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Results for “regulatory element transcripts”

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PubMed1995

The GATA-4 transcription factor transactivates the cardiac-specific troponin C promoter-enhancer in non-muscle cells.

The unique contractile phenotype of cardiac myocytes is determined by the expression of a set of cardiac-specific genes. By analogy to other mammalian developmental systems, it is likely that the coordinate expression of cardiac genes is controlled by lineage-specific transcription factors that interact with promoter and enhancer elements in the transcriptional regulatory regions of these genes. Here, we demonstrate that the slow/cardiac-specific troponin C (cTnC) enhancer contains a specific binding site for the lineage-restricted, zinc finger transcription factor, GATA-4 and that GATA-4 mRNA and protein is expressed in cardiac myocytes. In addition, GATA-4 binding sites were identified in several previously characterized cardiac-specific transcriptional regulatory elements. The cTnC GATA-4 binding site is required for transcriptional enhancer activity in primary cardiac myocytes. Moreover, the cTnC enhancer can be transactivated by over-expression of GATA-4 in non-cardiac muscle cells such as NIH 3T3 cells. Taken together, these results are consistent with a model in which GATA-4 functions to direct tissue-specific gene expression during mammalian cardiac development.

3T3 Cells↗

PubMedDate not supplied

A consideration of previously uncharacterized fowl poxvirus unidirectional and bidirectional late promoters for inclusion in homologous recombinant vaccines.

Because of the limited analysis of fowl poxvirus (FPV) promoters, expression of foreign proteins by recombinant FPV has usually been directed by heterologous vaccinia virus or synthetic poxvirus promoters. Thus, the impact of completely homologous recombinant virus vaccines has yet to be realized by the poultry industry. In an effort to increase the availability of such transcriptional regulatory elements, the modulation of gene expression by six previously uncharacterized FPV late promoters was examined. To simplify this comparison, each promoter region was separately coupled to the same reporter gene (lacZ) in individual plasmid constructs, and their activities in transfected, virus-infected cells were monitored. In each of the four selected unidirectional transcriptional regulatory elements as well as a 30-base pair representative of the bidirectional promoter region, the predicted temporal specificity of expressing at late stages of virus replicative cycle was verified. Stable lacZ gene transcripts arising from each plasmid varied less than threefold in quantity, whereas the amounts of beta-galactosidase product ranged within a 130-fold interval. Only the promoter that naturally regulates expression of the A type inclusion body protein gene directed production of beta-galactosidase at a level comparable with that associated with the strong vaccinia virus P11 promoter. Because one of the remaining unidirectional transcriptional regulatory elements, P174, was only 2.4-fold less efficient, both of these promoters, P174 and P190, should be satisfactory for directing the expression of poultry pathogen genes inserted into the genomes of FPV recombinant vaccines.

Animals↗

PubMed2003

A novel transcriptional element which regulates expression of the CYP2D4 gene by Oct-1 and YY-1 binding.

We first identified the transcriptional regulatory element of the CYP2D4 gene. CYP2D4 is of interest in brain pharmacology and physiology because this enzyme can be involved in the metabolism of endogenous and exogenous compounds, which act on the central nervous system. Transfection studies using a series of the CYP2D4 promoter luciferase constructs identified the transcriptional element of CYP2D4 in the sequence between nucleotides -116 and -90 (named the neural expression regulatory element, NERE). The nucleotide sequence of NERE was specific for the CYP2D4 gene. Within this region, two nuclear factor-binding sequences, Oct-1 and YY-1, were present. Oct-1 acts as the activator of the CYP2D4. The core sequence of the YY-1 binding motif partially overlapped that of the Oct-1 binding motif. YY-1 may act as the repressor of CYP2D4, which interferes with Oct-1 activation by its binding to NERE. We concluded that a novel transcriptional regulatory element NERE specifically regulates the expression of the CYP2D4. This regulation system may be involved in the unique distribution of this isoform, such as the expression in the brain.

Amino Acid Sequence↗

PubMed1983

Identification of transcriptional elements within the long terminal repeat of Rous sarcoma virus.

Transcriptional regulatory elements within the Rous sarcoma virus long terminal repeat were examined by the construction of a series of deletions and small insertions within the U3 region of the long terminal repeat. The analysis of these mutations in chicken embryo cells and COS cells permitted the identification of important transcriptional regulatory elements. Sequences within the region 31 to 18 base pairs upstream of the RNA cap site (-31 to -18), encompassing a TATA box-like sequence, function in the selection of the correct site of transcription initiation and, in addition, augment the efficiency of transcription. These sequences are essential for virus replication. Sequences within the region -79 to -59, overlapping a CAAT box-like sequence, are not required for virus replication and have no obvious effect on viral RNA transcription in the presence of an intact TATA box. However, in mutants lacking a functional TATA sequence, mutations in this region serve to decrease the efficiency of correct transcriptional initiation events.

