Double-stranded Probes Research Articles

Control of ACAT2 liver expression by HNF1

ACAT catalyzes the formation of cholesteryl esters from cholesterol and long-chain fatty acids. There are two known genes encoding the two ACAT enzymes, ACAT1 and ACAT2 (also known as Soat1 and Soat2). In adult humans, ACAT1 is present in most tissues, whereas ACAT2 is localized to enterocytes and hepatocytes. In this report, we elucidate the mechanisms that control the liver-specific expression of the human ACAT2 gene. We identified hepatic nuclear factor 1 (HNF1) as an important liver-specific trans-acting element for the human ACAT2 gene using the human hepatocellular carcinoma cell lines HuH7 and HepG2. Targeted deletion of the HNF1 binding site in the DNA sequence abolished not only the basal promoter function in HepG2 and HuH7 cells but also the induction of the ACAT2 promoter by HNF1. Electrophoretic mobility shift assay and chromatin immunoprecipitation assay demonstrated that the transcription factors HNF1alpha and HNF1beta interact with this region in the human ACAT2 gene in vitro and in vivo. These data indicate that a) the identified HNF1 binding site serves as a positive regulator sequence, b) the binding site is functionally active both in vivo and in vitro, and c) the transcription factors HNF1alpha and HNF1beta, which bind to this site, play an important part in the regulation of the human ACAT2 promoter.

Blockade of Nuclear Factor of Activated T Cells Activation Signaling Suppresses Balloon Injury-induced Neointima Formation in a Rat Carotid Artery Model

We have previously reported that nuclear factor of activated T cells (NFATs) play an important role in the regulation of vascular smooth muscle cell migration and proliferation by receptor tyrosine kinase and G protein-coupled receptor agonists, platelet-derived growth factor-BB and thrombin, respectively. To understand the role of NFATs in vascular disease, we have now studied the involvement of these transcription factors in neointima formation in a rat carotid artery balloon injury model. The levels of NFATc1 in injured right common carotid arteries were increased at 72 h, 1 week, and 2 weeks after balloon injury compared with its levels in uninjured left common carotid arteries. Intraperitoneal injection of cyclosporine A (CsA), a pharmacological inhibitor of the calcineurin-NFAT activation pathway, suppressed balloon injury-induced neointima formation by 40%. Similarly, adenoviral-mediated expression of GFPVIVIT, a competent peptide inhibitor of the calcineurin-NFAT activation pathway, in injured arteries also reduced neointima formation by about 40%. Furthermore, CsA and GFPVIVIT attenuated balloon injury-induced neointimal smooth muscle cell proliferation as determined by bromodeoxyuridine staining. Platelet-derived growth factor-BB induced the expression of COX-2 in cultured VSMC in a time- and NFAT-dependent manner. COX-2 expression was also increased in the right common carotid artery in a time-dependent manner after balloon injury as compared with its levels in uninjured left common carotid artery and both CsA and GFPVIVIT negated this response. Together these results for the first time demonstrate that NFATs play a critical role in neointima formation via induction of expression of COX-2.

Transcriptional Activity of the Paired-like Homeodomain Proteins CHX10 and VSX1

CHX10 and VSX1 are homeodomain (HD) proteins essential for normal retinal development. CHX10 is required first for retinal progenitor cell proliferation and later for bipolar cell differentiation, whereas VSX1 is important in the terminal differentiation of a subset of bipolar cells. Elucidating the transcriptional activity of CHX10 and VSX1 is required to understand how these factors control retinal development. We show that CHX10 and Vsx1 can function as transcriptional repressors. When tethered to a promoter by a heterologous LexA DNA-binding domain or its HD, CHX10 repressed multiple classes of activators in different immortalized cell lines. CHX10 blocked TATA-containing and TATA-less promoters, repressed at a distance, and inhibited a complex enhancer positioned upstream or downstream of the reporter gene, whereas retinoblastoma protein (RB) inhibited the downstream enhancer only. Interestingly, CHX10 mildly potentiated a subset of activators in chick neuronal cultures. Thus, CHX10 is both a versatile repressor and a context-specific weak activator. The CHX10 HD and CVC domains were sufficient for DNA binding and repression. VSX1 contains closely related homeo and CVC domains and, like CHX10, also repressed transcription. A VSX1 HD mutation, R166W, that impairs DNA binding and causes keratoconus in humans, hindered repressor function. Therefore, CHX10 and VSX1 may control retinal bipolar cell specification or differentiation by repressing genes required for the development of other cell types.

