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At least 1,099 records · Page 61Linked to original sources
PubMed2000

Gene structures of the human non-neuronal monoamine transporters EMT and OCT2.

Non-neuronal monoamine transporters OCT1, OCT2, and EMT, which are all members of the amphiphilic solute facilitator family, control signal transmission by removing released transmitters, such as dopamine, noradrenaline, adrenaline, 5-hydroxytryptamine, and histamine, from the extracellular space. In the current study, we have isolated human EMT (gene symbol SLC22A3) and OCT2 (SLC22A2) genes and report the gene and promoter organization. Both genes consist of 11 coding exons, with consensus GT/AG splice sites and conserved intron locations. The EMT gene is 77 kb, and the OCT2 gene is 45 kb in size. For the EMT gene, two transcription start points were identified by inverse polymerase chain reaction based on mRNA from Caki-1 cells. The EMT promoter, located within a CpG island, lacks a consensus TATA box but contains a prototypical initiator element and a number of potential binding sites for ubiquitous transcription factors Sp1 and NF-1. In contrast, the OCT2 promoter is not associated with a CpG island, contains a putative TATA box, and potential binding sites for specific transcription factors, such as HFH-8 and IK2. Since EMT and OCT2 may play important roles in catecholamine homeostasis and, as such, are candidate genes in human disease, the present results provide a basis for the analysis of genetic variation and the regulation of transcription.

Base Composition↗
PubMed1997

Murine laminin alpha3A and alpha3B isoform chains are generated by usage of two promoters and alternative splicing.

We already identified two distinct laminin alpha3A and alpha3B chain isoforms which differ in their amino-terminal ends and display different tissue-specific expression patterns. In this study we have investigated whether these two different isoforms are products of the same laminin alpha3 (lama3) gene and transcribed from one or two separate promoters. Genomic clones were isolated that encompass the sequences upstream to the 5' ends of both the alpha3A and the alpha3B cDNAs. Sequence analysis of the region upstream to the alpha3A open reading frame revealed the presence of a TATA box and potential binding sites for responsive elements. By primer extension analysis, the transcription start site of the alpha3B mRNA isoform was defined. The sequences upstream to the alpha3B mRNA transcription start site do not contain a TATA box near the transcription initiation sites, but AP-1, AP-2, and Sp1 consensus binding site sequences were identified. The genomic regions located immediately upstream of the alpha3A and alpha3B transcription start sites were shown to possess promoter activities in transfection experiments. In the promoter regions, response elements for the acute phase reactant signal and NF-interleukin 6 were found, and their possible relevance in the context of inflammation and wound healing is discussed. Our results demonstrate that the lama3 gene produces the two polypeptides by alternative splicing and contains two promoters, which regulate the production of the two isoforms alpha3A and alpha3B.

3T3 Cells↗
PubMed1994

Facilitated binding of TATA-binding protein to nucleosomal DNA.

BINDING of the TATA-binding protein (TBP) to the TATA box is required for transcription from many eukaryotic promoters in gene expression. Regulation of this binding is therefore likely to be an important determinant of promoter activity. Incorporation of the TATA sequence into nucleosomes dramatically reduces transcription initiation, presumably because of stereochemical constraints on binding of general transcription factors. Biochemical and genetic studies imply that cellular factors such as yeast SWI/SNF are required for activator function and might alter chromatin structure. One step that could be regulated during the activation process is TBP binding in chromatin 12, 13. We show here that binding of TBP to the TATA sequence is severely inhibited by incorporation of this sequence into a nucleosome. Inhibition can be overcome by ATP-dependent alterations in nucleosomal DNA structure mediated by hSWI/SNF, a putative human homologue of the yeast SWI/SNF complex. Additionally, the orientation of the TATA sequence relative to the surface of the histone core affects the access of TBP. We propose that the dynamic remodelling of chromatin structure to allow TBP binding is a key step in the regulation of eukaryotic gene expression.

Adenosine Triphosphatases↗
PubMed1988

Synergistic action of the glucocorticoid receptor with transcription factors.