Animals↗

PubMed1991

A variant octamer motif in a Xenopus H2B histone gene promoter is not required for transcription in frog oocytes.

Xenopus oocytes, arrested in G2 before the first meiotic division, accumulate histone mRNA and protein in the absence of chromosomal DNA replication and therefore represent an attractive biological system in which to examine histone gene expression uncoupled from the cell cycle. Previous studies have shown that sequences necessary for maximal levels of transcription in oocytes are present within 200 bp at the 5' end of the transcription initiation site for genes encoding each of the five major Xenopus histone classes. We have defined by site-directed mutagenesis individual regulatory sequences and characterized DNA-binding proteins required for histone H2B gene transcription in injected oocytes. The Xenopus H2B gene has a relatively simple promoter containing several transcriptional regulatory elements, including TFIID, CBP, and ATF/CREB binding sites, required for maximal transcription. A sequence (CTTTACAT) in the H2B promoter resembling the conserved octamer motif (ATTTGCAT), the target for cell-cycle regulation of a human H2B gene, is not required for transcription in oocytes. Nonetheless, substitution of a consensus octamer motif for the variant octamer element activates H2B transcription. Oocyte factors, presumably including the ubiquitous Oct-1 factor, specifically bind to the consensus octamer motif but not to the variant sequence. Our results demonstrate that a transcriptional regulatory element involved in lymphoid-specific expression of immunoglobulin genes and in S-phase-specific activation of mammalian H2B histone genes can activate transcription in nondividing amphibian oocytes.

Animals↗

PubMed1995

Human herpesvirus 6A ts suppresses both transformation by H-ras and transcription by the H-ras and human immunodeficiency virus type 1 promoters.

Human herpesvirus 6 strain U1102 (HHV-6A) was shown to contain a 1,473-bp functional transformation suppressor gene (ts). ts exhibited 24% identity and 51% similarity to adeno-associated virus type 2 Rep68/78. Like adeno-associated virus type 2 Rep68/78, HHV-6A ts suppressed H-ras transformation of NIH 3T3 cells. Suppression of H-ras transformation was eliminated by translation termination linker mutation at amino acid 25, 125, or 245. These data indicated the importance of the C-terminal portion of the ts protein. H-ras transformation was suppressed by ts only when H-ras was expressed by its endogenous H-ras promoter and not when it was expressed by the heterologous murine osteosarcoma virus long terminal repeat (LTR). Furthermore, ts suppressed chloramphenicol acetyltransferase (CAT) activity when the CAT gene was expressed from the H-ras promoter but not the murine osteosarcoma virus LTR promoter. Taken together, the data showed that ts suppressed H-ras transformation at the level of the H-ras promoter. To further identify the interaction of ts with transcriptional regulatory elements, the human immunodeficiency virus type 1 (HIV-1) LTR was used. This promoter was selected because it has well-defined transcriptional regulatory elements for both basal and activated transcription, because its activity is inhibited by the Rep68/78 gene, and because both HHV-6 and HIV-1 naturally infect CD4+ T cells in vivo and have been shown to infect the same cell in vitro. ts suppressed expression from both wild-type and upstream mutant HIV-1 LTR-CAT constructs. However, downstream HIV-1 TAR mutations reversed ts suppression, indicating that TAR is one of the critical elements involved. The data presented demonstrated that HHV-6A ts functionally suppressed H-ras transformation and HIV-1 LTR expression and thus that it may be useful in future gene therapy.

3T3 Cells↗

PubMed1997

Identification of essential cis-acting regulatory elements for transcription of the rat DAN gene.

The DAN gene was initially isolated as one of the genes whose expression is significantly decreased in a variety of transformed rat fibroblasts 3Y1 cells when compared with the parental 3Y1 cells. In the present study, we have isolated the genomic clone of the DAN gene from a 3Y1 genomic library and characterized the possible regulatory elements responsible for the transcription of the DAN gene. The transcription initiation site was determined by a primer extension experiment. Putative TATA and CAAT-like elements were present 31 and 358 bp upstream from the transcription start site, respectively. Transient transfection of a series of DAN-chloramphenicol acetyltransferase (CAT) gene constructs, which contain different portions of the 5'-flanking region (2,236 bp) of the DAN gene and the CAT gene, was used to localize a regulatory element. These experiments demonstrated the presence of the regions that regulate DAN gene expression positively (-57 to +118) and negatively (-1,232 to -636). The electrophoretic mobility-shift assays revealed that 3Y1 and SR-3Y1 nuclear extracts specifically interact with the positive (-57 to +118) and the negative (-1,226 to -987) regulatory regions, respectively.

