic enzymes has been documented [16][17][18]. Besides, DCA treatment resulted in significant decrease in cell motility, which further argues in favor of targeting PDK1 in RB. Earlier studies using RB tumor specimens showed an elevated expression of certain genes involved in metabolic reprogramming [19][20][21]. ...
Pyruvate dehydrogenase kinase 1 (PDK1), a key enzyme implicated in metabolic reprogramming of tumors, is induced in several tumors including glioblastoma, breast cancer and melanoma. However, the role played by PDK1 is not studied in retinoblastoma (RB). In this study, we have evaluated the expression of PDK1 in RB clinical samples, and studied its inhibition as a strategy to decrease cell growth and migration. We show that PDK1 is specifically overexpressed in RB patient samples especially in vitreous seeds and hypoxic regions and cell lines compared to control retina using immunohistochemistry and real-time PCR. Our results further demonstrate that inhibition of PDK1 using small molecule inhibitors dichloroacetic acid (DCA) and dichloroacetophenone (DAP) resulted in reduced cell growth and increased apoptosis. We also confirm that combination treatment of DCA with chemotherapeutic agent carboplatin further enhanced the therapeutic efficacy compared to single drug treatment. In addition, we observed changes in glucose uptake, lactate and reactive oxygen species (ROS) levels as well as decreased cell migration in response to PDK1 inhibition. Additionally, we show that DCA treatment led to inhibition of PI3K/Akt pathway and reduction in PDK1 protein levels. Overall, our data suggest that targeting PDK1 could be a novel therapeutic strategy for RB.
... Another example of transcriptional regulation of metabolism is the regulation of DHFR by E2F1 [119]. Noteworthy, we have recently shown that the activity of E2F1 is critically dependent on the presence of another lysine methyltransferase, Set7/9 [90]. ...
Cancer-related metabolism has recently emerged as one of the "hallmarks of cancer". It has several important features, including altered metabolism of glucose and glutamine. Importantly, altered cancer metabolism connects different biochemical pathways into the one fine-tuned metabolic network, which stimulates high proliferation rates and plasticity to malignant cells. Among the keystones of cancer metabolism are one-carbon metabolism and nucleotide biosynthesis, which provide building blocks to anabolic reactions. Accordingly, the importance of these metabolic pathways for anticancer therapy has well been documented by more than fifty years of clinical use of specific metabolic inhibitors - methotrexate and nucleotides analogs. In this review we discuss one-carbon metabolism and nucleotide biosynthesis as common and specific features of many, if not all, tumors. The key enzymes involved in these pathways also represent promising anti-cancer therapeutic targets. We review different aspects of these metabolic pathways including their biochemistry, compartmentalization and expression of the key enzymes and their regulation at different levels. We also discuss the effects of known inhibitors of these pathways as well as the recent data on other enzymes of the same pathways as perspective pharmacological targets.
... The promoter region of BCL11A remains to be characterized, however, the present work found transcription factors such as KLF6, ZNF300, ZNF219, ZBED4 and E2F1 as potential inducers of BCL11A transcription in cancer cells. Studies on KLF6, ZNF300, ZNF219, ZBED4 and E2F1 revealed that the binding sites of these transcription factors in human promoters can positively aff ect the BCL11A transcription [17][18][19][20][21] . In agreement with the role of BCL11A in breast cancer, KLF6 was reported to have signifi cant eff ect on breast cancer metastasis and associated with poor clinical outcomes [22] . ...