Steroid responsive elements (SRE) have been mapped at variable positions relative to the transcription start site and are often adjacent to binding sites of transcription regulatory proteins. In order to define the role of these transcriptional control sequences in the induction process, we inserted the previously defined 15-bp glucocorticoid response element (GRE) or 15-bp estrogen response element (ERE) immediately upstream of the TATA box of the thymidine kinase promoter, deleting all distal promoter elements. Both ERE and GRE confer inducibility by the respective hormone to the truncated promoter. These data suggest that the steroid receptor protein, possibly in conjunction with the TATA box binding protein, is able to form an active transcription complex. In contrast, the GRE when inserted 351 bp upstream of the start site of transcription of the tyrosine aminotransferase gene (TAT) is not capable of mediating hormone inducibility. Inducibility can be attained at this position by either two GREs or a single GRE in combination with a CCAAT motif. A cluster of point mutations in the CCAAT box abolishes hormone inducibility, strongly suggesting a synergistic action between the glucocorticoid receptor and the factor recognizing the CCAAT motif. The CCAAT box can be replaced by a CACCC box, an NF I and an SP1 binding site, thus demonstrating that synergistic action is not restricted to the CCAAT box binding protein. These combinations of a GRE with different transcription factor binding sites show a pronounced cell-type-dependent glucocorticoid induction of expression.

Animals↗
PubMed1988

The human beta-globin promoter; nuclear protein factors and erythroid specific induction of transcription.

We have shown that the promoter of the human beta-globin gene contains three regions in addition to the known CAC, CAAT and TATA box regions that are important for the induction of transcription in erythroid cells. By using DNaseI footprinting and gel mobility shift assays we were able to show that two of these regions bind the erythroid specific nuclear factor NF-E1 (and ubiquitous factors). The third region binds a ubiquitous CAAT-box factor (CP1). Deletion experiments suggest that only the combination of NF-E1 and CP1 binding sites, but not each of the sites alone, are capable of mediating the induction of transcription of a minimal (CAC, CAAT, TATA box) beta-globin promoter in mouse erythroleukaemia (MEL) cells.

Animals↗
PubMed2002

Contribution of Sp1 to initiation of transcription of angiotensinogen.

Several genetic polymorphisms have been identified in the proximal promoter of angiotensinogen ( AGT). Gene titration experiments in transgenic animals have demonstrated that small increases in the basal expression of AGT can lead to elevated blood pressure. The direct proof that promoter variants of AGT can lead to elevated blood pressure will ultimately require the development of specific animal models. Before such work can be contemplated, however, a formal understanding of the mechanisms controlling transcriptional activation of AGT needs to be developed. Analysis of DNA-protein interactions in vitro and transactivation experiments in cultured cells reveal the critical role of an Sp1 binding site immediately upstream of the TATA box of AGT in both mouse and human. Both sites are required for transcription initiation in the mouse. By contrast, a minimal human AGT promoter can initiate transcription in the absence of either this Sp1 site or the TATA box, albeit at a lower level. Further analysis and consideration of these interspecific differences will be essential for the development of meaningful animal models to probe the mechanism by which AGT may predispose to human essential hypertension.

Angiotensinogen↗
PubMed1990

Sequences downstream of the transcription initiation site modulate the activity of the murine dihydrofolate reductase promoter.

The murine dihydrofolate reductase gene is regulated by a bidirectional promoter that lacks a TATA box. To identify the DNA sequences required for dihydrofolate reductase transcription, the activities of various templates were determined by in vitro transcription analysis. Our data indicate that sequences both upstream and downstream of the transcription initiation site modulate the activity of the dihydrofolate reductase promoter. We have focused on two regions downstream of the transcription initiation site that are important in determining the overall efficiency of the promoter. Region 1, which included exon 1 and part of intron 1, could stimulate transcription when placed in either orientation in the normal downstream position and when inserted upstream of the transcription start site. This region could also stimulate transcription in trans when the enhancer was physically separate from the promoter. Deletion of region 2, spanning 46 nucleotides of the 5' untranslated region, reduced transcriptional activity by fivefold. DNase I footprinting reactions identified protein-binding sites in both downstream stimulatory regions. Protein bound to two sites in region 1, both of which contain an inverted CCAAT box. The protein-binding site in the 5' untranslated region has extensive homology to binding sites in promoters that both lack (simian virus 40 late) and contain (adenovirus type 2 major late promoter and c-myc) TATA boxes.