Animals↗

PubMed1988

A mammalian viral enhancer confers transcriptional regulation in yeast.

Transcriptional enhancers are DNA sequences that regulate RNA transcription from linked promoters by binding to cellular proteins (trans-activators). In the mammalian virus SV40, initiation of transcription is controlled, in part, by a strong 72-base pair enhancer. We show that yeast cells contain a factor that binds specifically to a key DNA motif in the SV40 enhancer, the P element that is essential for viral transcription in mammalian cells. The P element shows sequence similarity to a yeast DNA transcriptional regulatory element, GCN4, that controls transcription of genes that code for amino acid biosynthetic enzymes. Insertion of the SV40 enhancer or single or multiple copies of the P element itself upstream from the cytochrome Cyc-1 promoter places the hybrid viral-yeast transcription unit under metabolic control in yeast cells. These studies suggest that the SV40 P element and its complementary trans-activator represent a conserved transcriptional control mechanism that operates on widely divergent functions in evolution.

Base Sequence↗

PubMed2004

Java-based application framework for visualization of gene regulatory region annotations.

MOTIVATION: The genome sequences of several organisms are either complete, or being sequenced. Each genome needs to be integrated with various types of annotations, e.g. locations of genes, promoters and other functional elements such as transcriptional regulatory elements. A robust application framework will be useful for developing web-based applications to visualize various genome annotations. RESULTS: We developed genome data visualization toolkit (GDVTK) as an application framework that consists of a set of data structures and core classes, using Java technology. GDVTK is a sound framework for developing web-based applications to present the gene regulatory region annotations in visual form. The current version of GDVTK consists of eight packages and 38 Java classes that are portable, reusable and extensible for plugging in new data sources and models. We implemented GDVTK for visualization of promoter annotations in Mammalian Promoter Database (MPromDb), a web-based gene-regulatory information server. AVAILABILITY: GDVTK is available under GNU general public license. Source code and software documentation can be found at the URL http://bioinformatics.med.ohio-state.edu/GDVTK.

Computer Graphics↗

PubMed1998

Identification and characterization of potential cis-regulatory elements governing transcriptional activation of the rat tyrosine hydroxylase gene.

Tyrosine hydroxylase (TH) catalyzes the conversion of L-tyrosine to 3,4-dihydroxy-L-phenylalanine, which is the first and rate-limiting step in catecholamine biosynthesis. We have previously shown that the cyclic AMP response element (CRE), an essential promoter element for both basal and cyclic AMP-inducible TH transcription, activates the promoter activity in a distance-dependent manner. To identify further cis-regulatory elements controlling TH gene expression, we analyzed the potential regulatory sequences by several approaches. First, using transient transfection assays, we examined the cell-specific promoter activities of TH-reporter gene constructs and a dopamine beta-hydroxylase (DBH)-reporter construct containing the 5' upstream sequences of the rat TH and human DBH genes, respectively, that had been shown to direct tissue-specific reporter expression in transgenic mice experiments. Second, DNase I footprinting analysis of the 503-bp proximal area of the rat TH gene identified seven footprinted regions that encompass the putative cis-regulatory motifs, including the CRE (domain 1), Sp1 (domain III), Octamer (domain IV), AP1 (domain V), AP2 (domain VI), and two potentially novel sequence motifs (domains II and VII). Footprinting patterns at these sites by nuclear proteins from TH-positive and -negative cell lines appeared to be similar. Third, site-directed mutagenesis demonstrates that domain III, but not domain II, critically contributes to the TH promoter activity. Furthermore, electrophoretic mobility shift, competition, and supershift assays demonstrate that domain III is an authentic Sp1 site and that the transcription factor Sp1 interacts with it. This and previous results suggest that the CRE and Sp1 site may synergistically activate TH transcription in a promoter context-dependent manner.

3T3 Cells↗

PubMed2004

Some microsatellites may act as novel polymorphic cis-regulatory elements through transcription factor binding.

