SV40 Promoter Research Articles

Compactin and Simvastatin, but Not Pravastatin, Induce Bone Morphogenetic Protein-2 in Human Osteosarcoma Cells

Bone morphogenetic protein (BMP)-2, a member of the BMP family, plays an important role in osteoblast differentiation and bone formation. To discover small molecules that induce BMP-2, a luciferase reporter vector containing the 5′-flanking promoter region of the human BMP-2 gene was constructed and transfected into human osteosarcoma (HOS) cells. By the screening of an in-house natural product library with stably transfected HOS cells, a fungal metabolite, compactin, known as an inhibitor of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, was isolated. The stimulation of the promoter activity by compactin seemed to be specific for BMP-2 gene in HOS cells, since it had little effect on BMP-4 or SV40 promoter activity and the stimulation was not observed in Chinese hamster ovary (CHO) cells. RT-PCR analysis and alkaline phosphatase assay revealed that compactin induced an increase in the expression of BMP-2 mRNA and protein. Like compactin, simvastatin also activated the BMP-2 promoter, whereas pravastatin did not. The statin-mediated activation of BMP-2 promoter was completely inhibited by the downstream metabolite of HMG-CoA reductase, mevalonate, indicating that the activation was a result of the inhibition of the enzyme. These results suggest that statins, if they are selectively targeted to bone, have beneficial effects in the treatment of osteoporosis or bone fracture.

Identification of a cell type-specific silencer in the first exon of the His-1 gene

The His-1 gene is developmentally expressed in the murine choroid plexus but is silenced in the adult brain. To test the hypothesis that the gene contains cis-acting elements that contribute to this repression, we have analyzed segments of the proximal promoter for negative regulatory sequences by transient transfection analysis. The activity of the proximal promoter was moderately influenced by positively and negatively acting sequences located from −335 to −168 and −617 to −335, respectively. A strong His-1-positive regulatory element (HPRE, +18 to +29) was essential for maximal promoter activity and could also enhance the activity of the heterologous SV40 promoter in an orientation-dependent manner. The HPRE contains homology to the neuronal restrictive silencer element (NRSE) but interacted with nuclear proteins that were distinct from the NRSE-binding factor (NRSF). By contrast, a potent negative regulatory sequence (HNRE) was identified in the first exon that repressed either the His-1 or SV40 promoters by greater than 80%. This negative regulatory sequence interacted with nuclear proteins from cells that contain a silent His-1 gene but showed no interaction with nuclear proteins from cells that actively transcribe the endogenous gene. HNRE-mediated repression was orientation independent; most of this activity was mapped to a minimal 26-bp sequence. These findings suggest that the first exon of the His-1 gene contains a cell type-specific silencer that contributes to the regulation of His-1 transcription. J. Cell. Biochem. 76:615–624, 2000. © 2000 Wiley-Liss, Inc.

Erythroid Kruppel-like factor is recruited to the CACCC box in the beta-globin promoter but not to the CACCC box in the gamma-globin promoter: the role of the neighboring promoter elements.

The programmed expression of the five beta-like globin genes (epsilon, (A)gamma, (G)gamma, delta, and beta) is characterized by a series of switches that are developmentally regulated. The (A)gamma- and (G)gamma- (fetus) to beta-globin (adult) switch depends on transcription factor erythroid Krüppel-like factor (EKLF), which, like Sp1, binds to CACCC boxes. EKLF is essential for the expression of the beta-globin but not the gamma-globin gene. Because both gamma-globin and beta-globin promoters contain the CACCC box, and their promoter elements are similar, it is not known why the two promoters behave so differently. In this report, we searched for the functional differences between the two promoters by studying their ability to recruit EKLF. We used the in vivo PIN*POINT assay to show that EKLF is recruited to the beta-globin promoter but not to the gamma-globin promoter. We show that this selectivity is a result of differences in surrounding promoter elements and not CACCC box alone. One of the differences between the two promoters with a functional consequence is the CCTTG repeat that is present in the gamma-globin promoter but not in the beta-globin promoter. The repeat, when inserted in the beta-globin promoter, decreases EKLF recruitment to and activity of the beta-globin promoter, suggesting that the repeat functions as a suppressor element. The CCTTG repeat can also suppress the SV40 promoter in cis, and the suppressor factor binding to the repeat can be squelched with a plasmid containing a high copy number of the repeat. These findings may have implications in designing drug targets for treatment of beta-globin disorders.

