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Results for “regulatory element transcripts”

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PubMed1992

Intragenic regulatory elements contribute to transcriptional control of the neurofilament light gene.

To date, no DNA regions involved in the neuron-specific expression of the neurofilament light gene (NF-L) have been defined using transfection assays in cultured cells. To identify those regulatory regions in the human NF-L gene, we generated transgenic mice with a construct containing the basal NF-L promoter (-292 to +15) fused to the cat gene and with three DNA fragments of 21.5, 7.6 and 4.9 kb each, including NF-L with different lengths of either 5'- or 3'-flanking sequences. We show that the proximal NF-L 5' region (0.3 kb) constitutes a weak promoter and that it lacks information to confer neural specificity. However, appropriate expression in the nervous system occurred when this minimal promoter was combined with either 7.3 or 4.6 kb of NF-L sequences downstream from the transcription start point. We conclude that the intragenic NF-L region contains cis-acting elements conferring cell-type-specific regulation on the basal activity of the NF-L promoter. Interestingly, AP-2 motifs were found within homologously placed introns of all three NF genes, as well as in the promoter regulatory regions of many neuronal genes. We propose that the acquisition of introns by an ancestral intronless IF gene may have contributed to the emergence of a lineage of IF genes expressed in the nervous system.

Animals↗

PubMed1991

Existence of common homologous elements in the transcriptional regulatory regions of human nuclear genes and mitochondrial gene for the oxidative phosphorylation system.

Our previous study indicated that nuclear protein factors of HeLa cells specifically bind to three nuclear Mt elements, Mt1, Mt3, and Mt4, located in the 5'-flanking regions of the human nuclear genes for cytochrome c1 and for ubiquinone-binding protein, both of which are subunits of mitochondrial cytochrome bc1 complex (Suzuki, H., Hosokawa, Y., Toda, H., Nishikimi, M., and Ozawa, T. (1990) J. Biol. Chem. 265, 8159-8163). In this study, we examined whether the same nuclear factors could recognize a set of the Mt3 and Mt4 elements that were found in the displacement loop and the promoter region of mammalian mitochondrial genomes. Gel retardation experiments disclosed that the same nuclear protein factors specifically bind to those Mt elements in the human mitochondrial genome as well as to the nuclear Mt3 and Mt4 elements of the two genes, and that the coexistence of both the elements is required for the efficient binding. The nuclear protein factors which recognize the Mt elements located in the regulatory regions of the nuclear and mitochondrial genes may play an important role in coordinate expression of the two physically separated genes during mitochondrial biogenesis.

Base Sequence↗

PubMed2005

Identification of cis-regulatory elements involved in transcriptional regulation of the sea urchin SpFoxB gene.

The SpFoxB gene is transiently expressed first in the mesoderm, then in the endoderm and oral ectoderm during sea urchin gastrulation. Perturbations of a number of proteins involved in endomesoderm specification have been shown to alter the mRNA levels of SpFoxB, but the cis-regulatory elements required for expression of SpFoxB have not been examined. In order to investigate this, we have screened the SpFoxB gene for sequences that can drive its expression. Both positive and negative cis-regulatory elements were found to be present. An enhancer was found that contains four GATA sites and four YY1 sites clustered within 210 base pairs (bp), as well as three lef/tcf binding sites. Electrophoretic mobility shifts indicate that the lef/tcf sites bind a complex of proteins that include beta-catenin in early cleavage, but not during subsequent stages of development. The GATA and YY1 sites bind nuclear proteins prior to SpFoxB transcription, and this binding diminishes coincident with cessation of transcription. Deletion of the GATA/YY1 sites causes a significant decrease in transcription. The DNA binding site of the SpFoxB protein has been determined, and Fox binding sites are found within the 5' UTR of SpFoxB.

Animals↗

PubMed2004

Functional analysis of multiple transcription factor sites in a regulatory element of human epsilon-globin gene.