Although microsatellites with functional effects have been described, generally, these repeats are considered as "junk" DNA in the same way as other repetitive sequences. Our aim was to investigate if certain microsatellites can have a functional role as cis-regulatory elements. A database was created of all short tandem repeats, from 2 to 10 bases, located in the first 10-kb 5' of the transcription start sites of all annotated genes of the human genome. Of 114 microsatellites selected based on their size and location in the promoter, 51 were found to be polymorphic. Using electrophoretic mobility shift assay (EMSA), we studied five repetitive motifs and three displayed specific protein binding which were found in 12 of the polymorphic microsatellites. An interesting microsatellite is the CTC/GAG repeat which, as double-stranded (DS) DNA, bound specificity protein 1 (SP1) with high affinity, formed triplexes in vitro and displayed differences in SP1 binding and triplex formation capacity for repeats with distinct numbers of repeat units. Interestingly, the polypyrimidine strand of the repeat (CTC) bound other proteins such as polypyrimidine tract-binding protein 1 (PTBP1) as single-stranded (SS) DNA, and a model with two alternative DNA conformations is proposed for these repeats. Distinct protein binding to DS DNA was also observed for different numbers of AAACA and AAAAT repeats. Our results suggest that certain microsatellites may act as cis-regulatory elements, controlling gene expression through transcription factor binding and/or secondary DNA structure formation. Due to their high polymorphism and abundance, they might represent an important source of quantitative genetic variation.

Base Sequence↗

PubMed1996

Structural analysis of human natural resistance-associated macrophage protein 1 promoter.

Natural resistance-associated macrophage protein gene 1 (Nramp1) was isolated as a candidate for the mouse gene locus Lsh/Ity/Bcg, which regulates macrophage activation for antimicrobial activity against intracellular pathogens. To investigate the structural and functional organization of the human homologue, NRAMP1, the overlapping genomic clones encompassing NRAMP1 were isolated. These clones spanned approximately 35 kb in size and the nucleotide sequence of 3779 bp of the 5' flanking region has been determined. The transcription start site was mapped by primer extension analysis. A TATA box element and the transcription regulatory elements in the acute phase reaction were found.

Base Sequence↗

PubMed2001

Regulation of gene expression by GC-rich DNA cis-elements.

GC-rich DNA cis elements are important transcriptional regulatory elements present in the promoter, enhancer and locus control regions of many eukaryotic genes from several species. This review attempts to examine the structure, function and biological significance of GC-rich cis -regulatory elements and their cognate binding proteins, with a view to understanding their role in regulation of gene expression.

Animals↗

PubMed1991

Cross-talk between the virulence and phosphate regulons of Agrobacterium tumefaciens caused by an unusual interaction of the transcriptional activator with a regulatory DNA element.

Transcription of a virulence gene on the hairy-root-inducing plasmid A4, which is induced by plant factors in Agrobacterium tumefaciens, was also activated by phosphate limitation in both A. tumefaciens and Escherichia coli. The starting site of RNA synthesized under the two inducing conditions was the same, and an identical promoter was responsible for both inducible expressions. The response of the virulence gene to phosphate limitation did not require the positive regulator VirG for the virulence regulon, but depended entirely on the presence of PhoB protein, the positive regulator for the phosphate regulon. The DNA signal upstream of the virulence gene, which is targeted by the VirG protein, was recognized by the E. coli PhoB protein in vitro. These results indicate that cross-talk between the two regulons occurred during the recognition of a DNA signal by the regulatory protein.

Bacterial Proteins↗

PubMed2001

Solitary human endogenous retroviruses-K LTRs retain transcriptional activity in vivo, the mode of which is different in different cell types.

Solitary long terminal repeats (LTRs) of human endogenous retroviruses (HERVs), tens of thousands of which are spread all over the genome, contain a variety of potential transcription regulatory elements. Information on transcriptional behavior of individual solitary LTRs, however, is limited. We studied the transcriptional activity of several individual HERV-K LTRs in a variety of tissues and cell lines. The RT-PCR technique targeted at specific amplification of the U3 or U5 regions of individual LTRs together with their unique genomic flanks was used to estimate the content of each region in the transcripts. An unequal abundance of the U3 and U5 regions of the transcripts of the same LTR in different cells and tumors was observed. Each LTR is transcribed differently in different cells or tissues, and transcriptional behavior of different LTRs was different in the same cell line or tissue. The transcriptional status of LTRs varies in response to mitogenic and stress factors and in tumor tissues compared to normal counterparts. The LTRs thus seem to be the subjects of specific transcription regulation. The data obtained indicate that an appreciable fraction of the LTRs retained regulatory potential throughout millions of years of evolution and thus may contribute to the overall transcription regulatory network.

