E-box Motifs Research Articles

Formation and properties of hairpin and tetraplex structures of guanine-rich regulatory sequences of muscle-specific genes

Clustered guanine residues in DNA readily generate hairpin or a variety of tetrahelical structures. The myogenic determination protein MyoD was reported to bind to a tetrahelical structure of guanine-rich enhancer sequence of muscle creatine kinase (MCK) more tightly than to its target E-box motif [K. Walsh and A. Gualberto (1992) J. Biol. Chem., 267, 13714–13718], suggesting that tetraplex structures of regulatory sequences of muscle-specific genes could contribute to transcriptional regulation. In the current study we show that promoter or enhancer sequences of various muscle-specific genes display a disproportionately high incidence of guanine clusters. The sequences derived from the guanine-rich promoter or enhancer regions of three muscle-specific genes, human sarcomeric mitochondrial creatine kinase (sMtCK), mouse MCK and α7 integrin formed diverse secondary structures. The sMtCK sequence folded into a hairpin structure; the α7 integrin oligonucleotide generated a unimolecular tetraplex; and sequences from all three genes associated to generate bimolecular tetraplexes. Furthermore, two neighboring non-contiguous guanine-rich tracts in the α7 integrin promoter region also paired to form a tetraplex structure. We also show that homodimeric MyoD bound bimolecular tetraplex structures of muscle-specific regulatory sequences more efficiently than its target E-box motif. These results are consistent with a role of tetrahelical structures of DNA in the regulation of muscle-specific gene expression.

Transcriptional regulation of mouse 6-phosphogluconate dehydrogenase by ADD1/SREBP1c

6-Phosphogluconate dehydrogenase (6PGDH) constitutes the pentose phosphate pathway and produces NADPH. 6PGDH is also considered as a lipogenic gene since NADPH is a pivotal cofactor for lipogenesis. Thus, it is important to elucidate how 6PGDH is regulated by various signals related to energy homeostasis. Here, we provide several evidences that ADD1/SREBP1c regulates the expression of mouse 6PGDH gene. DNase I footprinting assay and point mutation studies revealed that the E-box (CANNTG) motif in the promoter of mouse 6PGDH is an important cis-regulatory element for ADD1/SREBP1c. 6PGDH mRNA is highly expressed in white adipose tissue and tightly modulated by nutritional status. Furthermore, we found that ADD1/SREBP1c mediates insulin-dependent 6PGDH expression and that PI3-kinase is an important linker for its regulation. Taken together, these data suggest that ADD1/SREBP1c is a key transcription factor for 6PGDH gene expression and would coordinate glucose metabolism and lipogenesis for energy homeostasis.

Roles for USF‐2 in lung cancer proliferation and bronchial carcinogenesis

The upstream stimulatory factors USF-1 and USF-2 dimerize to regulate transcription through E-box motifs in target genes. Although widely expressed, they can mediate tissue-specific transcription and we previously reported that USF-2 can enhance transcription of arginine vasopressin, a neuropeptide growth factor in small cell lung cancer. Here we determine the expression and role of USF-2 in lung cancer subtypes and examine USF-2 distribution in the bronchial epithelium. For a panel of 12 cell lines and 10 frozen human tumour samples, immunoblotting demonstrated that USF-2 expression was more frequent and abundant in small cell lung cancer than in non-small cell lung cancer. An immunohistochemical study of 108 formalin-fixed and paraffin-embedded human samples was undertaken to localize USF-2 expression and included 44 small cell and 32 non-small cell lung cancers, and 32 samples with bronchial dysplasia. USF-2 was restricted to ciliated cells in normal bronchial epithelium, but was more strongly expressed in dysplastic epithelium (72%) and certain lung cancer types, including small cell lung cancer (71%), squamous cell carcinoma (69%) and a large cell neuroendocrine carcinoma, but was less common in adenocarcinoma (11%). In a small series, expression was assessed adjacent to positively staining tumours; in phenotypically normal bronchial tissues, USF-2 was more highly expressed at 1 cm than at 5 cm from the tumour. Transient USF-2 overexpression in non-small cell lung cancer cell lines significantly stimulated in vitro cell proliferation; this response was most apparent for NCI-H460 (p < 0.005), reducing the mean cell doubling time from 19 to 16 h. Dominant-negative USF-2 mutants had no significant effect on cell growth. Taken together, these data suggest that USF-2 represents a relatively early molecular marker for the development of bronchial dysplasia and non-adenocarcinoma lung cancer. USF may also play a role in bronchial carcinogenesis, perhaps through promoting cell proliferation, although the genes through which this is regulated remain to be determined.

