Differences In Promoter Activity Research Articles

Quantification of Different Human Alpha Interferon Subtypes and Pegylated Interferon Activities by Measuring MxA Promoter Activation

Alpha interferons (alpha-IFNs) are potent biologically active proteins synthesized and secreted by somatic cells during viral infection. Quantification of alpha-IFN concentrations in biological samples is used for diagnosis. More recently, recombinant IFNs have been used as antiviral, antiproliferative, and immunomodulatory therapeutic agents, and particularly for the treatment of chronic hepatitis C virus infection. For this purpose, IFN has recently been coupled to polyethylene glycol (PEG) to improve the pharmacokinetic properties. The measure of alpha-IFN in biological samples from treated patients could be useful to ensure compliance to therapy and the true IFN activity in relation to viral decay during follow-up. In particular, it could be used to monitor the PEG-IFN concentration in patients treated for hepatitis C virus infection. The most frequently used test is a bioassay based on the antiviral property of the IFN, but the assay is not highly reproducible. Here, we present a reporter test based on MxA promoter activation of chloramphenicol acetyltransferase expression (Mx-CAT). MxA is an antiviral protein induced and tightly regulated by alpha-IFN. The Mx-CAT assay showed good reproducibility of 15% and was suitable to quantify PEG-IFN and numerous other alpha-IFN subtypes as well, despite a differential MxA promoter activation in relation with the subtype. A good correlation was obtained with the reporter assay and a commercial enzyme-linked immunosorbent assay on samples from treated patients. This test could be useful for monitoring IFN therapy of chronically infected hepatitis C virus-infected patients treated with the standard IFN, PEG-IFN, and probably forthcoming recombinant IFNs.

Identification of a New pebp2αA2 Isoform From Zebrafish runx2 Capable of Inducing Osteocalcin Gene Expression In Vitro

The zebrafish runx2b transcription factor is an ortholog of RUNX2 and is highly conserved at the structural level. The runx2b pebp2alphaA2 isoform induces osteocalcin gene expression by binding to a specific region of the promoter and seems to have been selectively conserved in the teleost lineage. RUNX2 (also known as CBFA1/Osf2/AML3/PEBP2alphaA) is a transcription factor essential for bone formation in mammals, as well as for osteoblast and chondrocyte differentiation, through regulation of expression of several bone- and cartilage-related genes. Since its discovery, Runx2 has been the subject of intense studies, mainly focused in unveiling regulatory targets of this transcription factor in high vertebrates. However, no single study has been published addressing the role of Runx2 in bone metabolism of low vertebrates. While analyzing the zebrafish (Danio rerio) runx2 gene, we identified the presence of two orthologs of RUNX2, which we named runx2a and runx2b and cloned a pebp2alphaA-like transcript of the runx2b gene, which we named pebp2alphaA2. Zebrafish runx2b gene and cDNA were isolated by RT-PCR and sequence data mining. The 3D structure of runx2b runt domain was modeled using mouse Runx1 runt as template. The regulatory effect of pebp2alphaA2 on osteocalcin expression was analyzed by transient co-transfection experiments using a luciferase reporter gene. Phylogenetic analysis of available Runx sequences was performed with TREE_PUZZLE 5.2. and MrBayes. We showed that the runx2b gene structure is highly conserved between mammals and fish. Zebrafish runx2b has two promoter regions separated by a large intron. Sequence analysis suggested that the runx2b gene encodes three distinct isoforms, by a combination of alternative splicing and differential promoter activation, as described for the human gene. We have cloned a pebp2alphaA-like transcript of the runx2b gene, which we named pebp2alphaA2, and showed its high degree of sequence similarity with the mammalian pebp2alphaA. The cloned zebrafish osteocalcin promoter was found to contain three putative runx2-binding elements, and one of them, located at -221 from the ATG, was capable of mediating pebp2alphaA2 transactivation. In addition, cross-species transactivation was also confirmed because the mouse Cbfa1 was able to induce the zebrafish osteocalcin promoter, whereas the zebrafish pebp2alphaA2 activated the murine osteocalcin promoter. These results are consistent with the high degree of evolutionary conservation of these proteins. The 3D structure of the runx2b runt domain was modeled based on the runt domain of mouse Runx1. Results show a high degree of similarity in the 3D configuration of the DNA binding regions from both domains, with significant differences only observed in non-DNA binding regions or in DNA-binding regions known to accommodate considerable structure flexibility. Phylogenetic analysis was used to clarify the relationship between the isoforms of each of the two zebrafish Runx2 orthologs and other Runx proteins. Both zebrafish runx2 genes clustered with other Runx2 sequences. The duplication event seemed, however, to be so old that, whereas Runx2b clearly clusters with the other fish sequences, it is unclear whether Runx2a clusters with Runx2 from higher vertebrates or from other fish.

