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Results for “Enhancer Elements, Genetic”

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PubMed2006

Non-identical patterns of proviral insertions around host transcription units in lymphomas induced by different strains of murine leukemia virus.

In a small sample of 57 retrovirus integration sites (RISs) isolated from 23 end-stage lymphomas induced in NMRI mice by the B-lymphotropic Akv wt or an enhancer mutant hereof, Akv1-99, we identified 14 novel RISs and defined 9 novel CISs (common insertion sites). Moreover, when comparing with RISs from tumors induced by the T-lymphomagenic SL3-3, we observed that SL3-3 targets RefSeq promoter regions with a significantly higher frequency than Akv/Akv1-99 and in an orientation-dependent way. Altogether, our results strongly emphasize the importance of host genetic background and virus type for retroviral insertion mutagenesis screens and suggest that different types of MLV may favor specific genomic regions and orientations in order exert optimal effect on target gene expression during lymphoma induction and development.

Animals↗

PubMed2005

Identification of an epithelial-specific enhancer regulating ESX expression.

The Ets transcription factor, ESX, exhibits a unique pattern of epithelial-restricted expression and transactivates genes involved in epithelial differentiation and cancer. The aim of this study was to determine the underlying genetic basis for epithelial-specific expression of ESX. We have identified a 30bp ESX enhancer sequence (EES) approximately 3 kb upstream of the proximal promoter. This region displays enhancer activity in an epithelial-specific manner and deletion of this region abrogates ESX gene transcription. An EES binding protein complex (EBC) was identified through electrophoretic mobility shift assays whose degree of EES binding correlated well with endogenous ESX levels in epithelial cells and was regulated by epithelial differentiation. Understanding the regulation of this element will lend insight into mechanisms of epithelial differentiation and the etiology of breast cancer and may provide novel targets for cancer therapeutic intervention.

Amino Acid Sequence↗

PubMed2025

p300/CBP is an essential driver of pathogenic enhancer activity and gene expression in Ewing sarcoma.

The t(11;22) translocation encodes the EWS::FLI1 fusion oncoprotein which is the primary driver of Ewing sarcoma. EWS::FLI1 creates unique, de novo pathogenic enhancers that drive gene expression and are a central mechanism of oncogenesis. Which chromatin regulatory proteins are critical to this mechanism is understudied. Here, we perform a comparative analysis of the function of the chromatin complexes MLL3/4 and p300/CBP in EWS::FLI1-mediated gene regulation. Using EWS::FLI1 degradation models, we define a subset of EWS::FLI1-sensitive enhancers whose activity correlates with p300/CBP function. We perturb both chromatin complexes to establish that in contrast to MLL3/4, p300/CBP is a critical regulator of EWS::FLI1-driven enhancer activity and downstream gene expression. We also show that p300/CBP small-molecule inhibition decelerates tumor growth in vivo. Our work highlights the context-dependent nature of chromatin protein activity at oncogenic enhancers and reveals p300/CBP as an important regulator of Ewing sarcoma.

Sarcoma, Ewing↗

PubMed2006

Single nucleotide polymorphism in 5'-flanking region reduces transcription of surfactant protein B gene in H441 cells.

Surfactant protein (SP)-B is expressed in a cell-specific manner and is essential for surfactant function and survival. Abnormal surfactant function occurs in humans and genetically engineered mice with SP-B levels well below 50% of normal. SP-B mRNA levels vary in fetal lung explants among individuals, possibly due to genetic variety. Polymorphisms within the SP-B gene have been described extensively; however, some of their functional relevance remains unclear. Mutations within the SP-B gene may affect mRNA content, but altered gene transcription or mRNA-stability has not been clearly demonstrated. We characterized a single nucleotide polymorphism (SNP) found in the upstream enhancer of SP-B, consisting of a single base pair change in the consensus sequence of the most downstream-located thyroid transcription factor 1 binding element in the upstream enhancer of the SP-B 5'-flanking region and located at position 384 upstream of the transcriptional start site of the SP-B gene. In a small patient population (n = 53) we found 70% were homozygous for the wild type (WT), one individual (2%) was homozygous for the polymorphism (Pm), and 28% were heterozygous. To further elucidate possible functions we performed electromobility shift assays with extracts from H441 cells that showed a reduced binding affinity of the mutated sequence compared with WT. In reporter gene assays the Pm caused a reduction of 53% in transcriptional activity compared with WT in transfected H441 cells. Stimulation of these constructs with retinoic acid resulted in enhanced reporter gene activity of both constructs. After stimulation the Pm still exhibited a reduced activity compared with the WT sequence. We conclude that the described SNP causes differences in SP-B transcriptional activity and thus may contribute to individually different SP-B mRNA levels.

