Competitive Electrophoretic Mobility Shift Assays Research Articles

DNA microarray analysis of global gene regulation by A-factor in Streptomyces griseus

A-factor (2-isocapryloyl-3R-hydroxymethyl-gamma-butyrolactone) is a microbial hormone that triggers morphological differentiation and secondary metabolism in Streptomyces griseus. The effects of A-factor on global gene expression were determined by DNA microarray analysis of transcriptomes obtained with the A-factor-deficient mutant DeltaafsA. A-factor was added at a concentration of 25 ng ml(-1) to mutant DeltaafsA at the middle of the exponential growth phase, and RNA samples were prepared from the cells grown after A-factor addition for a further 5, 15 and 30 min, and 1, 2, 4, 8 and 12 h. The effects of A-factor on transcription of all protein-coding genes of S. griseus were evaluated by comparison of the transcriptomes with those obtained from cells grown in the absence of A-factor. Analysis of variance among the transcriptomes revealed that 477 genes, which were dispersed throughout the chromosome, were differentially expressed during the 12 h after addition of A-factor, when evaluated by specific criteria. Quality threshold clustering analysis with regard to putative polycistronic transcriptional units and levels of upregulation predicted that 152 genes belonging to 74 transcriptional units were probable A-factor-inducible genes. Competitive electrophoretic mobility shift assays using DNA fragments including putative promoter regions of these 74 transcriptional units suggested that AdpA bound 37 regions to activate 72 genes in total. Many of these A-factor-inducible genes encoded proteins of unknown function, suggesting that the A-factor regulatory cascade of S. griseus affects gene expression at a specific time point more profoundly than expected.

Protein-coding gene promoters in Methanocaldococcus (Methanococcus) jannaschii

Although Methanocaldococcus (Methanococcus) jannaschii was the first archaeon to have its genome sequenced, little is known about the promoters of its protein-coding genes. To expand our knowledge, we have experimentally identified 131 promoters for 107 protein-coding genes in this genome by mapping their transcription start sites. Compared to previously identified promoters, more than half of which are from genes for stable RNAs, the protein-coding gene promoters are qualitatively similar in overall sequence pattern, but statistically different at several positions due to greater variation among their sequences. Relative binding affinity for general transcription factors was measured for 12 of these promoters by competition electrophoretic mobility shift assays. These promoters bind the factors less tightly than do most tRNA gene promoters. When a position weight matrix (PWM) was constructed from the protein gene promoters, factor binding affinities correlated with corresponding promoter PWM scores. We show that the PWM based on our data more accurately predicts promoters in the genome and transcription start sites than could be done with the previously available data. We also introduce a PWM logo, which visually displays the implications of observing a given base at a position in a sequence.

Point mutations within the streptococcal regulator of virulence (Srv) alter protein–DNA interactions and Srv function

Group A Streptococcus (GAS) possesses a complex regulatory system enabling the organism to colonize a range of physiologically distinct host sites. Within this network of regulators is the streptococcal regulator of virulence (Srv). Srv is a member of the CRP/FNR family of transcriptional regulators and is most similar to pleiotropic regulatory factor A (PrfA), a positive regulator of virulence in Listeria monocytogenes. Members of this family possess a characteristic C-terminal helix-turn-helix motif (HTH) that facilitates binding to DNA targets. Genome scanning identified four targets in GAS that were similar to the consensus DNA target recognized by PrfA. Furthermore, previous amino acid sequence alignments identified conserved residues within the Srv HTH which are necessary for function in PrfA and CRP. Here we investigated the ability of Srv to interact with DNA and evaluated the role of the HTH in this interaction. Purified recombinant Srv (rSrv) was found to co-purify with an untagged form of Srv. Glutaraldehyde cross-linking and gel-filtration chromatography indicated that this co-purification is likely due to the ability of Srv to oligomerize. Electrophoretic mobility shift assays (EMSAs) demonstrated that rSrv retarded the mobility of DNA targets and a supershift analysis confirmed the observation was rSrv-dependent. Competition EMSA indicated that rSrv had a higher relative affinity for the DNA targets studied than non-specific DNA. Site-directed mutagenesis of residues predicted to be in or near the HTH resulted in a decrease or abrogation of DNA binding. Complementation of MGAS5005Deltasrv with one of these site-directed mutants failed to restore wild-type SpeB activity. Taken together, these data suggest that the Srv HTH is necessary for DNA binding and Srv function.

