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PubMed1987

Properties of a distal regulatory element controlling transcription of the U2 small nuclear RNA.

The upstream region of human U2 genes contains a distal transcriptional control element, previously mapped between nucleotide (nt) positions -198 and -258 (Westin et al., 1984b). In the present study we show that it resembles transcriptional enhancers in being active even from a distance of 1.4 kb. However, in contrast to most other enhancers it functions unidirectionally in Xenopus laevis oocytes. The distal control element was further mapped by construction of truncated templates for U2 RNA transcription. The results showed that templates, which extended to either of nt positions -214 and -218, were inactive. Templates comprising sequences to nt positions -225 or -226 displayed an intermediate level of activity whereas templates which extend to nt -258 were fully active. It has previously been shown that the human U2 enhancer contains binding sites for the so-called octamer binding protein and for transcription factor Sp1 [Janson et al., Nucl. Acids Res. 15 (1987) 4997-5016]. The partially active templates included one binding site for the octamer binding protein, whereas the fully active template included, in addition, two Sp1 binding sites, thus indicating that these transcription factors are of importance for U2 RNA transcription. The structure of the enhancer was also probed by inserting a pair of complementary synthetic oligodeoxynucleotides which represented the region between nt positions -235 and -215 into a truncated template which lacked the enhancer. The oligodeoxynucleotide enhanced transcription to approximately 50% of the level obtained with templates extending to position -258.

Animals↗

PubMed2003

Inhibition of c-src transcription by mithramycin: structure-activity relationships of biosynthetically produced mithramycin analogues using the c-src promoter as target.

The aureolic acid antitumor antibiotic mithramycin (MTM) inhibits both cancer growth and bone resorption by cross-linking GC-rich DNA, thus blocking binding of Sp-family transcription factors to gene regulatory elements. Transcription of c-src, a gene implicated in many human cancers and required for osteoclast-dependent bone resorption, is regulated by the binding of Sp factors to specific elements in its promoter. Therefore, this gene represents an important anticancer target and a potential lead target through which MTM displays its so far uncharacterized action against osteoclastic bone resorption. Here we demonstrate, using DNA binding studies, promoter reporter assays, and RT-PCR, that MTM inhibits Sp binding to the c-src promoter region, thereby decreasing its expression in human cancer cells. Furthermore, selected mithramycin analogues, namely, premithramycin B, mithramycin SK, 7-demethylmithramycin, 4E-ketomithramycin, and 4C-ketodemycarosylmithramycin, generated through combinatorial biosynthesis, were compared with MTM for their ability to block Sp binding to the c-src promoter. Although most of the tested compounds lost their ability to bind to the DNA, alteration of the MTM 3-pentyl side chain led to a compound (mithramycin SK) with the same DNA binding specificity but with lower binding affinity than MTM. While this compound was comparable to MTM in promoter reporter, gene expression, and anticancer assays, given its weaker interaction with the DNA, it may be much less toxic than MTM. The results presented here supplement recent findings and, moreover, allow new conclusions to be made regarding both the structure-activity relationships, particularly with respect to the alkyl side chains, and the mechanism of action of aureolic acid drugs.

Antibiotics, Antineoplastic↗

PubMed2003

Evaluation of combined gene regulatory elements for transcriptional targeting of suicide gene expression to malignant melanoma.

Selective killing of tumors can be achieved by targeting the transcription of suicide genes via specific DNA control elements to malignant cells. Three different enhancer-promoter systems were constructed and evaluated for their capability to direct gene expression to melanoma. Two tissue-specific (tyrosine and MIA) promoters and one weak viral promoter were fused to multiple tandem copies of a melanocyte-specific enhancer element. Reporter gene assays revealed a maximum increase in transcription by combining each promoter with 3-4 copies of the enhancer and demonstrated that all enhancer-promoter combinations exhibited tissue-specific activity. Though this activity was still significantly less than that of the strong but unspecific cytomegalovirus (CMV) promoter. In contrast, when those combinations were employed to drive the expression of two suicide genes, encoding the diptheria toxin A chain (DT-A) and the prodrug-activating herpes simplex virus thymidine kinase (TK), respectively, only those constructs in which transcription was under control of tissue-specific promoter elements mediated selective killing of melanoma cells. This killing was in the range of cell death induced by CMV promoter activity. Our data indicate that the enhancer/tyrosinase and enhancer/MIA promoter constructs but not the viral promoter constructs can provide a valuable tool for selective suicide gene expression in melanoma.

