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Results for “regulatory element transcripts”

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PubMed1998

Differential regulation of basal and cyclic adenosine 3',5'-monophosphate-induced somatostatin gene transcription in neural cells by DNA control elements that bind homeodomain proteins.

A number of genes encoding neuropeptides are expressed in the peripheral and central nervous systems, in different endocrine organs, and in specialized cells distributed along the gastrointestinal tract. Whether expression of the same neuropeptide gene in different tissues is regulated by similar transcriptional mechanisms or by mechanisms that differ in a cell-specific manner remains unclear. We report on promoter studies on the regulation of the somatostatin gene in immortalized neural precursor cells derived from developing rat forebrain. Expression of the somatostatin gene in these cells was determined by RT-PCR/Southern blot analysis, by immunocytochemistry, and by RIA. We show that in cerebrocortical and hippocampal cells, expression of the somatostatin gene is regulated by several negative and positive DNA cis-regulatory elements located throughout the promoter region. The somatostatin cAMP-response element appears to play a prominent role in neural somatostatin gene expression by acting as a strong enhancer even in the absence of cAMP stimulation. Site-directed mutagenesis followed by transient transfection assays indicated that SMS-TAAT1, SMS-TAAT2, and SMS-UE, three previously identified homeodomain protein-binding regulatory elements that enhance transcription in pancreatic cells, act as repressors of transcription in neural cells. Electrophoretic mobility shifts assays indicate that those elements bind protein complexes that differ between neural and pancreatic cells. Our results support the notion that expression of the somatostatin gene in neural cells occurs via transcriptional mechanisms that are different from those regulating expression of the same gene in pancreatic cells.

Animals↗

PubMed1995

Cyclic AMP inhibits expression of the IL-2 gene through the nuclear factor of activated T cells (NF-AT) site, and transfection of NF-AT cDNAs abrogates the sensitivity of EL-4 cells to cyclic AMP.

cAMP inhibits PMA-induced IL-2 production at the transcriptional level in EL-4, a mouse lymphoma line. The region of the mouse IL-2 promoter covering positions from -321 to +46 relative to the transcription initiation site is required for activation by PMA and inhibition by cAMP. This region contains the nuclear factor of activated T cells (NF-AT), nuclear factor-kappa B (NF-kappa B), AP-1, and Oct binding sites, and the role of each element in responding to PMA and/or cAMP signals was characterized. The IL-2 promoter carrying mutations in each element reduced response to PMA while it retained sensitivity to cAMP, thereby suggesting that multiple elements contribute to positive and negative responses to PMA and cAMP, respectively. Using reporter plasmid carrying multiple copies of each element, we then found that the NF-AT construct was most effective in responding to PMA activation and to cAMP inhibition. Electrophoretic mobility shift assay revealed that, after exposure of cells to Bt2cAMP, NF-AT binding complex changed in amount or in mobility as a function of time. Furthermore, overexpression of the cytoplasmic component of NF-AT abrogated the inhibitory action of cAMP. These results indicate that the NF-AT site is a target of the inhibitory action of cAMP. In addition, binding of the NF-kappa B (p50/p65) heterodimer to the NF-kappa B site was inhibited by cAMP. Taken together, our data show that cAMP in EL-4 cells inhibits mouse IL-2 gene transcription through cis regulatory elements that include the NF-AT site as well as the NF-kappa B site.

Amino Acid Sequence↗

PubMed2002

Oncostatin M induced alpha1-antitrypsin (AAT) gene expression in Hep G2 cells is mediated by a 3' enhancer.

alpha(1)-Antitrypsin (AAT) is the major serine proteinase inhibitor (SERPIN A1) in human plasma. Its target proteinase is neutrophil elastase and its main physiological function is protection of the lower respiratory tract from the destructive effects of neutrophil elastase during an inflammatory response. Circulating levels of AAT rise 2-3-fold during inflammation and the liver produces most of this increase. The cytokines oncostatin M (OSM) and interleukin-6 have been shown to be mainly responsible for this effect, which is mediated via the interaction of cytokine-inducible transcription factors with regulatory elements within the gene. In the present study, we report for the first time that OSM stimulation of hepatocyte AAT occurs via an interaction between the hepatocyte promoter and an OSM-responsive element at the 3'-end of the AAT gene. This effect is mediated by the transcription factor signal transducer and activator of transcription 3 ('STAT 3') binding to an OSM-responsive element (sequence TTCTCTTAA), and this site is distinct from, but close to, a previously reported interleukin-6-responsive element.

