Positive Cis-acting Elements Research Articles

Identification and characterization of a novel enhancer for the human MCT-1 oncogene promoter.

Cloning and characterization of the promoter region for the MCT-1 oncogene is described. We used luciferase assays to identify cis-acting elements responsible for human MCT-1 promoter function. The MCT-1 promoter is TATA-less with a consensus initiator element located at the transcription start site and facilitated by two Sp1 sites that directs basal transcription. Deletion of a region of the MCT-1 promoter (-133 to -122) resulted in significant decrease in luciferase activity, suggesting that this region contains a positive cis-acting element. Using mobility shift assays with a 26-mer oligonucleotide, which contains this fragment and its flanking regions, we demonstrated the presence of sequence-specific DNA-binding protein in both Jurkat and Hela nuclear extracts that we designated as LMBF (for lymphoid MCT-1 binding factor). This 26-mer oligonucleotide containing the LMBF binding site is required for maximum transcriptional activity of the MCT-1 promoter. Although the 26-mer oligonucleotide contains a sequence with strong homology to a heat-shock factor consensus, competitive electrophoretic mobility shift assay (EMSA) analysis demonstrated that the binding protein is not a known member of heat shock family. Furthermore, this sequence when placed in reverse orientation downstream of the luciferase gene was able to enhance luciferase activity driven by a minimal promoter. These data are consistent with this sequence behaving as an enhancer. Finally, Southwestern blot analysis revealed a 96-kDa protein capable of binding a probe containing the LMBF binding site.

Encystation-specific regulation of the cyst wall protein 2 gene in Giardia lamblia by multiple cis-acting elements

Giardia lamblia, a worldwide cause of diarrhoea, must differentiate into environmentally resistant cysts for dissemination and completion of its life cycle. Although G. lamblia is an early diverging eukaryote, encystation involves many complex cellular changes including formation of the cyst wall that contains at least two cyst wall proteins, cyst wall proteins 1 and 2. Cwp genes are transcribed only during encystation. In this study, we examine the regulatory elements for the encystation-specific gene cwp2. The 64 bp immediately upstream of the cwp2 open reading frame (−64 to −1 relative to ATG) was shown to be sufficient for the encystation-specific expression of luciferase. To determine which region(s) within this 64 bp contributed to encystation-specific expression in vivo, a series of deletions were cloned into a Giardia luciferase expression vector and their ability to control encystation-specific expression of luciferase was assessed. Deletion of elements in the −64 to −23 region of the cwp2 promoter significantly increased expression of luciferase in vegetative trophozoites, suggesting that this area contains a negative cis-acting element. Deletions of elements from −23 to −10 led to decreased expression in encysting cells, suggesting that this region may contain positive cis-acting elements. When the A/T-rich initiator was deleted but the cis-acting elements (−64 to −10) were retained, encystation-specific expression of luciferase was maintained but an aberrant transcriptional start site was utilised. These results indicate that Giardia has developed a classic repressor mechanism(s) that allows tight, encystation-specific control by the cwp2 promoter.

Regulation of the intronic promoter of rat estrogen receptor alpha gene, responsible for truncated estrogen receptor product-1 expression.

We have characterized the intronic promoter of the rat estrogen receptor (ER) alpha gene, responsible for the lactotrope-specific truncated ER product (TERP)-1 isoform expression. Transcriptional regulation was investigated by transient transfections using 5'-deletion constructs. TERP promoter constructs were highly active in MMQ cells, a pure lactotrope cell line, whereas a low basal activity was detected in alphaT3-1 gonadotrope cells or in COS-7 monkey kidney cells. Serial deletion analysis revealed that 1) a minimal -693-bp region encompassing the TATA box is sufficient to allow lactotrope-specific expression; 2) the promoter contains strong positive cis-acting elements both in the distal and proximal regions, and 3) the region spanning the -1698/-1194 region includes repressor elements. Transient transfection studies, EMSAs, and gel shifts demonstrated that estrogen activates the TERP promoter via an estrogen-responsive element (ERE1) located within the proximal region. Mutation of ERE1 site completely abolishes the estradiol-dependent transcription, indicating that ERE1 site is sufficient to confer estrogen responsiveness to TERP promoter. In addition, ERalpha action was synergized by transfection of the pituitary-specific factor Pit-1. EMSAs showed that a single Pit-1 DNA binding element in the vicinity of the TATA box is sufficient to confer response by the TERP promoter. In conclusion, we demonstrated, for the first time, that TERP promoter regulation involves ERE and Pit-1 cis-elements and corresponding trans-acting factors, which could play a role in the physiological changes that occur in TERP-1 transcription in lactotrope cells.