B-cell lymphoma 11A, a transcriptional repressor, is highly expressed in triple negative breast cancer. The in vitro studies and animal models provide initial evidence suggesting that the knockdown of B-cell lymphoma 11A has a therapeutic eff ect on breast cancer. Defining the regulators driving the high expression of B-cell lymphoma 11A is important to understand its cancer-related functions. Among these regulators, transcription factors and microRNAs are critical for gene expression and associated with expression perturbations. Firstly, weidentifi ed the transcription factors that potentially interact with B-cell lymphoma 11A promoter. Based on bioinformatics prediction and multiple Omics datasets, two upregulated transcriptional activators Zinc Finger BED-Type Containing 4 and E2F Transcription Factor 1 in triple negative breast cancer were found to have seven sites within B-cell lymphoma 11A promoter. Secondly, we aimed to determine a putative set of microRNA that can mediate the post-transcriptional repression of B-cell lymphoma 11A. miR-513a-5p, miR-139-5p, miR-1179, miR-140-5p, and miR-542-3p, harboring at least one site of interaction with B-cell lymphoma 11A 3 untranslated region, were found inhibited in triple negative breast cancer. Taken together, the combinatorial regulation by transcription factors and microRNAs provide valuable information for further investigation on controlling the expression level of B-cell lymphoma 11A in triple negative breast cancer.
... E2F1 is a member of E2F transcription factors which act on gene promoters regulation during the cell cycle (Dyson, 1994;Dyson, 1998). It has been demonstrated that E2F1 is involved in the transcriptional regulation of several genes, whose products participate in cell cycle progression and DNA synthesis and whose expression is up-regulated at the cell cycle G1/S transition (Zwicker et al., 1997;Fry et al., 1999;Ohtani et al., 1999). Berteaux and his partners (Berteaux et al., 2005) found two E2F1 binding sites on H19 promoter, and the relationship between them was investigated. ...
H19 is an imprinted oncofetal gene, and loss of imprinting at the H19 locus results in over-expression of H19 in cancers. Aflatoxin B1(AFB1) is regarded as one of the most dangerous carcinogens. Exposure to AFB1 would most easily increase susceptibility to diseases such as hepatocellular carcinoma(HCC) but any possible relationship between AFB1 and H19 is not clear. In present study, we found that AFB1 could up-regulate the expression of H19 and promote cell growth and invasion by hepatocellular carcinoma HepG2 cells. Knocking down H19 RNA co ld reverse the effects of AFB1 on cell growth and invasion. In addition, AFB1 induced the expression of E2F1 and its knock-down could down-regulate H19 expression and suppress cell growth and invasion in hepatocellular carcinoma HepG2 cells. Furthermore, E2F1 over-expression could up-regulate H19 expression and promote cell growth and invasion, with binding to the H19 promoter being demonstrated by chromatin immunoprecipitation assays (ChIP). In summary, our results suggested that aflatoxin B1could promote cell growth and invasion in hepatocellular carcinoma HepG2 cells through actions on H19 and E2F1.
... Until recently, the central dogma related to mutations that facilitate neoplastic transformation by disabling the RB-E2F suppressive activity supported a scenario whereby loss of RB function resulted in transcriptional upregulation of cell-cycle mediators which in turn caused a shift in the diffusion of nutrients and biosynthetic enzyme equilibria. However, an early indication that RB-E2F might directly regulate metabolic enzyme expression came from the observation that two enzymes required for nucleotide synthesis, thymidine kinase (TK1) and dihydrofolate reductase (DHFR), are direct transcriptional targets of E2F family members (16)(17)(18)(19)(20)(21)(22)(23). Because RB suppresses the transcription of these nucleotide biosynthetic enzyme mRNAs via physical interaction with E2Fs, investigators postulated that RB might also suppress the transcription of mRNAs required to translate the multitude of regulators, transporters, and enzymes required for energetic and anabolic metabolism. ...
The discovery of the retinoblastoma (RB-1) gene as a tumor suppressor that is disrupted in a majority of human cancers either via direct or indirect genetic alterations has resulted in increased interest in its functions and downstream effectors. Although the canonical pathway that links this tumor suppressor to human cancers details its interaction with the E2F transcription factors and cell-cycle progression, recent studies have shown an essential role for RB-1 in the suppression of glycolytic and glutaminolytic metabolism. Characterization of the precise metabolic transporters and enzymes suppressed by the RB-E2F axis should enable the identification of small molecule antagonists that have selective and potent antitumor properties. Clin Cancer Res; 18(22); 6096-100. ©2012 AACR.