Animals↗

PubMed1986

Cellular promoters incorporated into the adenovirus genome: effects of viral regulatory elements on transcription rates and cell specificity of albumin and beta-globin promoters.

In the accompanying paper (Friedman et al., Mol. Cell. Biol. 6:3791-3797, 1986), hepatoma-specific expression of the rat albumin promoter within the adenovirus genome was demonstrated. However, the rate of transcription was very low compared with that of the endogenous chromosomal albumin gene. Here we show that in hepatoma cells the adenovirus E1A enhancer, especially in the presence of E1A protein, greatly stimulates transcription from the albumin promoter but not the mouse beta-globin promoter. This enhancer-dependent stimulation did not occur in myeloma cells in which a virus containing a immunoglobulin promoter and enhancer did function. These experiments suggest a limited distribution in cultured differentiated cells of cell-specific transcription factors. However, either the regulation of such cell-specific factors breaks down in other cultured cells, or strictly cell-specific factors are not at play in controlling cell-specific transcription, because HeLa cells could transcribe the albumin promoter from the same start site about 10% as well as hepatomas could and 293 cells could transcribe both albumin and globin promoters.

Adenovirus Early Proteins↗

PubMed2006

Epidermal growth factor receptor-induced circadian-time-dependent gene regulation in suprachiasmatic nucleus.

A significant functional role for epidermal growth factor receptor (EGFR) in the suprachiasmatic nucleus is suggested by recent findings that epidermal growth factor receptor and its ligand transforming growth factor-alpha are highly expressed in the suprachiasmatic nucleus. Recent studies indicate that epidermal growth factor receptor activation induces behavioral and physiological effects, strengthening the notion that epidermal growth factor receptor can modulate suprachiasmatic nucleus neural function and behavior. A global transcriptional profiling study is performed to investigate the gene expression response to epidermal growth factor receptor activation in the suprachiasmatic nucleus. The results indicate that all of the observed gene expression response is circadian-time dependent. The response included several genes encoding different neuropeptide receptors, ion channels and kinases. In order to hypothesize the transcription factors underlying the epidermal growth factor receptor response, different circadian-time-dependent gene expression groups were analyzed for enriched transcriptional regulatory elements in the promoters. The results indicate that several transcription factors such as Elk1 and cAMP-responsive element binding protein/activating transcription factor family, known to be 'input points' to the core clock network, are playing a role. Together, these results indicate that epidermal growth factor receptor has a circadian-time-dependent neuromodulatory function in the suprachiasmatic nucleus.

Animals↗

PubMed1989

Pancreatic beta-cell-type-specific expression of the rat insulin II gene is controlled by positive and negative cellular transcriptional elements.

The insulin gene is expressed almost exclusively in pancreatic beta-cells. The DNA sequences that control cell-specific expression are located upstream of the transcription initiation site. To identify the cis-acting transcriptional control regions within the rat insulin II gene that are responsible for this tissue-specific expression pattern, we constructed a series of 5'-flanking deletion mutants and analyzed their expression in vivo in transfected insulin-producing and -nonproducing cell lines. Pancreatic beta-cell-specific expression was shown to be controlled by enhancer sequences lying between nucleotides -342 and -91 relative to the transcription start site. The rat insulin II enhancer appears to be a chimera, composed of a number of distinct cis-acting DNA elements. Both positive and negative transcriptional regulatory elements appear to be responsible for this cell-type-specific expression. We have shown that expression from one element within the enhancer, which is found between nucleotides -100 and -91, is regulated by both positive- and negative-acting cellular transcription factors. Expression from chimeras containing only the enhancer element sequences from -100 to -91 were active only in insulin-producing cells, indicating that the positive-acting factor(s) required for this activity may be active only in beta-cells. In contrast to the enhancer region, the rat insulin II gene promoter did not appear to require cell-specific transcription factors. Promoter mutants with 5'-flanking sequences extending to nucleotides -90 and -73 were constitutively active in both insulin-producing and -nonproducing cells. These results suggest that rat insulin II gene transcription in pancreatic beta-cells is imparted by a combination of both negative- and positive-acting cellular factors interacting with the gene enhancer.