De novo deletion within the telomeric region flanking the human alpha globin locus as a cause of alpha thalassaemia.

We have identified and characterized a Scottish individual with alpha thalassaemia, resulting from a de novo 48 kilobase (kb) deletion from the telomeric flanking region of the alpha globin cluster which occurred as a result of recombination between two misaligned repetitive elements that normally lie approximately 83 kb and 131 kb from the 16p telomere. The deletion removes two previously described putative regulatory elements (HS-40 and HS-33) but leaves two other elements (HS-10 and HS-8) intact. Analysis of this deletion, together with eight other published deletions of the telomeric region, showed that they all severely downregulated alpha globin expression. Together they defined a 20.4-kb region of the human alpha cluster, which contains all of the positive cis-acting elements required to regulate alpha globin expression. Comparative analysis of this region with the corresponding segment of the mouse alpha globin cluster demonstrated conserved non-coding sequences corresponding to the putative regulatory elements HS-40 and HS-33. Although the role of HS-40 as an enhancer of alpha globin expression is fully established, these observations suggest that the role of HS-33 and other sequences in this region should be more fully investigated in the context of the natural human and mouse alpha globin loci.

Functional analysis of the larval serum protein gene promoter from silkworm, Bombyx mori.

The regulation region of larval serum protein gene, Bombyx mori. (BmLSP), consisting of the first intron, the first exon, the central promoter region and 5’-upstream region, is cloned from genomic DNA from the silkworm variety of Suju × Minghu. Using PCR and restriction endonuclease methods, a series of luciferase reporter plasmids, driven by different length of BmLSP promoters, are constructed. Via the transient expression system in BmN cells, the effects of the regulation elements and foreign insect hormones on the BmLSP promoter activity are investigated. The results demonstrate that the promoter activity of BmLSP is 5.8- or 4.4-fold higher than that of BmLSPs whose first intron or the element in 5’-upstream region harboring the homologous sequence with the first intron of light-chain fibroin gene (EHIF) is deleted, respectively, suggesting that both the first intron and EHIF contain the main positive cis-acting elements. However, the inactive mariner transposable element (MTE) in 5 ’-upstream region presents a negative effect. Furthermore, the effects of juvenile hormone analogue (JHA) on the BmLSP promoter activity show a typical dose-dependent manner, that is, low concentration treatments increase the BmLSP promoter activity and high concentration treatments decrease it. Meanwhile, insect ecdysone (MH) treatments present no significant effect.

Multiple Transcription Initiation Sites, Alternative Splicing, and Differential Polyadenylation Contribute to the Complexity of Human Neurofibromatosis 2 Transcripts

