E-box Binding Factor Research Articles

Expression Profiling of EMT Transcriptional Regulators ZEB1 and ZEB2 in Different Histopathological Grades of Oral Squamous Cell Carcinoma Patients.

Pakistan has a high burden of oral cancers, with a prevalence rate of around 9%. Oral Squamous Cell Carcinoma (OSCC) accounts for about 90% of oral cancer cases. Epithelial to Mesenchymal Transition (EMT) gets highly stimulated in tumor cells by adopting subsequent malignant features of highly invasive cancer populations. Zinc Finger E-Box binding factors, ZEB1 and ZEB2, are regulatory proteins that promote EMT by suppressing the adherent ability of cells transforming into highly motile cancerous cells. The present study aimed to analyze the expression of EMT regulators, ZEB1 and ZEB2, and their association with the clinicopathological features in different grades of OSCC patients. Tissue samples were collected for both case and control groups from the recruited study participants. Cancer tissues (cases) were collected from the confirmed OSCC patients, and healthy tissues (controls) were collected from third-molar dental extraction patients. The study participants were recruited with informed consent and brief demographic and clinical characteristics. The case group was further segregated with respect to the histological cancer grading system into well-differentiated (WD), moderately differentiated (MD), and poorly differentiated (PD) squamous cell carcinoma (SCC) groups. RNA was extracted from the tissue samples for expression profiling of ZEB1 and ZEB2 genes through quantitative real-time PCR (qRT-PCR). All of the recruited participants had a mean age of 46.55 ± 11.7 (years), with most of them belonging to Urdu speaking ethnic group and were married. The BMI (kg/m2) of the healthy participants was in the normal range (18-22 kg/m2). However, BMI was found to be reduced with the proliferation in the pathological state of cancer. The oral hygiene of patients was better than the healthy participants, possibly due to the strict oral hygiene practice concerns of consultants. Every recruited OSCC patient had one or multiple addiction habits for more than a year. Patients reported health frailty (46.6%), unhealed mouth sores (40%), swallowing difficulties and white/reddish marks (80%), and restricted mouth opening (64.4%). Furthermore, 82.2% of the recruited patients observed symptoms within 1-12 months, and buccal mucosa was the most exposed tumor site among 55.6% of the patients. Expression profiling of EMT regulators showed gradual over-expressions of ZEB1 (8, 20, and 42 folds) and ZEB2 (4, 10, and 18 folds) in respective histological cancer grades. High expressions of ZEBs have been significantly associated with cancer progression and poor health. However, no association was found between OSCC with other clinicopathological features when compared to healthy controls.

Is the Secret of VDAC Isoforms in Their Gene Regulation? Characterization of Human VDAC Genes Expression Profile, Promoter Activity, and Transcriptional Regulators

VDACs (voltage-dependent anion-selective channels) are pore-forming proteins of the outer mitochondrial membrane, whose permeability is primarily due to VDACs’ presence. In higher eukaryotes, three isoforms are raised during the evolution: they have the same exon–intron organization, and the proteins show the same channel-forming activity. We provide a comprehensive analysis of the three human VDAC genes (VDAC1–3), their expression profiles, promoter activity, and potential transcriptional regulators. VDAC isoforms are broadly but also specifically expressed in various human tissues at different levels, with a predominance of VDAC1 and VDAC2 over VDAC3. However, an RNA-seq cap analysis gene expression (CAGE) approach revealed a higher level of transcription activation of VDAC3 gene. We experimentally confirmed this information by reporter assay of VDACs promoter activity. Transcription factor binding sites (TFBSs) distribution in the promoters were investigated. The main regulators common to the three VDAC genes were identified as E2F-myc activator/cell cycle (E2FF), Nuclear respiratory factor 1 (NRF1), Krueppel-like transcription factors (KLFS), E-box binding factors (EBOX) transcription factor family members. All of them are involved in cell cycle and growth, proliferation, differentiation, apoptosis, and metabolism. More transcription factors specific for each VDAC gene isoform were identified, supporting the results in the literature, indicating a general role of VDAC1, as an actor of apoptosis for VDAC2, and the involvement in sex determination and development of VDAC3. For the first time, we propose a comparative analysis of human VDAC promoters to investigate their specific biological functions. Bioinformatics and experimental results confirm the essential role of the VDAC protein family in mitochondrial functionality. Moreover, insights about a specialized function and different regulation mechanisms arise for the three isoform gene.