Fetal Alz-50 clone 1 (FAC1) protein interacts with the Myc-associated zinc finger protein (ZF87/MAZ) and alters its transcriptional activity.

Transcription factors mediate their regulatory effects through interaction with DNA and numerous nuclear proteins. The fetal Alz-50 clone 1 (FAC1) protein, a novel DNA-binding protein with the capacity to repress transcription, is likely to function through a similar mechanism (1). Using the two-hybrid yeast screen, we have shown that FAC1 interacts with the myc-associated zinc finger protein (ZF87/MAZ). This association was confirmed in vitro with recombinant protein. The ZF87/MAZ interaction domain was mapped to the region containing a putative nuclear localization signal (NLS) and nuclear export sequence (NES) of FAC1, using deletion mutants of the FAC1 protein. FAC1, on the other hand, recognizes a conformational interface that includes the proline/alanine-rich domain of ZF87/MAZ and the first zinc finger. Cotransfection of NIH3T3 cells with ZF87/MAZ and a luciferase reporter containing the SV40 promoter and enhancer results in an increase in transcriptional activation, suggesting ZF87/MAZ is able to recognize its consensus binding site present in the SV40 promoter. Cotransfection with FAC1 reduces the level of ZF87/MAZ-induced activation of the SV40 promoter in a dose dependent manner. A mutant FAC1, lacking the ZF87/MAZ interaction domain, does not alter ZF87/MAZ activation of the SV40 promoter. These data demonstrate that interaction between FAC1 and ZF87/MAZ alters the transactivation capacity of ZF87/MAZ. By immunoblot analysis, FAC1 and ZF87/MAZ exhibit similar tissue distribution and co-localize to pathologic structures in Alzheimer's disease brain. Coexpression of FAC1 and ZF87/MAZ suggest that interaction of these two proteins will have biological implications for gene regulation in neurodegeneration.

Herpesvirus saimiri-based gene delivery vectors maintain heterologous expression throughout mouse embryonic stem cell differentiation in vitro.

In order to achieve a high efficiency of gene delivery into rare cell types like stem cells the use of viral vectors is presently without alternative. An ideal stem cell gene therapy vector would be able to infect primitive progenitor cells and sustain or activate gene expression in differentiated progeny. However, many viral vectors are inactivated when introduced in developing systems where cell differentiation occurs. To this end, we have developed a mouse in vitro model for testing herpesvirus saimiri (HVS)-based gene therapy vectors. We demonstrate here for the first time that HVS is able to infect totipotent mouse embryonic stem (ES) cells with high efficiency. We have transduced ES cells with a recombinant virus carrying the enhanced green fluorescent protein (EGFP) gene and the neomycin resistance gene (NeoR) driven by a CMV promoter and the SV40 promoter, respectively. ES cells maintain the viral episomal genome and can be terminally differentiated into mature haematopoietic cells. Moreover, heterologous gene expression is maintained throughout in vitro differentiation. Besides its obvious use in gene therapy, this unique expression system has wide ranging applications in studies aimed at understanding gene function and expression in cell differentiation and development.

The HTLV Receptor Is a Widely Expressed Protein

The receptor for human T-cell leukemia virus type 1 (HTLV-1) was found to be expressed on a broad range of cell lines derived from multiple species. Receptor expression was assessed using human immunodeficiency virus type 1 particles, pseudotyped with the HTLV-1 envelope glycoprotein, and expressing luciferase under the control of an SV40 enhancer and promoter. Infection by pseudotyped virus was blocked with neutralizing antibodies to HTLV-1, and infection was dependent on the presence of the cleavage and fusogenic sequences in the envelope protein precursor. Trypsin treatment of susceptible target lymphocytes reduced entry. Entry was partially resistant to ammonium chloride.