Recruitment of Rad50 and MCM Complexes on the Human Beta Globin Locus Control Region (LCR): A Novel Role for LCR Hypersensitive Site 4 (HS4).

The human beta globin locus control region (LCR) aids in the establishment of an open chromatin configuration thereby facilitating the expression of linked downstream globin genes in a temporal and spatial manner along the developmental ontogeny. The LCR also functions as a classical enhancer of the beta like globin genes. The four DNAase-I hypersensitive regions (HS 1–4) on the LCR are believed to act as nucleation centers for the assembly of multi-protein transcriptional complexes that interact with the distant gene promoters. Present work is focused on characterizing the protein complex assemblies on the HS4 region and delineating their function in the context of goblin gene regulation. Our finding shows an assembly of a 2–5 MDa complex on the HS4 locus that is both erythroid restricted and sequence specific. Electrophoretic mobility shift assays (EMSA) were performed using K562 and HeLa nuclear extracts and ∼35mer double stranded oligonucleotides with 7 bp overlaps tiling the entire core HS4 sequence. We detected prominent K562 specific bands formed with an AT rich 39 bp HS4 sequence. Sequence specificity of DNA-protein complex formation on this AT rich sequence was determined by competitive EMSA assays. Gel supershift assays with a wide range of antibodies and subsequent partial purification of this multiprotein complex followed by the LC-MS/MS analysis reveal the presence of a mini chromosome maintenance (MCM) complex that is associated with the DNA replication, DNA repair associated Rad50-Mre-11-NBS-1 complex, histone acetyl transferase p300 and DNA binding transcription factors ILF2 (NF45) and ILF3 (NF90). The association of these proteins was determined by co-immunoprecipitation experiments. Chromatin immunoprecipitation (ChIP) experiments demonstrate the in vivo recruitment of these proteins on the HS4 region of the LCR. We are currently working on further characterization and functional studies of this complex and its significance on the role of the LCR in DNA replication and repair.

Loss of TP53-DNA interaction induced by p.C135R in lung cancer

The p53 tumor suppressor gene (TP53; OMIM: 191170) plays an important role in tumorigenesis in lung epithelial cells. TP53 encodes a sequence-specific DNA-binding protein that regulates transcription of several genes in response to DNA damage promoting cell cycle arrest, DNA repair or apoptosis. A mutation does not necessarily alter the protein function and since not all altered tumor protein p53 (TP53) conformations lead to the same biological properties, we studied Cys135Arg TP53 gene mutation in squamous cell type of non-small cell lung cancers (NSCLCs), by polymerase chain reaction-single-strand conformation polymorphism (PCR-SSCP) and direct sequencing. Cys135Arg TP53 mutation, rare in databases (11/23544 in R11, IARC TP53 database), was detected. We chose p.C135R in order to examine DNA-TP53 interaction. A comparison with the wild-type after 1 nano-second molecular dynamic simulation analysis revealed a significant structural change (over 4A displacement) in the contact loop Lys-Ser-Val which lies upstream and next to the mutated site in the TP53, that sterically prevents its DNA-binding activity. Additionally, the mutation produced a change in the electrostatic potential surface of the protein in the same loop where the structural modification took place. To demonstrate the degree of loss of function, functional assays in yeast and bacteria with oligonucleotides for competitive electrophoretic mobility shift assays (EMSAs) were done proving that this mutation decreases TP53 ability to bind DNA of the TP53 response element from the human p21 gene. These results demonstrate that the amino acid change C135R in the human TP53 generates the loss of TP53 DNA-binding activity directly affecting its role as a transcription factor and suggests that this observation can explain part of the phenotype described in patients affected by this type of tumor.