Triple Vectors Expand AAV Transfer Capacity in the Retina.

Retinal gene transfer with adeno-associated viral (AAV) vectors holds great promise for the treatment of inherited retinal degenerations (IRDs). One limit of AAV is its transfer capacity of about 5 kb, which can be expanded to about 9 kb, using dual AAV vectors. This strategy would still not suffice for treatment of IRDs such as Usher syndrome type 1D or Alström syndrome type I (ALMS) due to mutations in CDH23 or ALMS1, respectively. To overcome this limitation, we generated triple AAV vectors, with a maximal transfer capacity of about 14 kb. Transcriptomic analysis following triple AAV transduction showed the expected full-length products along a number of aberrant transcripts. However, only the full-length transcripts are efficiently translated in vivo. We additionally showed that approximately 4% of mouse photoreceptors are transduced by triple AAV vectors and showed correct localization of recombinant ALMS1. The low-photoreceptor transduction levels might justify the modest and transient improvement we observe in the retina of a mouse model of ALMS. However, the levels of transduction mediated by triple AAV vectors in pig retina reached 40% of those observed with single vectors, and this bodes well for further improving the efficiency of triple AAV vectors in the retina.

A novel polymorphism in the PAI-1 gene promoter enhances gene expression. A novel pro-thrombotic risk factor?

IntroductionPlasminogen activator inhibitor-1 (PAI-1) is the major physiological inhibitor of tissue-type plasminogen activator in plasma and the most important regulator of the fibrinolytic pathway. The 4G/5G polymorphism (rs1799889) in the PAI-1 promoter is associated with altered PAI-1 transcription. We have identified a new 4G/5G allele, in which a T is inserted near the 4G tract or replaces a G in the 5G tract, forming a T plus 4G (T4G) region. Materials and MethodsThis new variant was first identified in two women, one had experienced juvenile myocardial infarction, the other repeated miscarriage; both had increased PAI-1 plasma activity. In view of the important influence of this promoter region on PAI-1 protein plasma level, we performed in vitro evaluation of the effects of the T4G variant on the transcription activity of the PAI-1 gene promoter. Results and ConclusionsIn silico prediction analysis showed that presence of the T4G allele disrupts the E-Box region upstream of the T4G variant, altering the affinity of the target sequence for E-Box binding factors like upstream stimulatory factor-1 (USF-1). Basal T4G promoter activity was 50% higher compared to 4G and 5G variants, but it was less stimulated by USF-1 overexpression. We also analyzed the effects of IL-1β and IL-6 on the PAI-1 promoter activity of our three constructs and showed that the T4G variant was less affected by IL-1β than the other variants. These findings indicate that the T4G variant may be a novel risk factor for thrombotic events.

Abstract A15: A reciprocal feedback loop between grainyhead-like-2 and ZEB1 controls EMT and tumor suppression

Abstract The oncogenic Epithelial-Mesenchymal Transition (EMT) enhances malignant progression by promoting invasion and survival of tumor cells. EMT is induced by microenvironmental factors including TGF-β and Wnt agonists, and transcription factors including the E-box binding factors Twist, Snail and ZEB. Previously, we reported that a member of the mammalian Grainyhead family of wound healing-regulatory transcription factors, Grainyhead-like-2 (GRHL2), suppresses EMT and restores sensitivity to anoikis by repressing ZEB1 expression and inhibiting TGF-β signaling. Here, we elucidate the functional relationship between GRHL2 and ZEB1 in EMT/MET and tumor biology. At least three homeodomain proteins, Six1, LBX1, and HoxA5, transactivated the ZEB1 promoter; for Six1, this occurred through direct protein-promoter interaction. GRHL2 altered the Six1-DNA complex, inhibiting this transactivation. Correspondingly, GRHL2 expression prevented tumor initiation in xenograft assays, sensitized breast cancer cells to paclitaxel, and suppressed the emergence of CD44highCD24low cells in a cell line where this marker set correlates with cancer stem cell phenotype. GRHL2 was down-regulated in recurrent mouse tumors that had evolved to an oncogene-independent, EMT-like state, supporting a role for GRHL2 down-regulation in this tumor phenotype transition modeling disease recurrence. The combination of TGF-β and Wnt activation repressed GRHL2 expression through direct interaction of ZEB1 with the GRHL2 promoter, inducing EMT. These observations indicate that a reciprocal feedback loop between GRHL2 and ZEB1 controls epithelial vs. mesenchymal phenotypes and EMT-driven tumor progression. Citation Format: Benjamin Cieply, Heide L. Ford, Joshua Farris, Steven Frisch. A reciprocal feedback loop between grainyhead-like-2 and ZEB1 controls EMT and tumor suppression. [abstract]. In: Proceedings of the Third AACR International Conference on Frontiers in Basic Cancer Research; Sep 18-22, 2013; National Harbor, MD. Philadelphia (PA): AACR; Cancer Res 2013;73(19 Suppl):Abstract nr A15.