The developmental control of the human epsilon-globin gene expression is mediated by transcriptional regulatory elements in the 5' flanking DNA of this gene. A previously identified negative regulatory element (-3028 to -2902 bp, termed epsilon-NRAII) was analyzed and one putative NF-kappaB site and two GATA sites locate at -3004 bp, -2975 bp and -2948 bp were characterized. Electrophoresis mobility shift assay (EMSA) showed that the putative NF-kappaB site was specifically bound by nuclear proteins of K562 cells. Data obtained from transient transfection showed that the expression of reporter gene could be upregulated about 50% or 100% respectively when epsilon-NRAII was inserted upstream of the SV40 promoter or epsilon-globin gene proximal promoter (-177 bp to +1 bp), suggesting that epsilon-NRAII might not be a classic silencer. Mutation in the putative NF-kappaB site or in the GATA site (at -2975 bp) slightly reduced the expression of reporter gene driven by SV40 promoter or epsilon-globin gene proximal promoter. However, the mutation of GATA site at -2948 bp remarkably reduced the reporter gene activity driven by SV40 promoter, but not by epsilon-globin gene proximal promoter. Further mutation analysis showed that the negative effect of mutation in GATA site at -2948 bp on SV40 promoter was not affected by the mutation of the putative NF-kappaB site, whereas it could be abolished by the mutation of GATA site at -2975 bp. Furthermore, the mutation of both GATA sites could synergistically reduce the reporter gene activity driven by epsilon-globin gene proximal promoter. Those results suggested that epsilon-NRAII might function differently on the SV40 promoter and epsilon-globin gene proximal promoter.

Binding Sites↗

PubMed1995

Interaction of the CCAAT displacement protein with shared regulatory elements required for transcription of paired histone genes.

The H2A and H2B genes of the Xenopus xlh3 histone gene cluster are transcribed in opposite directions from initiation points located approximately 235 bp apart. The close proximity of these genes to one another suggests that their expression may be controlled by either a single bidirectional promoter or by separate promoters. Our analysis of the transcription of histone gene pairs containing deletions and site-specific mutations of intergenic DNA revealed that both promoters are distinct but that they overlap physically and share multiple regulatory elements, providing a possible basis for the coordinate regulation of their in vivo activities. Using the intergenic DNA fragment as a probe and extracts from mammalian and amphibian cells, we observed the formation of a specific complex containing the CCAAT displacement protein (CDP). The formation of the CDP-containing complex was not strictly dependent on any single element in the intergenic region but instead required the presence of at least two of the three CCAAT motifs. Interestingly, similar CDP-containing complexes were formed on the promoters from the three other histone genes. The binding of CDP to histone gene promoters may contribute to the coordination of their activities during the cell cycle and early development.

Animals↗

PubMed1996

Regulatory elements that control transcription activation and unsaturated fatty acid-mediated repression of the Saccharomyces cerevisiae OLE1 gene.

In Saccharomyces cerevisiae, unsaturated fatty acids are formed from saturated acyl-CoA precursors by Ole1p, a delta-9 fatty acid desaturase. OLE1 mRNA levels are differentially regulated by the addition of saturated or unsaturated fatty acids to the growth medium. One component of this regulation system involves the control of OLE1 transcription. Saturated fatty acids induce a 1.6-fold increase in transcription activity, whereas a large family of unsaturated fatty acids repress OLE1 transcription as much as 60-fold. A deletion analysis of OLE1 promoter::lacZ fusion reporter genes identified a 111-base pair (bp) fatty acid-regulated (FAR) region approximately 580 bp upstream of the start codon that is essential for transcription activation and unsaturated fatty acid repression. Deletion of an 88-bp sequence within that region resulted in a complete loss in transcription activation and unsaturated fatty acid regulation. The 111-bp FAR element strongly activates transcription and confers unsaturated fatty acid regulation on a heterologous CYC1 promoter test plasmid. Essential elements required for unsaturated fatty acid repression of OLE1 were found in the 5 and 3 region of the 111-bp sequence. The FAR element-mediated activation and fatty acid repression of transcription was found to be closely tied to fatty acyl-CoA metabolism. Two fatty acid activation genes, FAA1 and FAA4, were found to be essential for unsaturated fatty acid repression of OLE1 through the FAR sequences. Disruption of either gene results in reduced levels of unsaturated fatty acid repression; disruption of both genes completely blocks the regulatory response. Acyl-CoA binding protein (ACBP) plays a role in determining the level of FAR element activated transcription. Disruption of the ACBP gene causes a >5-fold activation of OLE1 transcription and a similar increase in OLE1 mRNA levels. Unsaturated fatty acid repression of OLE1 transcription, however, is not affected by the disrupted ACBP gene. These studies show that promoter elements responsible for unsaturated fatty acid-mediated transcription repression are tightly linked to OLE1 activation sequences and that OLE1 transcription levels are closely tied to acyl-CoA metabolism.