Northern blot analysis has shown that the human neurofibromatosis type 2 (NF2) cDNA hybridizes to multiple RNA species. To examine whether these hybridizing RNA species represent NF2 transcripts, we cloned the complete NF2 cDNA by a combination of techniques: 5′ and 3′ rapid amplification of cDNA ends, RT–PCR, and searching and sequencing the NF2-related cDNA clones from the IMAGE consortium. We showed that human NF2 transcripts initiate at multiple positions. Analogous to those reported previously, NF2 transcripts undergo alternative splicing in the coding exons. We isolated eight alternatively spliced NF2 cDNA isoforms, including one that contains a new exon termed exon 2′, which potentially could encode proteins of different sizes. We assembled the overlapping cDNA fragments, and the longest NF2 cDNA, containing all 17 exons, consists of 6067 nucleotides, which is consistent with the size of the major RNA species hybridized to the NF2 probe. The cDNA has a 425-nucleotide 5′ untranslated region upstream from the ATG start codon, and a long 3′ untranslated region of 3869 nucleotides. We also isolated two shorter NF2 cDNAs that were terminated by different polyadenylation signal sequences, which indicates that differential usage of multiple polyadenylation sites also contributes to the complexity of human NF2 transcripts. By reference to the transcription initiation site mapped, we analyzed the 5′ flanking sequence of the human NF2 gene. Transient transfection analysis in human 293 kidney, SK-N-AS neuroblastoma, and NT2/D1 teratocarcinoma cells with NF2 promoter-luciferase chimeric constructs revealed a core promoter region extending 400 base pairs from the major transcription initiation site. Although multiple regions are required for full promoter activity, a site-directed mutagenesis experiment identified a GC-rich sequence (position −58 to −46), which could be bound by transcription factor Sp1, as a positive cis-acting regulatory element. Cotransfection studies in Drosophila melanogaster SL2 cells showed that Sp1 could activate the NF2 promoter through the GC-rich sequence.

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.

Transcriptional regulation of the human mu opioid receptor (hMOR) gene: evidence of positive and negative cis-acting elements in the proximal promoter and presence of a distal promoter.

The mu opioid receptor (MOR), the primary binding site for morphine, is an important target for treating pain and drug addiction. The MOR gene is tightly regulated at the level of transcription, and potential polymorphisms in its 5' regulatory region can cause individual variation in MOR gene expression, nociception, and opiate responses. To study the 5' regulatory region of the human MOR gene (hMOR), we further investigated our previous finding of two regulatory regions and have localized a 40-bp positive cis-acting element and a 35-bp negative cis-acting element that regulate hMOR transcription in SK-N-SH cells. Electromobility shift assays and methylation interference assay with the 40-bp probe suggested that protein contacts were made with the core recognition sequence GCC (-510 to -508). The 35-bp sequence (-694 to -660) was the hMOR homolog of the mMOR negative regulatory element, and it suppressed proximal promoter activity of the hMOR gene. Additionally, the presence of an hMOR distal promoter was confirmed using RT-PCR. However, the activity of the distal promoter construct (-2325 to -777) was weak compared with the activity of the proximal promoter construct (-776 to -212).

CD99 expression is positively regulated by Sp1 and is negatively regulated by Epstein-Barr virus latent membrane protein 1 through nuclear factor-κB

Epstein-Barr virus (EBV)-encoded latent membrane protein-1 (LMP1) is highly expressed in Hodgkin and Reed-Sternberg (H-RS) cells from patients with EBV-associated Hodgkin disease. It was previously demonstrated that CD99 can be negatively regulated by LMP1 at the transcriptional level, and the decreased expression of CD99 in a B lymphocyte cell line generates H-RS-like cells. In this study, detailed dissection of the CD99 promoter region was performed to search regulatory factor(s) involved in the expression of the gene. Using various mutant constructs containing deletions in the promoter region, it was revealed that the maximal promoter activity was retained on 5'-deletion to the position -137 from the transcriptional initiation site. Despite the presence of multiple putative Sp1-binding sites in the promoter region, the site located at -95 contributes heavily as a positive cis-acting element to its basal promoter activity. However, on examination of the involvement of the positive-acting Sp1-binding site of the promoter for the repressive activity of LMP1, it appeared to be dispensable. Instead, the repressive effect was mapped to the nuclear factor (NF)-kappaB activation domains in the cytoplasmic carboxyl terminus of LMP1 despite the absence of the NF-kappaB consensus sequences in the CD99 promoter region. Furthermore, the decreased CD99 promoter activity by LMP1 was markedly restored when NF-kappaB activity was inhibited. Taken together, these data suggest that Sp1 activates, whereas LMP1 represses, transcription from the CD99 promoter through the NF-kappaB signaling pathway, and they might aid in the understanding of the molecular mechanisms of viral pathogenesis in EBV-positive Hodgkin disease. (Blood. 2001;97:3596-3604)