Molecular pathogenesis of Gilbert's syndrome: decreased TATA-binding protein binding affinity of UGT1A1 gene promoter

Gilbert's syndrome is a congenital, nonhemolytic, unconjugated hyperbilirubinemia. The most common genotype of Gilbert's syndrome is the homozygous polymorphism, A(TA)7TAA, in the promoter of the gene for UDP-glucuronosyltransferase 1A1 (UGT1A1), with a thymine adenine insertion in the TATA-box-like sequence, which results in a decrease in UGT1A1 activity. The mechanism responsible for this decrease in UGT1A1 activity, however, has not been elucidated. To clarify the mechanism underlying this deficiency in UGT1A1 activity in patients with Gilbert's syndrome. The promoter activity assay using the wild-type A(TA)6TAA or the mutant A(TA)7TAA promoter and a luciferase reporter was performed in two different hepatoma cell lines. The binding affinity for a nuclear protein complex or for TATA-binding protein was evaluated by a competitive electophoretic mobility shift assay using wild-type or mutant TATA-box-like oligonucleotide probes and nuclear extract or TATA-binding protein. The formation of complexes between TATA-binding protein and wild-type or mutant oligonucleotide probes was also studied by a quantitive electophoretic mobility shift assay. A TA insertion in the TATA-box-like sequence of the promoter activity of UGT1A1 gene. A competitive electrophoretic mobility shift assay showed a decrease in nuclear protein complex binding affinity and TATA-binding protein binding affinity of the mutant TATA-box-like sequence A(TA)7TAA. When the mutants A(TA)5TAA and A(TA)8TAA were also compared, quantitative electrophoretic mobility shift assay demonstrated that the TATA-binding protein binding affinity progressively decreased as the number of TA repeats in the TATA-box-like sequence increased. TA insertion in the TATA-box-like sequence of the UGT1A1 promoter affected its binding affinity for TATA-binding protein, causing a decrease in its activity. This explains the pathogenesis of Gilbert's syndrome.

Identification and Functional Analysis of a Novel HumanCYP2E1Far Upstream Enhancer

Both transcriptional and post-transcriptional CYP2E1 regulatory mechanisms are known, resulting in 20-fold or greater variation in CYP2E1 expression. To evaluate functional regulatory elements controlling transcription, CYP2E1 promoter constructs were used to make adenovirus vectors containing CYP2E1 promoter-driven luciferase reporters for analyses in both primary human hepatocytes and HepG2 cells. A 1.2-kilobase pair portion of the CYP2E1 promoter was associated with 5- to 10-fold greater luciferase activity. This upstream region contained five direct repeats of 59 base pairs (bp) that increased thymidine kinase-driven luciferase reporter activity in HepG2 cells more than 5-fold, regardless of orientation. Electrophoretic mobility shift assays (EMSAs) identified sequence-specific nuclear protein binding to the 59-bp repeats that was dependent on a 17-bp sequence containing a canonical GATA binding site (WGATAR). Competitive and supershift EMSA identified the participation of GATA4, another GATA family member or GATA-like factor, and a third factor unrelated to the GATA family. Involvement of the tricho-rhino-phalangeal syndrome-1 factor, which also binds a GATA sequence, was eliminated. Rather, competitive EMSA using known binding sequences for the orphan nuclear receptors, steroidogenic factor-1 (or NR5A1), and fetoprotein transcription factor (or NR5A2) implicated an NR5A member in binding a sequence overlapping the canonical GATA. Chromatin immunoprecipitation assay demonstrated in vivo binding of NR5A2 to the enhancer sequence in human hepatocytes. The enhancer sequence is conserved within the human population but seems species-specific. The identification of this novel enhancer and its putative mechanism adds to the complexities of human CYP2E1 regulation.