Evolution of transcriptional enhancers in the immunoglobulin heavy-chain gene: functional characteristics of the zebrafish E?3? enhancer

Past studies of the channel catfish immunoglobulin heavy-chain (IgH) locus indicates that it lacks an Emu enhancer in the J(H)-Cmu1 intron but does have an enhancer, termed Emu3', in the mu-delta intergenic region. The positioning of the catfish enhancer downstream of the mu-chain exons is predicted to be unfavorable for antibody-affinity maturation in catfish, and would also have been an impediment to the evolution of class switch recombination, had it existed in early tetrapods. To determine if this downstream enhancer is a general feature of teleost fish, we have identified the location of the transcriptional enhancer in the zebrafish IgH locus. We find that zebrafish, like catfish, only have an Emu3'-like enhancer that has cross-species activity, but which is B-cell-specific in its activity. A 300-bp region of the zebrafish enhancer shares sequence homology with the core of the catfish Emu3', although there has been loss and gain of specific octamer enhancer motifs. Mutagenesis studies demonstrate that the zebrafish IgH enhancer depends on a pair of E-box motifs that are found in the enhancer core. Similarly spaced E-box motifs appear to exist in the Emu3' enhancer regions of other teleost fish, suggesting this is a common feature among fish IgH enhancers. We discuss how this distal positioning of the enhancer may influence affinity maturation in extant teleosts as well as the evolution of this process in the early tetrapods.

E-cadherin intron 2 contains cis-regulatory elements essential for gene expression

Cadherin-mediated cell-cell adhesion plays important roles in mouse embryonic development, and changes in cadherin expression are often linked to morphogenetic events. For proper embryonic development and organ formation, the expression of E-cadherin must be tightly regulated. Dysregulated expression during tumorigenesis confers invasiveness and metastasis. Except for the E-box motifs in the E-cadherin promoter, little is known about the existence and location of cis-regulatory elements controlling E-cadherin gene expression. We have examined putative cis-regulatory elements in the E-cadherin gene and we show a pivotal role for intron 2 in activating transcription. Upon deleting the genomic intron 2 entirely, the E-cadherin locus becomes completely inactive in embryonic stem cells and during early embryonic development. Later in development, from E11.5 onwards, the locus is activated only weakly in the absence of intron 2 sequences. We demonstrate that in differentiated epithelia, intron 2 sequences are required both to initiate transcriptional activation and additionally to maintain E-cadherin expression. Detailed analysis also revealed that expression in the yolk sac is intron 2 independent, whereas expression in the lens and the salivary glands absolutely relies on cis-regulatory sequences of intron 2. Taken together, our findings reveal a complex mechanism of gene regulation, with a vital role for the large intron 2.

E47, IRF-4, and PU.1 synergize to induce B-cell-specific activation of the class II transactivator promoter III (CIITA-PIII)

AbstractIn B cells, expression of CIITA and resulting major histocompatibility complex II (MHCII) is mediated exclusively by promoter III (CIITA-PIII) activation. Recent studies have established that CIITA-PIII also participates in the expression of CIITA in activated human T cells, dendritic cells, and monocytes. In this study we characterized the various regulatory elements and interacting factors of CIITA-PIII that account for specific activation in B lymphocytes. We identified 2 E-box motifs and an Ets/ISRE-consensus element (EICE) in CIITA-PIII as playing a crucial role in the B-cell-specific transcriptional regulation of CIITA. Abolishment of factor binding to these elements resulted in a strong reduction of CIITA-PIII activation in B cells only, whereas it did scarcely affect or not affect the activity of CIITA-PIII in activated T cells and monocytes. We show that in B cells, E47 and PU.1/IRF-4 interact with the E-box motifs and the EICE, respectively, and act synergistically in the activation of CIITA-PIII. Moreover, functional inhibition of either E47 or IRF-4 resulted in strong reduction of CIITA-PIII activity in B lymphocytes only. The finding that PU.1, IRF-4, and E47 play an important role in the B-cell-mediated activation of CIITA-PIII provides a link between antigen presentation functions and activation and differentiation events in B lymphocytes.