A novel EBNA-1 tag system for high level expression and efficient detection of fusion proteins in vitro and in vivo

A highly specific monoclonal antibody, which recognises an epitope between amino acids (aa) 408 and 446 of EBNA-1, was found to immunoprecipitate this protein with great efficiency. By adding a 39-aa tag, the BGLF4 protein product of EBV was shown to express in the cytoplasm of transfected cells. This EBNA-1 tag could be used for the detection of specific protein expression by immunoblotting, immunofluorescence and, especially, immunoprecipitation assays. A plasmid vector encoding this EBNA-1 tag sequence was therefore designed for efficient expression both in vitro and in vivo. The efficient translational start signal of black beetle virus (BBV) was placed under the control of the SV40 or T7 promoters, and the β-globin splicing signal used to enhance the transportation of mRNA from the nucleus to the cytoplasm.

Expression of human Wiskott-Aldrich syndrome protein in patients' cells leads to partial correction of a phenotypic abnormality of cell surface glycoproteins.

The Wiskott-Aldrich syndrome (WAS) is an uncommon X-linked recessive disease characterized by thrombocytopenia, eczema and immunodeficiency. The biochemical defect of this disorder primarily affects cells derived from bone marrow. To understand better the molecular mechanisms underlying this disease and to evaluate the possibility of correcting the genetic defects in hematopoietic cells, a Moloney murine leukemia virus (MoMLV)- based retroviral vector carrying a functional Wiskott-Aldrich syndrome protein (WASp) cDNA driven by an SV40 promoter (LNS-WASp) was constructed. A packaging cell line containing this vector produced a stable level of WAS protein and maintained a high titer of viral output. Epstein-Barr virus (EBV)-transformed B lymphoblastoid cell lines (B-LCL) from WAS patients, which lack expression of the WAS protein, were transduced by the LNS-WASp retroviral vector and showed expression of WASp by Western blot. Analysis of the O-glycan pattern on cell surface glycoproteins from WAS patients' B-LCL showed an altered glycosylation pattern, due to increased activity of beta-1, 6-N-acetylglucosaminyltransferase (C2GnT). Transduction by the retroviral vector carrying the functional WASp cDNA partially restored the abnormal glycosylation pattern, and was accompanied by a decreasing C2GnT activity. These findings imply a functional linkage between the WAS protein and the expression of the glycosyltransferase involved in the O-glycosylation, and also suggest a potential gene therapy via transferring a functional WASp cDNA into hematopoietic cells for Wiskott-Aldrich syndrome. Gene Therapy (2000) 7, 314-320.

Identification of a cell type-specific silencer in the first exon of theHis-1 gene

The His-1 gene is developmentally expressed in the murine choroid plexus but is silenced in the adult brain. To test the hypothesis that the gene contains cis-acting elements that contribute to this repression, we have analyzed segments of the proximal promoter for negative regulatory sequences by transient transfection analysis. The activity of the proximal promoter was moderately influenced by positively and negatively acting sequences located from -335 to -168 and -617 to -335, respectively. A strong His-1-positive regulatory element (HPRE, +18 to +29) was essential for maximal promoter activity and could also enhance the activity of the heterologous SV40 promoter in an orientation-dependent manner. The HPRE contains homology to the neuronal restrictive silencer element (NRSE) but interacted with nuclear proteins that were distinct from the NRSE-binding factor (NRSF). By contrast, a potent negative regulatory sequence (HNRE) was identified in the first exon that repressed either the His-1 or SV40 promoters by greater than 80%. This negative regulatory sequence interacted with nuclear proteins from cells that contain a silent His-1 gene but showed no interaction with nuclear proteins from cells that actively transcribe the endogenous gene. HNRE-mediated repression was orientation independent; most of this activity was mapped to a minimal 26-bp sequence. These findings suggest that the first exon of the His-1 gene contains a cell type-specific silencer that contributes to the regulation of His-1 transcription.

Active intracellular domain of Notch enhances transcriptional activation of CCAAT/enhancer binding protein beta on a rat pregnancy-specific glycoprotein gene.