Cadmium-responsive Element of the Human Heme Oxygenase-1 Gene Mediates Heat Shock Factor 1-dependent Transcriptional Activation

Transcription of a number of mammalian genes is activated by heavy metals, but mechanisms of signaling and transcriptional regulation are not well understood. From a comparison of heavy metal responses of several human genes, it was noted that the heme oxygenase-1 (HO-1) gene is quite similar in the spectrum of metal response and induction kinetics to the heat shock protein 70 (HSP70) gene, suggesting a common regulatory mechanism shared by these genes. The cadmium-responsive element (CdRE) known to be responsible for the metal regulation of ho-1 formed complexes with proteins from heavy metal-treated HeLa cells in an electrophoretic mobility shift assay (EMSA). These complexes were indistinguishable in mobility from those formed by the heat shock factor 1 (HSF1) and the heat shock element involved in hsp70 regulation, suggesting the involvement of HSF1 also in the CdRE complexes. Competitive EMSA and supershift analysis with an anti-HSF1 antibody revealed that HSF1 was in fact a component of the CdRE complexes. A fine analysis on the affinity of HSF1 to a series of mutant CdRE sequences showed that HSF1 recognizes a sequence motif TnCTAGA. Transient transfection analysis with overexpressed recombinant HSF1 demonstrated that CdRE has HSF1-dependent enhancer-like activity that requires direct binding of HSF1. In the absence of overexpressed HSF1, however, CdRE by itself was insufficient to mediate heavy metal-induced transcription, suggesting requirement of additional regulatory sequences. The finding that HSF1 is directly involved in the regulation of ho-1 with an anti-oxidative role revealed a new aspect of the biological defense mechanism.

The Anti-rheumatic Gold Salt Aurothiomalate Suppresses Interleukin-1β-induced Hyaluronan Accumulation by Blocking HAS1 Transcription and by Acting as a COX-2 Transcriptional Repressor

Gold compounds are among the oldest disease-modifying drugs and are still widely used today for treating rheumatoid arthritis. Despite decades of use, little is known about the mode of action of this class of drugs. Here we have demonstrated that aurothiomalate (AuTM) suppresses hyaluronan accumulation by blocking interleukin (IL)-1beta-induced hyaluronan synthase-1 transcription. We have further demonstrated that, in fibroblast-like synoviocytes (FLSs), AuTM acts as a specific COX-2 transcriptional repressor in that IL-1beta-induced COX-2 transcription is blocked, whereas COX-1 transcription and translation is unaffected. As a consequence, PGE2 levels released by FLS are dose-dependently reduced in cells exposed to AuTM. Of similar importance is the demonstration that AuTM does block NFkappaB-DNA interaction. In addition, two other transcription factors implicated in inflammatory events, namely AP-1 and STAT3, are blocked as well. The effect on NFkappaB likely explains the inhibition of COX-2 as well as that of HAS1, as both are genes that depend on the activation of NFkappaB. Interestingly, AuTM does not interfere with IL-1beta-induced IkappaB alpha degradation, in most cases a prerequisite for subsequent NFkappaB activation. Furthermore, evidence is presented that, in FLS, AuTM blocks NFkappaB-DNA interaction neither by binding to NFkappaB binding sites nor by interacting with activated NFkappaB proteins. Taken together, AuTM treatment of FLS blocks two of the most important proinflammatory events that are associated with rheumatoid arthritis. AuTM blocks the release of PGE2 and prevents the activation of NFkappaB, therefore blocking IL-1beta-induced hyaluronan accumulation and likely a series of other pro-inflammatory NFkappaB-dependent genes.

Investigating the basis of substrate recognition in the pC221 relaxosome

The nicking of the origin of transfer (oriT) is an essential initial step in the conjugative mobilization of plasmid DNA. In the case of staphylococcal plasmid pC221, nicking by the plasmid-specific MobA relaxase is facilitated by the DNA-binding accessory protein MobC; however, the role of MobC in this process is currently unknown. In this study, the site of MobC binding was determined by DNase I footprinting. MobC interacts with oriT DNA at two directly repeated 9 bp sequences, mcb1 and mcb2, upstream of the oriT nic site, and additionally at a third, degenerate repeat within the mobC gene, mcb3. The binding activity of the conserved sequences was confirmed indirectly by competitive electrophoretic mobility shift assays and directly by Surface Plasmon Resonance studies. Mutation at mcb2 abolished detectable nicking activity, suggesting that binding of this site by MobC is a prerequisite for nicking by MobA. Sequential site-directed mutagenesis of each binding site in pC221 has demonstrated that all three are required for mobilization. The MobA relaxase, while unable to bind to oriT DNA alone, was found to associate with a MobC–oriT complex and alter the MobC binding profile in a region between mcb2 and the nic site. Mutagenesis of oriT in this region defines a 7 bp sequence, sra, which was essential for nicking by MobA. Exchange of four divergent bases between the sra of pC221 and the related plasmid pC223 was sufficient to swap their substrate identity in a MobA-specific nicking assay. Based on these observations we propose a model of layered specificity in the assembly of pC221-family relaxosomes, whereby a common MobC:mcb complex presents the oriT substrate, which is then nicked only by the cognate MobA.