Helicobacter-induced Intestinal Metaplasia in the Stomach Correlates with Elk-1 and Serum Response Factor Induction of Villin

Chronic Helicobacter pylori infection results in serious sequelae, including atrophy, intestinal metaplasia, and gastric cancer. Intestinal metaplasia in the stomach is defined by the presence of intestine-like cells expressing enterocyte-specific markers, such as villin. In this study, we demonstrate that villin is expressed in intestine-like cells that develop after chronic infection with H. pylori in both human stomach and in a mouse model. Transfection studies were used to identify specific regions of the villin promoter that are inducible by exposure of the cells to H. pylori. We demonstrated that induction of the villin promoter by H. pylori in a human gastric adenocarcinoma cell line (AGS) required activation of the Erk pathway. Elk-1 and the serum response factor (SRF) are downstream transcriptional targets of the Erk pathway. We observed inducible binding of Elk-1 and the SRF after 3 and 24 h of treatment with H. pylori, suggesting that the bacteria alone are sufficient to initiate a cascade of signaling events responsible for villin expression. Thus, H. pylori induction of villin in the stomach correlates with activation and cooperative binding of Elk-1 and the SRF to the proximal promoter of villin.

EVI1 Abrogates Interferon-α Response by Selectively Blocking PML Induction

EVI1 is an oncogene frequently associated with chronic and acute myeloid leukemia. In hematopoietic cells, EVI1 impairs several pathways including proliferation, differentiation, and apoptosis. Interferon-alpha (IFN-alpha) is a powerful cytokine that controls the immune response and limits the expansion of several tissues including bone marrow. These properties contribute to the effectiveness of IFN-alpha in the treatment of many neoplastic disorders especially chronic myeloid leukemia. We report here that in murine hematopoietic progenitors the expression of EVI1 completely abrogates the antiproliferative and apoptotic effects of IFN-alpha. EVI1 does not repress the JAK/STAT signaling pathway or the activation of many IFN-responsive genes. On the contrary, EVI1 prolongs the phosphorylation of STAT1 and the activation of an IFN-dependent reporter gene. However, EVI1 specifically represses the IFN-dependent induction of the tumor suppressor PML and blocks the apoptotic pathways activated by PML. We show that the position of the ISRE, which is located within the first exon of PML, is critical to block PML induction by IFN-alpha. The relocation of the ISRE to a position upstream of the transcription start site is sufficient to re-establish the response to IFN in the presence of EVI1. Our data suggest that stabilized STAT1 phosphorylation and prolonged binding of the STAT1 complex to the first exon could impair PML transcription and inhibit the activation of PML-dependent apoptotic pathways resulting in loss of IFN response. These results point to a novel mechanism utilized by an oncogene to escape normal cell response to growth-controlling cytokines.

Post-transcriptional Regulation of Endothelial Nitric-oxide Synthase by an Overlapping Antisense mRNA Transcript

Endothelial nitric-oxide synthase (eNOS) mRNA levels are abnormal in diseases of the cardiovascular system, but changes in gene expression cannot be accounted for by transcription alone. We found evidence for the existence of an antisense mRNA (sONE) that is derived from a transcription unit (NOS3AS) on the opposite DNA strand from which the human eNOS (NOS3) mRNA is transcribed at human chromosome 7q36. The genes are oriented in a tail-to-tail configuration, and the mRNAs encoding sONE and eNOS are complementary for 662 nucleotides. The mRNA for sONE could be detected in a variety of cell types, both in vivo and in vitro, but not vascular endothelial cells. In contrast, expression of eNOS is highly restricted to vascular endothelium. Most surprisingly, interrogation of transcriptional events across NOS3/NOS3AS genomic regions, using single- and double-stranded probes for nuclear run-off analyses and chromatin immunoprecipitation-based assessments of RNA polymerase II distribution, indicated that NOS3 and NOS3AS gene transcription did not correlate with steady-state mRNA levels. We found strong evidence supporting a role for NOS3AS in the post-transcriptional regulation of NOS3 expression. RNA interference-mediated inhibition of sONE expression in vascular smooth muscle cells increased eNOS expression. Overexpression of sONE in endothelial cells blunted eNOS expression. Finally, the histone deacetylase inhibitor trichostatin A is known to regulate the expression of eNOS via a post-transcriptional mechanism. We found that trichostatin A treatment of vascular endothelial cells increased expression of sONE mRNA levels prior to the observed decrease in eNOS mRNA expression. Taken together, these results indicate that an antisense mRNA (sONE) participates in the post-transcriptional regulation of eNOS and provide a newer model for endothelial cell-specific gene expression.