Pregnancy-specific glycoproteins (PSGs) are primarily expressed in the placenta and become the major glycoproteins at term. To understand the regulation of PSG expression, we characterized the promoter elements of a rodent PSG gene, rnCGM3, and delineated three nuclear protein binding sites: FPI, -II, and -III in the 5'-flanking region of the gene. The FPII-binding factor is shown to be C/EBPbeta, which positively regulates rnCGM3 expression [Chen, H., et al. (1995) DNA Cell Biol. 14, 681-688]. In the current study, we used the yeast one-hybrid system to isolate transcription factors binding to the FPIII site and demonstrated that a rodent J kappa recombination signal sequence binding protein, rRBPJ kappa-2N, bound to the FPIII site. Electrophoretic mobility shift assay with rat placental nuclear proteins revealed a constitutive occupancy of the FPIII site by RBPJ kappa. By transient expression analyses, we demonstrated that rRBPJ kappa-2N repressed the expression from an FPIII-driven SV40 promoter. However, this repression effect was counteracted by the active intracellular domain of Notch (NotchIC). Using the native rnCGM3 promoter construct, we demonstrated that the promoter activity stimulated by C/EBP beta was also repressed by rRBPJ kappa-2N but enhanced by NotchIC. Additionally, we found that NotchIC can stimulate expression through another RBPJkappa site within the FPI site. A functional interaction between factors binding to the FPI, FPII, and FPIII sites is proposed.

Characterization of the c-specific promoter of the gene encoding human endothelin-converting enzyme-1 (ECE-1)

Human ECE-1 is expressed in four isoforms with different tissue distribution and its mRNA and protein levels are altered under certain pathophysiological conditions. To investigate the transcriptional regulation of ECE-1, we studied the regulatory region of ECE-1c, the major ECE-1 isoform. A genomic clone comprising the complete human ECE-1 gene including the putative ECE-1c-specific promoter was obtained. Up to 968 bp upstream of the putative c-specific translation initiation start codon and several serial deletion mutants were subcloned into a reporter vector and transfected into endothelial (BAEC, EA.hy926, ECV304) and epithelial (MDA MB435S, MCF7) cells, showing very strong promoter activity in comparison to the SV40 promoter and to the previously described ECE-1a and 1b promoters. Transfection of serial deletion mutants indicated two positive regulatory regions within the promoter (−142/−240 and −240/490) likely involved in binding GATA and ETS transcription factors. RNase protection assay (RPA) and 5′-RACE revealed multiple transcriptional start sites located at about −110, −140 and −350 bp. Site-directed mutagenesis demonstrated a crucial role for the E2F cis-element for basal ECE-1c promoter activity. Additionally, we found a correlation between isoform-specific ECE-1 mRNA levels and corresponding ECE-1a, 1b, 1c promoter activities.

Engineering CHO cells to overexpress a secreted reporter protein upon induction from mouse mammary tumor virus promoter

The mouse mammary tumor virus (MMTV) promoter is induced by the addition of a glucocorticoid hormone or analog such as dexamethasone. The hormone binds to its specific transcription factor, glucocorticoid receptor (GR), and the activated complex then binds to the glucocorticoid response elements (GREs) in the enhancer region of the MMTV promoter to induce the overexpression of downstream genes. We have constructed an expression vector for a reporter protein, secreted alkaline phosphatase (SEAP),controlled by the MMTV promoter and co-transfected this vector along with a GR expression cassette into Chinese hamster ovary (CHO) cells. High producers were cloned and grown in suspension cultures. A very high titer, over 0.4 mg/mL, of SEAP was obtained from this inducible overexpression system, about ten times that achievable with the same reporter protein using the strong constitutive SV40 promoter in CHO cells. A peak production rate of 187 pg SEAP per cell per day was observed within 3 days after induction, compared to the peak rate of 23 pg SEAP per cell per day expressed using the constitutive SV40 promoter. With the reduced or zero growth rate during the protein production phase, this novel MMTV overexpression system is highly suited for optimizing glycoprotein synthesis rates in high cell density fed-batch or perfusion bioreactors.