Sterol-responsive Element-binding Protein (SREBP) 2 Down-regulates ATP-binding Cassette Transporter A1 in Vascular Endothelial Cells: A NOVEL ROLE OF SREBP IN REGULATING CHOLESTEROL METABOLISM

ATP-binding cassette transporter A1 (ABCA1) is a pivotal regulator of cholesterol efflux from cells to apolipoproteins, whereas sterol-responsive element-binding protein 2 (SREBP2) is the key protein regulating cholesterol synthesis and uptake. We investigated the regulation of ABCA1 by SREBP2 in vascular endothelial cells (ECs). Our results showed that sterol depletion activated SREBP2 and increased its target, low density lipoprotein receptor mRNA, with a concurrent decrease in the ABCA1 mRNA. Transient transfection analysis revealed that sterol depletion decreased the ABCA1 promoter activity by 50%, but low density lipoprotein receptor promoter- and the sterol-responsive element-driven luciferase activities were increased. Overexpression of the N terminus of SREBP2 (SREBP2(N)), an active form of SREBP2, also inhibited the ABCA1 promoter activity. Functionally adenovirus-mediated SREBP2(N) expression increased cholesterol accumulation and decreased apoA-I-mediated cholesterol efflux. The conserved E-box motif was responsible for the SREBP2(N)-mediated inhibition since mutation of the E-box increased the basal activity of the ABCA1 promoter and abolished the inhibitory effect of SREBP2(N). Furthermore sterol depletion and SREBP2(N) overexpression induced the binding of SREBP2(N) to both consensus and ABCA1-specific E-box. Chromatin immunoprecipitation assay demonstrated that serum starvation enhanced the association of SREBP2 and the ABCA1 promoter in ECs. To correlate this mechanism pathophysiologically, we found that oscillatory flow caused the activation of SREBP2 and therefore attenuated ABCA1 promoter activity in ECs. Thus, this SREBP-regulated mechanism may control the efflux of cholesterol, which is a newly defined function of SREBP2 in ECs in addition to its role in cholesterol uptake and biosynthesis.

Upstream stimulatory factors stimulate transcription through E-box motifs in the PF4 gene in megakaryocytes

AbstractPlatelet factor 4 (PF4) is expressed during megakaryocytic differentiation. We previously demonstrated that the homeodomain proteins (myeloid ecotropic integra tion site 1 [MEIS1], Pbx-regulating protein 1 [PREP1], and pre-B-cell leukemia transcription factors [PBXs]) bind to the novel regulatory element tandem repeat of MEIS1 binding element [TME] and transactivate the rat PF4 promoter. In the present study, we investigated and identified other TME binding proteins in megakaryocytic HEL cells using mass spectrometry. Among identified proteins, we focused on upstream stimulatory factor (USF1) and USF2 and investigated their effects on the PF4 promoter. USF1 and 2 bound to the E-box motif in the TME and strongly transactivated the PF4 promoter. Furthermore, physiologic bindings of USF1 and 2 to the TME in rat megakaryocytes were demonstrated by the chromatin immunoprecipitation (ChIP) assay. Interestingly, the E-box motif in the TME was conserved in TME-like sequences of both the human and mouse PF4 promoters. USF1 and 2 also bound to the human TME-like sequence and transactivated the human PF4 promoter. Expressions of USF1 and 2 were detected by reverse-transcriptase–polymerase chain reaction (RT-PCR) in the human megakaryocytes derived from CD34+ cells. Thus, these studies demonstrate that the novel TME binding transcription factors, USF1 and 2, transactivate rat and human PF4 promoters and may play an important role in megakaryocytic gene expression.

Role of NF-kappaB and p38 MAP kinase signaling pathways in the lipopolysaccharide-dependent activation of heme oxygenase-1 gene expression.