Base Sequence↗

PubMed1987

trp operon induction during the expression in E. coli of two IFN-gamma sequences.

Two nucleotide sequences coding for mature human immune interferon (IFN-gamma) and differing from each other by nine N-terminal nucleotides were expressed in E. coli under the control of a trp promoter. The longer gene variant after the ATG initiatory codon contained a TGT TAC TGC sequence, which was absent in the shorter gene. When expressed in E. coli under the direction of identical transcription and translation regulatory elements, these genes showed different susceptibility to induction.

Base Sequence↗

PubMed1986

Characterization of a regulatory region of the steroid 21-hydroxylase gene.

Steroid 21-hydroxylase is expressed at high levels in the adrenal gland, where it is required for the synthesis of mineralocorticoids and glucocorticoids. The cis-acting nucleotide sequences required for expression of murine 21-hydroxylase were assessed by stable transfection of Y1 adrenocortical tumor cells with 21-hydroxylase genes containing progressive deletions of the 5' flanking sequences. Expression of authentic 21-hydroxylase transcripts was quantitated by S1 nuclease protection assays using total RNA from pools of transfectant colonies. Removal of sequences from 1,100 to 230 base pairs preceding the transcription initiation site did not significantly affect 21-hydroxylase transcription. However, an important regulatory element falls between 230 and 180 base pairs upstream of the transcription initiation site. Comparisons of the murine 5' flanking sequence with those of the human and bovine 21-hydroxylase genes revealed a 40-base pair region that is highly conserved among all three species. The conserved sequence in the murine gene occurs between 210 and 170 nucleotides 5' of the transcription initiation site; similar locations are seen in the human and bovine genes. These results thus define an important regulatory region which is required for expression of 21-hydroxylase in Y1 adrenal cells.

Adrenal Cortex Neoplasms↗

PubMed2026

Balancing growth and immunity of potato by humidity-dependent expression of a late blight resistance gene.

Inducible expression of resistance genes is an effective approach to balance plant growth and immunity, thus facilitating the development of disease-resistant crop cultivars. While pathogen-responsive and immunity-related promoters have been adopted for this purpose, alternative design strategies remain to be explored. High relative humidity (RH) has been recognized as a crucial permissive environmental condition for the occurrence of devastating plant diseases including tomato and potato late blight. Here, we identified humidity-activated cis-regulatory elements (HAEs) in Solanum lycopersicum through an integrative analysis of transcriptomics and chromatin accessibility data. Sequence homology-inferred HAEs in S. tuberosum can predict humidity-elicited changes in downstream gene expression. Transgenic S. tuberosum lines expressing a late blight resistance gene driven by an artificial humidity-inducible promoter containing a natural S. tuberosum HAE were generated. These transgenic lines exhibited comparable late blight resistance levels to the lines overexpressing the same resistance gene in controlled zoospore inoculation bioassays, while avoiding growth suppression and tuber yield penalties in common garden experiments. Our findings highlight the importance of plant cis-regulatory elements in the transcriptional responses to high RH and provide a proof-of-concept for a humidity-inducible environment-responsive resistance gene deployment strategy to engineer disease-resistant crop cultivars without compromising growth and yield.

Phytophthora infestans↗

PubMed2005

Regulation of lipid metabolism by soy protein and its implication in diseases mediated by lipid disorders.