ゲノムDNAの機能的配置と細胞核骨格構造

The nuclear matrix (nuclear scaffold), the RNA-protein skeleton of the nucleus, has a role in the organization and function of nuclear DNA. Nuclear processes associated with the nuclear matrix include transcription, replication, repair and splicing. We have purified a nuclear matrix protein, P130, which binds to several matrix attachment regions (MARs). Since the nucleotide sequence of P130 cDNA cloned by us was closely similar to that of matrin 3 cDNA cloned, except for two incorrect nucleotides within the matrin 3 coding region, and since the functions of matrin 3 were unknown, P130, referred to as P130/Mat3, was functionally characterized. The primary structure deduced for P130/Mat3 contained two DNA binding domains with C2H2-type zinc finger motif and two RNA binding domains. In addition, there were a nuclear localization signal and several phosphorylation sites for tyrosine or serine/threonine protein kinases, suggesting its multiple functions. MAR inserted upstream from the SV40 promoter in pMAR/luc assisted luciferase gene transcription in a transient expression system in Ac2F cells. Cotransfection of a plasmid carrying P130/Mat3 cDNA downstream from the CMV promoter into Ac2F cells produced this protein a level 4 times higher than that in wild-type Ac2F, causing 20 times higher luciferase activity from pMAR/luc than that induced by pMAR/luc alone. These findings indicated that MAR functions as a cis-element to which P130/Mat3 binds as one of the possible transactivators. Nuclear matrix proteins, which are tissue- and cell-type-specific, are altered with transformation and state of differentiation. We have shown that an MAR binding protein, P230, is detectable in rat hepatoma cells but not in normal liver, and suggested that this protein is a diagnostic and prognostic marker for liver cancer. It is clear that nuclear matrix proteins hold a considerable promise as diagnostic tools for pathologists. Present evidence, including our data, suggests that nuclear matrix proteins may be useful biomarkers of neoplastic disease in the serum, body fluids, and tissues. Nuclear matrix proteins are also potential candidates for the use as tumor prognostic factors and targets of anticancer drugs through apoptosis. We will discuss screening of drugs that interact with nuclear matrix proteins and influence nuclear events.

CaMV 35S Promoter Fragmentation Hotspot Confirmed, and it is Active in Animals

We would like to address the suggestion of Matzke et al. (1) and others that pararetroviral promoters such as the CaMV 35S are not exotic to plant genomes as they already contain many integrated pararetroviral sequences. The crucial question is whether the CaMV 35S promoter in transgenic constructs poses special risks. We would like to draw attention to several publications that are relevant to this issue. Kumpatla and Hall (2) analyzed a transgenic rice locus and confirmed that fragmentation and recombination occur frequently within the CaMV 35S promoter, but not in the wheat plant ubiquitin promoter used in another transgenic cassette. This indicates that the CaMV promoter is not like any other promoter. Six out of seven recombination junctions in the CaMV promoter map near the 19 basepair palindrome identified as a recombination hotspot by Kohli et al. (3). The conventional wisdom among plant molecular geneticists is that plant promoters, such as the CaMV 35S, are not active in animals (4). In fact, the CaMV 35S promoter was found to support high levels of reporter gene expression in mature Xenopus oocytes (5), and to give very efficient transcription in extracts of HeLa cell nuclei (6). The CaMV promoter worked at least as well as the SV40 promoter in Xenopus oocytes, and better than the major late promoter of the adenovirus-2 in HeLa cell extracts. So, while the CaMV is specific for plants in the cruciferae family, its isolated promoter is promiscuous across domains and kingdoms of living organisms. It is the genetic (and evolutionary) context that makes all the difference. There is no justification for claiming that the promoter in transgenic constructs is as safe as the promoter in the intact viral genome, nor to consider it equivalent to the promoter of proviral sequences in the plant genome.

Prostate-specific suicide gene therapy using the prostate-specific membrane antigen promoter and enhancer.