5' Flanking Region↗

PubMed1990

Novel and useful properties of a chimeric plant promoter combining CaMV 35S and MAS elements.

The CaMV 35S and Ti plasmid mannopine synthetase (mas) promoters are commonly used by plant genetic engineers. To combine their useful properties, we constructed hybrid promoters incorporating elements from both. These promoters were spliced to the beta-glucuronidase reporter gene and introduced into tobacco and tomato plants by Agrobacterium cocultivation. T1 and T2 transgenic plant populations transformed with different constructs were assayed for the marker enzyme. Comparisons were made based on the range of expression levels found for each promoter construct. We found that a hybrid promoter incorporating the mas region from +65 to -301 and the 35S enhancer region from -90 to -941 had new and interesting properties. This promoter, called Mac, expressed gus at a level three to five times that expressed by a double 35S promoter in the leaves, and 10 to 15 times in hypocotyls and roots. The Mac promoter, however, showed only marginal wound inducibility. Five- to seven-fold wound induction required the presence of the region from -301 to -613 of mas. Reiteration of the 35S enhancer region, from -90 to -430, behind the 35S TATA box region or the mas +65 to -301 region had a smaller effect on expression, ranging from equal to twice the level of the single enhancer control.

Cloning, Molecular↗

PubMed1991

The phosphorylated form of the enhancer-binding protein NTRC has an ATPase activity that is essential for activation of transcription.

The NTRC protein of enteric bacteria is an enhancer-binding protein that activates transcription in response to limitation of combined nitrogen. NTRC activates transcription by catalyzing formation of open complexes by RNA polymerase (sigma 54 holoenzyme form) in an ATP-dependent reaction. To catalyze open complex formation, NTRC must be phosphorylated. We show that phosphorylated NTRC has an ATPase activity, and we present biochemical and genetic evidence that NTRC must hydrolyze ATP to catalyze open complex formation. It is likely that all activators of sigma 54 holoenzyme have an ATPase activity.

Adenosine Triphosphatases↗

PubMed2003

Finding genes for SLE: complex interactions and complex populations.

Many years of work, multiplex family collection and endless genotyping finally give fruit. The original aim cannot be lost. The aim is not only to identify mutations involved in susceptibility for systemic lupus erythematosus (SLE) but to elucidate the disease pathogenesis as well. After genetics comes the biology. We review our recent findings on the genetics of lupus, provide possible mechanisms for disease susceptibility and present some facts on the problematic of identifying susceptibility mutations for complex diseases in complex human populations.

Antigens, CD↗

PubMed2004

Conserved enhancer in the serum response factor promoter controls expression during early coronary vasculogenesis.

Serum response factor (SRF) is a transcription factor required for mesoderm formation in the developing mouse embryo that is important for myogenic differentiation, including notably, the differentiation of the proepicardial organ (PEO) into coronary vascular cells during early development. To identify regulatory sequences that control SRF expression during early mouse development, we used a novel transgenic approach to study the role of conserved noncoding DNA sequences (CNCS) in the SRF gene. Embryonic stem (ES) cells containing a targeted single-copy of putative SRF regulatory sequences were used to directly generate transgenic embryos by tetraploid aggregation. Because the ES cell-derived targeted embryos are genetically equivalent, except for the putative regulatory sequence of interest, differences in transgene expression can be attributed directly to these sequences. Using this approach, we identified an E-box/Ets containing 270-bp cis-acting module in the SRF promoter that mediates expression in the PEO. Reporter transgenes containing this module express in derivatives of the PEO that give rise to the coronary vasculature, but do not express in the PEO-derived epicardium. These results are the first reported in vivo analysis of SRF regulatory elements that control expression during early development. Using this reporter module and this approach, it should be possible to begin to elucidate molecular mechanisms involved in the differentiation of coronary vasculature progenitor cells, as well as identify additional SRF regulatory elements important during mammalian development.