Animals↗

PubMed2005

Annotation of cis-regulatory elements by identification, subclassification, and functional assessment of multispecies conserved sequences.

An important step toward improving the annotation of the human genome is to identify cis-acting regulatory elements from primary DNA sequence. One approach is to compare sequences from multiple, divergent species. This approach distinguishes multispecies conserved sequences (MCS) in noncoding regions from more rapidly evolving neutral DNA. Here, we have analyzed a region of approximately 238kb containing the human alpha globin cluster that was sequenced and/or annotated across the syntenic region in 22 species spanning 500 million years of evolution. Using a variety of bioinformatic approaches and correlating the results with many aspects of chromosome structure and function in this region, we were able to identify and evaluate the importance of 24 individual MCSs. This approach sensitively and accurately identified previously characterized regulatory elements but also discovered unidentified promoters, exons, splicing, and transcriptional regulatory elements. Together, these studies demonstrate an integrated approach by which to identify, subclassify, and predict the potential importance of MCSs.

Animals↗

PubMed1997

A complex enhancer of the chicken beta A3/A1-crystallin gene depends on an AP-1-CRE element for activity.

PURPOSE: To define transcriptional regulatory elements of the chicken beta A3/A1-crystallin gene. METHODS: Reporter genes were made with fragments of the chicken beta A3/A1-crystallin gene fused to the bacterial gene encoding chloramphenicol acetyltransferase (CAT). The reporter plasmids were transfected into primary cultures of chicken-patched lens epithelium or fibroblast cells, and the CAT activity of cellular extracts was measured. The binding of lens nuclear proteins to beta A3/A1 sequences was tested in electrophoretic mobility shift assays. RESULTS: Sequences from -287 to -254 bp relative to the transcriptional start site function as an enhancer in transfected lens and nonlens cells. The length of a T-rich sequence downstream of the enhancer influences its activity. Minimal enhancer activity depends on sequences between -270 and -254 bp, and full activity requires additional upstream sequences. The minimal enhancer includes a consensus sequence (TGAGTCA) for basic region-leucine zipper (bZIP) proteins of the AP-1-CREB superfamily. Lens nuclear proteins bind the enhancer sequences to form several specific complexes, some of which are related antigenically to members of the AP-1 and CREB families of proteins. CONCLUSIONS: An enhancer of the chicken beta A3/A1-crystallin gene between -287 and -254 bp functions in both lens and nonlens cells and binds multiple nuclear proteins. Temporal and spatial regulation of beta A3/A1 expression in the lens may be regulated by the enhancer.

Animals↗

PubMed1997

Arabidopsis thaliana CBF1 encodes an AP2 domain-containing transcriptional activator that binds to the C-repeat/DRE, a cis-acting DNA regulatory element that stimulates transcription in response to low temperature and water deficit.

Recent efforts have defined a cis-acting DNA regulatory element in plants, the C-repeat/dehydration responsive element (DRE), that stimulates transcription in response to low temperature and water deficit. Here we report the isolation of an Arabidopsis thaliana cDNA that encodes a C-repeat/DRE binding factor, CBF1 (C-repeat/DRE Binding Factor 1). Analysis of the deduced CBF1 amino acid sequence indicates that the protein has a molecular mass of 24 kDa, a potential nuclear localization sequence, and a possible acidic activation domain. CBF1 also has an AP2 domain, which is a DNA-binding motif of about 60 aa present in the Arabidopsis proteins APETALA2, AINTEGUMENTA, and TINY; the tobacco ethylene response element binding proteins; and numerous other plant proteins of unknown function. The transcript levels for CBF1, which appears to be a single or low copy number gene, did not change appreciably in plants exposed to low temperature or in detached leaves subjected to water deficit. Binding of CBF1 to the C-repeat/DRE was demonstrated in gel shift assays using recombinant CBF1 protein expressed in Escherichia coli. Moreover, expression of CBF1 in yeast was found to activate transcription of reporter genes containing the C-repeat/DRE as an upstream activator sequence but not mutant versions of the DNA element. We conclude that CBF1 can function as a transcriptional activator that binds to the C-repeat/DRE DNA regulatory element and, thus, is likely to have a role in cold- and dehydration-regulated gene expression in Arabidopsis.