Heme oxygenase (HO)-1 is the inducible isoform of the rate-limiting enzyme of heme degradation, which is up-regulated by a host of stress stimuli. The bacterial cell membrane component lipopolysaccharide (LPS) is a prototypical activator of monocytic cells. Here, it is shown that LPS induced the endogenous HO-1 gene expression in RAW264.7 monocytic cells. To investigate the molecular mechanisms of HO-1 gene induction by LPS, we performed transfection experiments with reporter gene constructs containing sequences of the proximal rat HO-1 gene promoter. Deletion and mutation analysis indicated that a cyclic AMP response element/activator protein-1 site (-664/-657), but not an E-box motif (-47/-42), played a major role for LPS-dependent HO-1 gene induction. Up-regulation of HO-1 promoter activity by LPS was decreased by pharmacological nuclear factor-kappaB (NF-kappaB) inhibitors and by cotransfected expression vectors with dominant negative isoforms of NF-kappaB-inducing kinase, inhibitor of NF-kappaB (IkappaB) kinase beta, and IkappaBalpha. Moreover, the p38 mitogen-activated protein kinase (MAPK) inhibitor SB203580 and overexpressed dominant negative p38beta decreased, whereas dominant negative p38delta increased, LPS-dependent induction of HO-1 gene expression. The results suggest that the NF-kappaB and p38 MAPK signaling pathways mediate the LPS-dependent induction of HO-1 gene expression via DNA sequences of the proximal promoter region.

Genomic structure of the chicken slow skeletal muscle troponin T gene

Troponin T (TnT) is a key protein for Ca 2+-sensitive molecular switching of muscle contraction. In vertebrates, three TnT genes have been identified, which produce isoforms characteristic of cardiac, fast skeletal, and slow skeletal muscles through alternative splicing in a tissue-specific and developmentally regulated manner. The diversification of myofibers into forms with specific metabolic and contractile characteristics is thought to be closely associated with the differential expression of these TnT isoforms. Herein, we determined the nucleotide sequence of the chicken slow skeletal muscle TnT gene and its upstream region. The gene was simpler in structure than the two other chicken genes. The transcription initiation site was positioned 183 bp upstream of the 3′ end of exon 1. Alternative splicing of exon 5 using an internal acceptor site generated two distinct slow skeletal muscle troponin T (sTnT) transcripts. We identified possible regulatory elements, M-CAT-like, CACC-box, and E-box (E-box1 to E-box3) motifs in the upstream region and an E-box motif (E-box4) in exon 1.

Mechanisms of cholesterol and sterol regulatory element binding protein regulation of the sterol 12α-hydroxylase gene (CYP8B1)

Sterol 12α-hydroxylase (CYP8B1) is an obligatory enzyme for the synthesis of cholic acid and regulation of liver bile acid synthesis and intestine cholesterol absorption. The present study evaluates the roles for sterol regulatory element binding proteins (SREBPs) in the regulation of the CYP8B1 gene. Cholesterol feeding of mice and rats decreased the activity of CYP8B1, contrary to the up-regulation of cholesterol 7α-hydroxylase (CYP7A1). Cholesterol feeding also reduced mRNA levels for SREBP-1 but not for SREBP-2 in rat livers. Cholesterol and 25-hydroxycholesterol decreased the CYP8B1/luciferase reporter activity. Co-transfection of SREBP-1a and -1c stimulated CYP8B1 promoter activity, while SREBP-2 did not have any effects. Electrophoretic mobility shift assay and mutagenesis analyses identified several functional sterol regulatory elements (SRE) and E-box motifs in the rat CYP8B1 promoter. Our results indicate that SREBP-1a and -1c enhance transcription of the CYP8B1 gene through binding to SRE. Cholesterol loading reduces SREBP-1 mRNA expression in addition to reducing functional SREBP-1 protein, and results in decreasing CYP8B1 gene transcription.