Animals↗

PubMed1991

A potential approach for gene therapy targeting hepatoma using a liver-specific promoter on a retroviral vector.

Recent technological advances made in molecular biology and in vitro culture of human and other mammalian cells have led to broad medical and scientific acceptance of the feasibility of gene therapy for genetic diseases. Cancer might practically be one of the attractive targets for such therapy. For the treatment of cancer, it is important to manipulate the gene of interest such that it is expressed solely in cancer cells. We have developed a tissue-specific gene expression system, based on a tissue-specific promoter on a retroviral vector. A murine ecotropic retroviral vector was constructed in which the Escherichia coli beta-galactosidase gene served as a reporter; it was expressed under control of the albumin enhancer element and promoter. The tissue specificity of this vector was first assessed in vitro, and beta-galactosidase activity was detected exclusively in hepatoma cell lines. This recombinant retrovirus was injected directly into a subcutaneous tumor composed of transplantable murine MH-134 hepatoma cells, and expression of the gene was observed in vivo. Then this recombinant retrovirus was injected via the spleen or directly into the liver, resulting in the gene expression in dividing hepatocytes in partially hepatectomized mice, but not in nondividing hepatocytes in normal mice. Gene transfer specific to dividing hepatocytes and expression by means of retroviral vectors should possess high potential for selective elimination of hepatoma cells surrounded by nondividing normal hepatocytes.

Animals↗

PubMed1991

C-myc as an inducer of polycystic kidney disease in transgenic mice.

In this study, a genetic model of polycystic kidney disease (PKD) has been produced in transgenic mice bearing the murine c-myc gene driven by the SV40 enhancer and the adult beta-globin promoter. These animals reproducibly develop PKD and die of renal failure. The phenotype appears to result from the overexpression of c-myc in the renal tubular epithelium and consequent abnormal cell proliferation. These transgenic mice represent a genetic model of PKD which bears similarities to human autosomal dominant PKD (ADPKD) with respect to renal morphology, renal functional alterations and dominant transmission. Study of these transgenic mice may offer valuable insights into the pathogenesis of PKD.

Animals↗

PubMed1987

Prediction of 'hot spots' in SV40 enhancer and relation with experimental data.

A theoretical method for prediction of the points in the sequence which are relevant for its biological function, the so-called 'hot spots', is presented. This method is based on significant correlation between the spectrum of numerical presentation of any genetic sequence and its biological function. One number corresponds to the particular nucleotide, thus forming a numerical sequence. The 'hot spot' prediction has been tested on the SV40 enhancer as a model system. The SV40 enhancer was chosen because of existing detailed data about activities of systematically obtained mutants. These results have been compared with results obtained theoretically.

Base Sequence↗

PubMed2004

Reduced Myc overexpression and normal B-cell differentiation mediate resistance to avian leukosis virus lymphomagenesis.