Strand-specific fluorescence in situ hybridization: the CO-FISH family

The ability to prepare single-stranded chromosomal target DNA allows innovative uses of FISH technology for studies of chromosome organization. Standard FISH methodologies require functionally single-stranded DNAs in order to facilitate hybridization between the probe and the complementary chromosomal target sequence. This usually involves denaturation of double-stranded probes to induce temporary separation of the DNA strands. Strand-specific FISH (CO-FISH; Chromosome Orientation-FISH) involves selective removal of newly replicated strands from DNA of metaphase chromosomes which results in single-stranded target DNA. When single-stranded probes are then hybridized to such targets, the resulting strand-specific hybridization is capable of revealing a level of information previously unattainable at the cytogenetic level. Mammalian telomeric DNA consists of tandem repeats of the (TTAGGG) sequence, oriented 5′→3′ towards the termini of all vertebrate chromosomes. Based on this conserved structural organization, CO-FISH with a telomere probe reveals the absolute 5′→3′ orientation of DNA sequences with respect to the pter→qter direction of chromosomes. Development and various applications of CO-FISH will be discussed: detection of cryptic inversions, discrimination between telomeres produced by leading- versus lagging-strand synthesis, and replication timing of mammalian telomeres.

Positive and Negative Modulation of the Transcriptional Activity of the ETS Factor ESE-1 through Interaction with p300, CREB-binding Protein, and Ku 70/86

Epithelium-specific ETS (ESE)-1 is a prototypic member of a novel subset of the ETS transcription factor family that is predominantly expressed in cells of epithelial origin but can also be induced in other cell types including vascular endothelial and smooth muscle cells in response to inflammatory stimuli. To further define the molecular mechanisms by which the transcriptional activity of ESE-1 is regulated, we have focused our attention on identifying proteins that interact with ESE-1. We have determined that Ku70, Ku86, p300, and CREB-binding protein (CBP) are ESE-1 interacting proteins. The Ku proteins have previously been shown to bind to breaks in DNA where they function to recruit additional proteins that promote DNA repair. Interestingly, Ku70 and Ku 86 negatively regulate the transcriptional activity of ESE-1. Using a series of deletion constructs, we have determined that the Ku proteins bind to the DNA-binding domain of ESE-1. The Ku proteins inhibit the ability of ESE-1 to bind to oligonucleotide probes in gel mobility shift assays. The finding that Ku proteins can interact with other transcription factors and block their function has not been previously demonstrated. In contrast, co-transfection of p300 and CBP with ESE-1 enhances the transcriptional activity of ESE-1. Moreover, the induction of ESE-1 in response to inflammatory cytokine interleukin-1 is associated with a parallel increase of the expression of p300 in vascular endothelial cells, suggesting that in the setting of inflammation, the transcriptional activity of ESE-1 is positively modulated by interaction with the transcriptional co-activator p300. In summary, our results demonstrated that the activity of ESE-1 is positively and negatively modulated by other interacting proteins including Ku70, Ku86, p300, and CBP.