Prostate-specific membrane antigen (PSMA) is abundantly expressed in virtually 100% of prostate cancers and metastases. In addition, unlike prostate-specific antigen (PSA), PSMA is upregulated under conditions of androgen deprivation. Therefore, PSMA is an attractive therapeutic target for advanced prostate cancer. Recently, both the promoter and the enhancer driving prostate-specific expression of the PSMA gene were cloned. We describe here our analysis of the PSMA enhancer for the most active region(s) and present a way of using the enhancer in combination with the E. coli cytosine deaminase gene for suicide-driven gene therapy that converts the nontoxic prodrug 5-fluorocytosine (5-FC) into the cytotoxic drug 5-fluorouracil (5-FU) in prostate cancer cells. Deletion constructs of the full-length PSMA enhancer were subcloned into a luciferase reporter vector containing either the PSMA or SV-40 promoter. The most active portion of the enhancer was then determined via luciferase activity in the C4-2 cell line. We then replaced the luciferase gene with the E. coli cytosine deaminase gene in the subclone that showed the most luciferase activity. The specificity of this technique was examined in vitro, using the prostate cancer cell line LNCaP, its androgen-independent derivative C4-2, and a number of nonprostatic cell lines. The toxicity of 5-FC and 5-FU on transiently transfected cell lines was then compared. The enhancer region originally isolated from the PSMA gene was approximately 2 kb. Deletion constructs revealed that at least two distinct regions seem to contribute to expression of the gene in prostate cancer cells, and therefore the best construct for prostate-specific expression was determined to be 1, 648 bp long. The IC(50) of 5-FC was similar in all cell lines tested (>10 mM). However, transfection with the 1648 nt PSMA enhancer and the PSMA promoter to drive the cytosine deaminase gene enhanced toxicity in a dose-dependent manner more than 50-fold, while cells that did not express the PSMA gene were not significantly sensitized by transfection. Suicide gene therapy using the PSMA enhancer may be of benefit to patients who have undergone androgen ablation therapy and are suffering a relapse of disease.

The Leukemia-associated Protein Btg1 and the p53-regulated Protein Btg2 Interact with the Homeoprotein Hoxb9 and Enhance Its Transcriptional Activation

BTG1 and BTG2 belong to a family of functionally related genes involved in the control of the cell cycle. As part of an ongoing attempt to understand their biological functions, we used a yeast two-hybrid screening to look for possible functional partners of Btg1 and Btg2. Here we report the physical and functional association between these proteins and the homeodomain protein Hoxb9. We further show that Btg1 and Btg2 enhance Hoxb9-mediated transcription in transfected cells, and we report the formation of a Hoxb9.Btg2 complex on a Hoxb9-responsive target, and the fact that this interaction facilitates the binding of Hoxb9 to DNA. The transcriptional activity of the Hoxb9.Btg complex is essentially dependent on the activation domain of Hoxb9, located in the N-terminal portion of the protein. Our data indicate that Btg1 and Btg2 act as transcriptional cofactors of the Hoxb9 protein, and suggest that this interaction may mediate their antiproliferative function.

HeLa cell transformants overproducing mouse metallothionein show in vivo resistance to cis-platinum in nude mice.

Plasmid pSV2MT‐I encoding mouse metallothionein‐I (MT‐I) designed to be expressed under the control of an SV40 promoter was introduced into human HeLa S3 cells. Several transformants (HeLa/MTH) carrying multi‐copies of mouse MT‐I cDNA in their genomes were isolated. These transformants produced 4 to 20‐fold larger amounts of MT than their parent cells. The MT levels in HeLa/MTH were well correlated with the extent of resistance to cadmium, but not with that to cis‐platinum (cis‐DDP) in vitro. To study the role of MT in resistance to cis‐DDP in vivo, nude mice were inoculated subcutaneously with two independent HeLa/MTH clones. MT levels in these tumors were about 3‐fold higher than those in the parental cells. The growth of tumors derived from either HeLa/MTH clone was not inhibited in the presence of 15 μmol/kg of cis‐DDP, which completely inhibited the growth of tumors derived from the parental HeLa cells. These data strongly suggest that the elevated level of MT confers resistance to cis‐DDP in vivo but not in vitro. Thus, the results of this study indicate that in vitro determinations of the influence of MT on cis‐DDP resistance may underestimate its importance in in vivo situations.

Cloning and characterization of repressory and stimulatory DNA sequences upstream the Na/I-symporter gene promoter.

To investigate the existence of potential enhancer or silencer elements in the 5'-flanking region of the human Na+/I-symporter (NIS) gene, we cloned 2,512 bp of genomic DNA further upstream of the previously defined proximal promoter. When tested in reporter gene assays, this sequence had no transcriptional activity per se, but was able to repress the activity of the heterologous SV40 promoter. Conversely, when fused to the homologous NIS gene promoter and thus comprising 3,800 bp 5'-flanking region, the transcription of the proximal NIS promoter was stimulated in the human cell lines FTC-133 (from thyroid) and HeLa, but inhibited in the rat thyroid cell line FRTL-5. This might be due to differences between the upstream regions of the rat and human NIS gene. Comparative analysis with standard promoters (SV40) led to the conclusion that the 5'-flanking region of the human NIS gene also exhibited transcriptional activity in non-thyroid cells. Thyroid-stimulating hormone (TSH) had a moderately stimulating effect on the full length NIS reporter gene construct in FRTL-5 cells. This stimulation is presumably mediated by a putative cAMP responsive element found in the first half of the cloned sequence.