Analysis of Promoters Recognized by HrpL, an Alternative σ-Factor Protein from Pantoea agglomerans pv. gypsophilae

HrpL, an alternative sigma factor, activates the transcription of the Hrp regulon by its binding to a common "hrp box" promoter. Based on computational techniques, the hrp box previously was defined as a consensus bipartite cis element, 5'-GGAACC-N(15-16)-CCACNNA-3'. The present report combines a quantitative in vivo assay for measuring Hrp promoter activity with site-specific mutagenesis to analyze the effect of consensus and nonconsensus nucleotides on promoter activity. The analysis was carried out with Hop effectors of the tumorigenic bacterium Pantoea agglomerans pv. gypsophilae, in which HrpL is indispensable for gall formation. Mutational analysis indicates that the hrp box consensus can be divided into crucial and noncrucial nucleotides. The first 5 nucleotides (nt) of the--35 consensus motif (GGAAC) and the 3 nt of the--10 motif (ACNNA) are crucial, whereas other consensus and adjacent nonconsensus nucleotides exert a significant effect on the promoter's strength. With spacing of 13 or 17 nt between the two motifs, significant activity was still retained. Gel shift assays indicated that deletion of GG from the--35 consensus motif eliminated HrpL binding, whereas mutations in the--10 consensus motif or modification of the spacing, which eliminates promoter activity, did not elicit any effect. The degeneracy in Hrp promoters of four hrp and type III effector genes of P agglomerans pv. gypsophilae indicated significant differences in promoter activity, whereas increasing the promoter strength of the Hop effector, HsvG, resulted in overexpression of gall formation.

437. Alternative Strategies To Deliver Multiple shRNAs in Order to Simultaneously Inhibit Multiple Targets

RNA interference is a new promising technology for therapeutic drug development. It can be initiated either by chemically synthesized double-stranded RNA delivered to a cell, or by transcription of a short hairpin RNA (shRNA) in the cell nucleus from a DNA template. We are interested in developing RNAi therapies for HCV, which is difficult infection to treat due to the generation of viral escape mutants. RNAi is uniquely positioned to address this issue because it has the ability to simultaneously inhibit multiple viral targets. This can be accomplished by delivering several shRNAs that target different sequences of the viral genome. One approach to accomplish this is to include several promoter-shRNA cassettes in a single DNA vector. The disadvantages of this strategy are the possible interference of the promoters, potential differences in promoter activity, and vector size limitations. Alternatively, multiple shRNAs could be expressed from a single promoter as a long shRNA precursor. Processing of this pre-shRNA by Drosha/Dicer complex may result in the generation of multiple siRNAs; however it may be difficult to optimize the pre-shRNA sequences for correct processing, and there is the potential for toxic effects attributed to the generation of a long double-stranded RNA. In this work we present a third alternative to express multiple shRNAs from a single promoter which includes the tandem positioning of short shRNAs (19-20 nucl) separated by short spacers. We identified several individual shRNA sequences that efficiently inhibit replication of HCV in the replicon system. We then combined these validated shRNAs in a triple construct under the expression of a single promoter and demonstrated functional activity of each of the individual shRNAs against their cognate targets. Results comparing these different strategies and their implication for shRNA-based drug development will be discussed.