Phosphorylation of Conserved Casein Kinase Sites Regulates cAMP-response Element-binding Protein DNA Binding in Drosophila

The Drosophila homolog of cAMP-response element-binding protein (CREB), dCREB2, exists with serine 231, equivalent to mammalian serine 133, in a predominantly phosphorylated state. Thus, unlike the mammalian protein, the primary regulation of dCREB2 may occur at a different step from serine 231 phosphorylation. Although bacterially expressed dCREB2 bound cAMP-response element sites, protein from Drosophila extracts was unable to do so unless treated with phosphatase. Phosphorylation of recombinant protein by casein kinase (CK) I or II, but not calcium-calmodulin kinase II or protein kinase A, inhibited DNA binding. Up to four conserved CK sites likely to be phosphorylated in vivo were responsible for this effect, and these sites were phosphorylated by a kinase present in Drosophila cell extracts that biochemically resembles CKII. We propose that the relative importance of different signaling pathways in regulating CREB activity may differ between Drosophila and mammals. In Drosophila, the dephosphorylation of CK sites appears to be the major regulatory step, while phosphorylation of serine 231 is necessary but secondary.

Washing-free electrochemical DNA detection using double-stranded probes and competitive hybridization reaction.

A new electrochemical DNA detection method using double-stranded probes and competitive hybridization reaction offers highly selective discrimination of single base mismatch without post-hybridization washing.

The maize Single myb histone 1 gene, Smh1, belongs to a novel gene family and encodes a protein that binds telomere DNA repeats in vitro.

We screened maize (Zea mays) cDNAs for sequences similar to the single myb-like DNA-binding domain of known telomeric complex proteins. We identified, cloned, and sequenced five full-length cDNAs representing a novel gene family, and we describe the analysis of one of them, the gene Single myb histone 1 (Smh1). The Smh1 gene encodes a small, basic protein with a unique triple motif structure of (a) an N-terminal SANT/myb-like domain of the homeodomain-like superfamily of 3-helical-bundle-fold proteins, (b) a central region with homology to the conserved H1 globular domain found in the linker histones H1/H5, and (c) a coiled-coil domain near the C terminus. The Smh-type genes are plant specific and include a gene family in Arabidopsis and the PcMYB1 gene of parsley (Petroselinum crispum) but are distinct from those (AtTRP1, AtTBP1, and OsRTBP1) recently shown to encode in vitro telomere-repeat DNA-binding activity. The Smh1 gene is expressed in leaf tissue and maps to chromosome 8 (bin 8.05), with a duplicate locus on chromosome 3 (bin 3.09). A recombinant full-length SMH1, rSMH1, was found by band-shift assays to bind double-stranded oligonucleotide probes with at least two internal tandem copies of the maize telomere repeat, TTTAGGG. Point mutations in the telomere repeat residues reduced or abolished the binding, whereas rSMH1 bound nonspecifically to single-stranded DNA probes. The two DNA-binding motifs in SMH proteins may provide a link between sequence recognition and chromatin dynamics and may function at telomeres or other sites in the nucleus.

Prostaglandin E2 Stimulates Bone Sialoprotein (BSP) Expression through cAMP and Fibroblast Growth Factor 2 Response Elements in the Proximal Promoter of the Rat BSP Gene

Bone sialoprotein (BSP), an early marker of osteoblast differentiation, has been implicated in the nucleation of hydroxyapatite during de novo bone formation. Prostaglandin E2 (PGE2) has anabolic effects on proliferation and differentiation of osteoblasts via diverse signal transduction systems. Because PGE2 increases the proportion of functional osteoblasts in fetal rat calvarial cell cultures, we investigated the regulation of BSP, as an osteoblastic marker, by PGE2. Treatment of rat osteosarcoma UMR 106 cells with 3 microm, 300 nm, and 30 nm PGE2 increased the steady state levels of BSP mRNA about 2.7-, 2.5-, and 2.4-fold after 12 h. From transient transfection assays, the constructs including the promoter sequence of nucleotides (nt) -116 to +60 (pLUC3) were found to enhance transcriptional activity 3.8- and 2.2-fold treated with 3 microm and 30 nm PGE2 for 12 h. 2-bp mutations were made in an inverted CCAAT box (between nt -50 and -46), a cAMP response element (CRE; between nt -75 and -68), a fibroblast growth factor 2 response element (FRE; nt -92 to -85), and a pituitary-specific transcription factor-1 motif (between nt -111 and -105) within pLUC3 and pLUC7 constructs. Transcriptional stimulation by PGE2 was almost completed abrogated in constructs that included 2-bp mutations in either the CRE and FRE. In gel shift analyses an increased binding of nuclear extract components to double-stranded oligonucleotide probes containing CRE and FRE was observed following treatment with PGE2. These studies show that PGE2 induces BSP transcription in UMR 106 cells through juxtaposed CRE and FRE elements in the proximal promoter of the BSP gene.