APBP-1, a DNA/RNA-binding Protein, Interacts with the Chick Aggrecan Regulatory Region

Expression of the extracellular proteoglycan aggrecan is both cell-specific and developmentally regulated. Previous studies identified six functionally defined cis elements in the aggrecan promoter region which were shown to repress aggrecan gene expression (1). Using competition electrophoretic mobility shift assays (EMSAs) we have now identified in nuclear extracts a functional repressor cis element, (T/C)TCCCCT(A/C)RRC, which occurs at multiple locations within the chick aggrecan regulatory region. We purified the factor that binds to this cis element and established that it, APBP-1 (aggrecan promoter-binding protein-1), is a 19-kDa protein that has significant homology to CIRP (cold inducible RNA-binding protein). Recombinantly expressed APBP-1 mimics the native cis element-trans factor interaction in EMSAs. In situ hybridization demonstrates that aggrecan and APBP-1 RNA expression are restricted to complementary tissues in the developing limb, and Northern blot analysis of chick limb bud mRNA shows that APBP-1 mRNA expression is inversely correlated with aggrecan mRNA expression. Functional analyses by transient transfections and Northern blot analyses suggest APBP-1 has the capacity to repress aggrecan expression, indicating that this factor may be important regulator of aggrecan gene expression.

Activation of transcription factor AP-1 and mitogen-activated protein kinases in aniline-induced splenic toxicity

Signaling mechanisms in aniline-induced fibrogenic and/or tumorigenic response in the spleen are not known. Previous studies have shown that aniline exposure leads to iron accumulation and oxidative stress in the spleen, which may cause activation of redox-sensitive transcription factors and regulate the transcription of genes involved in fibrosis and/or tumorigenesis. To test this, male SD rats were treated with 0.5 mmol/kg/day aniline via drinking water for 30 days, and activation of transcription factor AP-1 was determined in the splenocyte nuclear extracts (NEs). AP-1 DNA-binding activity in the NEs of freshly isolated splenocytes from aniline-treated rats increased in comparison to the controls, as determined by electrophoretic mobility shift assay (EMSA). AP-1 binding was also determined in the NEs of cultured splenocytes (2 h and 24 h), which showed even a greater increase in binding activity at 2 h. The specificity of AP-1 binding for relevant DNA motifs was confirmed by competition EMSA and by supershift EMSA using antibodies specific to c-Jun and c-Fos. To further explore the signaling mechanisms in the AP-1 activation, phosphorylation patterns of mitogen-activated protein kinases (MAPKs) were pursued. Aniline exposure induced increases in the phosphorylation of the three classes of MAPKs: extracellular-signal-regulated kinase (ERK 1/2), c-Jun N-terminal kinase (JNK 1/2), and p38 MAPKs. Furthermore, TGF-beta1 mRNA expression showed a 3-fold increase in the spleens of aniline-treated rats. These observations suggest a strong association among MAPK phosphorylation, AP-1 activation, and enhanced TGF-beta1 gene expression. The observed sequence of events subsequent to aniline exposure could regulate genes that lead to fibrogenic and/or tumorigenic response in the spleen.