Two Adjacent cis-Regulatory Elements Are Required for Ecdysone Response of Ecdysone Receptor (EcR) B1 Transcription

Three distinct classes of nuclear receptors, EcR, E75, and HR3, are key regulators in the ecdysone-inducible gene activation cascade in insects. The transcription of these genes is induced by ecdysone (20E) differently, although the detailed mechanisms underlying their responses to 20E are largely unknown. We identified ecdysone response elements (EcREs) present in the promoters of genes coding BmEcR-B1, BmE75-A, and BHR3-B isoforms from Bombyx mori employing luciferase reporter assays in an ecdysteroid-responsive cultured cell line, NIAS-Bm-aff3 (aff3). The EcRE of BmEcR-B1 at −2800 comprises of two adjacent elements separated by 5 bp, E1 (15 bp) and E2 (21 bp), both of which are required for the 20E response. Further analysis using electrophoretic mobility shift assays showed that E1 binds to the EcR/USP heterodimer and that E2 may bind to the E-box (CACGTG) binding factor such as bHLH protein. The unique E1+E2-type EcRE is also detected in the promoter upstream regions of EcR-B1 from seven lepidopteran species studied. In contrast, both a 20 bp EcRE identified in the promoter of BmE75-A and a 18 bp EcRE identified in the BHR3-B promoter, contained only E1-type EcR/USP binding element but the E2 type element was not in the promoter regions of these genes. The combination of presence of the E2 element or other cis-regulatory elements in promoter regions explains the different 20E response of each class of nuclear receptor genes. Furthermore, the E1+E2 structure for EcR-B1 can be involved in a possible cross-talk between ecdysteroid and other regulatory pathways.

Characterization and regulation of the hb9/mnx1 beta-cell progenitor specific enhancer in zebrafish

Differentiation of insulin producing beta-cells is a genetically well defined process that involves functions of various conserved transcription factors. Still, the transcriptional mechanisms underlying specification and determination of beta-cell fate are poorly defined. Here we provide the description of a beta-cell progenitor specific enhancer as a model to study initial steps of beta-cell differentiation. We show that evolutionary non-conserved upstream sequences of the zebrafish hb9 gene are required and sufficient for regulating expression in beta-cells prior to the onset of insulin expression. This enhancer contains binding sites for paired-box transcription factors and two E-boxes that in EMSA studies show interaction with Pax6b and NeuroD, respectively. We show that Pax6b is a potent activator of endodermal hb9 expression and that this activation depends on the beta-cell enhancer. Using genetic approaches we show that pax6b is crucial for maintenance but not induction of pancreatic hb9 transcription. As loss of Pax6b or Hb9 independently results in the loss of insulin expression, the data reveal a novel cross-talk between the two essential regulators of early beta-cell differentiation. While we find that the known pancreatic E-box binding proteins NeuroD and Ngn3 are not required for hb9 expression we also show that removal of both E-boxes selectively eliminates pancreatic specific reporter expression. The data provide evidence for an Ngn3 independent pathway of beta-cell specification that requires function of currently not specified E-box binding factors.