854. Optimization of AAV Serotype and Promoter for Increased Distribution of Expression in Both Somatic and Central Nervous Systems of Mice

The characterization of adeno-associated virus (AAV) vector biodistribution, as measured by transgene expression, may be affected by many factors including the presence of viral receptor molecules on cell surfaces, cell-type related intracellular trafficking, the ability of the cell to form double-stranded DNA from the single-stranded AAV genome, and the cell-type specificity of the promoter. The optimal evaluation of biodistribution eliminates or controls as many of these variables as possible. We have approached this by using self-complementary AAV vectors (scAAV) and combiniations of promoters with activity in a very broad range of cell-types, thus narrowing the variable to the intrinsic capacity of the cells to take up and traffic the vector to the nuclei. Self-complementary AAV serotype 1 (AAV1) or 2 (AAV2) vectors (11011 DNase resistant particles) expressing green fluorescent protein (GFP) from either the human cytomegalovirus (CMV) promoter, or a murine small nuclear RNA promoter (U1a), were delivered into adult mice by an intravenous (IV) injection, 10 minutes after an IV infusion of mannitol. The mannitol enhances delivery of vector across the blood brain barrier. Transgene expression was detected by immunofluorescence in multiple somatic and CNS tissues using a polyclonal antibody against GFP, at 1 and 3 months postinjection. Both AAV1 and AAV2 vectors mediated transgene expression in liver, kidney, heart, spleen, intestine, skeletal muscles, and lung, as well as a global distribution of GFP-positive neuronal and non-neuronal cells in the brain and spinal cord. In the CNS, major targets of AAV1 were glial cells, while AAV2 targeted mainly neurons. Endothelial cells of capillaries in both somatic and CNS tissues were efficiently transduced by AAV1, but not AAV2. In kidney, AAV2, but not AAV1, targeted medullar tubule cells. In the white pulp of spleen, AAV1 trasduced lymphatic sheaths while AAV2 infected B-lymphocytes. Promoter activity also contributed to the observed biodistribution of AAV vector expression. The CMV promoter was more efficient than U1a in neurons of the CNS and in endothelial cells in all tissues. The U1a promoter functioned more efficiently in hepatocytes in liver and tubule cells in kidney. The U1a promoter, but not CMV, was active in lymphatic tissues in spleen, while CMV, but not U1a, functioned in glial cells in the CNS. Our observations provide a basis for matching the specific cell-type tropism of different AAV serotypes with the specific transcriptional activity of different promoters to control biodistribution. A cocktail of serotypes containing optimal promoters will afford the broadest vector expression for the treatment of conditions such as lysosomal storage diseases with widespread somatic and CNS pathology.

SLC18A2 promoter haplotypes and identification of a novel protective factor against alcoholism

The vesicular monoamine transporter 2 (VMAT2, SLC18A2) takes up cytosolic monoamines into intracellular secretory vesicles, preventing their neurotoxicity in the cytosol and discharging them into extracellular space by exocytosis. It has been shown that one-copy deletion of the VMAT2 gene increases locomotion activity significantly in response to drug treatments and dopamine neuron death rate in response to neurotoxin treatments in knockout mice. Little is known about promoter polymorphisms and their influence on SLC18A2 promoter activity. We have re-sequenced a 17.4 kb DNA in the SLC18A2 promoter region for Caucasians and revealed 47 polymorphisms that confer 13 haplotypes. One of the haplotypes reaches a frequency as high as 65%, likely due to positive selection. In vitro analysis showed a 20% difference in promoter activity between two frequent haplotypes and identified some of the polymorphisms that influence promoter activity. Four haplotype-defining single nucleotide polymorphisms (hdSNPs) can define the frequent haplotypes and by genotyping these hdSNPs, we find that haplotypes with -14234G and -2504C of SLC18A2 promoter region represent a protective factor against alcoholism (P = 0.0038 by Fisher's exact tests). Therefore, SLC18A2 promoter haplotypes defined here create a foundation for transcriptional characterization of individuality and for association study on monoamine-related human diseases.