Base Sequence↗

PubMed1988

The 5' flanking region of the gene for the Epstein-Barr virus-encoded nuclear antigen 2 contains a cell type specific cis-acting regulatory element that activates transcription in transfected B-cells.

We have recently identified the promoter that positions the initiation (cap) site for RNA encoding the Epstein-Barr virus (EBV) determined nuclear antigen 2 (EBNA2) in transfected COS-1 cells. The cells were transfected with recombinant vectors that contained the BamHI WYH region of the EBV genome. In order to delineate regulatory DNA sequences required for the expression of EBNA2 the 5' flanking region of the gene was linked to reporter genes in expression vectors and transfected into EBV genome-negative lymphoid DG75 cells. We demonstrate that several cis-acting elements contribute to a transcriptional enhancer activity found in the region between nucleotides-553 and -86 relative to the cap site. The enhancer was active in lymphoid DG75 cells but not in HeLa cells and stimulated transcription also from the heterologous thymidine kinase (TK) and beta-globin promoters. Nuclear extracts of lymphoid cells contained protein factors that bound to the enhancer. The in vitro introduction of a mutation in the enhancer sequence that substantially reduced the transcription stimulatory activity concurrently blocked the binding of one of the factors.

Animals↗

PubMed2000

Nutritional regulation of the fatty acid synthase promoter in vivo: sterol regulatory element binding protein functions through an upstream region containing a sterol regulatory element.

The transcription of fatty acid synthase (FAS), a central enzyme in de novo lipogenesis, is dramatically induced by fasting/refeeding and insulin. We reported that upstream stimulatory factor binding to the -65 E-box is required for induction of the FAS transcription by insulin in 3T3-L1 adipocytes. On the other hand, we recently found that two upstream 5' regions are required for induction in vivo by fasting/refeeding and insulin; one at -278 to -131 albeit at a low level, and the other at -444 to -278 with an E-box at -332 where upstream stimulatory factor functions for maximal induction. Here, we generated double transgenic mice carrying the chloramphenicol acetyltransferase reporter driven by the various 5' deletions of the FAS promoter region and a truncated active form of the sterol regulatory element (SRE) binding protein (SREBP)-1a. We found that SREBP participates in the nutritional regulation of the FAS promoter and that the region between -278 and -131 bp is required for SREBP function. We demonstrate that SREBP binds the -150 canonical SRE present between -278 and -131, and SREBP can function through the -150 SRE in cultured cells. These in vivo and in vitro results indicate that SREBP is involved in the nutritional induction of the FAS promoter via the -278/-131 region and that the -150 SRE is the target sequence.

3T3 Cells↗

PubMed1992

A single MEF-2 site is a major positive regulatory element required for transcription of the muscle-specific subunit of the human phosphoglycerate mutase gene in skeletal and cardiac muscle cells.