Animals↗
PubMed1986

Structure and function of the S1 nuclease-sensitive site in the adenovirus late promoter.

We analyzed an S1 nuclease-sensitive site present in supercoiled, but not linear, recombinant plasmids containing the adenovirus late promoter. S1 nicking was detected on both strands, primarily in the TATA box. Analysis of deletion mutants showed that sequences upstream of -47 and downstream of -12 are not required for S1 cutting. However, a number of different base substitution mutations in stretches of G residues upstream and/or downstream of the TATA box were sufficient to eliminate S1 cutting. When the transcriptional activities of these mutant promoters were assayed in vivo, six of seven mutants lacking the ability to form the S1-sensitive structure showed no reduction in transcriptional potential. In fact, several showed increased promoter activities. These data show that the S1 nuclease cutting site in the adenovirus late promoter has precise nucleotide sequence requirements for its formation. However, the ability of recombinant plasmids to adapt this conformation in vitro is not necessary for such plasmids to serve as templates for transcription in vivo.

Adenoviridae↗
PubMed2005

Transcriptional suppression of human microsomal triglyceride transfer protein by hypolipidemic insulin sensitizers.

Microsomal triglyceride transfer protein (MTP) catalyzes the assembly and secretion of liver triglyceride-rich lipoproteins. The human MTP (hMTP) promoter activity is reported here to be suppressed by HNF-4alpha ligand antagonists (e.g., Medica analogs) or by PPARgamma ligand agonists (e.g., thiazolidinediones), thus accounting for their hypolipidemic activity in humans. Suppression of liver hMTP by Medica analogs or by thiazolidinediones was mediated by the TAAA sequence that serves as non-canonical TATA box of the hMTP core promoter. MTP suppression was evident in the specific context of the wild type hMTP core promoter, but not in the context of the mutated rodent-conforming hMTP core promoter governed by a canonical TATA box conjoined with its proximal (-50/-38)DR-1 element. hMTP suppression by Medica analogs or thiazolidinediones mediated by hMTP TAAA was independent of HNF-4alpha or PPARgamma. hMTP suppression by Medica analogs, but not by thiazolidinediones, was further complemented by inhibition of HNF-4alpha transcriptional activity transduced by the distal (-83/-70)DR-1 element of hMTP promoter. hMTP promoter activity was unaffected by PPARalpha activation. Furthermore, in contrast to hMTP, the promoter activity of the rodent-conforming hMTP was robustly activated by Wy-14,643-activated PPARalpha or by thiazolidinedione-activated PPARgamma. Transcriptional activation by PPARalpha or PPARgamma of the rodent-conforming, but not the wild type hMTP gene promoter, resulted from the species-specific context of the respective proximal DR-1 elements. Hence, suppression of hMTP transcription by hypolipidemic insulin sensitizers requires the specific context of hMTP core promoter. In light of the species-specific context of MTP core promoters, the rodent MTP promoter may not substitute for the human promoter when searching for hypolipidemic MTP suppressors.

Animals↗
PubMed2003

The murine G+C-rich promoter binding protein mGPBP is required for promoter-specific transcription.