Avian leukosis virus (ALV) induces bursal lymphoma in tumor-susceptible chicken strains after proviral integration within the c-myc gene, and subsequent expansion of Myc-overexpressing lymphocytes within transformed follicles. Line 6(3) strain chickens are resistant to ALV tumorigenesis, largely failing to develop Myc-transformed follicles, although they show similar levels of ALV infection and integration as lymphoma-susceptible strains. Immunohistochemical analysis determined that the transformed follicles that do arise in lymphoma-resistant birds show much lower and more variable Myc overexpression than those of susceptible birds. This reduced Myc overexpression fails to block B-cell differentiation in resistant birds, while high Myc consistently blocks development at a late embryo stage in susceptible birds. This failure of Myc to block differentiation results in a normal pattern of posthatching bursal emigration in resistant transformed follicles, while transformed follicles of susceptible birds grow rapidly due to blocked emigration. Forced Myc overexpression produces transformed follicles in resistant birds, indicating that resistant lymphocytes can tolerate high Myc expression. The coding sequence and expression of the endogenous c-myc gene is the same in resistant and susceptible birds, suggesting that genetic resistance is instead mediated by reduced ALV LTR enhancer-driven transcription in the target lymphocytes of resistant birds.

Animals↗

PubMed1993

Gene therapy for neurologic disease.

Gene therapy is a potentially potent new method of treating a number of neurologic disorders previously considered refractory to current conventional therapeutic treatments. Numerous advances have been made in the construction of expression vectors, cellular and viral transgene carriers, and the characterization of target cells for neuronal gene therapy. Two primary approaches to nervous system gene transfer have emerged as a result of these advances. The in vivo approach concentrates on direct transfer of genetic material to cells in vivo using viral and chemical agents. The ex vivo approach relies on genetic transfer to cultured cells that are subsequently implanted into a host organism. Both of these methods have been used in preliminary experiments designed to test the efficacy of gene transfer strategies in the amelioration of nervous system dysfunction.

Adenoviridae↗

PubMed1997

Intestinal transcription and synthesis of apolipoprotein AI is regulated by five natural polymorphisms upstream of the apolipoprotein CIII gene.

To understand the factors contributing to the synthesis of human apolipoprotein AI (apoAI), relative apoAI synthesis was measured from endoscopic biopsy samples obtained from 18 healthy volunteers. The relative amount of apoAI synthesis was directly correlated with steady state intestinal apoAI mRNA levels and a 10-fold within-group variability was observed. Analysis of genomic DNA from the subjects revealed five polymorphic sites which defined two haplotypes in the intestinal enhancer region of the apoAI gene located upstream of the apolipoprotein CIII gene transcriptional start site (+ 1): (-641 C to A, -630 G to A, -625 T to deletion, -482 C to T, and -455 T to C). The population frequencies of the wild-type and mutant alleles were 0.53 and 0.44, respectively. Mean steady state apoAI mRNA levels and mean relative apoAI synthesis were 49 and 37% lower, respectively, in homozygotes for the mutant allele and 28 and 41% lower, respectively, in heterozygotes than in homozygotes for the wild-type allele (P < 0.05 for both). Site-directed mutants of apoAI gene promoter/reporter constructs containing the above mutations were transfected into Caco-2 cells and showed a 46% decrease in transcriptional activity compared with the wild type (P < 0.001); however, no significant differences were observed in HepG2 cells. Electrophoretic mobility shift assays showed that the mutated sequences from -655 to -610 bound Caco-2 cell nuclear protein(s) while the wild type did not. These results indicate that intestinal apoAI gene transcription and protein synthesis are genetically determined and are reduced in the presence of common mutations which induced binding of nuclear protein(s), possibly a transcriptional repressor.

Adult↗

PubMedDate not supplied

[Isolation of nucleotide sequences, possessing enhancer functions from the Saccharomyces cerevisiae genetic library].

We propose a method for isolation of enhancer-like sequences from a yeast genomic library. The method was used to identify DNA inserts capable of increasing bacterial bla gene expression when located downstream from the transcription initiation site. Plasmids carrying different fragments of one such insert were used to localize the enhancer-like function on a 1.2 kb fragment.

Bacteriophage lambda↗

PubMed1990

Enhancer sequences from Arabidopsis thaliana obtained by library transformation of Nicotiana tabacum.