In order to analyze the transcriptional regulation of the muscle-specific subunit of the human phosphoglycerate mutase (PGAM-M) gene, chimeric genes composed of the upstream region of the PGAM-M gene and the bacterial chloramphenicol acetyltransferase (CAT) gene were constructed and transfected into C2C12 skeletal myocytes, primary cultured cardiac muscle cells, and C3H10T1/2 fibroblasts. The expression of chimeric reporter genes was restricted in skeletal and cardiac muscle cells. In C2C12 myotubes and primary cultured cardiac muscle cells, the segment between nucleotides -165 and +41 relative to the transcription initiation site was sufficient to confer maximal CAT activity. This region contains two E boxes and one MEF-2 motif. Deletion and substitution mutation analysis showed that a single MEF-2 motif but not the E boxes had a substantial effect on skeletal and cardiac muscle-specific enhancer activity and that the cardiac muscle-specific negative regulatory region was located between nucleotides -505 and -165. When the PGAM-M gene constructs were cotransfected with MyoD into C3H10T1/2, the profile of CAT activity was similar to that observed in C2C12 myotubes. Gel mobility shift analysis revealed that when the nuclear extracts from skeletal and cardiac muscle cells were used, the PGAM-M MEF-2 site generated the specific band that was inhibited by unlabeled PGAM-M MEF-2 and muscle creatine kinase MEF-2 oligomers but not by a mutant PGAM-M MEF-2 oligomer. These observations define the PGAM-M enhancer as the only cardiac- and skeletal-muscle-specific enhancer characterized thus far that is mainly activated through MEF-2.

Animals↗

PubMed1997

Two different negative regulatory elements control the transcription of T-cell activation gene 3 in activated mast cells.

T-cell activation gene 3 (TCA3) encodes a beta-chemokine that is transcriptionally regulated in mast cells; the gene has a functional NF-kappaB element at positions -194 to -185. The 5'-flanking region of this gene is also known to have a negative regulatory region between -2057 and -1342. To characterize the negative regulatory elements (NREs), this region was sequenced and then digested by HindIII enzyme into two fragments, NRE-1 (-2057 to -1493) and NRE-2 (-1492 to -1342). Both NRE-1 and NRE-2 in the 5'-3' orientation inhibited chloramphenicol acetyltransferase (CAT)-protein synthesis by a TCA3-CAT construct transfected into mast cells that were then activated. Only NRE-1 inhibited CAT-protein synthesis in the 3'-5' orientation. Further deletion of the 5' region of NRE-1 partially abolished the inhibitory activity. Both NRE-1 and NRE-2 inhibited the activity of a CD20-CAT construct independent of cell activation. Electrophoretic mobility shift assays showed DNA-protein complex formation with subsequences (CCCCCATTCT) of NRE-1 (NRE-1a) and (CCATGA) of NRE-2 (NRE-2b). NRE-1a appears to be novel. NRE-2b is identical with a putative silencer motif in the alphaIIb integrin gene. Site-directed mutagenesis demonstrated that both NRE-1a and NRE-2b are important in the negative regulation of TCA3 promoter activity. In vivo ligation-mediated PCR footprinting of the NRE-2 region revealed protection between -1372 and -1354, which contains NRE-2b. The data thus demonstrate identity of a silencer motif, here termed NRE-2b, in both the alphaIIb integrin gene and the TCA3, and that this silencer region in mast cells is functional both in vivo and in vitro. Further, evidence is presented that the promoter for TCA3 contains a novel silencer motif, termed NRE-1a, characterized by a CT-rich sequence.

Animals↗

PubMed2003

Transcription regulatory cis-element (GCC)8 in the 5'-untranslated region of the gene for human very-low-density lipoprotein receptors.

The 5'-untranslated region of very-low-density lipoprotein (VLDL) receptor gene includes 2 groups of triplet repeats (GCC)n. Four repeats are localized near the promoter region in position 15.23 from the transcription initiation site. Eight repeats were detected in position 573.597. Sequence (GCC)8 in VLDL receptor gene forms specific complexes with nuclear proteins of HepG2 cells, the formation of these complexes depended on Zn(2+). Superexpression of the CGGBP-20 protein interacting with long sequences (GCC)n suppressed transcriptional activity of VLDL receptor gene. Removal of fragment (397.616) containing cis-element (GCC)8 from the 5'-untranslated region of VLDL receptor gene led to activation of the linked marker gene cat in Hutu80 cells, but did not abolish the repressor effect of CGGBP-20 protein. Our results suggest that (GCC)n-binding proteins differing from CGGBP-20 regulate activity of the VLDL receptor gene via cis-element (GCC)8.