Species‐Specific Regulation of the α‐2(I) Procollagen Gene by Proximal Promoter Elements

Transcriptional regulation of the human alpha 2(I) procollagen proximal promoter involves the interaction of trans-acting factors at the inverted CCAAT box (G/CBE) located at position -80 and an adjacent GGAGGCCC-box at -70. Both these elements have previously been shown to be essential for activity of the human promoter. This study investigated nucleotide differences at three sites (-74, -72 and -71) between the human and mouse promoters that were sufficient to abolish trans-acting factor binding with the mouse sequence (GGAGACGT). Two distinct DNA-protein interactions were detected on the human -107/+54 promoter fragment while a single interaction was observed at the equivalent mouse promoter. One of these factors is the CCAAT-binding factor (CBF) and it's binding was observed on both the human and mouse promoters. Although the GGAGGCCC DNA-binding element was not detected on the mouse promoter, GGAGGCC-binding proteins were present in mouse nuclear extracts as observed by their interaction with the human promoter. Functional analysis of the human and mouse -343/+54 and -107/+54 promoter regions revealed significant differences between species; the human constructs having higher activity than the mouse. The differences in promoter activity between species may in part be a result of the nucleotide differences in the GGAGGCCC-box. Mutations in this region of the human -107/+54 promoter prevented DNA-protein interaction and lowered promoter activity. These results support the hypothesis that the GGAGGCCC-box in the human alpha 2(1) procollagen promoter has a regulatory function and that there exists a species-specific difference in transcription factor binding and regulation of the gene.

Isolation, sequencing, and functional analysis of the TATA-less human ATPase II promoter

Multiple lines of evidence indicate that the P-type Mg 2+-ATPase, termed ATPase II, could play an important role in apoptosis. With the long-term objective of studying the regulation of this protein during apoptosis, we delineated the exon–intron organization of the human ATPase II gene (within chromosome 4). Subsequently, we used RNA ligase-mediated rapid amplification of cDNA ends to identify a major transcription start site at position −143 with respect to the translation start site. Luciferase reporter analysis of a 1.2-kb 5′-flanking sequence (−1222 to +94 with respect to the transcription start site) revealed strong promoter activity in three human cell lines, human oligodendroglioma (HOG), SHSY5Y (hybrid neuroblastoma), and EA.hy926 (endothelial cell line). Serial deletions from the 5′ end of this sequence up to nucleotide −291 yielded some decrease in activity only in the EA.hy926 cells. Further deletion to −217 caused a drastic decrease in activity in all three cell lines, but a −148 fragment showed preferential reduction in activity in the EA.hy926 cells. The promoter activity was nearly equal in two sequence variants of the promoter, one of which (designated as Variant 2) contained a 15-bp direct repeat within a GC-rich region. Additionally, there were several single base-pair changes from the sequence reported by the human genome project. Despite the presence of enhancer/repressor elements, such as Sp1 and NFκB, relatively small differences in promoter activity were observed in the three cell lines. However, it is likely that such sequence elements could cause major regulation of promoter activity in cells subjected to conditions that trigger apoptosis. The ATPase II promoter sequence will provide valuable clues to the regulation and role of the ATPase II protein.

Identification of alternative promoter usage for the matrix Gla protein gene

Recent cloning of the Xenopus laevis (Xl) matrix Gla protein (MGP) gene indicated the presence of a conserved overall structure for this gene between mammals and amphibians but identified an additional 5'-exon, not detected in mammals, flanked by a functional, calcium-sensitive promoter, 3042 bp distant from the ATG initiation codon. DNA sequence analysis identified a second TATA-like DNA motif located at the 3' end of intron 1 and adjacent to the ATG-containing second exon. This putative proximal promoter was found to direct transcription of the luciferase reporter gene in the X. laevis A6 cell line, a result confirmed by subsequent deletion mutant analysis. RT-PCR analysis of XlMGP gene expression during early development identified a different temporal expression of the two transcripts, strongly suggesting differential promoter activation under the control of either maternally inherited or developmentally induced regulatory factors. Our results provide further evidence of the usefulness of nonmammalian model systems to elucidate the complex regulation of MGP gene transcription and raise the possibility that a similar mechanism of regulation may also exist in mammals.