Amino Acid Sequence↗

PubMed1998

Repressor elements in the coding region of the human histone H4 gene interact with the transcription factor CDP/cut.

The coding region of the human histone H4 gene FO108 undergoes dynamic changes in chromatin structure that correlate with modifications in gene expression. Such structural alterations generally reflect transcription factor interactions with gene regulatory sequences. To test for regulatory elements within the coding region, we performed transient transfection experiments in HeLa cells using constructs with histone H4 sequences fused upstream of a heterologous thymidine kinase promoter and CAT reporter gene. H4 gene sequences from -10 to +210 repressed transcription 4.8-fold. Further deletion and mutational analysis delineated three repressor elements within this region. Using oligonucleotide competition analysis and specific antibody recognition in electrophoretic mobility shift assays, as well as methylation interference and DNase I footprinting analyses, we have identified the CCAAT displacement protein (CDP/cut) as the factor that interacts with these three repressor elements. CDP/cut binding to these repressor sites is proliferation-specific and cell-cycle-regulated, increasing in mid to late S phase. Our results indicate that the proximal 200 nucleotides of the histone H4-coding region contain transcriptional regulatory elements that may contribute to cell-cycle control of histone gene expression by interacting with repressor complexes containing CDP/cut homeodomain transcription factors.

Base Sequence↗

PubMed2003

A CRE and the region occupied by a protein induced by growth factors contribute to up-regulation of cyclin D1 expression in hepatocytes.

Induction of cyclin D1 expression is a critical feature of growth factor-induced cell proliferation in hepatocytes. To clarify the mechanisms regulating cyclin D1 gene expression, we isolated the rat cyclin D1 gene and analyzed the transcriptional regulatory elements in rat hepatoma cells and primary cultured rat hepatocytes. Two transcriptional regulatory regions were analyzed. One was mapped to a cAMP-responsive element (CRE) at position -41bp and was occupied by a CRE-binding protein (CREB), resulting in cyclin D1 expression. Another (CD1E0.7), located at -753bp, revealed high homology with binding sites for the Ets family, the hepatocyte nuclear factor-3beta, or the nuclear factor of activated T cells. However, CD1E0.7 did not interact with these nuclear factors and specific interaction with a protein extracted from growth factor-treated rat hepatocytes in primary cultures. These results indicate that CREB binds to the CRE and mediates activation of the cyclin D1 promoter, and suggest that CD1E0.7 may be possibly occupied by a protein induced by growth factors in hepatocytes.

5' Flanking Region↗

PubMed1992

[Effect of cyclic AMP and adenosine on hCG transcription by trophoblast cells].

It is well established that cAMP stimulates secretion of both alpha and beta subunits of hCG by trophoblasts. However, the molecular mechanism behind this phenomenon, especially that of beta subunit stimulation is still obscure. Moreover, while adenosine is known to play a pivotal role in the message transduction system of various cells, its effect on trophoblasts is not known. We therefore tried to answer these questions and found that cAMP stimulates hCG beta secretion by increasing transcription of its mRNA. However, no discrete cAMP responsive element like that seen in the hCG alpha gene seemed to be present in hCG beta transcriptional regulatory elements. It was assumed that hCG beta transcription is augmented indirectly by numerous trans acting elements which respond to cAMP. We have also shown that adenosine is a potent stimulator of hCG production. Its effect was synergistic with cAMP. It was therefore assumed that adenosine and cAMP are likely to act through different pathways.