The E-box Binding Factors Max/Mnt, MITF, and USF1 Act Coordinately with FoxO to Regulate Expression of Proapoptotic and Cell Cycle Control Genes by Phosphatidylinositol 3-Kinase/Akt/Glycogen Synthase Kinase 3 Signaling

Phosphatidylinositol (PI) 3-kinase/Akt signaling plays a critical role in cell proliferation and survival, partly by regulation of FoxO transcription factors. Previous work using global expression profiling indicated that inhibition of PI 3-kinase in proliferating cells led to induction of genes that promote cell cycle arrest and apoptosis. The upstream regulatory regions of these genes had binding sites not only for FoxO, but also for Myc/Max transcription factors. In the present study, we have addressed the role of Myc family members and related E-box-binding proteins in the regulation of these genes. Chromatin immunoprecipitations and RNA interference indicated that transcription was repressed by Max-Mnt-Sin3a-histone deacetylase complexes in proliferating cells. Inhibition of PI 3-kinase led to a loss of Max/Mnt binding and transcriptional induction by MITF and USF1, as well as FoxO. Both MITF and USF1 were activated by glycogen synthase kinase (GSK) 3, with GSK3 phosphorylation sites on USF1 identified as the previously described activating site threonine 153 as well as serine 186. siRNA against MITF as well as against FoxO3a protected cells from apoptosis following PI 3-kinase inhibition. These results define a novel E-box-regulated network that functions coordinately with FoxO to regulate transcription of apoptotic and cell cycle regulatory genes downstream of PI 3-kinase/Akt/GSK3 signaling.

Zinc finger E‐box binding factor 1 plays a central role in regulating Epstein‐Barr virus (EBV) latent‐lytic switch and acts as a therapeutic target in EBV‐associated gastric cancer

The role of Epstein-Barr virus (EBV) infection in gastric carcinogenesis remains largely unknown. The authors studied the effects of zinc finger E-box binding factor 1 (ZEB1) on latent-lytic switch of EBV infection in gastric cancer and explored the importance of EBV in gastric carcinogenesis. Loss or gain of ZEB1 function was obtained by ZEB1 small-interfering RNA (siRNA) knock-down or forced ZEB1 re-expression. Cell growth was evaluated by cell viability and colony formation assays, and the cell cycle was assessed by flow cytometry. EBV was detected using quantitative polymerase chain reaction (PCR) and in situ hybridization analyses. ZEB1 knock-down in a latent EBV-infected gastric cancer cell line (YCC10) increased lytic gene BamHI W leftward reading frame 1 (BZLF1) expression and decreased the expression of latent gene EB nuclear antigen 1 (EBNA1) concomitant with the inhibition of cell viability (P < .05) and S-phase DNA synthesis (P < .01). ZEB1 depletion combined with ganciclovir revealed a further reduction in cell viability (P < .001). ZEB1 knock-down induced cell apoptosis and the up-regulation of caspase 3 and poly(adenosine diphosphate-ribose) polymerase cleavage. Conversely, ectopic overexpression of ZEB1 in a lytic EBV-infected gastric cancer cell line (AGS-EBV) inhibited BZLF1 promoter (Zp) activity, BZLF1 expression, and apoptosis and promoted cell growth. EBV infection was detected in 11.3% (80 of 711) of gastric cancers. The presence of EBV was associated with age, men, and intestinal type cancer. ZEB1 was confirmed as a key mediator of the latent-lytic switch of EBV-associated gastric cancer, a distinct subtype with different clinicopathologic features. The current results indicated that inhibition of ZEB1 may be a potential target for EBV-associated gastric cancer therapy.

M1912 Role of Zinc Finger E-Box Binding Factor 1 Modulating Latent-Lytic Switch of Epstein-Barr Virus in Gastric Cancer