5' Untranslated Regions↗

PubMed1998

Potential regulatory elements for germline transcription in or near murine Sgamma1.

We were interested in identifying cis-acting elements that regulate germline transcription and switch recombination of heavy chain genes. The murine gamma1 heavy chain gene includes two DNase I hypersensitive sites, which may represent protein:DNA interactions important for germline transcription and switch recombination. One DNase hypersensitive site is at the promoter/I exon boundary (termed 'Site I'); we localized a second pair of DNase hypersensitive sites to just 5' of the Sgamma1 region (termed 'Site II'). The DNA region of hypersensitive Site II includes a NF-kappaB/Rel binding site and a STAT6 binding site. It is noteworthy that NF-kappaB and STAT6 are induced by the same agents (CD40 ligation and IL-4 respectively) that stimulate germline transcription and switch recombination of the murine gamma1 gene. Transgenes with the gamma1 promoter region (DNase hypersensitive Site I), Igamma1 and DNase I hypersensitive Site II expressed germline transcripts with correct regulation, including IL-4 inducibility. However, the level of stable transcripts produced by the transgenes was much lower than that of the endogenous gamma1 gene, a complete 17 kb gamma1 transgene or a derivative of the 17 kb gamma1 transgene that lacked most of Cgamma1. The promoter/Igamma1/Site II transgenes lacked Sgamma1 and we found that gamma1 transgenes that lacked only Sgamma1 also expressed germline transcripts with proper regulation, but at a low level. This suggested that the Sgamma1 region includes positive elements for regulation of the amount of germline transcripts.

Animals↗

PubMed1993

Organization, sequence, and expression of the murine S100 beta gene. Transcriptional regulation by cell type-specific cis-acting regulatory elements.

The organization, sequence, and transcriptional regulation of expression of the murine S100 beta gene are reported. The gene is approximately 9 kilobase pairs in length and is composed of three exons and two introns. The deduced murine S100 beta protein sequence differs from the human S100 beta protein by only 1 amino acid. The murine S100 beta gene contains a TATA box (AATAA) and a reverse CCAAT box (ATTGG) located at 30 nucleotides and 92 nucleotides upstream of the cap site, respectively. A 149-base pair DNA fragment (-157/-9) spanning the TATA box and the reverse CCAAT box functions as a promoter. The murine S100 beta promoter drives a 4-fold higher level of transcription in glial (C6) than in non-glial (3T3) cells, suggesting the existence of a potential cell type-specific regulatory element within the promoter region. The 5'-flanking region suppresses transcription from the homologous S100 beta as well as the heterologous SV40 promoters in an orientation-independent fashion. However, the 5'-flanking region exhibits cell type specificity when suppressing the S100 beta promoter-dependent transcription, indicating its involvement in the cell type-specific expression of S100 beta gene. In order to map cell type-specific regulatory elements, transcription analyses of various deletions of the 5'-region were carried out in C6 and 3T3 cells. Two cell type-specific negative regulatory elements, one active in non-glial cells and another active in glial cells, were mapped to the regions -1552/-1234 and -1234/-551, respectively. A strong negative regulatory element and a relatively weak negative element were located in the regions -551/-157 and -1669/-1552, respectively. The murine S100 beta gene is under complex transcriptional regulation involving tonic negative control exerted by combination of multiple cis-acting regulatory elements including cell type-specific elements.

Amino Acid Sequence↗

PubMed1990

Interleukin-1-induced suppression of type II collagen gene transcription involves DNA regulatory elements.