Adenosine↗

PubMed2000

COMPEL: a database on composite regulatory elements providing combinatorial transcriptional regulation.

COMPEL is a database on composite regulatory elements, the basic structures of combinatorial regulation. Composite regulatory elements contain two closely situated binding sites for distinct transcription factors and represent minimal functional units providing combinatorial transcriptional regulation. Both specific factor-DNA and factor-factor interactions contribute to the function of composite elements (CEs). Information about the structure of known CEs and specific gene regulation achieved through such CEs appears to be extremely useful for promoter prediction, for gene function prediction and for applied gene engineering as well. The structure of the relational model of COMPEL is determined by the concept of molecular structure and regulatory role of CEs. Based on the set of a particular CE, a program has been developed for searching potential CEs in gene regulatory regions. WWW search and browse routines were developed for COMPEL release 3.0. The COMPEL database equipped with the search and browse tools is available at http://compel.bionet.nsc.ru/. The program for prediction of potential CEs of NFAT type is available at http://compel.bionet.nsc. ru/FunSite.html and http://transfac.gbf.de/dbsearch/funsitep/ s_comp.html

Database Management Systems↗

PubMed2005

Direct isolation and identification of promoters in the human genome.

Transcriptional regulatory elements play essential roles in gene expression during animal development and cellular response to environmental signals, but our knowledge of these regions in the human genome is limited despite the availability of the complete genome sequence. Promoters mark the start of every transcript and are an important class of regulatory elements. A large, complex protein structure known as the pre-initiation complex (PIC) is assembled on all active promoters, and the presence of these proteins distinguishes promoters from other sequences in the genome. Using components of the PIC as tags, we isolated promoters directly from human cells as protein-DNA complexes and identified the resulting DNA sequences using genomic tiling microarrays. Our experiments in four human cell lines uncovered 252 PIC-binding sites in 44 semirandomly selected human genomic regions comprising 1% (30 megabase pairs) of the human genome. Nearly 72% of the identified fragments overlap or immediately flank 5' ends of known cDNA sequences, while the remainder is found in other genomic regions that likely harbor putative promoters of unannotated transcripts. Indeed, molecular analysis of the RNA isolated from one cell line uncovered transcripts initiated from over half of the putative promoter fragments, and transient transfection assays revealed promoter activity for a significant proportion of fragments when they were fused to a luciferase reporter gene. These results demonstrate the specificity of a genome-wide analysis method for mapping transcriptional regulatory elements and also indicate that a small, yet significant number of human genes remains to be discovered.

Chromatin Immunoprecipitation↗

PubMed2002

Genomic organization and characterization of the promoter of rat malonyl-CoA decarboxylase gene.

Malonyl-CoA decarboxylase (MCD) catalyzes the decarboxylation of malonyl-CoA, an elongating agent for fatty acid synthesis and also known as a fuel-sensing mediator. In order to elucidate the genome organization, we isolated a 2020 bp rat MCD cDNA from rat brain cDNA library and isolated the corresponding rat genomic clones from the rat genomic PAC library. Sequencing and comparison of these clones showed that the MCD genome consists of five exons and four introns spanning approximately 17 kb. The proximal upstream region is GC-rich, lacks a TATA box, and contains a variety of putative transcriptional regulatory elements within 2 kb. A major transcriptional initiation site was identified by a primer extension at a site 157 nucleotides upstream of the translational initiation site. To investigate the transcriptional regulation of MCD, a series of 5'-deletion constructs of the 5'-flanking region were generated and cloned upstream from the luciferase reporter gene. By comparing promoter activity in CV-1 cells, we suggest that an area of -15 bp 5' from the first exon acted as a basal promoter for MCD and that there are positive cis-regulatory elements in the region from -55 to -325 bp and negative regulator elements in the region -1380 to -2240 bp.

5' Flanking Region↗

PubMed2005

Pre-mRNA processing enhancer (PPE) elements from intronless genes play additional roles in mRNA biogenesis than do ones from intron-containing genes.