The evidences of human Orphan Receptor COUP-TFII inhibiting telomerase activity through decreasing hTERT transcription

Activation of hTERT, the human telomerase catalytic subunit, has been implicated as the critical event in triggering telomerase activity of cancer cells, but the details of regulation of hTERT need to be elucidated. By screening HeLa cDNA library with hTERT promoter-based yeast one-hybrid assay, one of positive clones, which potentially interacted with hTERT promoter, contained the full sequences of COUP-TFII cDNA. EMSA showed that the prepared His-tagged COUP-TFII could firmly bind to −201 to +35 fragment of hTERT promoter. The precise binding sites were confirmed by DNase I footprint analysis and proved to involve the E-box motif of hTERT promoter. Luciferase reporter assays indicated that COUP-TFII could suppress the transcription of hTERT promoter, and the inhibition to some extent could be reversed by co-transfection of c-myc. Stable introduction of COUP-TFII into HeLa cells decreased both endogenous hTERT expression and telomerase activity. The results suggested that the human COUP-TFII could specifically interact with hTERT promoter, in part via the E-box, and suppress the expression of hTERT.

2S storage protein gene of Douglas-fir: characterization and activity of promoter in transgenic tobacco seeds

To date a few sequences regulating expression of conifer seed-specific genes have been reported. To characterize Douglas-fir ( Pseudotsuga menziesii [Mirb] Franco) 2S albumin storage protein genes, a genomic DNA sequence containing upstream promoter sequences was isolated by screening a Douglas-fir genomic library. Sequence analysis of the Douglas-fir gPm2S1 promoter revealed the presence of RY-repeated elements (GCATGC), and multiple E-box motifs (CANNTG) and ACGT-core elements, features characteristic of 2S storage protein genes in angiosperms. When fused to the GUS reporter gene, the 1.16 kb Douglas-fir 2S promoter sequence was sufficient to direct transient expression in both developing Douglas-fir embryos and maternally derived haploid megagametophytes. Analysis of this promoter construct in transgenic tobacco showed that expression was restricted to embryo and endosperm in developing seeds and was not detected in vegetative tissues of two-week-old seedlings. These results strongly suggest that both structural and regulatory elements as well as upstream signaling components controlling the expression of 2S albumin genes are highly conserved during evolution.

980. Evaluation of the Tumor Specificity of a Conditionally Replicative Adenovirus Controlled by a Modified Human Core Telomerase Promoter

Background: Conditionally replicative adenoviruses (CRADs) are innovative anticancer agents with potential advantages over conventional adenoviral vectors due to their oncolytic effect and the ability to expand into the tumors. A strategy to achieve tumor specificity is to use tumor-selective promoters to control the expression of early viral genes essential for replication, such as E1A. The telomerase reverse transcriptase (hTERT) core promoter has been recently used to construct new CRADs because it is silenced in normal cells and active in 85% of malignancies such as liver, lung, gastric, breast, bladder, and prostate cancer. Further studies showed that the addition of 3 E-box motifs enhances the specificity of this sequence (255-4DEB promoter).

RNA interference of achaete-scute homolog 1 in mouse prostate neuroendocrine cells reveals its gene targets and DNA binding sites.

We have previously characterized a transgenic mouse model (CR2-TAg) of metastatic prostate cancer arising in the neuroendocrine (NE) cell lineage. Biomarkers of NE differentiation in this model are expressed in conventional adenocarcinoma of the prostate with NE features. To further characterize the pathways that control NE proliferation, differentiation, and survival, we established prostate NE cancer (PNEC) cell lines from CR2-TAg prostate tumors and metastases. GeneChip analyses of cell lines harvested at different passages, and as xenografted tumors, indicated that PNECs express consistent features ex vivo and in vivo and share a remarkable degree of similarity with primary CR2-TAg prostate NE tumors. PNECs express mAsh1, a basic helix-loop-helix (bHLH) transcription factor essential for NE cell differentiation in other tissues. RNA interference knockdown of mAsh1, GeneChip comparisons of treated and control cell populations, and a computational analysis of down-regulated genes identified 12 transcriptional motifs enriched in the gene set. Affected genes, including Adcy9, Hes6, Iapp1, Ndrg4, c-Myb, and Mesdc2, are enriched for a palindromic E-box motif, CAGCTG, indicating that it is a physiologically relevant mAsh1 binding site. The enrichment of a c-Myb binding site and the finding that c-Myb is down-regulated by mAsh1 RNA interference suggest that mAsh1 and c-Myb are in the same signaling pathway. Our data indicate that mAsh1 negatively regulates the cell cycle (e.g., via enhanced Cdkn2d, Bub1 expression), promotes differentiation (e.g., through effects on cAMP), and enhances survival by inhibiting apoptosis. PNEC cell lines should be generally useful for genetic and/or pharmacologic studies of the regulation of NE cell proliferation, differentiation, and tumorigenesis.