... As shown inFig. 5B, Smad7 caused a significant transcript decrease in the dihydrofolate reductase (Dhfr) gene, which is known to be responsive to E2F-1 via an E2F-binding sequence (Blake and Azizkhan, 1989; DeGregori et al., 1997; Fry et al., 1997; Fry et al., 1999). In contrast, K359A Smad7, which is unable to bind to E2F-1, had little effect. ...
... Lower arrows (P1 and P3; P2 and P6) indicate the positions of primer sets for PCR. The upper arrow (labeled as Dhfr) represents the major transcription start site Fry et al., 1999). A putative PCR fragment obtained from a control distal region (316 bp) and that from an E2F site-containing region (629 bp) proximal to the start site are shown as gray bars. ...
... At 72 h after serial introduction of the retroviral vectors, the levels of Flag-E2F-1 and Smad7 proteins were indeed substantial, but only slightly influenced by one another (Fig. 6A). While overexpression of E2F-1 is known to stimulate the activity of genes harboring E2F-responsive elements (Fry et al., 1999), the proliferation of cells expressing exogenous E2F-1 was comparable to that of control cells placed in 10% FBS for 72 h (Fig. 6B). In contrast to proliferation-inhibited cells expressing solely Smad7, cells expressing E2F-1 and Smad7 exhibited an almost normal proliferation with nontransformed phenotypes (Fig. 6B). ...
Smad family proteins are essential intracellular mediators that regulate transforming growth factor-b (TGF-b) ligand signaling. In response to diverse stimuli, Smad7 is rapidly expressed and acts as a cytoplasmic inhibitor that selectively interferes with signals elicited from TGF-b family receptors. In addition, earlier works have indicated that retrovirally transduced Smad7 induces long-lasting cell proliferation arrest in a variety of mesenchymal cells through down-regulation of G1 cyclins. However, the molecular mechanisms underlying the cytostatic effects of Smad7 remain unknown. We show here that Smad7 can form a complex with endogenous histone deacetylase proteins HDAC-1 and HDAC-3 in NIH 3T3 mouse fibroblast cells. By contrast, forced expression of a dominant-negative variant of HDAC-1 efficiently protected cells against Smad7 proliferation inhibition, suggesting that Smad7 depends on the deacetylase activity of its associated HDAC-1 to arrest the cell cycle. Furthermore, Smad7 caused HDAC-1 bind to E2F-1 to form a ternary complex on chromosomal DNA containing an E2F-binding motif and leading to repression in the activity of the E2F target genes. Smad7 mutations that prevented its binding to either HDAC-1 or E2F-1 resulted in a significant decrease in Smad7-mediated inhibition of cell proliferation. The present results strongly suggest that nuclear Smad7 is a transcriptional corepressor for E2F, providing a molecular basis for the Smad7-induced arrest of the cell cycle.
... Previous studies examined its function in different blood cells and found that TR4 bound to the CD36 promoter in macrophages (Xie, et al., 2009) binding sites in K562 cells; see Figure 8 for the binding patterns of TR4 across the entire chromosome 12 in all four cell types. stimulate transcription by interaction with the basal transcription machinery (Fry, et al., 1999;Xu, et al., 2007). In contrast, other transcription factors, such as GATA1 or TCF4 (TCF7L2), show significant binding to sites often located more than 10 kb away from the gene that they regulate (Blahnik, et al., 2010;Fujiwara, et al., 2009), suggesting that these factors may regulate transcription by looping mechanisms. ...