The Aspergillus nidulans multimeric CCAAT binding complex AnCF is negatively autoregulated via its hapB subunit gene

Cis-acting CCAAT elements are frequently found in eukaryotic promoter regions. Many of them are bound by conserved multimeric complexes. In the fungus Aspergillus nidulans the respective complex was designated AnCF ( A. nidulans CCAAT binding factor). AnCF is composed of at least three subunits designated HapB, HapC and HapE. Here, we show that the promoter regions of the hapB genes in both A. nidulans and Aspergillus oryzae contain two inversely oriented, conserved CCAAT boxes (box α and box β). Electrophoretic mobility shift assays (EMSAs) using both nuclear extracts and the purified, reconstituted AnCF complex indicated that AnCF binding in vitro to these boxes occurs in a non-mutually exclusive manner. Western and Northern blot analyses showed that steady-state levels of HapB protein as well as hapB mRNA were elevated in hapC and hapE deletion mutants, suggesting a repressing effect of AnCF on hapB expression. Consistently, in a hapB deletion background the hapB-lacZ expression level was elevated compared with the expression in the wild-type. This was further supported by overexpression of hapB using an inducible alcA-hapB construct. Induction of alcA-hapB expression strongly repressed the expression of a hapB-lacZ gene fusion. However, mutagenesis of box β led to a fivefold reduced expression of a hapB-lacZ gene fusion compared with the expression derived from a wild-type hapB-lacZ fusion. These results indicate that (i) box β is an important positive cis-acting element in hapB regulation, (ii) AnCF does not represent the corresponding positive trans-acting factor and (iii) that AnCF is involved in repression of hapB.

Transcriptional Activation of the Human Hematopoietic Prostaglandin D Synthase Gene in Megakaryoblastic Cells: ROLES OF THE Oct-1 ELEMENT IN THE 5′-FLANKING REGION AND THE AP-2 ELEMENT IN THE UNTRANSLATED EXON 1

The human hematopoietic prostaglandin D synthase (H-PGDS) gene is highly expressed in human megakaryoblastic cells, in which phorbol ester induces its expression. We characterized the promoter activity of the 5'-flanking region and the untranslated exon 1 (-1044 to +290) of the human H-PGDS gene in human megakaryoblastic Dami cells. Transient expression analysis using the luciferase reporter gene revealed that the 5'-flanking region and the untranslated exon 1 were sufficient for efficient expression of the H-PGDS gene in Dami cells, but not in monocytic U937 cells. Deletion and site-directed mutagenesis of the Oct-1 element in the 5'-flanking region decreased the promoter activity by approximately 30% compared with that of the entire region from -1044 to +290. An electrophoretic mobility shift assay demonstrated that Oct-1 specifically bound to the promoter region. Interestingly, even only untranslated exon 1 (+1 to +290) showed approximately 60% of the promoter activity of the entire region from -1044 to +290. Site-directed mutagenesis of the AP-2 element within the untranslated exon 1 abolished the basal promoter activity as well as its phorbol ester-mediated up-regulation. In AP-2-deficient HepG2 cells, the H-PGDS promoter activity was enhanced by coexpression with AP-2alpha. These findings indicate that the Oct-1 element in the 5'-flanking region acts as a positive cis-acting element and that the AP-2 element in the untranslated exon 1 is crucial for both basal and phorbol ester-mediated up-regulation of human H-PGDS gene expression in megakaryoblastic Dami cells.