MOLECULAR MECHANISMS REGULATING HUMANCYP4B1LUNG-SELECTIVE EXPRESSION

Lung-selective cytochrome P450 expression is well recognized; however, little is known regarding regulatory mechanisms. To address this knowledge gap, transient expression of CYP4B1/luciferase constructs was used to identify a proximal, positively acting regulatory element, position -139 to -45, that functioned in all cells examined; a negatively acting element, position -457 to -216, that only functioned in HepG2 hepatoblastoma cells; and a distal, positively acting element, position -1087 to -1008, that functioned in A549 or BEAS-2B lung-derived cells but not HepG2 cells or 293 kidney-derived cells. Competitive electrophoretic mobility shift assays further localized specific A549, but not HepG2, nuclear protein binding to two sites within the distal element, CYP4B1 position -1052 to -1042 and -1026 to -1008. Several potential lung-selective transcription factor recognition sequences were identified within these elements. However, attempts to identify specific factor(s) were unsuccessful. In contrast, in vitro DNA/protein binding assays combined with transient expression and mutagenesis studies identified two functional Sephadex protein/Krüppel-like factor families of transcription factor sites within the proximal element (position -118 to -114 and position -77 to -73) that bound both Sephadex protein 1 (Sp1) and Sephadex protein 3 (Sp3) in vitro. Furthermore, Sp1-dependent synergistic regulation was observed in A549 cells involving the proximal and distal regulatory elements. Chromatin immunoprecipitation assays demonstrated binding of neither Sp1 nor Sp3 to the CYP4B1 proximal element in human liver tissue, whereas selective Sp1 binding was observed in human lung tissue. Thus, the composite findings are consistent with both the proximal Sp1 elements and the distal regulatory element acting to synergistically control CYP4B1 lung-selective expression.

SP1 AND SP3 REGULATE BASAL TRANSCRIPTION OF THE HUMAN CYP2F1 GENE

Selective transcription of the human CYP2F1 gene in lung tissues may control the susceptibilities of this organ to diverse pneumotoxicants and lung carcinogens. However, the mechanisms responsible for CYP2F1 organ-selective transcription have not been elucidated. The objectives of the current studies were to identify and characterize basal transcription elements within the TATA-less promoter region of CYP2F1. Four putative Sp1-like sites were identified in the CYP2F1 promoter. Competitive electrophoretic mobility shift assay analysis with mutated oligonucleotide probes and lung A549 cell nuclear extract, along with supershift studies using antibodies to either Sp1 or Sp3 proteins, demonstrated that all four sites formed three specific protein-DNA complexes. Mutations in any of the four core Sp1-like motifs abolished protein-DNA binding. Western blot analysis of both human tissues and cells showed that Sp1 was considerably higher in lung than liver and that Sp3 was much higher in liver than lung. Promoter activation of a luciferase reporter construct was sequentially increased by addition of each of the four Sp1-like motifs in lung A549 cells but not in liver HepG2 cells. Cotransfection of a Sp1 expression vector with the reporter construct dramatically increased luciferase activity in either A549 cells or Sp1-deficient Drosophila Schneider line 2 (SL-2) cells. However, similar cotransfections with an Sp3 expression vector failed to increase activity. Cotransfection of both the Sp1 and Sp3 expression vectors considerably decreased Sp1-mediated activity in A549 cells and abolished activity in SL-2 cells. Thus, these studies demonstrated that four Sp1-dependent proximal promoter elements drive organ-selective CYP2F1 gene transcription, and that Sp1 and Sp3 factors interact to modulate constitutive CYP2F1 transcription in lung cells.

Induction of the RelB NF-κB Subunit by the Cytomegalovirus IE1 Protein Is Mediated via Jun Kinase and c-Jun/Fra-2 AP-1 Complexes