Most mRNA-encoding genes require introns for efficient expression in high eukaryotes. However, mRNAs can efficiently accumulate in the cytoplasm without intron excision if they contain cis-acting elements such as the post-transcriptional regulatory element (PRE) of hepatitis B virus (HBV), the constitutive transport element (CTE) of Mason-Pfizer monkey virus (MPMV), or the pre-mRNA processing enhancer (PPE) of herpes simplex virus' thymidine kinase (HSV-TK) gene. We compared the activities of these viral elements, the Rev-responsive element (RRE) of the human immunodeficiency virus (HIV), and the human c-Jun gene's enhancer (CJE), an element newly identified here, to enable expression of an intronless variant of the human beta-globin gene. The PRE, PPE and CJE from naturally intronless genes, but not the CTE or RRE from intron-containing genes, significantly enhanced stability, 3' end processing and cytoplasmic accumulation. When the transcripts included the beta-globin gene's first intron, the PRE, PPE and CJE still enhanced mRNA biogenesis, in some cases without intron excision. Thus, elements enabling stability, 3' end formation and nucleocytoplasmic export, not the presence of introns or their excision per se, are necessary for mRNA biogenesis. While the CTE and RRE primarily enhance nucleocytoplasmic export, PPE-like elements from naturally intronless genes facilitate polyadenylation as well.

Active Transport, Cell Nucleus↗

PubMed2006

Different levels of transcriptional regulation due to trophic constraints in the reduced genome of Buchnera aphidicola APS.

Symbiotic associations involving intracellular microorganisms and animals are widespread, especially for species feeding on poor or unbalanced diets. Buchnera aphidicola, the obligate intracellular bacterium associated with most aphid species, provides its hosts with essential amino acids (EAAs), nutrients in short supply in the plant phloem sap. The Buchnera genome has undergone severe reductions during intracellular evolution. Genes for EAA biosynthesis are conserved, but most of the transcriptional regulatory elements are lost. This work addresses two main questions: is transcription in Buchnera (i) regulated and (ii) scaled to aphid EAA demand? Two microarray experiments were designed for profiling the gene expression in Buchnera. The first one was characterized by a specific depletion of tyrosine and phenylalanine in the aphid diet, and the second experiment combined a global diminution of EAAs in the aphid diet with a sucrose concentration increase to manipulate the aphid growth rate. Aphid biological performance and budget analysis (the balance between EAAs provided by the diet and those synthesized by Buchnera) were performed to quantify the nutritional demand from the aphids toward their symbiotic bacteria. Despite the absence of known regulatory elements, a significant transcriptional regulation was observed at different levels of organization in the Buchnera genome: between genes, within putative transcription units, and within specific metabolic pathways. However, unambiguous evidence for transcriptional changes underpinning the scaling of EAA biosynthesis to aphid demand was not obtained. The phenotypic relevance of the transcriptional response from the reduced genome of Buchnera is addressed.

Amino Acids, Essential↗

PubMed1987

Promoter selection in human mitochondria involves binding of a transcription factor to orientation-independent upstream regulatory elements.

Selective transcription of human mitochondrial DNA requires a transcription factor (mtTF) in addition to an essentially nonselective RNA polymerase. Partially purified mtTF is able to sequester promoter-containing DNA in preinitiation complexes in the absence of mitochondrial RNA polymerase, suggesting a DNA-binding mechanism for factor activity. Functional domains, required for positive transcriptional regulation by mtTF, are identified within both major promoters of human mtDNA through transcription of mutant promoter templates in a reconstituted in vitro system. These domains are essentially coextensive with DNA sequences protected from nuclease digestion by mtTF-binding. Comparison of the sequences of the two mtTF-responsive elements reveals significant homology only when one sequence is inverted; the binding sites are in opposite orientations with respect to the predominant direction of transcription. Thus mtTF may function bidirectionally, requiring additional protein-DNA interactions to dictate transcriptional polarity. The mtTF-responsive elements are arrayed as direct repeats, separated by approximately 80 bp within the displacement-loop region of human mitochondrial DNA; this arrangement may reflect duplication of an ancestral bidirectional promoter, giving rise to separate, unidirectional promoters for each strand.

DNA, Mitochondrial↗