Identification of functional SNPs in the 5-prime flanking sequences of human genes

BackgroundOver 4 million single nucleotide polymorphisms (SNPs) are currently reported to exist within the human genome. Only a small fraction of these SNPs alter gene function or expression, and therefore might be associated with a cell phenotype. These functional SNPs are consequently important in understanding human health. Information related to functional SNPs in candidate disease genes is critical for cost effective genetic association studies, which attempt to understand the genetics of complex diseases like diabetes, Alzheimer's, etc. Robust methods for the identification of functional SNPs are therefore crucial. We report one such experimental approach.ResultsSequence conserved between mouse and human genomes, within 5 kilobases of the 5-prime end of 176 GPCR genes, were screened for SNPs. Sequences flanking these SNPs were scored for transcription factor binding sites. Allelic pairs resulting in a significant score difference were predicted to influence the binding of transcription factors (TFs). Ten such SNPs were selected for mobility shift assays (EMSA), resulting in 7 of them exhibiting a reproducible shift. The full-length promoter regions with 4 of the 7 SNPs were cloned in a Luciferase based plasmid reporter system. Two out of the 4 SNPs exhibited differential promoter activity in several human cell lines.ConclusionsWe propose a method for effective selection of functional, regulatory SNPs that are located in evolutionary conserved 5-prime flanking regions (5'-FR) regions of human genes and influence the activity of the transcriptional regulatory region. Some SNPs behave differently in different cell types.

Promoter analyses of SCC antigen genes

SCC antigen (SCCA) has been used as a tumor marker for squamous cell carcinoma. Analyses of the SCCA1 and SCCA2 genes, which are almost identical, and their promoters have been reported. Recently it was found that both SCCAs were stimulated by interleukin (IL)-4 and IL-13. Here we analyzed the promoter activity of both SCCAs in the 5′-flanking region, exon 1, and intron 1 to evaluate a putative STAT6 binding site. The addition of intron 1 to the luciferase assay constructs including the 5′-flanking region significantly augmented the promoter activity of both SCCA1 and SCCA2. Furthermore, deletion analyses of intron 1 revealed that a 50-bp fragment of intron 1 that includes putative STAT6 binding site was responsible for the increased promoter activity. Although the sequences of SCCA1 and SCCA2 are very similar in the 5′-flanking region, the analysis of the −337 single nucleotide polymorphism of SCCA2 indicated that this polymorphism may underlie the difference in promoter activity between SCCA1 and SCCA2.

Identification of cis-Active Elements in Ustilago maydis mig2 Promoters Conferring High-Level Activity During Pathogenic Growth in Maize

The Ustilago maydis mig2 cluster comprises five highly homologous genes that display a pronounced plant-specific expression profile. A 350-bp mig2-5 promoter fragment contained all elements sufficient to confer differential promoter activity. Mutational analysis of this region, fused to the green fluorescent protein reporter gene, allowed dissecting core promoter elements required for high-level promoter activity from elements conferring inducible expression in planta. In particular, the presence of several 5'-CCA-3' motifs within a short stretch of the mig2-5 promoter was decisive for inducible promoter activity. On this basis, we reconstituted an artificial promoter whose inducible activity specifically relied on multiple CCA motifs. In addition, we identified a novel mig2 homologous gene, mig2-6, that is not part of the mig2 cluster, but displayed the strongest differential expression profile among mig2 genes. The deletion of all six mig2 genes did not compromise the ability to induce tumor formation in infected maize plants. Comparative sequence analysis including the mig2-6 promoter revealed an over-representation of the consensus motif 5'-MNMNWNCCAMM-3'. We discuss putative transcriptional activators involved in mig2 regulation.