Background and Aims: Recent studies have shown epsterin-Barr virus (EBV) infection is related to the development of gastric cancer. In particular, latent EBV infection usually established persistently and promoted tumor progression. The zinc finger E-box binding factor 1 (ZEB1) mediated transcriptional silencing of intermediate-early genes and induced EBV infection from lytic to latent stage. We aim to study the effect of ZEB1 on modulating latent-lytic switch in gastric cancer, and to explore the potential of ZEB1 as a novel molecular target for the intervention of EBV associated Gastric Cancer (EBVaGc). Method Gastric cancer cell-lines with latent (YCC10 cell-line) and lytic (AGS-EBV infected cell-line) EBV infection were used in this study. Loss or gain function of ZEB1 was obtained by ZEB1 siRNA knockdown or forced re-expression experiments, respectively. The modulation of the intermediateearly gene and apoptotic mediators were assessed by real time RT-PCR and Western blot. Cell growth was evaluated by cell viability assay and colony formation assay, and cell cycle by flow cytometry analysis. Results: siRNA-mediated knockdown of ZEB1 in latent EBV infected cell-line YCC10 triggered a 3-fold enhanced expression of BZLF1, an intermediateearly gene of EBV, but a 5-fold reduction in expression of EBV nuclear antigen 1 (EBNA1), a gene that plays essential roles in enabling the replication and persistence of EBV genomes in latently infected cells and activating EBV latent gene expression. By doing so, knockdown of ZEB1 in YCC10 significantly inhibited cell viability (100% to 84%, P<0.01), suppressed cell proliferation as evidenced by reduced S phase cells (P=0.005), caused cell cycle arrest in G2M phase (P=0.006) and induced cell apoptosis (4.6±0.15% to 7.1±1.36%, P<0.05), The induction of cell apoptosis is confirmed by the cleavage of caspase-3 and PARP. On the other hand, over-expression of ZEB1 in lytic EBV infected cell line AGS-EBV through transient transfection significantly increased cell viability (P<0.01), induced S phase cell number (32.9±1.17% to 40.3±23.89%, P<0.05) and promoted colony formation (P<0.01). Conclusion: ZEB1 is involved in the latent-lytic switch of EBV associated gastric cancer. Inhibition of ZEB1 may be a promising approach for the treatment of EBVaGc.

M1914 Mechanisms by Which the New Reconstruction Procedure Exerts Beneficial Effects After Total Gastrectomy

Background and Aims: Recent studies have shown epsterin-Barr virus (EBV) infection is related to the development of gastric cancer. In particular, latent EBV infection usually established persistently and promoted tumor progression. The zinc finger E-box binding factor 1 (ZEB1) mediated transcriptional silencing of intermediate-early genes and induced EBV infection from lytic to latent stage. We aim to study the effect of ZEB1 on modulating latent-lytic switch in gastric cancer, and to explore the potential of ZEB1 as a novel molecular target for the intervention of EBV associated Gastric Cancer (EBVaGc). Method Gastric cancer cell-lines with latent (YCC10 cell-line) and lytic (AGS-EBV infected cell-line) EBV infection were used in this study. Loss or gain function of ZEB1 was obtained by ZEB1 siRNA knockdown or forced re-expression experiments, respectively. The modulation of the intermediateearly gene and apoptotic mediators were assessed by real time RT-PCR and Western blot. Cell growth was evaluated by cell viability assay and colony formation assay, and cell cycle by flow cytometry analysis. Results: siRNA-mediated knockdown of ZEB1 in latent EBV infected cell-line YCC10 triggered a 3-fold enhanced expression of BZLF1, an intermediateearly gene of EBV, but a 5-fold reduction in expression of EBV nuclear antigen 1 (EBNA1), a gene that plays essential roles in enabling the replication and persistence of EBV genomes in latently infected cells and activating EBV latent gene expression. By doing so, knockdown of ZEB1 in YCC10 significantly inhibited cell viability (100% to 84%, P<0.01), suppressed cell proliferation as evidenced by reduced S phase cells (P=0.005), caused cell cycle arrest in G2M phase (P=0.006) and induced cell apoptosis (4.6±0.15% to 7.1±1.36%, P<0.05), The induction of cell apoptosis is confirmed by the cleavage of caspase-3 and PARP. On the other hand, over-expression of ZEB1 in lytic EBV infected cell line AGS-EBV through transient transfection significantly increased cell viability (P<0.01), induced S phase cell number (32.9±1.17% to 40.3±23.89%, P<0.05) and promoted colony formation (P<0.01). Conclusion: ZEB1 is involved in the latent-lytic switch of EBV associated gastric cancer. Inhibition of ZEB1 may be a promising approach for the treatment of EBVaGc.