Transcription of genes encoding pregnancy-specific glycoproteins is regulated by negative promoter-selective elements

The human pregnancy-specific glycoprotein (PSG) genes comprise a family of 11 highly conserved members whose expression is maximal in placental cells and marginal in other cell types. We have investigated here the molecular basis of PSG regulation by analysing a large regulatory region of the PSG-5 gene in cells that do and do not express these genes. The promoter region (-254 to -43), which does not contain a TATA-box, large GC-rich sequences or a classical initiator, was active in all cell types analysed. Additional upstream sequences up to position -3204 repressed promoter activity. Two independent repressor regions were identified and found to operate effectively in HeLa, COS-7 and HTR8/SVneo placental cells. More significantly, these negatively acting modules failed to repress a heterologous TATA-containing thymidine kinase promoter. Detailed transcriptional and DNA–protein analyses of the proximal repressor region (-605 to -254) revealed the presence of both negative and positive cis-acting elements. Disruption of the repressive functions resulted in an enhanced transcription of the reporter constructs. In conclusion, these results demonstrate that PSG-5 gene transcription is highly repressed by promoter-selective negative regulatory regions and the relief of repression allows enhanced PSG-5 gene transcription irrespective of the cell type. Furthermore, our findings suggest that PSG genes are expressed mainly through a derepression mechanism.

Transcription of genes encoding pregnancy-specific glycoproteins is regulated by negative promoter-selective elements

The human pregnancy-specific glycoprotein (PSG) genes comprise a family of 11 highly conserved members whose expression is maximal in placental cells and marginal in other cell types. We have investigated here the molecular basis of PSG regulation by analysing a large regulatory region of the PSG-5 gene in cells that do and do not express these genes. The promoter region (-254 to -43), which does not contain a TATA-box, large GC-rich sequences or a classical initiator, was active in all cell types analysed. Additional upstream sequences up to position -3204 repressed promoter activity. Two independent repressor regions were identified and found to operate effectively in HeLa, COS-7 and HTR8/SVneo placental cells. More significantly, these negatively acting modules failed to repress a heterologous TATA-containing thymidine kinase promoter. Detailed transcriptional and DNA-protein analyses of the proximal repressor region (-605 to -254) revealed the presence of both negative and positive cis-acting elements. Disruption of the repressive functions resulted in an enhanced transcription of the reporter constructs. In conclusion, these results demonstrate that PSG-5 gene transcription is highly repressed by promoter-selective negative regulatory regions and the relief of repression allows enhanced PSG-5 gene transcription irrespective of the cell type. Furthermore, our findings suggest that PSG genes are expressed mainly through a derepression mechanism.

Cloning and Characterization of the 5′-Flanking Region for the Mouse Phospholipase C-δ1 Gene

To date, little is known about the molecular mechanisms controlling the regulation of phospholipase C-δ1 (PLC-δ1) gene expression. To understand the mechanisms responsible for the regulation of PLC-δ1 gene expression, the 5′-flanking region of the mouse PLC-δ1 gene was isolated from a mouse genomic DNA library. Primer extension analysis revealed that there is a single transcriptional start site located at 127 bases upstream from the translation start codon in the mouse PLC-δ1 gene. DNA sequence analysis showed that the sequence around the transcriptional start site is very GC-rich and has no TATA or CAAT boxes. Transient expression of a luciferase reporter gene under the control of serially deleted 5′-flanking sequences revealed that the 160-base-pair region from −622 to −462 upstream of the transcriptional start site includes a positive cis-acting element(s) for the efficient expression of the PLC-δ1 gene. Gel retardation analysis suggests that multiple transcription factors bind to separate sites on the promoter region. Based on these results, our study suggests that the minimal essential region located at −622 to +70 is fully sufficient to confer high-level transcriptional activity and contains high-affinity binding elements for multiple transcription factors.