The Id2 Transcriptional Repressor Is Induced by Follicle-stimulating Hormone and cAMP

Id (inhibitor of DNA binding/differentiation) proteins repress differentiation and promote cell division by dimerizing with and inhibiting the action of basic helix-loop-helix transcription factors including those that bind to E-box motifs. Of the four characterized Id proteins, only Id2 is found in the nucleus of Sertoli cells that support the development of spermatozoa in the testis. Differential display analysis of rat primary Sertoli cell mRNA identified Id2 as being inducible by forskolin, a stimulator of cAMP production. Northern blot analysis confirmed that Id2 mRNA expression peaked in Sertoli cells 6-12 h after stimulation with forskolin or follicle-stimulating hormone (FSH), the major physiological stimulator of cAMP in Sertoli cells. Similarly, Id2 promoter activity in Sertoli cells was induced after forskolin or FSH stimulation as well as by overexpression of protein kinase A. Forskolin induction of the Id2 promoter required sequences located between positions -122 and -82. Protein(s) of 40-45 kDa were found to bind two activated transcription factor/cAMP-response element-like sites and a GATA motif within the regulatory region. The induction of the Id2 gene by FSH corresponded with a decrease in protein binding to an E-box consensus motif and decreased E-box-mediated transcription. Together, these findings raise the possibility that FSH-mediated induction of Id2 and resultant inhibition of basic helix-loop-helix transcription factor-regulated genes in Sertoli cells may contribute to the regulation of spermatogenesis.

Physical and Functional Interactions between USF and Sp1 Proteins Regulate Human Deoxycytidine Kinase Promoter Activity

Deoxycytidine kinase (EC 2.7.1.74, dCK) is central to drug activity of anticancer and antiviral agents such as cytosine arabinoside (araC) and gemcitabine. HepG2 hepatocellular carcinoma cells were used to study the transcriptional regulation of dCK. 5'-Deletion and site-directed mutagenesis of the dCK upstream region (positions -464 to -27) confirmed the importance of two GC-boxes (positions -317 to -309 and -213 to -206) and two E-boxes (positions -302 to -297 and -278 to -273). In vitro electromobility shift assays with HepG2 nuclear extracts and in vivo chromatin immunoprecipitation assays with HepG2 chromatin extracts confirmed the presence of bound Sp1/Sp3 and USF1/2. Co-transfections in HepG2 cells showed that USF1 and USF2a stimulated and Sp1 repressed promoter activity from a dCK-luciferase reporter gene construct. In Sp- and USF-null Drosophila Mel-2 cells, both Sp1 and USF1 stimulated dCK promoter activity in a dose-dependent manner, however, both Sp3 and USF2a were effectively inert. Combined Sp1 and USF1 showed additive transactivation at lower concentrations of Sp1. Sp1 was inhibitory at higher levels. Stimulation by combined USF1/USF2a with Sp1 was similar to that for USF1 alone with Sp1, whereas transactivation by Sp1 and USF2a without USF1 was synergistic. Physical interactions between USF and Sp proteins were confirmed by immunoprecipitations with Sp- and USF-specific antibodies. Domain mapping of USF1 and USF2a localized the functional interactions between USF and Sp proteins to the DNA binding domain of USF. Identifying the physical and functional interactions between Sp and USF proteins may lead to a better understanding of the basis for differential expression of the dCK gene in tumor cells and may foster strategies for up-regulating dCK gene expression and improving chemotherapy with araC and gemcitabine.