Demonstration of differential virulence gene promoter activation in vivo in Bordetella pertussis using RIVET

Bordetella pertussis, the etiologic agent of whooping cough, causes disease by employing an array of virulence factors controlled by the BvgA-BvgS two-component signal transduction system. Regulation by this system has been extensively characterized in vitro, where bvg-activated genes are repressed in a process known as phenotypic modulation. Differential regulation of these genes by the response regulator BvgA results in promoters that are activated early, middle, or late after being released from modulation. However, the in vivo environmental signal and regulation pattern has not been described. In order to investigate BvgAS-mediated regulation of B. pertussis virulence factors in vivo using the mouse aerosol challenge model, we have adapted the recombinase-based in vivo technology (RIVET) system for use in B. pertussis. We have demonstrated that these strains show resolution during in vitro growth under non-modulating conditions. In addition, we have demonstrated that modulating strains by growth on media containing MgSO4 does not affect virulence in the mouse aerosol challenge model. We have therefore used the RIVET system to reveal the time-course of gene expression in vivo for selected B. pertussis virulence factors (cya, fha, prn and ptx). Our data indicate that this method can be effectively used to monitor and compare in vivo and in vitro gene expression in B. pertussis, and that temporal regulation patterns previously observed in vitro are mirrored in vivo.

P0358 FUNCTIONAL ANALYSIS OF THE TRANSCRIPTIONAL REGULATION OF THE HUMAN BILE SALT EXPORT PUMP GENE

Introduction: The bile salt export pump, BSEP, encoded by ABCB11, is the major bile salt transporter of human liver. ABCB11 is mutated in a form of low gamma-GT progressive familial intrahepatic cholestasis (PFIC), known as BSEP deficiency. It has been documented that transcription of ABCB11 is positively regulated by bile salts. However, little is known about which other transcription factors are involved in controlling the expression of the human gene, as well as the effect of exogenous compounds on its transcription. This study was designed to investigate these questions. Methods: 5′ and 3′ Rapid Amplification of cDNA Ends (RACE) were performed using total RNA from normal liver and various cholestatic livers as a template. Various ABCB11 promoter fragments were PCR amplified from genomic DNA and cloned into pGEM-Teasy by T-A cloning for sequencing purposes. They were then sub-cloned to the Firefly luciferase vector pGL3-basic (Promega). 2.7 mug of each promoter construct was transfected into 9 ×105 HepG2, HEK 293, and CaCo2 cells in triplicate using Tfx-20 (Promega), with a lipid:DNA ratio of 3:1. 0.3 μg of the Renilla luciferase vector pRL-TK was co-transfected with each sample to serve as a transfection efficiency control. In the case of HepG2 cells, 50 muM oestrogen or progesterone was added to the culture medium. Cells were harvested after 48 hours and the Dual-Luciferase Reporter Assay System (Promega) was used to determine the values of Firefly and Renilla luciferase in each sample. Protein concentrations were determined using a Bradford assay (Bio-Rad). Results were presented as Firefly luciferase units corrected to Renilla luciferase units per mg of protein. Results: 5′ RACE identified 3 transcription start sites, two of which are novel. 3′ RACE yielded products which on sequencing determined that the ABCB11 polyadenylation site is 1.365 kb downstream from the published mRNA sequence. The ABCB11 promoter fragment/Firefly luciferase constructs identified a putative silencer in the −845 to 670 bp region in HepG2 cells. The addition of oestrogen and progesterone caused no decrease in promoter activity in these cells. However, differential promoter activity was detected in CaCo2 and HEK293 cells. Conclusion: These preliminary findings are in keeping with both enhancer and silencer elements in the ABCB11 promoter. DNA footprinting and EMSA are underway to identify transcriptionally active molecules binding to the promoter. The effect of drugs on promoter activity is also being assessed. This work provides an insight into the transcriptional regulation of ABCB11 under normal and cholestatic conditions and an experimental system to assess the effect of ABCB11 promoter sequence variants found in cholestatic patients.