With the recent, rapid progress in high throughput DNA sequencing technology, deep sequencing has made it possible to perform unbiased genome-wide protein-DNA interaction studies (ChIP-Seq) and transcript expression (RNA-Seq) profiling. My thesis focuses on the data analysis and software development for ChIP-Seq and RNA-Seq. The second chapter of my dissertation focuses on applying bioinformatics approaches to integrate sequencing and phylogenetic conservation data to help to predict the genome position of a Gata3 enhancer element that is active exclusively in the T cell lineage. I also performed in vivo imaging to confirm the T cell specificity of the regulatory element. The third chapter consists of a ChIP-Seq study in which I mapped the in vivo binding sites of TR4, an orphan nuclear receptor, in four human ENCODE (ENCyclopedia Of DNA Elements) consortium cell lines. From these data, I discovered that TR4 preferentially binds to and is predicted to regulate genes that play crucial roles in RNA transcription and processing. In the fourth chapter, I performed an analysis of RNA-seq data from differentiating human CD34+ hematopoietic progenitor cells derived from the (fetal) umbilical cord and (adult) bone marrow. Analysis results revealed potential novel isoforms of several known erythroid regulatory proteins and these potential isoforms are supported by the identification of cloned ESTs in the sequence databases. In Chapter 5, I developed a bioinformatics software pipeline to prioritize ChIP-Seq peaks by incorporating external annotation data to facilitate more efficient downstream validation experiments. The developed ChIP-Seq pipeline estimates and adjusts for variation among biological replicates and incorporates peak location relative to gene structure to prioritize resulting peaks. It was then utilized to illustrate its performance with highly localized transcription factor binding (narrow peaks) and more broadly peaked histone modification profiles from ChIP-Seq data. Taken together, we have demonstrated that an integrative approach incorporating greater functional annotation advances our ability to effectively utilize and interpret data from massively parallel sequencing experiments of protein-DNA interactions. Finally, chapter 6 provides an overall summary and conclusions from my research with ChIP-Seq and RNA-Seq data.
... Our data demonstrate that the carboxyterminal transactivation domain was essential for E2F1 suppression of the AR promoter ( Figure 3) and therefore supports two possible models of AR repression. Several proteins are known to bind to this region and regulate transcription including CREBbinding protein [56], MDM2 [57] and TRRAP/Tip60 complex and non-specific (IgG) ChIPed DNA from BPH-1 cells using primers that flank sites A, B, and C. Primers flanking a region in the PS2 (targeted DNMT1) promoter and ABCB1 (non-targeted DNMT1) region were used as ChIP controls. Data is representative of the mean from 3 qPCR reactions and shown as a percent of input with the standard error indicated. ...
Although androgen receptor (AR) function has been extensively studied, regulation of the AR gene itself has been much less characterized. In this study, we observed a dramatic reduction in the expression of androgen receptor mRNA and protein in hyperproliferative prostate epithelium of keratin 5 promoter driven E2F1 transgenic mice. To confirm an inhibitory function for E2F1 on AR transcription, we showed that E2F1 inhibited the transcription of endogenous AR mRNA, subsequent AR protein, and AR promoter activity in both human and mouse epithelial cells. E2F1 also inhibited androgen-stimulated activation of two AR target gene promoters. To elucidate the molecular mechanism of E2F-mediated inhibition of AR, we evaluated the effects of two functional E2F1 mutants on AR promoter activity and found that the transactivation domain appears to mediate E2F1 repression of the AR promoter. Because DNMT1 is a functional intermediate of E2F1 we examined DNMT1 function in AR repression. Repression of endogenous AR in normal human prostate epithelial cells was relieved by DNMT1 shRNA knock down. DNMT1 was shown to be physically associated within the AR minimal promoter located 22 bps from the transcription start site; however, methylation remained unchanged at the promoter regardless of DNMT1 expression. Taken together, our results suggest that DNMT1 operates either as a functional intermediary or in cooperation with E2F1 inhibiting AR gene expression in a methylation independent manner.