Tip60 Is Regulated by Circadian Transcription Factor Clock and Is Involved in Cisplatin Resistance

Histone modification is important for maintaining chromatin structure and function. Recently, histone acetylation has been shown to have a critical regulatory role in both transcription and DNA repair. We report here that expression of histone acetyltransferase (HAT) genes is associated with cisplatin resistance. We found that Tip60 is overexpressed in cisplatin-resistant cells. The expression of two other HAT genes, HAT1 and MYST1, did not differ between drug-sensitive and -resistant cells. Knockdown of Tip60 expression rendered cells sensitive to cisplatin but not to oxaliplatin, vincristine, and etoposide. Tip60 expression is significantly correlated with cisplatin sensitivity in human lung cancer cell lines. Interestingly, the promoter region of the Tip60 gene contains several E boxes, and its expression was regulated by the E-box binding circadian transcription factor Clock but not by other E-box binding transcription factors such as c-Myc, Twist, and USF1. Hyperacetylation of H3K14 and H4K16 was found in cisplatin-resistant cells. The microarray study reveals that several genes for DNA repair are down-regulated by the knockdown of Tip60 expression. Our data show that HAT gene expression is required for cisplatin resistance and suggest that Clock and Tip60 regulate not only transcription, but also DNA repair, through periodic histone acetylation.

Transcription Factor PU.1 Controls Transcription Start Site Positioning and Alternative TLR4 Promoter Usage

Human and mouse show markedly different sensitivities toward bacterial endotoxins, and recent evidence suggests that a species-specific regulation of the lipopolysaccharide receptor Toll-like receptor 4 (Tlr4) may contribute to this phenomenon. To gain further insight into mechanisms of Tlr4 regulation, we conducted a detailed in vivo and in vitro study of the murine Tlr4 gene, including analysis of transcription start site location, transcription factor occupancy, and activities of its proximal regulatory sequences. Our analyses identified a PU.1-dependent myeloid promoter, which is conserved between humans and mouse. We also identified an additional, distal promoter, located approximately 200 bp upstream of the myeloid-specific promoter, which is a functional target of E-box binding factors. In contrast to humans, where non-myeloid cells utilize both promoters, the distal Tlr4 promoter initiates all Tlr4 transcripts in murine non-myeloid cells, indicating that species-specific differences in TLR4 mRNA regulation may primarily exist in non-myeloid cell types. Interestingly, PU.1 null murine myeloid progenitor cells predominantly use the distal promoter, and the conditional induction of PU.1 expression in these cells leads to the rapid switch of transcription initiation to the proximal myeloid promoter. This indicates a direct role for PU.1 in determining the transcriptional start site and in recruiting the basal transcription machinery to myeloid promoters.

Tubular Epithelial Cell Dedifferentiation Is Driven by the Helix-Loop-Helix Transcriptional Inhibitor Id1

In the fibrotic kidney, tubular cells undergo epithelial-to-mesenchymal transition (EMT), a phenotypic conversion that is characterized by sequential loss of epithelial markers and gain of mesenchymal features. For understanding of the molecular mechanism that governs this process, a high-throughput gene expression microarray analysis was used to identify the critical genes in the initial phase of the TGF-beta1-mediated EMT. Inhibitor of differentiation-1 (Id1), a dominant negative antagonist of the basic helix-loop-helix transcription factors, was found to be induced rapidly in human proximal tubular epithelial cells after TGF-beta1 treatment. This induction of Id1 depended on intracellular Smad signaling. Ectopic expression of Id1 suppressed epithelial E-cadherin and zonula occludens-1 expression. Id1 physically formed complex with basic helix-loop-helix transcription factor HEB (Hela E-box binding factor), sequestered its ability to bind to E-box, and repressed the trans-activation of E-cadherin promoter. However, overexpression of Id1 failed to induce alpha-smooth muscle actin, matrix metalloproteinase-2, fibronectin, and integrin-linked kinase (ILK), indicating its inability to confer a complete EMT. Overexpression of ILK or inhibition of ILK activity had no effect on Id1 induction by TGF-beta1, suggesting that Id1 and ILK have independent roles in epithelial dedifferentiation and EMT. In vivo, Id1 was induced exclusively in the degenerated, dilated renal tubular epithelium after unilateral ureteral obstruction. These studies identify Id1 transcriptional inhibitor as a crucial player in mediating cell dedifferentiation of renal tubular epithelium and suggest that EMT is a multistep process in which loss of epithelial adhesion does not necessarily lead to an autonomous mesenchymal transition.