Functional characterization of MDR3 promoter mutations detected in intrahepatic cholestasis of pregnancy

Background Intrahepatic cholestasis of pregnancy (ICP) is a liver disorder associated with risk of intrauterine fetal death and prematurity. There is increasing evidence that genetically determined dysfunction in the canalicular ABC transporter MDR3 (multidrug resistance protein 3) is a risk factor for ICP. The aim of this study was to functionally characterize newly detected MDR3 promoter variants in ICP patients. Methods Luciferase reporter constructs of the human MDR3 5'-regulatory regions were generated by PCR and ligated into the pGL3-Basic vector. DNA from three different ICP patients (covering the wildtype sequence and eight promoter mutations, three of which were ICP-specific: intron -4: C(-10)T; exon-3: A(-358)G; intron-3: C(-31)T) was used. The human hepatocyte derived cell lines Huh7 and HepG2 were transiently transfected with reporter constructs to assay for promoter activity. Results In both cell lines, no differences in promoter activity of the mutated constructs were observed with respect to the promoter containing the reference sequence. In HepG2 cells, activity of the two mutated constructs amounted to 96% and 103% of reference activity, respectively. In Huh-7 cells activity were 90% and 85% of reference activity, respectively. Conclusion Results indicate that the tested promoter mutations do not change the activity of the MDR3-promoter. They therefore do not seem to play a pathophysiological role in ICP development. Clinical Pharmacology & Therapeutics (2004) 75, P16–P16; doi: 10.1016/j.clpt.2003.11.061

Left-handedly curved DNA introduced into episomes can activate transcription in a TATA box-dependent manner

Using a transient transfection assay system, we have recently demonstrated that curved DNA that mimics a negative supercoil can activate transcription by influencing chromatin structure. To know whether this phenomenon is general, we investigated the effect of the DNA curvature on transcription in an episomal assay system. It was confirmed that the left-handedly curved DNA can activate transcription even in the episomal system. Furthermore, it was found that conversion of the TATA sequence in the reporter constructs from the original non-canonical sequence to the canonical one considerably elevated transcription from the control template and, as the result, diminished the difference in promoter activity between the control reporter and the reporters containing the left-handedly curved DNA, indicating that transcriptional activation by the DNA curvature is more evident when the TATA box is not optimized.

Transcriptional regulation of the human LINE-1 retrotransposon L1.2B.

Although LINE-1 (L1) sequences constitute the most important family of retrotransposons in the human genome, their transcriptional regulation is poorly understood. Specifically, their unusual internal promoter is incompletely characterized. Current promoter prediction programs fail to identify the promoter in the 5'UTR of the active LINE-1 element L1.2B. Experimental investigation of this promoter using reporter gene assays in various human and murine cell types confirmed that the promoter consists of two segments, and demonstrated that the distal portion is essential for cell-type-independent activity. No differences in promoter activity were found between normal and transformed cells. The complete promoter was shown to possess approximately 20% of the activity of the strong early promoter of cytomegalovirus, and to be capable of directing the expression of levels of p53 sufficient to kill normal and transformed human cells. Thus, active LINE-1 elements contain highly active promoters capable of driving cell-type-independent expression, which are of potential use in mammalian expression constructs. In vitro methylation of the promoter at HpaII sites decreased its activity independently of cell type, but this repression was alleviated in MBD2-/- cells. Surprisingly, mutation of specific HpaII sites was also found to reduce promoter activity. Thus, efficient repression of the L1.2B promoter by DNA methylation may involve MBD2 binding, but at least one HpaII site also appears to be involved specifically in transcriptional activation. Since neither promoter activity nor the efficiency of repression by methylation differed between normal and tumor cells, the re-activation of LINE-1 sequences observed in tumor cells is probably caused by hypomethylation of the promoter.

Identification of a short PIASx gene promoter that directs male germ cell-specific transcription in vivo

PIASx gene encodes two SUMO E3 ligases that are highly expressed in the testis. We have isolated and analyzed the promoter of the murine PIASx gene. Electrophoretic mobility shift assays with testicular nuclear extracts showed that the proximal promoter forms a major DNA–protein complex containing Sp1, Sp2, and Sp3 transcription factors. Reporter gene assays in cultured cells indicated that a fragment comprising nucleotides from −168 to +76 relative to transcription start site is sufficient for basal promoter activity in cultured cells, but these in vitro assays failed to reveal clear differences in promoter activity between testis- and non-testis-derived cell lines. Interestingly, however, the proximal promoter encompasses the elements necessary for a testis-specific transcription in vivo, as it directed β-galactosidase expression exclusively to male germ cells in transgenic mice. In conclusion, we have characterized the minimal PIASx promoter that can be used for highly specific targeting of transgene expression to male germ cells.