Soybeans have a high-quality protein that has been consumed for approximately 5000 years in Oriental countries. The awareness that soy products are healthy has increased their consumption in Western countries. Substantial data from epidemiological surveys and nutritional interventions in humans and animals indicate that soy protein reduces serum total and low-density lipoprotein (LDL) cholesterol and triglycerides as well as hepatic cholesterol and triglycerides. This review examines the evidence on the possible mechanisms for which soy protein has beneficial effects in diabetes, obesity and some forms of chronic renal disease. Consumption of soy protein due to low methionine content reduces serum homocysteine concentration, decreasing the risk of acquiring a cardiovascular disease. On the other hand, soy protein reduces the insulin/glucagon ratio, which in turn down-regulates the expression of the hepatic transcription factor sterol regulatory element binding protein (SREBP)-1. The reduction of this factor decreases the expression of several lipogenic enzymes, decreasing in this way serum and hepatic triglycerides as well as LDL cholesterol and very LDL triglycerides in diabetes and obesity, reducing lipotoxicity in the liver. Soy protein intake also reduces hepatic lipotoxicity by maintaining the number of functional adipocytes, preventing the transfer of fatty acids to extra adipose tissues. Furthermore, soy protein isoflavones stimulate the transcription factor SREBP-2, increasing serum cholesterol clearance. The reduction of serum cholesterol and triglyceride concentrations by soy protein intake produces beneficial effects in the kidney preventing the inflammatory response, increasing the renal flow by releasing endothelial nitric oxide (NO) synthase from the caveolae, facilitating the synthesis of NO. Thus, soy protein consumption may reduce the clinical and biochemical abnormalities in diseases mediated by lipid disorders.

Adipocytes↗

PubMed1995

Use of the Escherichia coli uidA gene as a reporter in Methanococcus voltae for the analysis of the regulatory function of the intergenic region between the operons encoding selenium-free hydrogenases.

The Escherichia coli beta-glucuronidase gene uidA was linked to a region of the Methanococcus voltae genome containing the putative promoter of a gene for a DNA-binding protein and introduced into the M. voltae chromosome. It was found that the enzyme was expressed in the cells in easily measurable amounts. The reporter gene was then placed under the control of the intergenic region found between two divergently transcribed gene groups encoding selenium-free hydrogenases, which are measurably transcribed only after selenium depletion. This region is supposed not only to contain the divergent promoters governing the transcription of the hydrogenase genes but also cis regulatory elements necessary for the negative transcriptional regulation in which selenium is involved. It was shown that the intergenic region functioned as a promoter region for the reporter gene in either orientation. The additional finding that beta-glucuronidase expression was dependent on selenium depletion localizes the cis regulatory elements to the intergenic region between the two hydrogenase operons.

Base Sequence↗

PubMed1993

Regulatory elements involved in tax-mediated transactivation of the HTLV-I LTR.

HTLV-I is the etiologic agent of adult T-cell leukemia. In this study, we investigated the regulatory elements and cellular transcription factors which function in modulating HTLV-I gene expression in response to the viral transactivator protein, tax. Transfection experiments into Jurkat cells of a variety of site-directed mutants in the HTLV-1 LTR indicated that each of the three motifs A, B, and C within the 21-bp repeats, the binding sites for the Ets family of proteins, and the TATA box all influenced the degree of tax-mediated activation. Tax is also able to activate gene expression of other viral and cellular promoters. Tax activation of the IL-2 receptor and the HIV-1 LTR is mediated through NF-kappa B motifs. Interestingly, sequences in the 21-bp repeat B and C motifs contain significant homology with NF-kappa B regulatory elements. We demonstrated that an NF-kappa B binding protein, PRDII-BF1, but not the rel protein, bound to the B and C motifs in the 21-bp repeat. PRDII-BF1 was also able to stimulate activation of HTLV-I gene expression by tax. The role of the Ets proteins on modulating tax activation was also studied. Ets 1 but not Ets 2 was capable of increasing the degree of tax activation of the HTLV-I LTR. These results suggest that tax activates gene expression by either direct or indirect interaction with several cellular transcription factors that bind to the HTLV-I LTR.

Base Sequence↗

PubMed1994

The basic helix-loop-helix protein upstream stimulating factor regulates the cardiac ventricular myosin light-chain 2 gene via independent cis regulatory elements.