Effect of inducers on the interaction of the positive and negativecis-acting elements of theCYP2B2 gene with rat liver nuclear proteins

A study was made of the interaction of the negative (−127…−160) and positive (−69…−98)cis-acting elements of the minimal promoter of the cytochrome P4502B2 gene with nuclear proteins of rats injected with phenobarbital and triphenyldioxane. On evidence of the gel retardation assay, binding of nuclear proteins to the negative element is suppressed, and that to the positive one is enhanced 6–9 h after injection. Binding to both elements is maximal 18–48 h after injection and decreases to the control level 72 h after injection. It is assumed that, when derepressed, the negative element acts as a positive one. Thein vitro binding proved to correlate with changes in theCYP2B1/2 mRNA content. The data obtained with triphenyldioxane testify to the involvement of the proximal promoter in the inducer-dependent activation of cytochrome P4502B2.

Negative Regulation of Rat GST-Ya Gene via Antioxidant/Electrophile Response Element Is Directed by a C/EBP-like Site

The present studies were conducted to evaluate functional interactions between aryl hydrocarbon and antioxidant/electrophile response elements (AhRE and ARE/EpRE, respectively) in transcriptional regulation of the rat (r)GST-Ya gene. Transient transfection of an AhRECAT reporter construct into vascular smooth muscle cells (vSMCs) or HepG2 cells showed that benzo(a)pyrene (BaP) (0.3–30 μM) or 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) (0.1–10 nM), but not hydrogen peroxide (H2O2) (100–400 μM), increased gene transcription. ARE/EpRE did not mediate gene inducibility by any of the chemicals in vSMCs but increased transcription in HepG2 cells treated with BaP or H2O2, but not TCDD. Gene inducibility in response to all chemicals was repressed in both cell types transfected with a 1.6CAT full-length promoter construct containing the AhRE and ARE/EpRE in genomic context. Site-directed mutagenesis of 1.6CAT showed that a CCAAT/enhancer-binding protein (C/EBP)-like site within the ARE/EpRE directed negative regulation of the rGST-Ya gene in vSMCs and HepG2 cells. These results show that ARE/EpRE in rGST-Ya does not function as a positive cis-acting regulatory element in all cell types, and that in the context of the full-length rGST-Ya promoter a C/EBP-like site directs negative regulation of the gene by BaP and related chemicals.

Structural and Functional Characterization of the 5′-Flanking Region of the Rat and Human Cytochrome P450 2E1 Genes: Identification of a Polymorphic Repeat in the Human Gene

Cytochrome P450 2E1 (CYP2E1) is a toxicologically very important enzyme with a high extent of interindividual variability in expression. We sequenced and characterized the 5′-flanking region of the human and rat CYP2E1 genes. The identity between the human and rat sequences (−3.8 kb to +1 kb) was generally between 35 and 60%, and the most similar regions were found in the proximal part of the sequence. Two more distant regions at −1.6 to −2.0 kb and −2.5 to −2.8 kb in the human sequence were also found to have high identity to the rat sequence. A polymorphic repeat sequence in the human gene was found between −2178 to −1945 bp. The common allele (CYP2E1*1C) contained 6 repeats (each 42–60 bp long) and the rare allele (CYP2E1*1D) had 8 repeats with an allele frequency of 1% among Caucasians and 23% among Chinese. The CYP2E1 5′-flanking regions of the human (−3712 bp to +10 bp) and rat (−3685 bp to +28 bp) genes were ligated in front of a luciferase reporter gene and transfected into rat hepatoma Fao and human hepatoma B16A2 cells. Important species specificity was noted in the control of gene expression and regions of negative and positive cis-acting elements were localized. No difference was seen in the constitutive expression between the two polymorphic forms. The importance of this repeat polymorphism for high and low inducible CYP2E1 phenotypes is discussed.