Endogenous Retroviruses↗

PubMed1997

Conserved structure, regulatory elements, and transcriptional regulation from the GATA-1 gene testis promoter.

Transcription factor GATA-1 was first identified in erythroid cells, but was later shown to also be expressed in Sertoli cells of the mouse testis. GATA-1 transcription in testis initiates from a different first exon (exon IT) than the erythroid mRNA (transcribed from exon IE). To begin to address the question of how expression of GATA-1 might be differentially regulated in Sertoli and erythroid cells, we have cloned and determined the structure of the IT promoters of both the rat and mouse GATA-1 genes. The transcription regulatory mechanism(s) controlling the synthesis of exon IT-derived mRNA was investigated by transfection of wild-type and mutant reporter genes, with and without co-transfected GATA factor expression plasmids, into either fibroblasts or Sertoli cell lines. Two GATA binding sites in the IT promoter were found to be required for GATA factor-mediated activation in fibroblasts: GATA-IT-directed reporter gene expression was activated only after co-transfection with GATA-1, implying that transcriptional activation of GATA-1 in the testis might be at least partially mediated through these GATA regulatory elements. We also found that the endogenous GATA-1 gene was silent in primary culture and two different Sertoli cell lines, and that the repression of co-transfected GATA-1 reporter genes could not be relieved by forced expression of GATA-1 in Sertoli cells. Thus the GATA-IT promoter may be under the control of a regulatory network in Sertoli cells which involves both positive and negative regulation of transcription, and conserved GATA motifs found in the IT promoter may be required for transducing these effects.

Animals↗

PubMed2005

Characterization of the proximal enhancer element and transcriptional regulatory factors for murine recombination activating gene-2.

Recombination-activating gene (RAG)-1 and RAG-2 are essential for V(D)J recombination and are expressed specifically in lymphoid cells. We previously identified two putative enhancer elements, the proximal and distal enhancers, located at -2.6 and -8 kb, respectively, 5' upstream of mouse RAG-2, and characterized the distal enhancer element in detail. In this study, to characterize the proximal enhancer in vitro as well as in vivo, we first defined a 170-bp core enhancer element within the proximal enhancer (Ep) and determined its activity in various cells. Ep conferred enhancer activity only in B-lymphoid cell lines, but not in T- or non-lymphoid cell lines. Analysis of the transgenic mice carrying an EGFP reporter gene linked with Ep revealed that Ep activated the transcription of the reporter gene in bone marrow and spleen, but not in thymus or non-lymphoid tissues. Ep was active in both B220+IgM- and B220+IgM+ subpopulations in the bone marrow and in the B220+ subpopulation in the spleen. Using electrophoretic mobility shift assays and mutational assays, we found that Ikaros and CCAAT/enhancer binding protein cooperatively bind Ep and function as the transcription factors responsible for B cell-specific enhancer activity. These results demonstrate the role of Ep as a cis-regulatory enhancer element for RAG-2-specific expression in B-lymphoid lineages.

Animals↗

PubMed1990

Regulatory elements mediating transcription from the Drosophila melanogaster actin 5C proximal promoter.

The major cytoskeletal actin gene of Drosophila melanogaster, the actin 5C gene, has two promoters, the proximal one of which controls constitutive synthesis of actin in all growing tissues. To locate regulatory elements required for constitutive activity of the proximal promoter, mutants of this promoter were fused to the bacterial chloramphenicol acetyltransferase gene and assayed for transient expression activity in cultured Drosophila embryonic Schneider line 2 cells. An essential regulatory element has been located 313 base pairs upstream from the cap site. Deletion of this element lowered expression to one-third of the wild-type level. The element has the sequence AAGTTGTAGTTG, as shown by protein-binding footprinting with the reagent methidiumpropyl-EDTA-Fe(II). This element is probably not a general one, since it was not detected in a search of the published 5'-flanking sequences of 27 Drosophila genes. In addition to this regulatory element, there are five GAGA elements in the actin 5C proximal promoter, some or all of which are essential for the promoter activity as shown by an in vivo competition assay. Although this promoter has no classical TATA element, there is an essential promoter region about 35 base pairs upstream from the cap site that could be a TATA surrogate. The promoter also shows sequences homologous to the alcohol dehydrogenase factor 1-binding site and to the core of the vertebrate serum response element, but mutations of these sites did not affect promoter activity in transient expression assays.

Actins↗