Previous studies have documented that 250 bp of the rat cardiac ventricular myosin light-chain 2 (MLC-2v) promoter is sufficient to confer cardiac muscle-specific expression on a luciferase reporter gene in both transgenic mice and primary cultured neonatal rat myocardial cells. Utilizing ligation-mediated PCR to perform in vivo dimethyl sulfate footprinting, the present study has identified protein-DNA interaction within the position from -176 to -165. This region, identified as MLE1, contains a core sequence, CACGTG, which conforms to the consensus E-box site and is identical to the upstream stimulating factor (USF)-binding site of the adenovirus major late promoter. Transient assays of luciferase reporter genes containing point mutations of the site demonstrate the importance of this cis regulatory element in the transcriptional activation of this cardiac muscle gene in ventricular muscle cells. The protein complex that occupies this site is capable of binding to HF-1a and PRE B sites which are known to be required for cardiac muscle-specific expression of rat MLC-2v and alpha-myosin heavy-chain genes, respectively. This study provides direct evidence that USF, a member of the basic helix-loop-helix leucine zipper family, binds to MLE1, HF-1a, and PRE B sites and suggests that it is a component of protein complexes that may coordinately control the expression of MLC-2v and alpha-myosin heavy-chain genes. The current study also provides evidence that USF can positively and negatively regulate the MLC-2v gene via independent cis regulatory elements.

Animals↗

PubMed2002

IRF-8/ICSBP and IRF-1 cooperatively stimulate mouse IL-12 promoter activity in macrophages.

IRF-8/ICSBP and IRF-1 are IRF family members whose expression is induced in response to IFN-gamma in macrophages. IL-12 is a cytokine produced in macrophages that plays a critical role in host defense. IFN-gamma and bacterial lipopolysaccharide (LPS) induce IL-12p40 transcription, which is necessary for the production of IL-12. We have previously shown that IL-12p40 expression is impaired in ICSBP-deficient mice and that transfection of ICSBP together with IRF-1 can activate IL-12p40 expression in mouse macrophage cells. To further study the role of ICSBP and IRF-1, we investigated murine IL-12p40 promoter activity in the macrophage cell line RAW 264.7. We show here that co-transfection of ICSBP and IRF-1 synergistically stimulates IL-12 promoter activity to a level comparable to that induced by IFN-gamma/LPS. Mutation of the Ets or NFkappaB site previously shown to be important for IL-12p40 transcription did not abolish the activation by ICSBP and IRF-1. However, mutation of the ISRE-like site found downstream from the NFkappaB and C/EBP sites abrogated the activation by ICSBP and IRF-1. Together, these results indicate that ICSBP and IRF-1 cooperatively stimulate murine IL-12 transcription through a novel regulatory element in the murine promoter.

Animals↗

PubMed2002

Transvection in Drosophila.

Pairing-dependent interallelic complementation was first described for the Ultrabithorax gene of the bithorax-complex in Drosophila by Lewis and cited as an example of a new phenomenon that Lewis called the "trans-vection effect." Several different kinds of pairing-dependent gene expression have been observed in Drosophila, and it is now clear that a variety of different molecular mechanisms probably underlie the changes in gene expression that are observed after disrupting chromosome pairing. Transvection in the bithorax-complex appears to result from the ability of cis-regulatory elements to regulate transcription of the promoter on the homologous chromosome. The same phenomenon appears to be responsible for pairing-dependent interallelic complementation at numerous other genes in Drosophila. Some transvection effects are dependent on the presence of wild-type or specific mutant forms of the protein encoded by the zeste trans-regulatory gene, but other transvection effects are zeste-independent. The ease with which chromosome aberrations can disrupt transvection also varies widely among different genes.

Alleles↗

PubMed2000

Neuronal localization of sterol regulatory element binding protein-1 in the rodent and primate brain: a light and electron microscopic immunocytochemical study.