Interleukin-1 is a proinflammatory polypeptide that influences cartilage macromolecular degradation and synthesis. Since previous studies have suggested that interleukin-1 may inhibit type II collagen synthesis, we have studied the mechanism of inhibition of type II collagen synthesis by interleukin-1. When rabbit articular chondrocytes were treated with purified recombinant interleukin-1 beta or macrophage-conditioned medium, the synthesis and assembly of type II collagen into the extracellular matrix were greatly reduced. The inhibition was concentration-dependent and occurred within 10 h of treatment with interleukin-1, with greater inhibition occurring at 30 h. The reduced level of collagen synthesis correlated with a reduction in the steady-state mRNA levels coding for type II collagen, as measured by a Northern blot analysis. This further correlated with a reduction in the transcription of type II collagen gene, as determined by nuclear run-on experiments. Finally, transfection studies using plasmid constructs containing DNA regulatory sequences from the type II gene, coupled to a reporter gene (CAT), revealed that in comparison to control chondrocytes, interleukin-1 treated cells showed a reduced level of CAT activity. These studies demonstrate that the inhibition of collagen type II synthesis by interleukin-1 is due to a reduction in the transcription of the type II collagen gene and that the reduction in gene transcription involves DNA regulatory sequences that determine type II collagen gene expression.

Animals↗

PubMed1993

Separable regulatory elements governing myogenin transcription in mouse embryogenesis.

Expression of the myogenic helix-loop-helix (HLH) protein myogenin in muscle cell precursors within somites and limb buds is among the earliest events associated with myogenic lineage determination in vertebrates. Mutations in the myogenin promoter that abolish binding sites for myogenic HLH proteins or myocyte enhancer factor-2 (MEF-2) suppressed transcription of a linked lacZ transgene in subsets of myogenic precursors in mouse embryos. These results suggest that myogenic HLH proteins and MEF-2 participate in separable regulatory circuits leading to myogenin transcription and provide evidence for positional regulation of myogenic regulators in the embryo.

Animals↗

PubMed1989

A point mutation in the CYC1 UAS1 creates a new combination of regulatory elements that activate transcription synergistically.

Dissection of the upstream activation site 1 (UAS1) of the yeast CYC1 gene showed that the A and B regions respond individually to regulation by the HAP1 protein, and that a point mutation in the B region converts this region to a translation upstream factor (TUF)-regulated element. Combinatorial analyses revealed that the transacting factors involved with these wild-type and mutant UAS1 target sites combine to activate transcription in a synergistic manner. Furthermore, combinations of heterologous factors, made possible by the point mutation, create a new specificity of regulation that differs from regulation by any one factor individually.

Base Sequence↗

PubMed2000

Functional analysis of heme regulatory elements of the transcriptional activator Hap1.

Heme regulation of the activity of diverse proteins was thought to be mediated by heme-responsive motifs (HRMs). The yeast transcriptional activator Hap1 contains seven HRMs: HRM1-7. Three copies of a 17-amino-acid repeat are also located in the region encompassing HRM1 to -6. We examined the effects of these HRMs and repeats on heme regulation of Hap1 activity by deletion analysis and by Ala substitutions of key residues. We found that the effect of mutation or deletion of one HRM or 17-amino-acid repeat on Hap1 heme responsiveness is different from the effect of mutation or deletion of another HRM or repeat. Our data suggest that HRM7 plays a dominant role in mediating heme activation of Hap1 in heme-sufficient cells while HRM1-6 may scavenge heme and cause a low level of Hap1 activation in heme-deficient cells. These results may help in understanding the roles of HRMs in other hemoproteins.

Amino Acid Motifs↗

PubMed2004

Regulatory elements involved in transcription of the human NeuAcalpha2,3Galbeta1,3GalNAcalpha2,6-sialyltransferase (hST6GalNAc IV) gene.

We previously cloned and characterized the promoter region of the human NeuAcalpha2,3Galbeta1,3GalNAcalpha2,6-sialyltransferase (hST6GalNAc IV) gene [Kim et al. (2003)]. In the present study, we identified a region of 294 bp upstream of exon 1 of the gene that produced maximal transcriptional activity in human Jurkat T cells. Site-directed mutagenesis and transient transfection assays demonstrated that Sp1 and MZF1 elements in this region were required for the promoter activity. Further analysis by electrophoretic mobility shift assays using specific competitors and antibody revealed that Sp1 and MZF1 nuclear proteins interacted with these elements. These results indicate that Sp1 and MZF1 are involved in the transcriptional regulation of the hST6GalNAc IV gene in Jurkat T cells.

Binding Sites↗