Analysis of the VMD2 Promoter and Implication of E-box Binding Factors in Its Regulation

The retinal pigment epithelium (RPE) is crucial for the normal development and function of retinal photo-receptors, and mutations in several genes that are preferentially expressed in the RPE have been shown to cause retinal degeneration. We analyzed the 5'-up-stream region of human VMD2, a gene that is preferentially expressed in the RPE and, when mutated, causes Best macular dystrophy. Transgenic mouse studies with VMD2 promoter/lacZ constructs demonstrated that a-253 to +38 bp fragment is sufficient to direct RPE-specific expression in the eye. Transient transfection assays using the D407 human RPE cell line with VMD2 promoter/luciferase reporter constructs identified two positive regulatory regions, -585 to -541 bp for high level expression and -56 to -42 bp for low level expression. Mutation of a canonical E-box located in the -56 to -42 bp region greatly diminished luciferase expression in D407 cells and abolished the bands shifted with bovine RPE nuclear extract in electrophoretic mobility shift assays. Independently a candidate approach was used to select microphthalmia-associated transcription factor (MITF) for testing because it is expressed in the RPE and associated with RPE abnormalities when mutated. MITF-M significantly increased luciferase expression in D407 cells in an E-box-dependent manner. These studies define the VMD2 promoter region sufficient to drive RPE-specific expression in the eye, identify positive regulatory regions in vitro, and suggest that MITF as well as other E-box binding factors may act as positive regulators of VMD2 expression.

Identification of E-box factor TFE3 as a functional partner for the E2F3 transcription factor.

Various studies have demonstrated a role for E2F proteins in the control of transcription of genes involved in DNA replication, cell cycle progression, and cell fate determination. Although it is clear that the functions of the E2F proteins overlap, there is also evidence for specific roles for individual E2F proteins in the control of apoptosis and cell proliferation. Investigating protein interactions that might provide a mechanistic basis for the specificity of E2F function, we identified the E-box binding factor TFE3 as an E2F3-specific partner. We also show that this interaction is dependent on the marked box domain of E2F3. We provide evidence for a role for TFE3 in the synergistic activation of the p68 subunit gene of DNA polymerase alpha together with E2F3, again dependent on the E2F3 marked box domain. Chromatin immunoprecipitation assays showed that TFE3 and E2F3 were bound to the p68 promoter in vivo and that the interaction of either E2F3 or TFE3 with the promoter was facilitated by the presence of both proteins. In contrast, neither E2F1 nor E2F2 interacted with the p68 promoter under these conditions. We propose that the physical interaction of TFE3 and E2F3 facilitates transcriptional activation of the p68 gene and provides strong evidence for the specificity of E2F function.

ZEB negatively regulates the lytic-switch BZLF1 gene promoter of Epstein-Barr virus.

Epstein-Barr virus (EBV) is a human herpesvirus capable of establishing a latent state in B lymphocytes. The product of the immediate-early BZLF1 gene, Zta, is a transcriptional transactivator essential for viral DNA amplification and virion production. Previously, we identified a negative cis-acting element within the BZLF1 promoter termed ZV. ZV contains the sequence 5'-CAGGTA-3' located at nucleotides -17 to -12 relative to the transcription initiation site. It sequence specifically binds a cellular factor, ZVR. Based on sequence binding specificity, we postulated that ZVR may be zinc finger E-box binding factor (ZEB) or a related zinc finger/homeodomain family member. We show here by immunoshift assays that ZVR and human ZEB specifically cross-react with an antibody to deltaEF1, the chicken homolog of ZEB. Competition electrophoretic mobility shift assays confirmed that ZEB binds to the ZV element with the same binding specificity as ZVR. Overexpression of ZEB in either B-lymphocytic DG75 cells or mammary epithelial MCF-7 cells repressed Zta-induced activation of the BZLF1 promoter four- to fivefold via the ZV site. Thus, we conclude that the previously identified cellular repressor ZVR is, in fact, ZEB. We also present evidence that other cellular factors likely affect the transcriptional activity of ZEB. Lastly, we identify a ZEB-binding site within the promoter of the lytic BRLF1 gene of EBV. We postulate that ZEB likely plays an important role in regulating the life cycle of EBV.