Sterol regulatory element binding proteins are membrane-bound transcription factors that activate expression of several genes controlling cellular cholesterol and fatty acid homeostasis. The present study aimed to investigate the in vivo expression of sterol regulatory element binding protein-1 in the normal rodent and primate brain, and in the brain in Niemann-Pick type C disease mice. These mutant animals have lysosomal cholesterol accumulation and progressive neurodegeneration caused by an inactivating mutation of the NPC1 gene whose protein product functions in vesicular lipid trafficking. Western blot analysis of rat hippocampal homogenates with an affinity purified rabbit polyclonal antibody directed against an internal epitope of sterol regulatory element binding protein-1 identified a major 68,000 mol. wt protein consistent with the amino-terminal, transcriptionally active fragment of sterol regulatory element binding proteins-1. Immunocytochemically, this antibody revealed dense sterol regulatory element binding protein-1 staining of nuclei and light staining of the cytoplasm of cells in the neocortex and hippocampus in the rat, mouse and monkey brain. By electron microscopy of immunogold-labeled brain sections, these densely labeled cells were found to be neurons. In contrast, normal glial cells had little or no sterol regulatory element binding protein-1 immunoreactivity even at a developmental stage (postnatal day 9) which coincides with active myelination in the rat brain. Also, in contrast to the normal mouse brain, Niemann-Pick type C mice showed reduced staining of cortical and hippocampal neuronal nuclei. Since sterol regulatory element binding protein-1 has been shown to be a transcriptional regulator of fatty acid synthesis in vivo, the current findings of a predominantly neuronal nuclear expression of the 68,000 mol. wt transcriptionally active fragment of sterol regulatory element binding protein-1 highlights the established role of phospholipid metabolites and other fatty-acid containing lipids in neuronal signal transduction and other neuronal functions. Reduced sterol regulatory element binding protein-1 expression in neurons in Niemann-Pick type C may reflect a deficiency in fatty acid synthesis that could contribute to the neuronal dysfunction in this disorder.

Animals↗

PubMed1996

Characterization of cardiac gene cis-regulatory elements in the early stages of chicken heart morphogenesis.

To study transcriptional regulation during early stages of cardiogenesis, stage 10-17 chicken embryo hearts were transfected efficiently within the intact embryo by application of plasmid DNA complexed to liposomes. Viral regulatory sequences and the skeletal alpha-actin, SERCA2 and ANF promoters activated expression of reporter genes in the primitive heart tube. Deletion and mutation analysis of the skeletal alpha-actin promoter revealed the importance of CArG cis-regulatory elements in enhancing transcription of the gene during early heart development. These results demonstrate the utility of this method for the identification of gene regulatory elements that specify the cardiac phenotype during early stages of heart morphogenesis.

Actins↗

PubMed2005

The repressor DREAM acts as a transcriptional activator on Vitamin D and retinoic acid response elements.

DREAM (downstream regulatory element antagonist modulator) is a transcriptional repressor, which binds DREs (downstream response elements) in a Ca2+-regulated manner. The DREs consist of core GTCA motifs, very similar to binding motifs for non-steroid nuclear receptors. In this work, we find that DREAM stimulates basal and ligand-dependent activation of promoters containing vitamin D and retinoic acid response elements (VDREs and RAREs), consisting of direct repeats of the sequence AGT/GTCA spaced by 3 or 5 nt, respectively. Stimulation occurs when the element is located upstream, but not downstream, the transcription initiation site. Activation requires both Ca2+ binding to the EF-hands and the leucine-charged domains (LCDs), analogous to those responsible for the interaction of the nuclear receptors with coregulators. Further more, DREAM can bind both 'in vitro' and in chromatin immunoprecipitation assays to these elements. Importantly, 'in vivo' binding is only observed in vitamin D- or RA-treated cells. These results show that DREAM can function as an activator of transcription on certain promoters and demonstrate a novel role for DREAM acting as a potential modulator of genes containing binding sites for nuclear receptors.

Calcium-Binding Proteins↗

PubMed1993

Transcriptional activation of type I collagen genes by ascorbic acid 2-phosphate in human skin fibroblasts and its failure in cells from a patient with alpha 2(I)-chain-defective Ehlers-Danlos syndrome.