Upstream stimulatory factor but not c-Myc enhances transcription of the human polymeric immunoglobulin receptor gene

Secretory antibodies protect mucosal surfaces from ingested, inhaled and sexually transmitted pathogens. The polymeric immunoglobulin receptor (pIgR) transports antibodies across mucosal epithelia into external secretions. We and others have identified a region of the human polymeric immunoglobulin receptor gene (locus PIGR) that is sufficient for basal transcriptional activity. An E-Box motif, which binds transcription factors of the basic helix-loop-helix/leucine zipper (bHLH/zip) family, was identified as a major regulatory element in the PIGR gene promoter. Transient transfection of PIGR promoter reporter plasmids in intestinal epithelial cell (IEC) lines suggested that the transcription factors upstream stimulatory factor (USF) and c-Myc may exert opposing effects on PIGR promoter activity. Mutations within and flanking the E-Box that favored USF binding enhanced promoter activity, while mutations that favored c-Myc binding reduced promoter activity. Ectopic expression of USF1 or USF2 enhanced PIGR promoter activity, while exogenous c-Myc did not. Electrophoretic mobility shift assays (EMSA) demonstrated that USF1 and USF2 bound to the E-Box motif as homo- and heterodimers. Chromatin immunoprecipitation (ChIP) demonstrated that USF proteins bind the PIGR promoter in vivo, which is enriched in acetylated histones. E-Box motifs are commonly observed in promoters of genes that are highly expressed in the human colon. Genes that are down-regulated in colorectal cancer, including PIGR, frequently have non-canonical E-Boxes, while genes that are up-regulated in colorectal cancer generally have canonical E-Boxes. The results of our experiments may shed light on the mechanisms of dysregulated expression of pIgR in inflammatory bowel disease and colorectal cancer, diseases associated with aberrant expression of c-Myc.

Transcription factor USF, expressed during the entire phase of varicella-zoster virus infection, interacts physically with the major viral transactivator IE62 and plays a significant role in virus replication.

The expression of the genes of varicella-zoster virus (VZV) is regulated by self-encoded viral as well as cellular transcription factors. A potential candidate with an ability to influence the transcription of VZV genes is USF (upstream stimulatory factor), which recognizes the consensus E-box motif. Quantitative RT-PCR and immunoblot assays indicate stable expression of both USF1 and USF2 throughout infection. It was also found that USF binds to a variety of E-boxes (consensus and closely related motifs) within the promoters of ORF 8/9 (two elements), ORF 22 and ORF 67. Co-immunoprecipitation experiments and His-tag protein affinity pull-down assays indicate that a direct physical interaction occurs between USF and the major virus transactivator IE62. To study the general effects of USF in the replication of VZV, a cell line expressing a dominant-negative form of USF (A-USF), which inhibits binding of USF to its recognition sites, was created. A significant decrease in virus replication was detected when this cell line was infected with cell-free virus, indicating that USF is an important cellular factor that regulates the transcription of VZV genes.

MYCN Enhances P-gp/ MDR1 Gene Expression in the Human Metastatic Neuroblastoma IGR-N-91 Model

Despite intensive high-dose chemotherapy and autologous hematopoietic stem cell transplantation, disseminated neuroblastoma (NB) frequently proves to be chemosensitive but not chemocurable, and more often so in NB-presenting MYCN amplification. To assess the direct relationship between the MYCN oncogene and chemoresistance acquisition during NB metastatic dissemination, we have studied MYCN and MDR1 genes using the human IGR-N-91 ectopic xenograft metastatic model. This characterized experimental in vitro model includes human neuroblasts derived from a subcutaneous primary tumor xenograft, disseminated blood cells, myocardium, and bone marrow (BM) metastatic cells. All IGR-N-91-derived neuroblasts harbor a consistent MYCN genomic content but, unlike primary tumor xenograft, BM, and myocardium, human neuroblasts elicit a concomitant increase in MYCN and MDR1 transcripts levels, consistent with chemoresistance phenotype and active P-gp. In contrast, no variation of MRP1 transcript level was associated with the metastatic process in this model. Using an MDR1 promoter-CAT construct, we have shown that the MycN protein activates MDR1 transcription both in exogenous transient MYCN-transfected SK-N-SH cells and in endogenous BM metastatic neuroblasts with an increase in the MYCN transcript level. Band-shift experiments indicate that IGR-N-91 cells enriched with the MycN transcription factor do bind to two E-box motifs localized within the MDR1 promoter. Overall, our data indicate that MYCN overexpression increment contributes to the acquired drug resistance that occurs throughout the NB metastatic process.