We recently demonstrated that the cytomegalovirus (CMV) immediate-early 1 (IE1) protein induces transcription of the gene encoding the RelB NF-kappaB subunit. The mechanism of this activation has been explored here. We report that the induction of the relB promoter by IE1 protein is mediated via activation of JNK and AP-1. The region controlling relB promoter induction was mapped to the upstream approximately 600-bp region between -1694 and -1096 bp. IE1 stimulated AP-1 activity in NIH 3T3 cells. Competition electrophoretic mobility shift assay (EMSA) confirmed the presence of one bona fide AP-1 element centered at -1503 bp. Introduction of a G-to-C mutation in the AP-1 binding site within the distal region of the relB promoter eliminated its activation by IE1 in both NIH 3T3 fibroblasts and vascular smooth muscle cells (SMCs). Supershift EMSA identified c-Jun, Fra-2, and c-Fos in AP-1 binding complexes in IE1 transfected NIH 3T3 cells. IE1 induced c-Jun phosphorylation, and treatment with SP600125, a selective JNK inhibitor, as well as overexpression of JNK-binding domain of JIP1, blocked IE1-mediated induction of AP-1 and relB promoter activity in NIH 3T3 cells and SMCs. Ectopic expression of c-Jun plus Fra-2, but not c-Fos, induced relB promoter activity. The relB promoter has two proximal NF-kappaB elements, and c-Jun/Fra-2 worked in synergy with p50/p65 NF-kappaB complexes. Overall, these findings demonstrate for the first time the role of AP-1 in transcriptional regulation of a gene encoding an NF-kappaB subunit, and its involvement in induction of RelB activity by the CMV IE1 protein.

Calcyclin (S100A6) expression is stimulated by agents evoking oxidative stress via the antioxidant response element

Calcyclin (S100A6) is a cell-specific, calcium binding protein of the S100 family whose expression is augmented in many types of cancer. By means of luciferase activity assays, RT-PCR and Northern blot hybridization, we established that transcription of S100A6 gene is increased by agents known to evoke oxidative stress. Mutation of the antioxidant response element (ARE) located at position −290/−281 of the calcyclin gene promoter, and overlapping the E-box sequence recognized by the upstream stimulatory factor (USF), led to inhibition of calcyclin gene promoter activity stimulated by cadmium ions. Electrophoretic mobility shift assays (EMSA) with the −302/−260 calcyclin gene promoter fragment revealed, apart from USF binding, the presence of another protein complex (N) shown by competitive EMSA to be bound to ARE. DNA affinity chromatography followed by Western blot showed the binding of Nrf2 transcription factor to the immobilized calcyclin gene promoter fragment and concomitant appearance of complex N in EMSA of the eluted fractions. The results indicate that agents evoking oxidative stress activate calcyclin gene via the ARE sequence in its promoter.

Cloning and characterization of a novel human zinc finger gene, hKid3, from a C 2H 2-ZNF enriched human embryonic cDNA library

To investigate the zinc finger genes involved in human embryonic development, we constructed a C 2H 2-ZNF enriched human embryonic cDNA library, from which a novel human gene named hKid3 was identified. The hKid3 cDNA encodes a 554 amino acid protein with an amino-terminal KRAB domain and 11 carboxyl-terminal C 2H 2 zinc finger motifs. Northern blot analysis indicates that two hKid3 transcripts of 6 and 8.5 kb express in human fetal brain and kidney. The 6 kb transcript can also be detected in human adult brain, heart, and skeletal muscle while the 8.5 kb transcript appears to be embryo-specific. GFP-fused hKid3 protein is localized to nuclei and the ZF domain is necessary and sufficient for nuclear localization. To explore the DNA-binding specificity of hKid3, an oligonucleotide library was selected by GST fusion protein of hKid3 ZF domain, and the consensus core sequence 5′-CCAC-3′ was evaluated by competitive electrophoretic mobility shift assay. Moreover, The KRAB domain of hKid3 exhibits transcription repressor activity when tested in GAL4 fusion protein assay. These results indicate that hKid3 may function as a transcription repressor with regulated expression pattern during human development of brain and kidney.

Development and Refinement of Pregnane X Receptor (PXR) DNA Binding Site Model Using Information Theory: INSIGHTS INTO PXR-MEDIATED GENE REGULATION