L-Ascorbic acid 2-phosphate (Asc 2-P), a long-acting vitamin C derivative, stimulated transcription of genes for pro alpha 1(I) and pro alpha 2(I) collagen in normal human skin fibroblasts after 8 h of treatment in the absence or in the presence of cycloheximide, indicating Asc 2-P stimulates transcription of type I collagen genes in the absence of protein synthesis. The transcriptional rate in these cells reached the maximum value after 40 h of treatment, and at that time it was three to four times higher than that of the control cells cultured in the absence of Asc 2-P. Steady-state levels of mRNAs for pro alpha 1(I) and pro alpha 2(I) chains were also increased to be three to four times higher than the control levels by treatment of the cells with Asc 2-P for 72 h. When the fibroblasts obtained from a patient with Ehlers-Danlos syndrome were treated with Asc 2-P, the derivative also stimulated transcription of the gene for pro alpha 1(I) chain and accumulation of mRNA for pro alpha 1(I) chain. On the other hand, Asc 2-P failed to stimulate transcription of the pro alpha 2(I) gene or an increase in mRNA for pro alpha 2(I) chain. Sodium ascorbate showed effects quite similar to those of Asc 2-P, when fibroblasts obtained from a normal control or the patient were cultured for 16 h with it. These results indicate the existence of cis-regulatory elements responsible for transcriptional activation by Asc 2-P or ascorbic acid in pro alpha 1(I) and pro alpha 2(I) genes of normal fibroblasts. These data also suggest some defect(s) of these elements in the pro alpha 2(I) gene of the patient with Ehlers-Danlos syndrome.

Ascorbic Acid↗

PubMed2004

Selecting transpositions using phage P1 headful packaging: new markerless transposons for functionally mapping long-range regulatory sequences in bacterial artificial chromosomes and P1-derived artificial chromosomes.

New Tn10 minitransposons were constructed to functionally map long-range transcription regulatory sequences in bacterial artificial chromosomes (BACs) and P1-derived artificial chromosomes (PACs). Each contained a wild-type loxP site but, significantly, contained no mammalian or bacterial genes and/or promoter elements within the transposed portion of DNA. In contrast to loxP transposons described previously, the new ones do not introduce transcription regulatory elements capable of interfering with those endogenous to the BAC clone in functional mapping studies. Progressive deletions from the loxP end of genomic DNA were efficiently generated using these transposons, and a series of truncations generated in a green fluorescence protein (GFP)-BAC fusion clone unambiguously identified three new long-range enhancer sequences functionally in the Nkx2-5 gene in transgenic mice. Insertions of these new transposons lacking antibiotic resistance genes into a BAC or PAC were indirectly selected by their ability to delete enough DNA from the clone so as to enable its packaging within a P1 phage head with both loxP sites intact for subsequent recovery of the large plasmid. The outcome of such an indirect mode of selection is both desirable and undesirable. First, because the screen is not antibiotic resistance marker dependent, the same transposon can be used to generate nested deletions efficiently in both BACs and PACs. Second, deletions through intrainsert recombinations unrelated to loxP/Cre also get packaged and recovered, and size analyses of the BAC/PAC vector band after NotI digestion is indispensable to identify authentic loxP/Cre deletions. The procedure nevertheless offers a potential approach to map recombinogenic sequences in BACs and PACs.

Animals↗

PubMedDate not supplied

Repair-modification and evolution of the eukaryotic genome organization.

For a complete reconstruction of the damaged unmethylated islands, in theory, the conventional excision-repair is sufficient. For a complete reconstruction of the damaged methylated domains, a coupling has to take place involving the excision-repair (able to reestablish their ATGC-language) plus the DNA-methylase (able to reestablish their modified ATGC5mC-language). This coupling, defined as "repair-modification," is essentially functioning during the S-phase, because the DNA-polymerase beta (pol beta) is active during the whole cell cycle, whereas the DNA-methylase (met) is active in S and appears to be repressed or inactive during the major part of G1 and during the phases G2 and M. Consequently, after damage, some silent genes might become expressed during these phases, if it is true that DNA methylation is inversely proportional to transcription. Repair-modification should, therefore, exert a continuous differential pressure on evolution of given parts of the genome, when they are methylated to a different extent. According to Darwinian concepts, repair-modification would lead to a high variability, especially of uncoding DNA sequences (if hypermethylated), whereas on the basis of this variability, selection might favor transposition of specific regulatory elements into given transcriptional units. In these, the conservative nature of the coding elements (if unmethylated) would obviously be ensured by the conventional excision-repair.

Animals↗