Identification and characterization of the MDR1 promoter-enhancing factor 1 (MEF1) in the multidrug resistant HL60/VCR human acute myeloid leukemia cell line.

In this report, the molecular mechanisms involved in the overexpression of MDR1 mRNA in the multidrug resistant variant of the HL60 human acute myeloid leukemia cell line, HL60/VCR, were investigated. RT-PCR and nuclear run-on assays revealed that the expression of MDR1 mRNA is regulated by increased transcriptional initiation in HL60/VCR cells. Transient transfections with a 241 bp MDR1 promoter (spanning the -198 to +43 region) DNA fragment/pGL3-basic plasmid construct resulted in about 6-fold increased luciferase activity in HL60/VCR but not in HL60 cells. Moreover, ds CAAT-oligomer from the MDR1 promoter cloned upstream of the SV-40 promoter in the pGL3-promoter plasmid caused about a 7-fold increase in luciferase activity compared with plasmid constructs containing CAAT-deleted, GC-box, and nonspecific oligomers in HL60/VCR transfectants. These results were confirmed by transfecting HL60/VCR cells with the pGL3-basic plasmid containing a 237 bp mutated MDR1 proximal promoter lacking the CAAT sequence in which no change in luciferase activity was observed. However, a 5-6-fold increase in luciferase activity was measured in these cells when transfected with the wt MDR1 promoter DNA/pGL3-basic plasmid constructs. These results show that the CAAT-region is involved in upregulating the MDR1 promoter in HL60/VCR cells. A nuclear factor binding to the CAAT-region of the MDR1 promoter specifically was detected in electrophoretic mobility shift assays (EMSAs) in HL60/VCR but not in HL60 extracts. Two MDR1 promoter-associated polypeptides with molecular masses of about 130 and 162 kDa were identified in HL60/VCR cells by electroelution, specific DNA-affinity chromatography, and silver staining. Interestingly, cross-linking and Southwestern analysis indicate that only the 130 kDa protein, which we refer to as MDR1-promoter enhancing factor 1 (MEF1), has a strong DNA-binding ability, interacting with the 5'-GTCAATCC-3' element of the MDR1 promoter, as determined by DNase I protection assay. These data reveal that MEF1 upregulates the MDR1 promoter activity.

Heterogeneous nuclear RNA-ribonucleoprotein F binds to DNA via an oligo(dG)-motif and is associated with RNA polymerase II.

The heterogeneous nuclear ribonucleoprotein F (hnRNP-F) is one of the constituents of the splicing-related hnRNP complex. Recent studies suggest that pre-mRNA modification and splicing factors are associated with transcriptional initiation factors and RNA polymerase II (RNA pol II) at a promoter, implying that pre-mRNA-engaged factors might be associated with a promoter. We isolated a cDNA of rat hnRNP-F and expressed the recombinant protein. HnRNP-F selectively bound to oligo(dG) in constructions with other oligohomonucleotides. The ssDNA of an SV40 promoter sequence having the GC-boxes was bound to hnRNP-F, while hnRNP-F bound to the (G/C)-stretch as dsDNA. Consequently, hnRNP-F was designated as an oligo(dG)-binding protein. None of the RNA-binding domains (RBDs) in hnRNP-F were critical for the oligo(dG)-binding. In contrast, the GY-rich region that exists between RBD-II and -III exhibited an oligo(dG)-binding ability. We suggest that the GY-rich region is a novel DNA-binding motif. HnRNP-F was shown to contain two types of binding motifs for RNA and DNA. Additionally, we found that hnRNP-F was co-precipitated with RNA pol II. Interestingly, the RNA pol II holoenzyme also contained hnRNP-F. The tissue distribution profile of hnRNP-F was similar to that of both RNA pol II and TBP. The above results suggest that hnRNP-F is associated with a transcriptional initiation apparatus that includes RNA pol II. The DNA-binding ability of hnRNP-F might facilitate the entry of pre-mRNA modification/splicing factors at a promoter.