Analysis of IL-10 gene variants in rheumatoid arthritis

IL-10 is known for its anti-inflammatory and regulatory function on different cell types involved in the pathogenesis of rheumatoid arthritis. Taking advantage of a stable intermediate phenotype (IL-10 induced by lipopolysaccharidestimulated blood cultures), large interindividual differences in IL-10 production were found. In two separate large studies including a total of > 50 twins, 70% of the variation between individuals could be explained by genetic factors. Differences in IL-10 protein production were present at the level of mRNA production, and analysis of the half-life of mRNA indicated that differences in transcription accounted for the interindividual differences. To test whether the different haplotypes were transcribed at a different rate, allele-specific transcript quantification was performed, and preliminary data indicated that some donors exhibited a 1:1 region of the respective mRNA species, and some donors a 1:1.2 ratio. To determine the length of the genomic region associated with different production of IL-10, eight single nucleotide polymorphisms (SNPs) and two CA repeats in the IL-10 gene, and six SNPs in the IL-10 homologues IL-19, IL-20 and IL-24 were analysed in 53 families in relation to IL-10 production. The preliminary data suggest that the region associated with differences in IL-10 production is a region of limited size around the IL-10 gene. Transient transfection assays of the -1413 to +31 fragments of the different haplotypes of the 5' region of the IL-10 gene in the myeloid cell lines U937 and Monomac6, in the B-cell line RAJI and in the T-cell line Jurkat showed no difference in promoter activity in response to stimulation for any of the haplotypes. However, a SNP outside this region, the -A2849G SNP, was associated with differences in IL-10 production. Next, a higher rate of joint destruction was observed in patients with the genotype associated with high IL-10 production (mean joint damage score of 20 vs 7; P < 0.001), most probably due to higher autoantibody titers in patients with the high IL-10 genotype.

Polymorphisms in the Promoter of the Human APP Gene

Missense mutations in the amyloid precursor protein (APP) gene cause early-onset Alzheimer disease (AD). However, little is known regarding the effects of polymorphisms in regulatory sequences of APP on AD susceptibility. To identify polymorphisms in the APP promoter, to test these for associations with AD, and to assess their influence on APP promoter activity in transfected cells. Community study of 1013 people of white, African American, or Caribbean Hispanic ethnicity, 65 years and older, residing in northern Manhattan. The diagnosis of AD was established by stringent criteria, with multiple follow-up examinations over 7 years. We identified 2 polymorphisms in the APP promoter: a rare G-->C variant at -9 and a frequent G-->C variant at +37 relative to the transcription start site. The +37C allele was most frequent in African American patients (18% frequency), followed by Caribbean Hispanic patients (10%) and white patients of European descent (3%). This allele was overrepresented among patients with AD compared with elderly controls (odds ratio [OR], 1.57; 95% confidence interval [CI], 1.08-2.27 in the combined ethnic groups), but this was not significant after adjusting for age, sex, and education (OR, 1.41; 95% CI, 0.93-2.12). A stronger association was found in participants lacking any apolipoprotein-E epsilon4 allele (OR, 2.12; 95% CI, 1.36-3.32 [univariate analysis]; OR, 2.08; 95% CI, 1.26-3.45 after adjusting for age, sex, and education). The -9C allele was not frequent enough to be evaluated for a disease association. Both variants were tested in promoter-reporter assays in U-87 glioma cells, and no differences in promoter activity were detected. The -9G/C and +37G/C APP promoter polymorphisms are unlikely to contribute strongly to AD susceptibility or to cause major differences in APP expression, but the +37C allele warrants further study for association with AD in larger population samples.