The archetypal TATA-box deficient G+C-rich promoter of the murine adenosine deaminase gene (Ada) requires a 48-bp minimal self-sufficient promoter element (MSPE) for function. This MSPE was used to isolate a novel full-length cDNA clone that encodes a 66-kDa murine G+C-rich promoter binding protein (mGPBP). The mGPBP mRNAs are ubiquitously expressed as either 3.0- or 3.5-kb forms differing in 3' polyadenylation site usage. Purified recombinant mGPBP, in the absence of any other mammalian cofactors, binds specifically to both the murine Ada gene promoter's MSPE and the nonhomologous human Topo IIalpha gene's G+C-rich promoter. In situ binding assays, immunoprecipitation, and Western blot analyses demonstrated that mGPBP is a nuclear factor that can form complexes with TATA-binding protein, TFIIB, TFIIF, RNA polymerase II, and P300/CBP both in vitro and in intact cells. In cotransfection assays, increased mGPBP expression transactivated the murine Ada gene's promoter. Sequestering of GPBP present in HeLa cell nuclear extract by immunoabsorption completely and reversibly suppressed extract-dependent in vitro transcription from the murine Ada gene's G+C-rich promoter. However, transcription from the human Topo IIalpha gene's TATA box-containing G+C-rich promoter was only partially suppressed and the adenovirus major late gene's classical TATA box-dependent promoter is totally unaffected under identical assay conditions. These results implicate GPBP as a requisite G+C-rich promoter-specific transcription factor and provide a mechanistic basis for distinguishing transcription initiated at a TATA box-deficient G+C-rich promoter from that initiated at a TATA box-dependent promoter.

Adenosine Deaminase↗
PubMed2004

WITHDRAWN: Silencing of human methionine adenosyltransferase 1A expression by methylation of the coding region.

Two genes (MAT1A and MAT2A) encode for the essential enzyme methionine adenosyltransferase (MAT). MAT1A is silenced in hepatocellular carcinoma (HCC), and absence of MAT1A leads to spontaneous development of HCC in mice. Here we investigated the role of methylation in regulating MAT1A expression. There are three MspI/HpaII sites from -1913 to +160 of the human MAT1A gene (numbered relative to the translational start site) at position -977, +10, and +88. Bisulfite treatment and DNA sequencing, and Southern blot analysis showed that methylation at +10 and +88, but not -977, correlated with lack of MAT1A expression. MAT1A promoter construct methylated at -977, +10 or +88 position has 0.7-fold, 3-fold, and 1.6-fold lower promoter activity, respectively. Methylation at -977 and +10 did not inhibit the promoter more than methylation at +10 alone; while methylation at +10 and +88 resulted in a 6-fold reduction of promoter activity. The promoter activity is not affected if these sites are mutated and cannot be methylated. Reactivation of MAT1A correlated with demethylation of +10 and +88. DNase I footprinting analysis using the probe containing nucleotides -346 to +160 and human liver nuclear extract or recombinant TATA binding protein (TBP) showed that HpaII methylation at +10 and +88 prevented TBP binding to the TATA box, but not if the sites were mutated. ChIP analysis confirmed TBP binding to MAT1A only in MAT1A expressing cells. Collectively our data support the novel finding that methylation of the MAT1A coding region can influence TBP binding to the TATA box and shut down gene transcription.

Journal Article↗
PubMed1988

Isolation and characterization of the human thyrotropin beta-subunit gene. Differences in gene structure and promoter function from murine species.

The human thyrotropin beta-subunit gene was isolated and characterized from two genomic libraries and found to contain three exons separated by two introns of 3.9 and 0.45 kilobase pairs. Exons 2 and 3 in the mouse thyrotropin beta-subunit gene are not found in humans due to a lack of consensus sequences important in exon splicing. Moreover, using primer extension, RNA sequencing, and S1 nuclease analysis, we determined, in a thyrotropin-producing pituitary adenoma, that exon 1 in humans contains only one transcriptional start site and is 37 base pairs in length. This is unlike both the rat and mouse thyrotropin beta-subunit genes which contain two transcriptional start sites. Changes in the genomic structure of the more 5' "TATA box" and surrounding "CAAT box" might explain why the more 5' start site in humans is apparently not utilized. Moreover, the first exon in humans is longer than the corresponding exon in murine species presumably due to a 9-base pair insertion between the TATA box and transcriptional start site (37 versus 27 nucleotides). Thus, while alternative exon splicing and differential start site utilization in response to thyroid hormone may be important in the regulation of murine thyrotropin beta-subunit genes, they are not found in man.

Adenoma↗
PubMed1987

Metal regulatory elements of the mouse metallothionein-I gene.