The pregnane X receptor (PXR) acts as a receptor to induce gene expression in response to structurally diverse xenobiotics through binding as a heterodimer with the 9-cis retinoic acid receptor (RXR) to enhancers in target gene promoters. We identified and estimated the affinities of novel PXR/RXR binding sites in regulated genes and additional genomic targets of PXR with an information theory-based model of the PXR/RXR binding site. Our initial PXR/RXR model, the result of the alignment of 15 previously characterized binding sites, was used to scan the promoters of known PXR target genes. Sites from these genes, with information contents of >8 bits bound by PXR/RXR in vitro, were used to revise the information weight matrix; this procedure was repeated by screening for progressively weaker binding sites. After three iterations of refinement, the model was based on 48 validated PXR/RXR binding sites and has an average information content (Rsequence) of 14.43 +/- 3.21 bits. A scan of the human genome predicted novel PXR/RXR binding sites in the promoters of UGT1A3 (19.78 bits at -8040 and 16.37 bits at -6930) and UGT1A6 (12.74 bits at -9216), both of which were identified previously as targets for PXR. These sites were subsequently demonstrated to specifically bind PXR/RXR in competition electrophoretic mobility shift assays. A strong PXR site was also predicted upstream of the CASP10 gene (18.69 bits at -7872) and was validated by binding studies and reporter assays as a PXR responsive element. This suggests that the PXR-mediated response extends beyond genes involved in drug biotransformation and transport.

Regulation of the Cell Type-specific Dentin Sialophosphoprotein Gene Expression in Mouse Odontoblasts by a Novel Transcription Repressor and an Activator CCAAT-binding Factor

Dentin sialophosphoprotein (DSPP) is an extracellular matrix protein that is cleaved into dentin sialoprotein (DSP) and dentin phosphoprotein (DPP) with a highly restricted expression pattern in tooth and bone. Mutations of the DSPP gene are associated with dentin genetic diseases. Regulation of tissue-specific DSPP expression has not been described. To define the molecular basis of this cell-specific expression, we characterized the promoter responsible for the cell-specific expression of the DSPP gene in odontoblasts. Within this region, DNase I footprinting and electrophoretic mobility shift assays delineated one element that contains an inverted CCAAT-binding factor site and a protein-DNA binding site using nuclear extracts from odontoblasts. A series of competitive electrophoretic mobility shift assay analyses showed that the protein-DNA binding core sequence, ACCCCCA, is a novel site sufficient for protein binding. These two protein-DNA binding sequences are conserved at the same proximal position in the mouse, rat, and human DSPP gene promoters and are ubiquitously present in the promoters of other tooth/bone genes. Mutations of the CCAAT-binding factor binding site resulted in a 5-fold decrease in promoter activity, whereas abolishment of the novel protein-DNA binding site increased promoter activity by about 4.6-fold. In contrast to DSPP, expression levels of the novel protein were significantly reduced during odontoblastic differentiation and dentin mineralization. The novel protein was shown to have a molecular mass of 72 kDa. This study shows that expression of the cell type-specific DSPP gene is mediated by the combination of inhibitory and activating mechanisms.

Sp1/Sp3 and the myeloid zinc finger gene MZF1 regulate the human N-cadherin promoter in osteoblasts

To determine the molecular mechanisms by which N-cadherin transcription is regulated, we cloned and sequenced a 3681-bp of the 5′-flanking region of the human N-cadherin gene. Deletion analysis of the proximal region identified a minimal 318-bp region with strong promoter activity in human osteoblasts. The cryptic promoter is characterized by high GC content and a GA-rich binding core that may bind zing finger transcription factors. Electrophoretic mobility shift assays (EMSA), competition and supershift EMSA revealed that an Sp1/Sp3 binding site acts as a basal regulatory element of the promoter in osteoblasts. Incubation of osteoblast nuclear extracts with −163/−131 wild-type probe containing the GA-rich binding core revealed another specific complex, which was not formed with a −163/−131 probe mutated in the GA repeat. EMSA identified the nuclear factor involved as myeloid zinc finger-1 (MZF1). Mutation analysis showed that Sp1/Sp3 and MZF1 binding sites contribute to basal promoter activity. Cotransfection analyses showed that Sp1 and MZF1 overexpression increases whereas Sp3 antagonizes Sp1-induced N-cadherin promoter activity in osteoblasts. RT-PCR analysis showed that human osteoblastic cells express MZF1 and that Sp1/MZF1 overexpression increased N-cadherin expression. These results indicate that Sp1/Sp3 and MZF1 are important transcription factors regulating N-cadherin promoter activity and expression in osteoblasts.