Identification of Novel Domains within Sox-2 and Sox-11 Involved in Autoinhibition of DNA Binding and Partnership Specificity

Sox transcription factors play key regulatory roles throughout development, binding DNA through a consensus (A/T)(A/T)CAA(A/T)G sequence. Although many different Sox proteins bind to this sequence, it has been observed that gene regulatory elements are commonly responsive to only a small subset of the entire family, implying that regulatory mechanisms exist to permit selective DNA binding and/or transactivation by Sox family members. To identify and explore the mechanisms modulating gene activation by Sox proteins further, we compared the function of Sox-2 and Sox-11. This led to the discovery that Sox proteins are regulated differentially at multiple levels, including transactivation, protein partnerships with Pit-Oct-Unc (POU) transcription factors, and DNA binding autoregulation. Specifically, we determined that Sox-11 activates transcription more strongly than Sox-2 and that the transactivation domain of Sox-11 is primarily responsible for this capability. Additionally, we demonstrate that the Sox-11 DNA binding domain is responsible for selective cooperation with the POU factor Brn-2. This requirement cannot be replaced by the DNA binding domain of Sox-2, indicating that the DNA binding domain of Sox proteins is critical for Sox-POU partnerships. Interestingly, we have also determined that a conserved domain of Sox-11 has the novel capability of autoinhibiting its ability to bind DNA in vitro and to activate gene expression in vivo. Our findings suggest that the autoinhibitory domain can repress promiscuous binding of Sox-11 to DNA and plays an important role in regulating the recruitment of Sox-11 to specific genes.

Excision of misincorporated ribonucleotides in DNA by RNase H (type 2) and FEN-1 in cell-free extracts.

Misincorporated ribonucleotides in DNA will cause DNA backbone distortion and may be targeted by DNA repair enzymes. Using double-stranded oligonucleotide probes containing a single ribose, we demonstrate a robust activity in human, yeast, and Escherichia coli cell-free extracts that nicks 5' of the ribose. The human and yeast extracts also make a subsequent cut 3' of the ribonucleotide releasing a ribonucleotide monophosphate. The resulting 1-nt gap is an ideal substrate for polymerase and ligase to complete a proposed repair sequence that effectively replaces the ribose with deoxyribose. Screening of yeast deletion mutant cells reveals that the initial nick is made by RNase H(35), a RNase H type 2 enzyme, and the second cut is made by Rad27p, the yeast homologue of human FEN-1 protein. RNase H type 2 enzymes are present in all kingdoms of life and are evolutionarily well conserved. We knocked out the corresponding rnhb gene in E. coli and show that extracts from this strain lack the nicking activity. Conversely, a highly purified archaeal RNase HII type 2 protein has a pronounced activity. To study substrate specificity, extracts were made from a yeast double mutant lacking the other main RNase H enzymes [RNase H1 and RNase H(70)], while maintaining RNase H(35). It was found that a single ribose is preferred as substrate over a stretch of riboses, further strengthening a proposed role of this enzyme in the repair of misincorporated ribonucleotides rather than (or in addition to) processing RNADNA hybrid molecules.

Oligonucleotide Fishing for STAT6: Cross-Talk between IL-4 and Chemokines

Signal transducer and activator of transcription 6 (STAT6) is essential for the biological activities of interleukin-4 (IL-4) and the development of allergic responses in mice. Here we report on a sensitive and specific assay for STAT6 activation in response to IL-4. We took advantage of double-stranded oligonucleotide probes containing a STAT6-binding gene-sequence from the promotor of the immunoglobulin heavy chain germline Ε transcript to study the IL-4-induced DNA binding of STAT6. Using these probes, we show that repeated adjacent STAT6-binding sites result in enhanced STAT6-DNA binding. Moreover, the distance between the binding sites is critical for STAT-DNA binding, i.e. STAT6 binding is decreased at distances above 20 nucleotides between neighbouring binding sites. Using this assay to study cross-talk between IL-4 and chemokines, we provide evidence that MIP-1β and MIG inhibit IL-4-induced STAT6 activation, whereas other chemokines and cytokines do not. In conclusion, our data show that oligonucleotide fishing is a supplementary tool for studying cytokine cross-talk at a genomic level.