The promoter of the mouse metallothionein-I (mMT-I) gene contains multiple metal regulatory elements (MREs) which allow transcription of the gene to be induced by heavy metals. Insertion into the promoter of the TK gene of two or more synthetic MREs enables that gene to respond to heavy metals. We tested each of the MREs from the mMT-I promoter in this assay, and found four to be functional. We have commenced a systematic analysis of single nucleotide changes within an MRE, to determine the contribution of each nucleotide. The MRE core sequence in which single nucleotide changes can abolish function is TGCRCNCG; certain changes outside this sequence have lesser effects. MREs can act cooperatively with distal promoter elements of the TK gene, but in the presence of just a TATA-box they function as heavy-metal dependent promoter elements. Experiments to determine the effect of spacing suggest a range of at least 90 bp for interaction of two MREs, but the range for efficient stimulation of transcription from a TATA-box appears to be shorter. Stimulation of MT gene transcription by heavy metals is probably mediated by heavy metal-activated regulatory proteins binding cooperatively to the multiple MREs.

Animals↗
PubMed1989

Nuclear factor I represses the reverse-oriented transcription from the adenovirus type 5 DNA terminus.

The promoter activity of a cloned inverted terminal repeat (ITR) of human adenovirus (Ad) type 5 was found to be oriented in the opposite direction with respect to that of DNA replication in a cell-free transcription system using HeLa nuclear extracts. The major transcript was initiated outside the Ad sequence about 30 nucleotides downstream from the putative TATA-box located between nucleotide positions 9-18 of Ad 5 left terminus. Competitive transcription experiments using double-stranded oligonucleotides and isolated nuclear factor I revealed that nuclear factor I and its cognate binding site located upstream of the putative TATA-box are involved in negative regulation of the transcription from ITR promoter.

Adenoviruses, Human↗
PubMed1991

Cloning of several lignin peroxidase (LIP)-encoding genes: sequence analysis of the LIP6 gene from the white-rot basidiomycete, Phanerochaete chrysosporium.

A Phanerochaete chrysosporium BKMF1767 genomic library, constructed in the BamHI site of vector YRp12, was screened with the lignin peroxidase(LIP)-encoding cDNAs, CLG4 and CLG5, that have been shown to encode LIP2 (previously H2) and LIP6 (previously H10), respectively. Six distinct LIP genomic clones, designated pGLG1, pGLG2, pGLG3, pGLG4, pGLG5, and pGLG6, were isolated in this study. Probe CLG4 hybridized only to pGLG1. Probe CLG5 gave intense hybridization to pGLG2 and weaker hybridization to clones pGLG3 through pGLG6, but showed little or no hybridization to pGLG1. These results, in agreement with previous biochemical results, indicate the existence of LIP gene subfamilies. The limits and transcriptional orientation of the LIP gene in each clone were determined. The sequence data showed that pGLG2 contains the LIP6 gene, which encodes a protein identical in amino acid (aa) composition to that encoded by CLG5. It contains a leader sequence of 27 aa and a mature protein of 344 aa (Mr 36,607). Archetypal TATA-box-like and CAAT-box-like sequences in the 5'-noncoding region are located 51 and 97 nt upstream from the cDNA start point, respectively. S1 nuclease analysis of the 5' region of LIP6 revealed two transcription start points 8 nt apart downstream from the TATA box. Comparison of the sequence of LIP6 with its corresponding cDNA CLG5 showed that the gene contains nine small introns which range in size from 50 to 62 bp. These introns contained consensus splice junction sequences similar to those reported in other fungal and yeast introns.

Amino Acid Sequence↗
PubMed2000

Selective recognition of consecutive G sequence in double-stranded DNA by a zinc(II)-macrocyclic tetraamine complex appended with an anthraquinone.