In this paper we report on the use of a bidirectional enhancer cloning vehicle to isolate and characterize new enhancer sequences from Arabidopsis thaliana. A library of A. thaliana genomic Sau3A segments was constructed in Escherichia coli in the binary plasmid enhancer cloning vehicle pROA97. The T-DNA based vector carries abbreviated TATA regions from the cauliflower mosaic virus 35S transcription unit upstream of two genes. The library was transferred via triparental mating into Agrobacterium tumefaciens. The neomycin phosphotransferase II gene was used for selection of kanamycin-resistant transformed tobacco callus cells. Approximately 1100 transgenic plants were regenerated and assayed for expression of the E. coli beta-glucuronidase (GUS) gene in leaves, stems, roots, or seeds. Plasmids carrying putative enhancer sequences were rescued from the genomes of transgenic plants and the cloned sequences were assayed for enhancer function in genetic selection experiments. Plants were regenerated from the kanamycin-resistant calli obtained in the secondary transformation experiments. Histochemical analysis of GUS activity in the leaf, stem, and root tissues of transgenic plants showed a variety of expression patterns. The DNA sequences are presented of five Arabidopsis segments which confer enhancer function.

Base Sequence↗

PubMed2004

Increased frequency of the immunoglobulin enhancer HS1,2 allele 2 in coeliac disease.

BACKGROUND: Coeliac disease (CD) is characterized by increased immunological responsiveness to ingested gliadin in genetically predisposed individuals. This genetic predisposition is not completely defined. A dysregulation of immunoglobulins (Ig) is present in CD: since antiendomysium antibodies (anti-EMA) are of the IgA class. One polymorphic enhancer within the locus control region (LCR) of the immunoglobulin heavy chain cluster at the 3' of the C alpha-1 gene was investigated. The correlation of the penetrance of the four different alleles of the HS1,2-A enhancer of the LCR-1 3' to C alpha-1 in CD patients compared to a control population was analysed. METHODS: A total of 115 consecutive CD outpatients, on a gluten-free diet, and 248 healthy donors, age- and sex-matched, from the same geographical area were enrolled in the study. HS1,2-A allele frequencies were investigated by nested polymerase chain reaction (PCR). RESULTS: The frequency of allele 2 of the enhancer HS1,2-A gene was increased by 30.8% as compared to the control frequency. The frequency of homozygosity for allele 2 was significantly increased in CD patients. Crude odds ratio (OR) showed that those with 2/2 and 2/4 (OR 2.63, P < 0.001 and OR 2.01, P = 0.03) have a significantly higher risk of developing the disease. In contrast, allele 1/2 may represent a protective genetic factor against CD (OR 0.52, P = 0.01). CONCLUSIONS: These data provide further evidence of a genetic predisposition in CD. Because of the Ig dysregulation in CD, the enhancer HS1,2-A may be involved in the pathogenesis.

Adult↗

PubMed2006

The ATP-sensitive potassium (KATP) channel-encoded dSUR gene is required for Drosophila heart function and is regulated by tinman.

The homeobox transcription factor Tinman plays an important role in the initiation of heart development. Later functions of Tinman, including the target genes involved in cardiac physiology, are less well studied. We focused on the dSUR gene, which encodes an ATP-binding cassette transmembrane protein that is expressed in the heart. Mammalian SUR genes are associated with K(ATP) (ATP-sensitive potassium) channels, which are involved in metabolic homeostasis. We provide experimental evidence that Tinman directly regulates dSUR expression in the developing heart. We identified a cis-regulatory element in the first intron of dSUR, which contains Tinman consensus binding sites and is sufficient for faithful dSUR expression in the fly's myocardium. Site-directed mutagenesis of this element shows that these Tinman sites are critical to dSUR expression, and further genetic manipulations suggest that the GATA transcription factor Pannier is synergistically involved in cardiac-restricted dSUR expression in vivo. Physiological analysis of dSUR knock-down flies supports the idea that dSUR plays a protective role against hypoxic stress and pacing-induced heart failure. Because dSUR expression dramatically decreases with age, it is likely to be a factor involved in the cardiac aging phenotype of Drosophila. dSUR provides a model for addressing how embryonic regulators of myocardial cell commitment can contribute to the establishment and maintenance of cardiac performance.

ATP-Binding Cassette Transporters↗