The DEXD/H-box RNA helicase RHII/Gu is a co-factor for c-Jun-activated transcription.

Tandem affinity purification (TAP) and mass spectrometric peptide sequencing showed that the DEAD-box RNA helicase RHII/Gu is a functional interaction partner of c-Jun in human cells. The N-terminal transcription activation region of, c-Jun interacts with a C-terminal domain of RHII/Gu. This interaction is stimulated by anisomycin treatment in a manner that is concurrent with, but independent of, c-Jun phosphorylation. A possible explanation for this effect is provided by the observation that RHII/Gu translocates from nucleolus to nucleoplasm upon anisomycin or UV treatment or when JNK signaling is activated by overexpression of a constitutively active form of MEKK1 kinase. Several experiments show that the RNA helicase activity of RHII/Gu supports c-Jun-mediated target gene activation: dominant-negative forms of RHII/Gu, as well as a neutralizing antibody against the enzyme, significantly interfered with c-Jun target gene activity but not with transcription in general. These findings clarify the mechanism of c-Jun-mediated transcriptional regulation, and provide evidence for an involvement of RHII/Gu in stress response and in RNA polymerase II-catalyzed transcription in mammalian cells.

A new class of homogeneous nucleic acid probes based on specific displacement hybridization.

We have developed a new class of probes for homogeneous nucleic acid detection based on the proposed displacement hybridization. Our probes consist of two complementary oligodeoxyribonucleotides of different length labeled with a fluorophore and a quencher in close proximity in the duplex. The probes on their own are quenched, but they become fluorescent upon displacement hybridization with the target. These probes display complete discrimination between a perfectly matched target and single nucleotide mismatch targets. A comparison of double-stranded probes with corresponding linear probes confirms that the presence of the complementary strand significantly enhances their specificity. Using four such probes labeled with different color fluorophores, each designed to recognize a different target, we have demonstrated that multiple targets can be distinguished in the same solution, even if they differ from one another by as little as a single nucleotide. Double-stranded probes were used in real-time nucleic acid amplifications as either probes or as primers. In addition to its extreme specificity and flexibility, the new class of probes is simple to design and synthesize, has low cost and high sensitivity and is accessible to a wide range of labels. This class of probes should find applications in a variety of areas wherever high specificity of nucleic acid hybridization is relevant.

Clinicopathological study on TTV infection in hepatitis of unknown etiology

To investigate the state of infection, replication site, pathogenicity and clinical significance of transfusion transmitted virus (TTV) in patients with hepatitis, especially in patients of unknown etiology. Liver tissues taken from 136 cases of non-A non-G hepatitis were tested for TT virus antigen and nucleic acid by in situ hybridization (ISH) and nested-polymerase chain reaction (PCR). Among them, TT virus genome and its complemental strand were also detected in 24 cases of autopsy liver and extrahepatic tissues with ISH. Meanwhile, TTV DNA was detected in the sera of 187 hepatitis patients by nested-PCR. The pathological and clinical data of the cases infected with TTV only were analyzed. In liver, the total positive rate of TTV DNA was 32.4% and the positive signals were located in the nuclei of hepatocytes. In serus, TTV DNA was detected in 21.4% cases of hepatitis A-G, 34.4% of non-A non-G hepatitis and 15% of healthy donors. The correspondence rate of TTV DNA detection between liver tissue with ISH and sera with PCR was 63.2% and 89.3% in the same liver tissues by ISH and by PCR, respectively. Using double-strand probes and single-strand probes designed to detect TTV genome, the correspondence rate of TTV DNA detected in liver and extrahepatic tissues was 85.7%. Using single-strand probes, TTV genome could be detected in liver and extrahepatic tissues by PCR, but its complemental strands (replication strands) could be observed only in livers. The liver function of most cases infected with TTV alone was abnormal and the liver tissues had different pathological damage such as ballooning, acidophilia degeneration, formation of apoptosis bodies and focus of necrosis, but the inflammation in the lobule and portal area was mild. The positive rate of TTV DNA among cases of hepatitis was higher than that of donors, especially in patients with non-A non-G hepatitis, but most of them were coinfected with other hepatitis viruses. TTV can infect not only hepatocytes, but also extrahepatic tissues. However, the chief replication place may be liver. The infection of TTV may have some pathogenicity. Although the pathogenicity is comparatively weak, it can still damage the liver tissues. The lesions in acute hepatitis (AH) and chronic hepatitis (CH) are mild, but in severe hepatitis (SH), it can be very serious and cause liver function failure, therefore, we should pay more attention to TTV when studying the possible pathogens of so-called "liver hepatitis of unknown etiology".