Regulation of the Uncoupling Protein-2 Gene in INS-1 β-Cells by Oleic Acid

Current evidence suggests that uncoupling protein-2 (UCP2) is a regulator of insulin secretion. It is also known that chronic exposure of pancreatic islets to free fatty acids (FFAs) blunts glucose-stimulated insulin secretion and is accompanied by elevated levels of UCP2. However, the mechanisms regulating expression of UCP2 in beta-cells are unknown. Here, we show that UCP2 mRNA and protein levels were increased after a 48-h exposure of INS-1(832/13) beta-cells to oleic acid (0.5 mm) by activation of the UCP2 promoter. Furthermore, progressive deletions of the mouse UCP2 promoter (from -7.3 kb to +12 bp) indicated that an enhancer region (-86/-44) was responsible for both basal and FFA-stimulated UCP2 gene transcription. This enhancer contains tightly clustered Sp1, sterol regulatory element (SRE), and double E-Box elements. While all three sequence motifs were required for basal activity of the UCP2 promoter, the mutations in either the SRE or the E-Box elements eliminated the response to FFAs. The SRE and sterol regulatory element binding protein-1 (SREBP1) appear to be crucial for the response of the UCP2 gene to FFAs, since overexpression of the nuclear forms of the SREBPs increased UCP2 promoter activity by 7-10-fold and restored the ability of E-Box mutants to respond to oleic acid. These data support a model in which SREBP is the major modulator of UCP2 gene transcription by FFA, while E-Box binding factors play a supportive role.

Downstream E-box-mediated regulation of the human telomerase reverse transcriptase (hTERT) gene transcription: evidence for an endogenous mechanism of transcriptional repression.

Regulation of the hTERT gene encoding the telomerase catalytic subunit plays an important role in human cell senescence, immortalization, and carcinogenesis. By examining the activity of various deleted or mutated hTERT promoter fragments, we show that an E-box element downstream of the transcription initiation site is critical to differential hTERT transcription between the telomerase/hTERT-positive renal cell carcinoma cell line (RCC23) and its telomerase/hTERT-negative counterpart containing a transferred, normal chromosome 3 (RCC23+3). This E-box element mediated repression of hTERT transcription in RCC23+3 but not in RCC23. A copy number-dependent enhancement of the repression suggested active repression, rather than loss of activation, in RCC23+3. Endogenous expression levels of c-Myc or Mad1, which could activate or repress hTERT transcription when overexpressed, did not account for the differential hTERT transcription. Gel mobility shift assays identified the upstream stimulatory factors (USFs) as a major E-box-binding protein complex in both RCC23 and RCC23+3 and, importantly, detected an RCC23+3-specific, E-box-binding factor that was distinct from the USF and Myc/Mad families. The E-box-mediated repression was also active in normal human fibroblasts and epithelial cells and inactive in some, but not all, telomerase/hTERT-positive cancer cells. These findings provide evidence for an endogenous, repressive mechanism that actively functions in telomerase/hTERT-negative normal cells and becomes defective during carcinogenic processes, e.g., by an inactivation of the telomerase repressor gene on chromosome 3.

Okadaic acid increases ARNT homodimer transactivation potential.

The human aryl hydrocarbon nuclear translocator (hARNT) protein belongs to the family of basic helix-loop-helix (bHLH) PAS transcription factors and regulates a range of cellular processes by either homodimerizing or heterodimerizing with other bHLH-PAS proteins. hARNT has been shown to be almost exclusively phosphorylated on serine residues. However, regulation of hARNT with respect to phosphorylation remains poorly understood. The phosphatase inhibitor okadaic acid was used to explore whether a change in hARNT phosphorylation status could influence hARNT homodimer activity. The hARNT homodimer has been shown to bind to E boxes and E-box binding factors are believed to be important in the regulation of cell differentiation and proliferation. Okadaic acid significantly increased hARNT-mediated class B, E-box-driven reporter activity in COS-1 cells, transiently expressing hARNT without affecting hARNT protein levels. This alteration in hARNT-mediated class B, E-box-driven reporter activity correlates with an observed increase in [32P]orthophosphate incorporation into hARNT. Treatment with okadaic acid resulted in a 12-fold increase in [32P]orthophosphate incorporation into hARNT that was transiently expressed in COS-1 cells; an increase in the number of tryptic phosphopeptides generated from hARNT digests on two-dimensional phosphopeptide maps was also observed. Despite the significant increase in [32P]orthophosphate incorporation into hARNT, serine remained the predominantly phosphorylated residue. Clearly, increased serine phosphorylation does not appear to negatively regulate hARNT homodimerization or transactivation potential. These results demonstrate that increased hARNT homodimer signaling in COS-1 cells may result from a direct change in hARNT phosphorylation status.