A zinc (II) complex with a macrocyclic tetraamine appended with an anthraquinone ((9,10-anthraquinon-2-yl)methyl-1,4,7,10-tetraazacyclododecane, ZnL, anthraquinonyl-cyclen) selectively recognizes consecutive G sequence in double-stranded DNA. The affinity of the Zn2+-anthraquinonyl-cyclen to consecutive dG groups in DNA was disclosed by comparison of K(app) values (=[DNA-bound ZnL]/[uncomplexed ZnL][uncomplexed nucleobase in DNA]) determined by the UV spectrophotometric titrations at pH 8, I=0.1 (NaNO3), and 25 degrees C for poly(dG) x poly(dC) (K(app) = 1.5 x 10(5) M(-1)), poly(dG-dC)2 (2.8 x 10(4) M(-1)), poly(dA-dT)2 (4.3 x 10(4) M(-1)), and calf thymus DNA (2.8 x 10(4) M(-1)). The corresponding K(app) values with the Zn2+-free ligand were 5.3 x 10(3) M(-1), 7.4 x 10(3) M(-1), 7.4 x 10(3) M(-1), and 5.9 x 10(3) M(-1), respectively. The selective recognition of consecutive G sequence was concluded from the DNase I footprinting of SV40 early promotor DNA fraction (197 bp) containing a TATA box and six GC boxes. The present finding is in remarkable contrast to the previous selective T-recognition by Zn2+-cyclen complexes appended with acridine, quinoline(s), and naphthalene(s) [J. Am. Chem. Soc. 121 (1999) 5426]. While the Zn2+-acridinyl-cyclen inhibited TATA binding protein from interacting with a TATA box consensus DNA [J. Inorg. Biochem. 79 (2000) 253], the present Zn2+-anthraquinonyl-cyclen inhibited the Sp1 transcriptional factor protein from interacting with a GC box-consensus DNA.

Anthraquinones↗
PubMed2001

Isolation and characterization of the murine transforming growth factor-beta2 promoter.

This report describes the isolation and characterization of the 5' flanking region of the murine transforming growth factor beta-2 (TGF-beta2) gene. A genomic clone containing the promoter region of the gene was isolated after screening a bacteriophage P1 genomic library. The resulting clone was sequenced and compared to promoters for the human and chicken TGF-beta2 genes. The sequence located near the transcription start site is highly conserved. It includes a TATA box, an E-box, and a largely conserved CRE/ATF site. A series of murine TGF-beta2 promoter/reporter constructs was generated to identify regulatory regions of the gene. As in the case of the human TGF-beta2 gene, sequences just upstream of the TATA box, including the CRE/ATF site, actively stimulate the murine TGF-beta2 promoter. However, unlike the human TGF-beta2 gene, the 5' flanking region of the murine TGF-beta2 gene contains a long alternating purine/pyrimidine repeat that unexpectedly exerts a strong positive effect on its promoter. This is of particular interest since alternating purine/pyrimidine repeats in other promoters have been observed to be inhibitory.

Animals↗
PubMed1997

Protease footprinting analysis of ternary complex formation by human TFIIA.

Transcription factor (TF) IIA performs two important regulatory functions during RNA polymerase II transcription: it is required for efficient binding of TFIID to a core promoter and it mediates the effects of upstream activators, both through direct interaction with the TATA box binding protein (TBP). To begin studying how TFIIA mediates these effects, we used a highly sensitive protease footprinting methodology to identify surfaces of human TFIIA participating in TFIIA x TBP x TATA ternary complex formation. Chymotrypsin and proteinase K cleavage patterns of TFIIA bearing a 32P-end-labeled gamma subunit revealed that amino acids 59-73 were protected from cleavage both in the context of an immobilized ternary complex and in a binary complex with TBP alone. In contrast, amino acids 341-367 in the beta portion of a 32P-labeled alpha-beta subunit were protected in the ternary but not in the binary complex, implying that those residues interact with promoter DNA. The regions of human TFIIA identified by protease footprinting are homologous to and encompass the yeast TFIIA residues that contact TBP and DNA in the recently solved crystal structure of the yeast ternary complex. The conservation of the regions and residues mediating complex formation implies that yeast and human TFIIA employ the same mechanism to stabilize the binding of TFIID to a core promoter.

Binding Sites↗