Microarray Analysis Detects Novel Pax3 Downstream Target Genes

Pax3 is a transcription factor that is required for the development of embryonic neural tube, neural crest, and somatic derivatives. Our previous study (Mayanil, C. S. K., George, D., Mania-Farnell, B., Bremer, C. L., McLone, D. G., and Bremer, E. G. (2000) J. Biol. Chem. 275, 23259-23266) reveals that overexpression of Pax3 in a human medulloblastoma cell line, DAOY, resulted in an up-regulation in alpha-2,8-polysialyltransferase (STX) gene expression and an increase in polysialic acid on neural cell adhesion molecule. This finding suggests that STX might be a previously undescribed downstream target of Pax3. Because Pax3 is important in diverse cellular functions during development, we are interested in the identification of additional downstream targets of Pax3. We utilized oligonucleotide arrays and RNA isolated from stable Pax3 transfectants to identify potential target genes. A total of 270 genes were altered in the Pax3 transfectants as compared with the vector control and parental cell line. An independent analysis by cDNA expression array and real-time quantitative polymerase chain reaction of several genes confirmed the changes observed by the oligonucleotide microarray data. Of the genes that displayed significant changes in expression, several contain paired and homeodomain binding motifs of Pax3 in their promoter regions. Using promoter-luciferase reporter transfection assays and electromobility shift assays, we showed at least one previously undescribed downstream target, STX, to be a biological downstream target of Pax3. Thus we report several previously undescribed candidate genes to be potential downstream targets of Pax3.

A Novel Zinc Finger Protein TReP-132 Interacts with CBP/p300 to Regulate Human CYP11A1 Gene Expression

The human CYP11A1 gene is expressed specifically in steroidogenic tissues and encodes cytochrome P450scc, which catalyzes the first step in steroid synthesis. A region of the 5'-flanking DNA of the gene from nucleotides -155 to -131 (-155/-131) is shown to activate transcription in steroidogenic human placental JEG-3 (1) and adrenal NCI-H295 cells. Using this region of the gene as probe, a cDNA clone of 4.4 kilobase pairs was isolated by screening JEG-3 cell and human placental cDNA expression libraries. The open reading frame encodes three zinc fingers of the C(2)H(2) subtype, and separate regions rich in glutamate, proline, and glutamine, which are indicative of a DNA-binding protein involved in gene transcription. Expression of the cDNA in vitro and in HeLa cells yields a protein of 132 kDa, which concurs with the predicted size. Northern blot analysis demonstrate expression of two TReP-132 transcripts of 4.4 and 7.5 kilobase pairs in the thymus, adrenal cortex, and testis; and expression is also found in the steroidogenic JEG-3, NCI-H295, and MCF-7 cell lines. Immunocytochemistry analysis demonstrates localization of the HA-tagged TReP-132 protein in the nucleus. The expression of exogenous TReP-132 in HeLa cells was demonstrated to interact with the -155/-131 region in bandshift analysis. Transfection of the cDNA in placental JEG-3 and adrenal NCI-H295 cells increases expression of a reporter construct controlled by the P450scc gene 5'-flanking region from nucleotides -1676 to +49. Moreover, a chimeric protein generated by fusion of TReP-132 with the Gal4 DNA-binding domain was able to significantly increase promoter activity of a reporter construct via Gal4-binding sites upstream of the E1b minimal promoter. Coexpression of CREB-binding protein (CBP)/p300 with TReP-132 has an additive effect on promoter activity, and the proteins were demonstrated to interact physically. Thus, these results together indicate the isolation of a novel zinc-finger transcriptional regulating protein of 132 kDa (TReP-132) involved in the regulation of P450scc gene expression.