Transcriptional Regulation of the Schizosaccharomyces pombe Malic Enzyme Gene, mae2

The NAD-dependent malic enzyme from Schizosaccharomyces pombe catalyzes the oxidative decarboxylation of L-malate to pyruvate and CO2. Transcription of the S. pombe malic enzyme gene, mae2, was studied to elucidate the regulatory mechanisms involved in the expression of the gene. No evidence for substrate-induced expression of mae2 was observed in the presence of 0.2% L-malate. However, transcription of mae2 was induced when cells were grown in high concentrations of glucose or under anaerobic conditions. The increased levels of malic enzyme may provide additional pyruvate or assist in maintaining the redox potential under fermentative conditions. Deletion and mutation analyses of the 5'-flanking region of the mae2 gene revealed the presence of three novel negative cis-acting elements, URS1, URS2, and URS3, that seem to function cooperatively to repress transcription of the mae2 gene. URS1 and URS2 are also present in the promoter region of the S. pombe malate transporter gene, suggesting co-regulation of their expression. Furthermore, two positive cis-acting elements in the mae2 promoter, UAS1 and UAS2, show homology with the DNA recognition sites of the cAMP-dependent transcription factors ADR1, AP-2, and ATF (activating transcription factor)/CREB (cAMP response element binding).

Cloning and functional characterization of the human norepinephrine transporter gene promoter

The norepinephrine transporter (NET) plays a critical role in brain norepinephrine homeostasis and is a target for antidepressants and drugs of abuse. We have analyzed the 5'-flanking regulatory region of the human NET gene (SLC6A2). Primer extension and 5' RACE revealed a single transcription start site, alternative splicing of exon 1 due to alternate splice donor usage, and a variable splice acceptor of intron 1. A TA-rich motif 35 bp upstream of the transcription start and several potential binding sites for transcription factors including a cAMP response element (CRE)-like motif are present in the 5'-flanking region. A 4.0-kb fragment, which had been fused to the luciferase reporter gene and transiently expressed in a NET+ cell line, displayed both constitutive and inducible promoter activity. Functional analysis by serial deletions revealed several clusters of cell-selective enhancer elements. Our findings indicate that (1) the NET gene promoter is active in NET-expressing cells and the information contained within approximately 4 kb of the 5'-flanking sequence is required to confer its cell-selective expression, (2) the expression of NET is regulated by a combination of positive cis-acting elements operating through a basal promoter defined by a TA-rich motif, and (3) the promoter responds to cAMP-dependent induction. Fusion of the human NET gene promoter to selected genes will facilitate their cell-selective expression in gene transfer strategies.

Cloning and Characterization of the 5′-Flanking Region for the Human Topoisomerase III Gene

The human DNA topoisomerase III (hTOP3) gene encodes a topoisomerase homologous to the Escherichia coli DNA topoisomerase I subfamily. To understand the mechanisms responsible for regulating hTOP3 expression, we have cloned the 5'-flanking region of the gene coding for the hTOP3 and analyzed its promoter activity. The presence of a single transcription initiation site was suggested by primer extension analysis. The hTOP3 gene promoter is moderately high in GC content and lacks a canonical TATA box, suggesting that hTOP3 promoter has overall similarity to promoters of a number of housekeeping genes. Examination of the promoter sequence indicated the presence of four Sp-1 consensus binding sequences and a putative initiator element surrounding the transcription initiation site. Transient expression of a luciferase reporter gene under the control of serially deleted 5'-flanking sequences revealed that the 52-base pair region from -326 to -275 upstream of the transcription initiation site includes a positive cis-acting element(s) for the efficient expression of hTOP3 gene. On the basis of gel mobility shift and supershift assays, we demonstrated that both YY1 and USF1 transcription factors can bind to the 52-base pair region. When HeLa cells were transiently transfected with a mutant construct which had disabled both YY1- and USF1-binding sites, the luciferase activity was greatly reduced, suggesting that these binding elements play a functional role in the basal activation of the hTOP3 promoter. Transfection studies with mutations that selectively impaired YY1 or USF1 binding suggested that both YY1 and USF1 function as activators in the hTOP3 promoter.